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20.3E: Antigen-Presenting Cells

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Antigen presentation is a process by which immune cells capture antigens and then enable their recognition by T cells.

Learning Objectives

• Describe the role of antigen-presenting cells
• The host’s cells express “self” antigens that identify them as such. These antigens are different from those in bacteria (“non-self” antigens) and in virus-infected host cells (“missing-self”).
• Antigen presentation consists of pathogen recognition, phagocytosis of the pathogen or its molecular components, processing of the antigen, and then presentation of the antigen to naive T cells.
• The T cell receptor is restricted to recognizing antigenic peptides only when bound to appropriate molecules of the major histocompatibility complex (MHC), also known in humans as human leukocyte antigen (HLA).
• Helper T cells recieve antigens from MHC II on an APC, while cytotoxic T cells recieve antigens from MHC I. Helper T cells present their antigen to B cells as well.Dendritic cells, B cells, and macrophages play a major role in the innate response, and are the primary antigen-presenting cells (APC).
• APCs use toll-like receptors to identify PAMPS and DAMPs, which are signs of an infection and may be processed into antigen peptides if phagocytized. Most APCs cannot tell the difference between different types of antigens like B and T cells can.
• damage-associated molecular pattern : Protein or nucleic acid based signs of pathogen induced damage. Protein DAMPs may be phagocytized and processed for antigen presentation.
• cytotoxic : A population of T cells specialized for inducing the deaths of other cells.

Antigen presentation is a process in the body’s immune system by which macrophages, dendritic cells and other cell types capture antigens, then present them to naive T-cells. The basis of adaptive immunity lies in the capacity of immune cells to distinguish between the body’s own cells and infectious pathogens. The host’s cells express “self” antigens that identify them as belonging to the self. These antigens are different from those in bacteria (“non-self” antigens) or in virally-infected host cells (“missing-self”). Antigen presentation broadly consists of pathogen recognition, phagocytosis of the pathogen or its molecular components, processing of the antigen, and then presentation of the antigen to naive (mature but not yet activated) T cells. The ability of the adaptive immune system to fight off pathogens and end an infection depends on antigen presentation.

Antigen Presenting Cells

Antigen Presenting Cells (APCs) are cells that capture antigens from within the body, and present them to naive T-cells. Many immune system cells can present antigens, but the most common types are macrophages and dendritic cells, which are two types of terminally differentiated leukocytes that arise from monocytes. Both of these APCs perform many immune functions that are important for both innate and adaptive immunity, such as removing leftover pathogens and dead neutrophils after an inflammatory response. Dendritic cells (DCs) are generally found in tissues that have contact with the external environment (such as the skin or respiratory epithelium) while macrophages are found in almost all tissues. Some types of B cells may also present antigens as well, though it is not their primary function.

APCs phagocytize exogenous pathogens such as bacteria, parasites, and toxins in the tissues and then migrate, via chemokine signals, to lymph nodes that contain naive T cells. During migration, APCs undergo a process of maturation in which they digest phagocytized pathogens and begin to express the antigen in the form of a peptide on their MHC complexes, which enables them to present the antigen to naive T cells. The antigen digestion phase is also called “antigen processing,” because it prepares the antigens for presentation. This MHC:antigen complex is then recognized by T cells passing through the lymph node. Exogenous antigens are usually displayed on MHC Class II molecules, which interact with CD4+ helper T cells.

This maturation process is dependent on signaling from other pathogen-associated molecular pattern (PAMP) molecules (such as a toxin or component of a cell membrane from a pathogen) through pattern recognition receptors (PRRs), which are received by Toll-like receptors on the DC’s body. They may also recognize damage-associated molecular pattern (DAMP) molecules, which include degraded proteins or nucleic acids released from cells that undergo necrosis. PAMPs and DAMPS are not technically considered antigens themselves, but instead are signs of pathogen presence that alert APCs through Toll-like receptor binding. However if a DC phagocytzes a PAMP or DAMP, it could be used as an antigen during antigen presentation. APCs are unable to distinguish between different types of antigens themselves, but B and T cells can due to their specificity.

Antigen Presentation

T cells must be presented with antigens in order to perform immune system functions. The T cell receptor is restricted to recognizing antigenic peptides only when bound to appropriate molecules of the MHC complexes on APCs, also known in humans as Human leukocyte antigen (HLA).

Several different types of T cell can be activated by APCs, and each type of T cell is specially equipped to deal with different pathogens, whether the pathogen is bacterial, viral or a toxin. The type of T cell activated, and therefore the type of response generated, depends on which MHC complex the processed antigen-peptide binds to.

MHC Class I molecules present antigen to CD8+ cytotoxic T cells, while MHC class II molecules present antigen to CD4+ helper T cells. With the exception of some cell types (such as erythrocytes), Class I MHC is expressed by almost all host cells. Cytotoxic T cells (also known as TC, killer T cell, or cytotoxic T-lymphocyte (CTL)) are a population of T cells that are specialized for inducing the death of other cells. Recognition of antigenic peptides through Class I by CTLs leads to the killing of the target cell, which is infected by virus, intracytoplasmic bacterium, or are otherwise damaged or dysfunctional. Additionally, some helper T cells will present their antigen to B cells, which will activate their proliferation response.

Antigen presentation : In the upper pathway; foreign protein or antigen (1) is taken up by an antigen-presenting cell (2). The antigen is processed and displayed on a MHC II molecule (3), which interacts with a T helper cell (4). In the lower pathway; whole foreign proteins are bound by membrane antibodies (5) and presented to B lymphocytes (6), which process (7) and present antigen on MHC II (8) to a previously activated T helper cell (10), spurring the production of antigen-specific antibodies (9).

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BiteSized Immunology: Systems & Processes

Antigen Processing and Presentation

In order to be capable of engaging the key elements of adaptive immunity (specificity, memory, diversity, self/nonself discrimination), antigens have to be processed and presented to immune cells. Antigen presentation is mediated by MHC class I molecules , and the class II molecules found on the surface of antigen-presenting cells (APCs) and certain other cells.

MHC class I and class II molecules are similar in function: they deliver short peptides to the cell surface allowing these peptides to be recognised by CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively. The difference is that the peptides originate from different sources – endogenous, or intracellular , for MHC class I; and exogenous, or extracellular for MHC class II. There is also so called cross-presentation in which exogenous antigens can be presented by MHC class I molecules. Endogenous antigens can also be presented by MHC class II when they are degraded through autophagy.

MHC class I presentation

MHC class I molecules are expressed by all nucleated cells. MHC class I molecules are assembled in the endoplasmic reticulum (ER) and consist of two types of chain – a polymorphic heavy chain and a chain called β2-microglobulin. The heavy chain is stabilised by the chaperone calnexin , prior to association with the β2-microglobulin. Without peptides, these molecules are stabilised by chaperone proteins : calreticulin, Erp57, protein disulfide isomerase (PDI) and tapasin. The complex of TAP, tapasin, MHC class I, ERp57 and calreticulin is called the peptide-loading complex (PLC). Tapasin interacts with the transport protein TAP (transporter associated with antigen presentation) which translocates peptides from the cytoplasm into the ER. Prior to entering the ER, peptides are derived from the degradation of proteins, which can be of viral- or self origin. Degradation of proteins is mediated by cytosolic- and nuclear proteasomes, and the resulting peptides are translocated into the ER by means of TAP. TAP translocates peptides of 8 –16 amino acids and they may require additional trimming in the ER before binding to MHC class I molecules. This is possibly due to the presence of ER aminopeptidase (ERAAP) associated with antigen processing.

It should be noted that 30–70% of proteins are immediately degraded after synthesis (they are called DRiPs – defective ribosomal products, and they are the result of defective transcription or translation). This process allows viral peptides to be presented very quickly – for example, influenza virus can be recognised by T cells approximately 1.5 hours post-infection. When peptides bind to MHC class I molecules, the chaperones are released and peptide–MHC class I complexes leave the ER for presentation at the cell surface. In some cases, peptides fail to associate with MHC class I and they have to be returned to the cytosol for degradation. Some MHC class I molecules never bind peptides and they are also degraded by the ER-associated protein degradation (ERAD) system.

There are different proteasomes that generate peptides for MHC class-I presentation: 26S proteasome , which is expressed by most cells; the immunoproteasome, which is expressed by many immune cells; and the thymic-specific proteasome expressed by thymic epithelial cells.

Antigen presentation

On the surface of a single cell, MHC class I molecules provide a readout of the expression level of up to 10,000 proteins. This array is interpreted by cytotoxic T lymphocytes and Natural Killer cells, allowing them to monitor the events inside the cell and detect infection and tumorigenesis.

MHC class I complexes at the cell surface may dissociate as time passes and the heavy chain can be internalised. When MHC class I molecules are internalised into the endosome, they enter the MHC class-II presentation pathway. Some of the MHC class I molecules can be recycled and present endosomal peptides as a part of a process which is called cross-presentation .

The usual process of antigen presentation through the MHC I molecule is based on an interaction between the T-cell receptor and a peptide bound to the MHC class I molecule. There is also an interaction between the CD8+ molecule on the surface of the T cell and non-peptide binding regions on the MHC class I molecule. Thus, peptide presented in complex with MHC class I can only be recognised by CD8+ T cells. This interaction is a part of so-called ‘three-signal activation model’, and actually represents the first signal. The next signal is the interaction between CD80/86 on the APC and CD28 on the surface of the T cell, followed by a third signal – the production of cytokines by the APC which fully activates the T cell to provide a specific response.

MHC class I polymorphism

Human MHC class I molecules are encoded by a series of genes – HLA-A, HLA-B and HLA-C (HLA stands for ‘Human Leukocyte Antigen’, which is the human equivalent of MHC molecules found in most vertebrates). These genes are highly polymorphic, which means that each individual has his/her own HLA allele set. The consequences of these polymorphisms are differential susceptibilities to infection and autoimmune diseases that may result from the high diversity of peptides that can bind to MHC class I in different individuals. Also, MHC class I polymorphisms make it virtually impossible to have a perfect tissue match between donor and recipient, and thus are responsible for graft rejection.

MHC class II presentation

MHC class II molecules are expressed by APCs, such as dendritic cells (DC), macrophages and B cells (and, under IFNγ stimuli, by mesenchymal stromal cells, fibroblasts and endothelial cells, as well as by epithelial cells and enteric glial cells). MHC class II molecules bind to peptides that are derived from proteins degraded in the endocytic pathway. MHC class II complexes consists of α- and β-chains that are assembled in the ER and are stabilised by invariant chain (Ii). The complex of MHC class II and Ii is transported through the Golgi into a compartment which is termed the MHC class II compartment (MIIC). Due to acidic pH, proteases cathepsin S and cathepsin L are activated and digest Ii, leaving a residual class II-associated Ii peptide (CLIP) in the peptide-binding groove of the MHC class II. Later, the CLIP is exchanged for an antigenic peptide derived from a protein degraded in the endosomal pathway. This process requires the chaperone HLA-DM, and, in the case of B cells, the HLA-DO molecule. MHC class II molecules loaded with foreign peptide are then transported to the cell membrane to present their cargo to CD4+ T cells. Thereafter, the process of antigen presentation by means of MHC class II molecules basically follows the same pattern as for MHC class I presentation.

As opposed to MHC class I, MHC class II molecules do not dissociate at the plasma membrane. The mechanisms that control MHC class II degradation have not been established yet, but MHC class II molecules can be ubiquitinised and then internalised in an endocytic pathway.

MHC class II polymorphism

Like the MHC class I heavy chain, human MHC class II molecules are encoded by three polymorphic genes: HLA-DR, HLA-DQ and HLA-DP. Different MHC class II alleles can be used as genetic markers for several autoimmune diseases, possibly owing to the peptides that they present.

Module 20: The Immune System

Antigen-presenting cells, learning outcomes.

• Describe the structure and function of antigen-presenting cells

Unlike NK cells of the innate immune system, B cells (B lymphocytes) are a type of white blood cell that gives rise to antibodies, whereas T cells (T lymphocytes) are a type of white blood cell that plays an important role in the immune response. T cells are a key component in the cell-mediated response—the specific immune response that utilizes T cells to neutralize cells that have been infected with viruses and certain bacteria. There are three types of T cells: cytotoxic, helper, and suppressor T cells. Cytotoxic T cells destroy virus-infected cells in the cell-mediated immune response, and helper T cells play a part in activating both the antibody and the cell-mediated immune responses. Suppressor T cells deactivate T cells and B cells when needed, and thus prevent the immune response from becoming too intense.

An antigen is a foreign or “non-self” macromolecule that reacts with cells of the immune system. Not all antigens will provoke a response. For instance, individuals produce innumerable “self” antigens and are constantly exposed to harmless foreign antigens, such as food proteins, pollen, or dust components. The suppression of immune responses to harmless macromolecules is highly regulated and typically prevents processes that could be damaging to the host, known as tolerance.

The innate immune system contains cells that detect potentially harmful antigens, and then inform the adaptive immune response about the presence of these antigens. An antigen-presenting cell (APC) is an immune cell that detects, engulfs, and informs the adaptive immune response about an infection. When a pathogen is detected, these APCs will phagocytose the pathogen and digest it to form many different fragments of the antigen. Antigen fragments will then be transported to the surface of the APC, where they will serve as an indicator to other immune cells. Dendritic cells are immune cells that process antigen material; they are present in the skin (Langerhans cells) and the lining of the nose, lungs, stomach, and intestines. Sometimes a dendritic cell presents on the surface of other cells to induce an immune response, thus functioning as an antigen-presenting cell. Macrophages also function as APCs. Before activation and differentiation, B cells can also function as APCs.

After phagocytosis by APCs, the phagocytic vesicle fuses with an intracellular lysosome forming phagolysosome. Within the phagolysosome, the components are broken down into fragments; the fragments are then loaded onto MHC class I or MHC class II molecules and are transported to the cell surface for antigen presentation, as illustrated in Figure 1. Note that T lymphocytes cannot properly respond to the antigen unless it is processed and embedded in an MHC II molecule. APCs express MHC on their surfaces, and when combined with a foreign antigen, these complexes signal a “non-self” invader. Once the fragment of antigen is embedded in the MHC II molecule, the immune cell can respond. Helper T- cells are one of the main lymphocytes that respond to antigen-presenting cells. Recall that all other nucleated cells of the body expressed MHC I molecules, which signal “healthy” or “normal.”

Figure 1. An APC, such as a macrophage, engulfs and digests a foreign bacterium. An antigen from the bacterium is presented on the cell surface in conjunction with an MHC II molecule Lymphocytes of the adaptive immune response interact with antigen-embedded MHC II molecules to mature into functional immune cells.

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Cancer immune escape: the role of antigen presentation machinery

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• Published: 09 April 2023
• Volume 149 , pages 8131–8141, ( 2023 )

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• Anoop Kallingal   ORCID: orcid.org/0000-0002-9613-3259 1 ,
• Mateusz Olszewski   ORCID: orcid.org/0000-0002-1952-4985 1 ,
• Natalia Maciejewska   ORCID: orcid.org/0000-0001-9942-285X 1 ,
• Wioletta Brankiewicz   ORCID: orcid.org/0000-0002-8314-0775 1 , 2 &
• Maciej Baginski 1  

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The mechanisms of antigen processing and presentation play a crucial role in the recognition and targeting of cancer cells by the immune system. Cancer cells can evade the immune system by downregulating or losing the expression of the proteins recognized by the immune cells as antigens, creating an immunosuppressive microenvironment, and altering their ability to process and present antigens. This review focuses on the mechanisms of cancer immune evasion with a specific emphasis on the role of antigen presentation machinery. The study of the immunopeptidome, or peptidomics, has provided insights into the mechanisms of cancer immune evasion and has potential applications in cancer diagnosis and treatment. Additionally, manipulating the epigenetic landscape of cancer cells plays a critical role in suppressing the immune response against cancer. Targeting these mechanisms through the use of HDACis, DNMTis, and combination therapies has the potential to improve the efficacy of cancer immunotherapy. However, further research is needed to fully understand the mechanisms of action and optimal use of these therapies in the clinical setting.

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Introduction

The role of antigen presentation in cancer immune cell escape is a complex and multifaceted topic that has been the subject of much research in recent years. Antigen presentation is the process by which cells in the immune system display foreign molecules, such as those from pathogens or cancer cells, on their surface for recognition by other immune cells (Zitvogel and Kroemer 2018 ). In the context of cancer, antigen presentation plays a crucial role in the ability of the immune system to identify and target cancer cells. However, cancer cells can evade the immune system by various mechanisms, including downregulating or losing the expression of the proteins recognized by the immune cells as antigens, a process known as an immune escape (Beatty and Gladney 2015 ). The process of antigen presentation begins with the cancer cells expressing proteins on their surface, which are then recognized by specialized immune cells called antigen-presenting cells (APCs) (Mpakali and Stratikos 2021 ). These APCs, such as dendritic cells, then internalize the cancer cell proteins and degrade them into smaller peptides. These peptides are then displayed on the surface of the APC, along with particular proteins called major histocompatibility complex (MHC) molecules (Blum et al. 2013 ). The MHC molecules act as a bridge between the cancer cell proteins and the immune cells responsible for recognizing and attacking cancer cells, called T cells. The T cells have T cell receptors (TCRs) that can recognize the cancer cell proteins displayed on the MHC molecules (Alberts et al. 2002 ). When a T cell recognizes a cancer cell protein displayed on an APC, it becomes activated and begins to divide and differentiate into specialized cells that can attack and destroy the cancer cells (Messerschmidt et al. 2016 ). Cancer cells can evade the immune system by downregulating or losing the expression of the proteins recognized by the immune cells as antigens (Beatty and Gladney 2015 ). This can happen by mutations in the cancer cells that affect the expression of these proteins or by the cancer cells creating an immunosuppressive microenvironment that prevents the immune cells from recognizing and attacking the cancer cells (Brody 2016 ). Some cancer cells can produce molecules called immune checkpoint inhibitors that bind to and inhibit the activity of T cells, preventing them from recognizing and attacking cancer cells (Lao et al. 2022 ).

Additionally, cancer cells can recruit immune cells that promote immune suppression, such as regulatory T cells and myeloid-derived suppressor cells, which further dampen the immune response against cancer (Brody 2016 ). Cancer cells can also evade the immune system by changing the location of the antigens within the cell, called the abscopal effect, where the cancer cells move the antigens to the inside of the cell, making them invisible to the immune system (Beatty and Gladney 2015 ; Alfonso et al. 2020 ). Recent research has shown that targeting the mechanisms of antigen presentation and immune escape can be an effective strategy for treating cancer. For example, drugs that block immune checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1/PD-L1, have been approved for use in several types of cancer and have shown promising results in clinical trials (Seidel et al. 2018 ; Rotte 2019 ). In a snapshot, antigen presentation plays a crucial role in the ability of the immune system to identify and target cancer cells. Understanding the mechanisms of antigen presentation and immune escape is crucial for developing effective cancer immunotherapies.

Immune system and cancer

The immune system plays a crucial role in the development and progression of cancer (Gonzalez et al. 2018 ). Cancer cells develop from normal cells and can evade the immune system through various mechanisms; one of them is a process known as an immune escape. The immune system can recognize and target cancer cells through immunosurveillance. This process involves specialized immune cells, such as T cells and natural killer cells, that can detect and destroy cancer cells (Marcus et al. 2014 ; Gonzalez et al. 2018 ). The immune system also plays a role in shaping the microenvironment of the tumour. Tumour-associated macrophages, dendritic cells, and Treg cells are some of the cells found in the tumour microenvironment and play a role in cancer progression (Anderson and Simon 2020 ). Tumour-associated macrophages and dendritic cells can promote cancer cell growth by secreting factors that promote angiogenesis and inhibiting T cell activity. On the other hand Treg cells can suppress the immune response against cancer by inhibiting the activation and proliferation of T cells (Baay et al. 2011 ).

Another important mechanism in cancer progression is the ability of cancer cells to evade the immune system by downregulating or losing the expression of the proteins recognized by the immune cells as antigens (Dhatchinamoorthy et al. 2021 ). Recent research has shown that targeting the mechanisms of antigen presentation and immune escape can be an effective strategy for treating cancer. For example, drugs that block immune checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1/PD-L1, have been approved for use in several types of cancer and have shown promising results in clinical trials (Wojtukiewicz et al. 2021 ; Xiang et al. 2022 ; Sové et al. 2022 ). The immune system plays a crucial role in the development and progression of cancer. Understanding the mechanisms of immunosurveillance, immune escape, and the immune system's role in shaping the tumour microenvironment is crucial for developing effective cancer immunotherapies. Immune-based therapies, such as cancer vaccines and checkpoint inhibitors, have shown great promise in treating cancer and are expected to play a significant role in cancer treatment.

Immune checkpoints and immune evasion in cancer

Cancer immune evasion refers to the ability of cancer cells to evade detection and destruction by the immune system (Vinay et al. 2015 ). This complex process involves multiple mechanisms that enable cancer cells to evade the immunosurveillance mechanisms of the body (Messerschmidt et al. 2016 ).

Immune checkpoints are molecules or pathways that regulate the activation and function of the immune system. Immune checkpoint inhibitors are a class of drugs that block the function of these checkpoints, thereby enhancing the immune response against cancer cells (He and Xu 2020 ). One of the most well-known immune checkpoint pathways is the CTLA-4 pathway (Buchbinder and Desai 2016 ). CTLA-4 is a protein expressed on the surface of T cells that acts as an inhibitory receptor, blocking the activation and proliferation of T cells (Parry et al. 2005 ). Anti-CTLA-4 therapies, such as ipilimumab, act by binding to and blocking the function of CTLA-4, thereby enhancing the immune response against cancer cells (Callahan et al. 2010 ). Another critical immune checkpoint pathway is the PD-1/PD-L1. PD-1 is a protein expressed on the surface of T cells that interacts with PD-L1, which is expressed on the surface of cancer cells. This interaction blocks the activation and proliferation of T cells, allowing cancer cells to evade the immune response (Han et al. 2020 ). Anti-PD-1/PD-L1 treatments, such as nivolumab and pembrolizumab, work by binding to and inhibiting the interaction of PD-1 and PD-L1, increasing the immune response against cancer cells (Fessas et al. 2017 ) (Fig.  1 ).

Immune checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1/PD-L1 drugs, enhance the immune response against cancer by blocking immune checkpoint pathways. Other checkpoint pathways, such as LAG-3 and TIGIT, are being investigated as potential targets for cancer therapy and may have synergistic effects when combined with other checkpoint inhibitors

Other immune checkpoint pathways, such as LAG-3 and TIGIT, are also being investigated as potential targets for cancer therapy. LAG-3 (lymphocyte activation gene 3) is a protein that binds to MHC class II molecules and regulates T cell activation and exhaustion (Ge et al. 2021 ; Huo et al. 2022 ). TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a protein that binds to both T cells and immune cells and regulates T cell activation and function. Preclinical research has demonstrated a significant impact of these pathways, and clinical trials are currently being conducted to explore their potential as therapeutic cancer targets (Yue et al. 2022 ). LAG-3 and TIGIT have a unique mechanism of action compared to other immune checkpoint inhibitors, such as PD-1 and CTLA-4, and may have a synergistic effect when combined with these drugs. This could potentially lead to improved efficacy and reduced side effects. In preclinical studies, TIGIT and LAG-3 inhibitors are effective in combination with PD-1 inhibitors in various cancer models, such as melanoma, lung cancer, and ovarian cancer (De Sousa et al. 2018 ; Seidel et al. 2018 ; Willsmore et al. 2021 ).

Antigen presentation in cancer

Antigen processing and presentation are crucial mechanisms by which the immune system recognizes and targets cancer cells. This process involves the recognition of cancer cell-associated antigens by APCs and their subsequent presentation on the surface of these cells in a form that can be recognized by T cells (Mpakali and Stratikos 2021 ). The antigen processing and presentation process begins with the internalization of cancer cell-associated antigens by APCs (Blum et al. 2013 ; Lee et al. 2020 ). Once inside the cell, the antigens are degraded into small peptides by a complex of enzymes called the proteasome. These peptides are then transported to the endoplasmic reticulum, complex with MHC molecules (Rock et al. 2010 ). MHC molecules are specialized proteins that are essential for the recognition of antigens by T cells. There are two main types of MHC molecules: MHC class I and MHC class II. MHC class I molecules are expressed on the surface of all nucleated cells, including cancer cells, and present peptides derived from intracellular antigens. On the other hand, MHC class II molecules are expressed primarily on the surface of APCs and present peptides derived from extracellular antigens (Wieczorek et al. 2017 ).

The MHC-peptide complex is then transported to the cell surface, where it can be recognized by T cells. T cells have specialized T cell receptors (TCRs) that recognize the MHC-peptide complex (Alberts et al. 2002 ). When a T cell recognizes a cancer cell-associated antigen displayed on an APC, it becomes activated and begins to divide and differentiate into specialized cells that can attack and destroy the cancer cells (Kunimasa and Goto 2020 ). However, cancer cells can evade the immune system by downregulating or losing the expression of the proteins recognized by the immune cells as antigens. This can happen by mutations in the cancer cells that affect the expression of these proteins or by the cancer cells creating an immunosuppressive microenvironment that prevents the immune cells from recognizing and attacking the cancer cells (Beatty and Gladney 2015 ) (Fig.  2 ). Many reports have shown that cancer cells can also evade the immune system by altering their ability to process and present antigens. For example, some cancer cells can downregulate the expression of MHC molecules, making them invisible to the immune system (Mittal et al. 2014 ; Reeves and James 2017 ; Kulkarni et al. 2019 ). Cancer cells can also interfere with the activity of the proteasome, thereby preventing the degradation of cancer cell-associated antigens (Mittal et al. 2014 ; Reeves and James 2017 ; Kulkarni et al. 2019 ).

APCs internalize cancer cell-associated antigens and degrade them into small peptides, which are then presented on the surface of APCs as MHC-peptide complexes that can be recognized by T cells. Cancer cells can evade the immune system by downregulating or losing the expression of antigen proteins, altering their ability to process and present antigens, or creating an immunosuppressive microenvironment

MHC 1 in antigen presentation

Major histocompatibility complex class I (MHC-I) molecules play a critical role in antigen presentation. These molecules are expressed on the surface of all nucleated cells, including cancer cells, and are responsible for the presentation of peptides derived from intracellular antigens to CD8 + T cells, also known as cytotoxic T cells (van den Elsen 2011 ; Wang et al. 2019 ). The MHC-I molecule comprises two main components: the heavy chain, encoded by the HLA gene, and the beta-2-microglobulin (β2m), a non-polymorphic component. The heavy chain comprises three main domains: the α1, α2, and α3. The α1 and α2 domains bind the MHC-I molecule to the peptide, while the α3 domain is responsible for interacting with the CD8 T-cell receptor (Cruz-Tapias et al. 2013 ). The process of MHC-I presentation begins with the internalization of antigens by the cell. Once an antigen enters a cell, a group of enzymes called the proteasome breaks it down into a little peptide.

Peptide loading delivers these peptides to the endoplasmic reticulum, where they interact with the MHC-I molecule. The MHC-I-peptide complex is then transported to the cell surface, where it can be recognized by CD8 + T cells (Hewitt 2003 ). The binding of the peptide to the MHC-I molecule is mediated by the peptide-binding groove, which is composed of the α1 and α2 domains. The peptide-binding groove can only bind to peptides that are 8–10 amino acids long. Once the peptide is bound to the MHC-I molecule, it is transported to the cell surface (Fig.  3 ) (Zacharias and Springer 2004 ). Downregulating or removing proteins that express antigens allows cancer cells to evade the immune system. The ability of cancer cells to process and present antigens on MHC-I molecules can change if they develop an immunosuppressive microenvironment or experience protein expression mutations. Understanding the mechanisms of MHC-I presentation in cancer is crucial for developing effective cancer immunotherapies.

MHC-I antigen presentation. MHC-I molecules on the cell surface present intracellular antigen peptides to CD8 + T cells. Cancer cells can evade the immune system by downregulating antigen expression or altering antigen processing and presentation on MHC-I

Immunopeptidome and cancer

The immunopeptidome is the set of peptides presented by MHC molecules on the surface of cells (Yewdell 2022a ). These peptides are derived from the degradation of intracellular proteins and are essential for recognizing cancer cells by the immune system. The study of the immunopeptidome, also known as peptidomics, has revealed insights into the mechanisms of cancer immune evasion and has potential applications in cancer diagnosis and treatment (Synowsky et al. 2017 ; Yewdell 2022b ). One of the critical roles of the immunopeptidome in cancer is its ability to identify unique peptides specific to cancer cells. These cancer-specific peptides, also known as neoantigens, can be used to develop personalized cancer vaccines targeting the unique mutations in an individual's cancer. Neoantigen-based vaccines have shown promising results in clinical trials and are expected to play an essential role in the future of cancer immunotherapy (D’Amico et al. 2022 ; Ouspenskaia et al. 2022 ). Another essential role of the immunopeptidome in cancer is its ability to provide insights into the mechanisms of cancer immune evasion. The study of the immunopeptidome can reveal which proteins are being presented by MHC molecules and which are not, providing insight into the mechanisms of cancer immune evasion (León-Letelier et al. 2022 ). The immunopeptidome can also provide valuable information for cancer diagnosis, such as immunopeptidome-based cancer diagnostics, tumour-associated antigen (TAA) testing, MHC class I tetramer staining and mass spectrometry-based peptidomics. Additionally, the study of the immunopeptidome can provide insights into the progression of cancer and the response to treatment by monitoring changes in the peptides presented by MHC molecules (Dersh et al. 2021 ).

Tumor antigen expression, presentation and control

The control of tumour antigen expression and presentation is a critical aspect of cancer biology that significantly impacts the immune system's ability to recognize and target cancer cells (Whiteside 2006 ). Tumours evade immune recognition through various mechanisms, such as the downregulation of antigens recognized by immune cells, the creation of an immunosuppressive microenvironment, and interaction with immune checkpoint pathways. Tumour antigens are molecules expressed on the surface of cancer cells and recognized by the immune system as foreign (Fig.  4 ).

Tumors can evade detection and destruction by the immune system, thereby allowing for uncontrolled growth and progression. This process is referred to as immune evasion and is a complex mechanism that involves the downregulation or loss of antigens recognized by immune cells, the creation of an immunosuppressive microenvironment, and interaction with immune checkpoint pathways

Cancer cells can regulate tumour antigen expression via epigenetics, like DNA structure changes (methylation, histone modification). They can also reduce antigen expression, hide from the immune system, and inhibit antigen-presenting cells/T cells (TGF-beta, IL-10) from suppressing immune response.(Gibney and Nolan 2010 ). Another mechanism by which cancer cells can control the expression of tumour antigens is through the manipulation of the proteasome and the MHC molecules (Boulpicante et al. 2020 ). The proteasome is a complex of enzymes responsible for degrading intracellular proteins, including tumour antigens, into peptides that MHC molecules can present. Cancer cells can interfere with the activity of the proteasome, thereby preventing the degradation of cancer cell-associated antigens and avoiding the presentation of the antigens on the MHC molecules (Chen et al. 2022 ). Cancer cells can also downregulate the expression of MHC molecules, thus making them invisible to the immune system and avoiding antigen presentation, or manipulate the structure of the MHC molecules, such as altering the peptide binding affinity, which can prevent the presentation of the cancer-associated antigens (Hewitt 2003 ; Rock et al. 2010 ; Blum et al. 2013 ).

Epigenetic modulation of immunotherapy

One mechanism by which cancer cells can control the expression of tumour antigens is through epigenetic regulation. Epigenetics refers to the regulation of gene expression through changes in the structure of DNA, such as methylation and histone modification, rather than changes in the genetic code itself (Gibney and Nolan 2010 ). Cancer cells can alter the epigenetic landscape to downregulate the expression of tumour antigens, making them invisible to the immune system. Cancer cells can also secrete factors that inhibit the activity of antigen-presenting cells and T cells, such as TGF-beta and IL-10, which further suppress the immune response (Thepmalee et al. 2018 ). Epigenetic modulation of antitumor immunity has been an active area of research in recent years and has been found to have potential applications in cancer immunotherapy (Gibney and Nolan 2010 ). Cancer cells' manipulation of the epigenetic landscape has been shown to play a critical role in suppressing the immune response against cancer. By targeting these mechanisms, it is possible to improve the efficacy of cancer immunotherapy (Liu et al. 2022a ). One way in which epigenetic modulation can be targeted is through the use of histone deacetylase inhibitors (HDACis). HDACis are a class of drugs that inhibit the activity of histone deacetylases, enzymes that remove acetyl groups from histones, leading to the repression of gene expression. HDACis have been shown to enhance the maturation of dendritic cells and increase the presentation of tumour antigens, thus enhancing the immune response against cancer (Gryder et al. 2012 ).

Another way to target epigenetic modulation is through DNA methyltransferase inhibitors (DNMTis) (Hu et al. 2021 ). DNMTis are a class of drugs that inhibit the activity of DNA methyltransferases, enzymes that add methyl groups to DNA, leading to the repression of gene expression. DNMTis have been shown to increase the expression of genes involved in the immune response, such as MHC molecules, and modulate the expression of genes involved in immune evasions, such as PD-L1 (Dan et al. 2019 ) (Fig.  5 ). The combination therapies that combine epigenetic modulation with other immunotherapeutic strategies, such as checkpoint inhibitors, have also yielded promising results in clinical trials. For example, combining HDACis with PD-1/PD-L1 inhibitors has enhanced the response to treatment in multiple cancer types (Mazzone et al. 2017 ; Liu et al. 2022b ).

Diagram illustrating the epigenetic regulation of chromatin accessibility and gene expression in cells. Nucleosomes, formed by DNA wrapped around histone octamers, are depicted as blue cylinders. Epigenetic modifications are depicted as dynamic interactions between chromatin components and enzymes, including histone methylation/demethylation, histone acetylation/deacetylation, and DNA methylation. Chromatin remodelling also plays a role in regulating gene expression

It is important to note that while the use of these epigenetic modulation therapies has shown promising results in preclinical and clinical studies, more research is needed to fully understand the mechanisms of action and optimal use in the clinical setting. Further research is also needed to understand these therapies' potential side effects and long-term safety.

Antigen presentation machinery components, modulation and their defects

The antigen processing machinery (APM) plays a critical role in developing an effective antitumor immune response (Maggs et al. 2021 ). The APM is a group of cellular structures and molecules responsible for processing and presenting APCs to T cells. Defects in the APM can compromise the ability of the immune system to recognize and respond to cancer cells, leading to the development of tumours that evade destruction by the immune system (Mpakali and Stratikos 2021 ). The major components of the APM include proteasomes, which are responsible for the degradation of proteins into peptides; TAP (transporter associated with antigen processing), which transports the peptides from the cytosol to the endoplasmic reticulum (ER); and MHC (major histocompatibility complex) molecules, which present the peptides on the surface of APCs to T cells. A growing body of evidence suggests that defects in the APM can contribute to cancer development. For example, mutations in the genes encoding the proteasomes or TAP can reduce the ability to generate peptides that can be presented on MHC molecules (Reiman et al. 2007 ). This can limit the ability of the immune system to recognize and respond to cancer cells. Additionally, defects in MHC molecules can result in a decreased ability to mount an immune response against certain infections and cancer (Charles et al. 2001 ; Dassa 2003 ).

Cancer cells can modulate antigen presentation in several ways to evade recognition and destruction by the immune system. Cancer cells can do this by deregulation of MHC molecules; Cancer cells can reduce the expression of MHC molecules on their surface, making them less visible to T cells and harder to target. Disruption of antigen processing; Cancer cells can interfere with the normal processing of antigens within the cell, making it harder for APCs to present them on MHC molecules. Production of immunosuppressive molecules; Cancer cells can produce molecules that suppress the immune response, such as TGF-beta and IDO, making it harder for T cells to recognize and attack cancer cells. Recruitment of immune-suppressive cells; Cancer cells can recruit immune cells that suppress the immune response, such as Tregs and MDSCs, to the tumour microenvironment (Vinay et al. 2015 ; Parcesepe et al. 2016 ; Mergener and Peña-Llopis 2022 ).

Defects in any of these components can result in a compromised immune response. For example, mutations in MHC molecules can result in a condition called MHC deficiency, which leads to a decreased ability to mount an immune response against certain infections. Similarly, TCR defects can result in T cell dysfunction and increased susceptibility to infections. Defects in the antigen presentation machinery can significantly impact the immune system's ability to recognize and respond to cancer cells, and understanding these defects can inform the development of new immunotherapies for cancer (Mpakali and Stratikos 2021 ). The development of immunotherapies for cancer has been a promising approach to targeting tumours that evade destruction by the immune system. These therapies aim to re-activate the patient's immune system to recognize and attack cancer cells. This can include checkpoint inhibitors, which block the immune-suppressive signals emitted by cancer cells and allow T cells to recognize and attack the tumour, and CAR T-cell therapy, which genetically modifies a patient's T cells to recognize and attack cancer cells (Filley et al. 2018 ).

Neoantigens in cancer immunotherapy

Neoantigens are a class of tumour-specific antigens generated by genetic mutations in cancer cells. They are not present in normal cells and, thus, represent a unique target for cancer immunotherapy. Identifying and characterising neoantigens have led to the development of new immunotherapeutic strategies for cancer treatment (Zhu and Liu 2021 ). The process of neoantigen identification begins with the sequencing of a patient's tumour and normal DNA (Zhu and Liu 2021 ). Algorithms are then used to identify potential neoantigens based on their predicted binding to MHC molecules and their potential to be presented on the cell surface. These potential neoantigens are further validated through functional assays, such as T-cell assays, to confirm their ability to elicit a T-cell response (Garcia-Garijo et al. 2019 ; Zaidi et al. 2020 ). Once identified, neoantigens can be used to develop personalized cancer vaccines (Blass and Ott 2021 ). These vaccines can target specific mutations in an individual's tumour and stimulate an immune response against cancer cells. The vaccines can be either ex vivo, where T cells are extracted from the patient, genetically modified to recognize the neoantigens, and then re-infused back into the patient or in vivo, where the patient is administered with the neoantigen peptides (Xie et al. 2023 ).

Recent clinical trials have demonstrated the safety and efficacy of personalized neoantigen cancer vaccines (Fritah et al. 2022 ). The results have shown that these vaccines can induce antitumor T-cell responses and result in durable clinical responses in a subset of patients with advanced cancer. Additionally, a combination of neoantigen vaccine with checkpoint inhibitors has shown to be more effective in inducing antitumor T-cell response and, in some cases, led to complete remission of the disease (Liao and Zhang 2021 ). Furthermore, the identification of neoantigens has also led to the development of neoantigen-targeting T-cell therapies, such as CAR-T cell therapy. In this approach, T cells are genetically modified to express a CAR specific for a neoantigen and then re-infused back into the patient. These therapies have shown effective in inducing long-lasting responses in patients with advanced cancer (Wang and Cao 2020 ).

The antigen processing and presentation mechanisms play a critical role in the immune system's recognition and targeting of cancer cells. Cancer cells can avoid immune detection by downregulating or losing the expression of proteins recognised as antigens, creating an immunosuppressive microenvironment, and altering their ability to process and present antigens. The study of the immunopeptidome, or peptidomics, has provided insights into the mechanisms of cancer immune evasion and has potential applications in cancer diagnosis and treatment. One mechanism by which cancer cells can control the expression of tumour antigens is through epigenetic regulation, such as methylation and histone modification; cancer cells can alter the epigenetic landscape to downregulate the expression of tumour antigens, making them invisible to the immune system. Additionally, cancer cells can manipulate the microenvironment, interfere with the activity of the proteasome and MHC molecules, and downregulate the expression of MHC molecules to avoid the presentation of antigens. Recent advances in cancer genomics and molecular biology have allowed the identification of unique antigens present in cancer cells but not in normal cells, known as "neoantigens." These neoantigens can be used to develop cancer vaccines and CAR-T cell therapy that target the specific mutations present in an individual's tumour, leading to the re-activation of the patient's immune system to recognize and attack cancer cells. Targeting the epigenetic mechanisms that cancer cells use to evade the immune system can improve cancer immunotherapy, such as using HDACis, DNMTis, and combination therapies. However, it's important to note that more research is needed to fully understand the mechanisms of action and optimal use of these therapies in the clinical setting. In snapshot, controlling tumour antigen expression and presentation is a critical aspect of cancer biology that significantly impacts the immune system's ability to recognize and target cancer cells. Understanding these mechanisms is crucial for developing effective cancer immunotherapies that target the mechanisms of antigen expression and presentation in cancer cells and for a better understanding of the epigenetic modulation of antitumor immunity for improved cancer immunotherapy.

Data availability

All data generated or analysed during this study are included in this published article.

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Kallingal, A., Olszewski, M., Maciejewska, N. et al. Cancer immune escape: the role of antigen presentation machinery. J Cancer Res Clin Oncol 149 , 8131–8141 (2023). https://doi.org/10.1007/s00432-023-04737-8

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Innate Immunity: Phagocytes and Antigen Presentation

The immune system Immune system The body's defense mechanism against foreign organisms or substances and deviant native cells. It includes the humoral immune response and the cell-mediated response and consists of a complex of interrelated cellular, molecular, and genetic components. Primary Lymphatic Organs is equipped with a varied repertoire of defense mechanisms Defense mechanisms Defense mechanisms are normal subconscious means of resolving inner conflicts between an individual's subjective moral sense and their thoughts, feelings, or actions. Defense mechanisms serve to protect the self from unpleasant feelings (anxiety, shame, and/or guilt) and are divided into pathologic, immature, mature, neurotic, and other types. Defense Mechanisms against pathogens. Functionally, the immune system Immune system The body's defense mechanism against foreign organisms or substances and deviant native cells. It includes the humoral immune response and the cell-mediated response and consists of a complex of interrelated cellular, molecular, and genetic components. Primary Lymphatic Organs is differentiated into the innate and adaptive components. Innate immunity, the 1st protective layer of defense, is a system that recognizes threatening microbes, distinguishes self-tissues from pathogens, and subsequently eliminates the foreign invaders. The response is nonspecific and uses different layers of protection: barriers such as the skin Skin The skin, also referred to as the integumentary system, is the largest organ of the body. The skin is primarily composed of the epidermis (outer layer) and dermis (deep layer). The epidermis is primarily composed of keratinocytes that undergo rapid turnover, while the dermis contains dense layers of connective tissue. Skin: Structure and Functions , pattern recognition receptors Receptors Receptors are proteins located either on the surface of or within a cell that can bind to signaling molecules known as ligands (e.g., hormones) and cause some type of response within the cell. Receptors (PRRs) as well as circulating proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis (e.g., complement) that relay signals of a threat, and immune cells that help eliminate the microbe. Pathogen-associated molecular patterns Pathogen-Associated Molecular Patterns Sepsis and Septic Shock ( PAMPs PAMPs Sepsis and Septic Shock ) in microorganisms and damage-associated molecular patterns Damage-Associated Molecular Patterns Sepsis and Septic Shock (DAMPs) from injured tissues are identified, and the appropriate cells are recruited. Involved cells include phagocytes and accessory cells. The offending pathogens are engulfed by phagocytes for destruction. In antigen-presenting cells Antigen-presenting cells A heterogeneous group of immunocompetent cells that mediate the cellular immune response by processing and presenting antigens to the T-cells. Traditional antigen-presenting cells include macrophages; dendritic cells; langerhans cells; and B-lymphocytes. Follicular dendritic cells are not traditional antigen-presenting cells, but because they hold antigen on their cell surface in the form of immune complexes for b-cell recognition they are considered so by some authors. Adaptive Immune Response (the most potent of which is the dendritic cell), parts of the pathogen material or peptides are transported to the cell surface. Through a unique antigen-loading mechanism specific to MHC I or II, the processed antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination peptides are then presented to the appropriate T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified - cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions , leading to T-cell activation. This interaction links innate immunity with adaptive immunity.

Last updated: Apr 18, 2023

Components of the Innate Immune System

Professional phagocytes, accessory cells, antigen presentation, clinical relevance.

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Immune system Immune system The body’s defense mechanism against foreign organisms or substances and deviant native cells. It includes the humoral immune response and the cell-mediated response and consists of a complex of interrelated cellular, molecular, and genetic components. Primary Lymphatic Organs

The immune system Immune system The body’s defense mechanism against foreign organisms or substances and deviant native cells. It includes the humoral immune response and the cell-mediated response and consists of a complex of interrelated cellular, molecular, and genetic components. Primary Lymphatic Organs provides defense (immunity) against invading pathogens ranging from viruses Viruses Minute infectious agents whose genomes are composed of DNA or RNA, but not both. They are characterized by a lack of independent metabolism and the inability to replicate outside living host cells. Virology to parasites, and components are interconnected by blood and the lymphatic circulation Circulation The movement of the blood as it is pumped through the cardiovascular system. ABCDE Assessment .

There are 2 lines of defense Lines of Defense Inflammation (that overlap):

• Innate immunity (which is nonspecific) 
• Cell-mediated immunity Cell-mediated immunity Manifestations of the immune response which are mediated by antigen-sensitized T-lymphocytes via lymphokines or direct cytotoxicity. This takes place in the absence of circulating antibody or where antibody plays a subordinate role. Squamous Cell Carcinoma (SCC) : adaptive response in the cells/tissues involving the T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions
• Humoral immunity: adaptive response in the fluids (humoral) involving B cells B cells Lymphoid cells concerned with humoral immunity. They are short-lived cells resembling bursa-derived lymphocytes of birds in their production of immunoglobulin upon appropriate stimulation. B cells: Types and Functions and immunoglobulins Immunoglobulins Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins: Types and Functions

Innate versus adaptive immunity

Related videos, innate immune response innate immune response immunity to pathogens is divided into innate and adaptive immune responses. the innate immune response is the 1st line of defense against a variety of pathogens, including bacteria, fungi, viruses, and parasites. in essentially the same form, the innate type of immunity is present in all multicellular organisms. innate immunity: barriers, complement, and cytokines.

• 1st line of defense (mechanical, chemical, and biologic)
• Define and line body surfaces
• Secrete substances to remove and reduce pathogens
• Pathogen-associated molecular patterns Pathogen-Associated Molecular Patterns Sepsis and Septic Shock ( PAMPs PAMPs Sepsis and Septic Shock ): structures conserved among microbial species
• Damage-associated molecular patterns Damage-Associated Molecular Patterns Sepsis and Septic Shock (DAMPs), or alarmins: endogenous molecules released from damaged cells
• Retinoic acid–inducible gene Gene A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms. Basic Terms of Genetics (RIG) I–like receptors Receptors Receptors are proteins located either on the surface of or within a cell that can bind to signaling molecules known as ligands (e.g., hormones) and cause some type of response within the cell. Receptors ( RLRs RLRs Innate Immunity: Barriers, Complement, and Cytokines )  
• NOD-like receptors NOD-like receptors Innate Immunity: Barriers, Complement, and Cytokines ( NLRs NLRs Innate Immunity: Barriers, Complement, and Cytokines )
• C-type lectin receptors C-type lectin receptors Innate Immunity: Barriers, Complement, and Cytokines ( CLRs CLRs Innate Immunity: Barriers, Complement, and Cytokines ) 
• AMPs AMPs Innate Immunity: Barriers, Complement, and Cytokines
• Collectins Collectins A class of c-type lectins that target the carbohydrate structures found on invading pathogens. Binding of collectins to microorganisms results in their agglutination and enhanced clearance. Collectins form trimers that may assemble into larger oligomers. Each collectin polypeptide chain consists of four regions: a relatively short n-terminal region, a collagen-like region, an alpha-helical coiled-coil region, and carbohydrate-binding region. Innate Immunity: Barriers, Complement, and Cytokines
• Lectins Lectins Proteins that share the common characteristic of binding to carbohydrates. Some antibodies and carbohydrate-metabolizing proteins (enzymes) also bind to carbohydrates, however they are not considered lectins. Plant lectins are carbohydrate-binding proteins that have been primarily identified by their hemagglutinating activity (hemagglutinins). However, a variety of lectins occur in animal species where they serve diverse array of functions through specific carbohydrate recognition. Innate Immunity: Barriers, Complement, and Cytokines
• Pentraxins Pentraxins Innate Immunity: Barriers, Complement, and Cytokines
• Antimicrobial oligosaccharides Antimicrobial oligosaccharides Innate Immunity: Barriers, Complement, and Cytokines
• Occurs in response to infection or injury
• Results in the cardinal signs ( swelling Swelling Inflammation , redness Redness Inflammation , heat Heat Inflammation , and pain Pain An unpleasant sensation induced by noxious stimuli which are detected by nerve endings of nociceptive neurons. Pain: Types and Pathways ) 
• Cytokines Cytokines Non-antibody proteins secreted by inflammatory leukocytes and some non-leukocytic cells, that act as intercellular mediators. They differ from classical hormones in that they are produced by a number of tissue or cell types rather than by specialized glands. They generally act locally in a paracrine or autocrine rather than endocrine manner. Adaptive Immune Response (functions include recruitment Recruitment Skeletal Muscle Contraction , cell-to-cell communication Communication The exchange or transmission of ideas, attitudes, or beliefs between individuals or groups. Decision-making Capacity and Legal Competence , and antiviral Antiviral Antivirals for Hepatitis B , antibacterial Antibacterial Penicillins , and antifungal Antifungal Azoles actions)
• Chemokines Chemokines Class of pro-inflammatory cytokines that have the ability to attract and activate leukocytes. They can be divided into at least three structural branches: c; cc; and cxc; according to variations in a shared cysteine motif. Adaptive Cell-mediated Immunity (cell migration)
• Complement system Complement system Serum glycoproteins participating in the host defense mechanism of complement activation that creates the complement membrane attack complex. Included are glycoproteins in the various pathways of complement activation (classical complement pathway; alternative complement pathway; and lectin complement pathway). Innate Immunity: Barriers, Complement, and Cytokines (complements (assists in) eliminating the microbes)
• Some PRRs (e.g., AMPs AMPs Innate Immunity: Barriers, Complement, and Cytokines ) are also involved in pathogen elimination Elimination The initial damage and destruction of tumor cells by innate and adaptive immunity. Completion of the phase means no cancer growth. Cancer Immunotherapy .
• Cellular response: Various cells (e.g., phagocytes) are recruited and participate in microbial killing.
• The immune response is terminated when there is no longer a need ( homeostasis Homeostasis The processes whereby the internal environment of an organism tends to remain balanced and stable. Cell Injury and Death ).

Cells of the innate immune system Immune system The body’s defense mechanism against foreign organisms or substances and deviant native cells. It includes the humoral immune response and the cell-mediated response and consists of a complex of interrelated cellular, molecular, and genetic components. Primary Lymphatic Organs

• Granulocytes Granulocytes Leukocytes with abundant granules in the cytoplasm. They are divided into three groups according to the staining properties of the granules: neutrophilic, eosinophilic, and basophilic. Mature granulocytes are the neutrophils; eosinophils; and basophils. White Myeloid Cells: Histology : professional phagocytes (neutrophils, monocytes/macrophages), eosinophils, basophils, mast cells
• Megakaryocytes → platelets Platelets Platelets are small cell fragments involved in hemostasis. Thrombopoiesis takes place primarily in the bone marrow through a series of cell differentiation and is influenced by several cytokines. Platelets are formed after fragmentation of the megakaryocyte cytoplasm. Platelets: Histology
• B cells B cells Lymphoid cells concerned with humoral immunity. They are short-lived cells resembling bursa-derived lymphocytes of birds in their production of immunoglobulin upon appropriate stimulation. B cells: Types and Functions and T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions (adaptive immunity)
• Natural killer (NK) cells (mostly innate immune response Innate Immune Response Immunity to pathogens is divided into innate and adaptive immune responses. The innate immune response is the 1st line of defense against a variety of pathogens, including bacteria, fungi, viruses, and parasites. In essentially the same form, the innate type of immunity is present in all multicellular organisms. Innate Immunity: Barriers, Complement, and Cytokines )
• NK– T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions (bridge innate and adaptive immunity)
• Individually, the cells have varying functions and targets in the immune response.
• Phagocytosis: Microbes or damaged particles are engulfed and digested.
• Antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination presentation: performed by dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions , macrophages, and B cells B cells Lymphoid cells concerned with humoral immunity. They are short-lived cells resembling bursa-derived lymphocytes of birds in their production of immunoglobulin upon appropriate stimulation. B cells: Types and Functions , facilitating antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination recognition by adaptive immunity

Stem cells differentiate into 2 pathways: Myeloid pathways produce erythrocytes, platelets, and cells of the innate immune response. Lymphoid pathways produce the cells of adaptive response and natural killer cells.

Phagocytes “eat” the foreign material, and help detect, clear, and repair damaged tissue, recognizing pathogens via PRRs or opsonization (by complement or immunoglobulins Immunoglobulins Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins: Types and Functions ).

Neutrophils

• 1st cells to be recruited into sites of infection
• N-formyl bacterial oligopeptide 
• Complement-derived C5a 
• Leukotriene Leukotriene Asthma Drugs B4 (secreted by numerous immune cells)
• Phagocytosis and production of reactive oxygen species Reactive oxygen species Molecules or ions formed by the incomplete one-electron reduction of oxygen. These reactive oxygen intermediates include singlet oxygen; superoxides; peroxides; hydroxyl radical; and hypochlorous acid. They contribute to the microbicidal activity of phagocytes, regulation of signal transduction and gene expression, and the oxidative damage to nucleic acids; proteins; and lipids. Nonalcoholic Fatty Liver Disease ( respiratory burst Respiratory burst A large increase in oxygen uptake by neutrophils and most types of tissue macrophages through activation of an NADPH-cytochrome b-dependent oxidase that reduces oxygen to a superoxide. Individuals with an inherited defect in which the oxidase that reduces oxygen to superoxide is decreased or absent often die as a result of recurrent bacterial infections. Leukocyte Adhesion Deficiency Type 1 ) that are cytotoxic Cytotoxic Parvovirus B19 to bacterial pathogens
• Neutrophil cytoplasmic granule proteases Proteases Proteins and Peptides : neutrophil elastase Elastase A protease of broad specificity, obtained from dried pancreas. Molecular weight is approximately 25, 000. The enzyme breaks down elastin, the specific protein of elastic fibers, and digests other proteins such as fibrin, hemoglobin, and albumin. Proteins and Peptides and cathepsin G
• Production of cytokines Cytokines Non-antibody proteins secreted by inflammatory leukocytes and some non-leukocytic cells, that act as intercellular mediators. They differ from classical hormones in that they are produced by a number of tissue or cell types rather than by specialized glands. They generally act locally in a paracrine or autocrine rather than endocrine manner. Adaptive Immune Response such as tumor Tumor Inflammation necrosis Necrosis The death of cells in an organ or tissue due to disease, injury or failure of the blood supply. Ischemic Cell Damage factor ( TNF TNF Tumor necrosis factor (TNF) is a major cytokine, released primarily by macrophages in response to stimuli. The presence of microbial products and dead cells and injury are among the stimulating factors. This protein belongs to the TNF superfamily, a group of ligands and receptors performing functions in inflammatory response, morphogenesis, and cell proliferation. Tumor Necrosis Factor (TNF) ) 
• Use of extracellular strands of chromatin Chromatin The material of chromosomes. It is a complex of dna; histones; and nonhistone proteins found within the nucleus of a cell. DNA Types and Structure laced with antimicrobial proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis (neutrophil extracellular traps (NETs)) that catch and kill pathogens

Neutrophil: Granulocyte with multilobed nucleus and fine faintly pink granules

Mechanism of neutrophil extracellular trap (NET) release: Neutrophils are stimulated by contact with bacteria, protozoa, fungi (yeast and hyphae forms), which leads to: (a) Ultrastructural alterations of nuclear shape with chromatin decondensation and a swollen and fragmented nuclear membrane, which allows the association of granules and cytoplasmic proteins with the chromatin, and (b) release of extracellular structures consisting of a DNA backbone, decorated with histones, neutrophil, granular, and cytoplasmic proteins (NETs), which ensnare and kill microorganisms.

Monocytes/macrophages

• Some remain as monocytes, picking up and bringing antigens to the lymph nodes Lymph Nodes They are oval or bean shaped bodies (1 – 30 mm in diameter) located along the lymphatic system. Lymphatic Drainage System: Anatomy .
• When monocytes migrate out of the circulation Circulation The movement of the blood as it is pumped through the cardiovascular system. ABCDE Assessment and go to tissues, monocytes differentiate into macrophages.
• Lymph Lymph The interstitial fluid that is in the lymphatic system. Secondary Lymphatic Organs node, spleen Spleen The spleen is the largest lymphoid organ in the body, located in the LUQ of the abdomen, superior to the left kidney and posterior to the stomach at the level of the 9th-11th ribs just below the diaphragm. The spleen is highly vascular and acts as an important blood filter, cleansing the blood of pathogens and damaged erythrocytes. Spleen: Anatomy , bone marrow Bone marrow The soft tissue filling the cavities of bones. Bone marrow exists in two types, yellow and red. Yellow marrow is found in the large cavities of large bones and consists mostly of fat cells and a few primitive blood cells. Red marrow is a hematopoietic tissue and is the site of production of erythrocytes and granular leukocytes. Bone marrow is made up of a framework of connective tissue containing branching fibers with the frame being filled with marrow cells. Bone Marrow: Composition and Hematopoiesis , perivascular connective tissue Connective tissue Connective tissues originate from embryonic mesenchyme and are present throughout the body except inside the brain and spinal cord. The main function of connective tissues is to provide structural support to organs. Connective tissues consist of cells and an extracellular matrix. Connective Tissue: Histology , and serous cavities
• In other tissues: lung ( alveolar macrophages Alveolar macrophages Round, granular, mononuclear phagocytes found in the alveoli of the lungs. They ingest small inhaled particles resulting in degradation and presentation of the antigen to immunocompetent cells. Acute Respiratory Distress Syndrome (ARDS) ), liver Liver The liver is the largest gland in the human body. The liver is found in the superior right quadrant of the abdomen and weighs approximately 1.5 kilograms. Its main functions are detoxification, metabolism, nutrient storage (e.g., iron and vitamins), synthesis of coagulation factors, formation of bile, filtration, and storage of blood. Liver: Anatomy ( Kupffer cells Kupffer cells Specialized phagocytic cells of the mononuclear phagocyte system found on the luminal surface of the hepatic sinusoids. They filter bacteria and small foreign proteins out of the blood, and dispose of worn out red blood cells. Benign Liver Tumors ), bone Bone Bone is a compact type of hardened connective tissue composed of bone cells, membranes, an extracellular mineralized matrix, and central bone marrow. The 2 primary types of bone are compact and spongy. Bones: Structure and Types ( osteoclasts Osteoclasts A large multinuclear cell associated with the bone resorption. An odontoclast, also called cementoclast, is cytomorphologically the same as an osteoclast and is involved in cementum resorption. Bones: Development and Ossification ), CNS ( microglia Microglia The third type of glial cell, along with astrocytes and oligodendrocytes (which together form the macroglia). Microglia vary in appearance depending on developmental stage, functional state, and anatomical location; subtype terms include ramified, perivascular, ameboid, resting, and activated. Microglia clearly are capable of phagocytosis and play an important role in a wide spectrum of neuropathologies. They have also been suggested to act in several other roles including in secretion (e.g., of cytokines and neural growth factors), in immunological processing (e.g., antigen presentation), and in central nervous system development and remodeling. Nervous System: Histology cells), and synovium (type A lining cells)
• Also differentiate into dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions during inflammation Inflammation Inflammation is a complex set of responses to infection and injury involving leukocytes as the principal cellular mediators in the body’s defense against pathogenic organisms. Inflammation is also seen as a response to tissue injury in the process of wound healing. The 5 cardinal signs of inflammation are pain, heat, redness, swelling, and loss of function. Inflammation
• Respond briskly to pathogens, facilitated by high density of surface PRRs
• Use NO to kill pathogens, and also produce large amounts of cytokines Cytokines Non-antibody proteins secreted by inflammatory leukocytes and some non-leukocytic cells, that act as intercellular mediators. They differ from classical hormones in that they are produced by a number of tissue or cell types rather than by specialized glands. They generally act locally in a paracrine or autocrine rather than endocrine manner. Adaptive Immune Response
• Antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination presentation to lymphocytes Lymphocytes Lymphocytes are heterogeneous WBCs involved in immune response. Lymphocytes develop from the bone marrow, starting from hematopoietic stem cells (HSCs) and progressing to common lymphoid progenitors (CLPs). B and T lymphocytes and natural killer (NK) cells arise from the lineage. Lymphocytes: Histology (stimulating the adaptive immune response Adaptive immune response Immune responses against pathogens are divided into the innate and adaptive immune response systems. The adaptive immune response, also called the acquired immune system, consists of 2 main mechanisms: the humoral- and cellular-mediated immune responses. Adaptive Immune Response )
• Play a role in iron Iron A metallic element with atomic symbol fe, atomic number 26, and atomic weight 55. 85. It is an essential constituent of hemoglobins; cytochromes; and iron-binding proteins. It plays a role in cellular redox reactions and in the transport of oxygen. Trace Elements homeostasis Homeostasis The processes whereby the internal environment of an organism tends to remain balanced and stable. Cell Injury and Death

Monocyte development starts from hematopoietic stem cells (HSCs) and progresses through stages to the colony-forming unit granulocyte-macrophage (CFU-GM): The 1st monocyte precursor is the monoblast, which has a round or oval nucleus. The promonocyte follows and has a convoluted nucleus. The monocyte arises with an indented nucleus and is released from the bone marrow to become a macrophage in the tissues.

Dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions

• Most potent antigen-presenting cells Antigen-presenting cells A heterogeneous group of immunocompetent cells that mediate the cellular immune response by processing and presenting antigens to the T-cells. Traditional antigen-presenting cells include macrophages; dendritic cells; langerhans cells; and B-lymphocytes. Follicular dendritic cells are not traditional antigen-presenting cells, but because they hold antigen on their cell surface in the form of immune complexes for b-cell recognition they are considered so by some authors. Adaptive Immune Response
• Name derived from presence of dendritic (branching) extensions
• Arise from bone marrow Bone marrow The soft tissue filling the cavities of bones. Bone marrow exists in two types, yellow and red. Yellow marrow is found in the large cavities of large bones and consists mostly of fat cells and a few primitive blood cells. Red marrow is a hematopoietic tissue and is the site of production of erythrocytes and granular leukocytes. Bone marrow is made up of a framework of connective tissue containing branching fibers with the frame being filled with marrow cells. Bone Marrow: Composition and Hematopoiesis
• Link innate and adaptive immunity by antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination presentation and release chemokines Chemokines Class of pro-inflammatory cytokines that have the ability to attract and activate leukocytes. They can be divided into at least three structural branches: c; cc; and cxc; according to variations in a shared cysteine motif. Adaptive Cell-mediated Immunity , which attract T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions and B cells B cells Lymphoid cells concerned with humoral immunity. They are short-lived cells resembling bursa-derived lymphocytes of birds in their production of immunoglobulin upon appropriate stimulation. B cells: Types and Functions when a pathogen is detected.
• Also called conventional dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions
• Can be interstitial dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions (in blood and interstices of lung, heart, kidney) or Langerhans dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions
• Lymphoid lineage
• Plasmacytoid dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions primarily reside in and recirculate through lymphoid organs Lymphoid organs A system of organs and tissues that process and transport immune cells and lymph. Primary Lymphatic Organs .
• Inefficient antigen-presenting cells Antigen-presenting cells A heterogeneous group of immunocompetent cells that mediate the cellular immune response by processing and presenting antigens to the T-cells. Traditional antigen-presenting cells include macrophages; dendritic cells; langerhans cells; and B-lymphocytes. Follicular dendritic cells are not traditional antigen-presenting cells, but because they hold antigen on their cell surface in the form of immune complexes for b-cell recognition they are considered so by some authors. Adaptive Immune Response but massively produce type I interferon ( IFN IFN Interferon (IFN) is a cytokine with antiviral properties (it interferes with viral infections) and various roles in immunoregulation. The different types are type I IFN (IFN-ɑ and IFN-β), type II IFN (IFN-ɣ), and type III IFN (IFN-ƛ). Interferons ) when viral infections Infections Invasion of the host organism by microorganisms or their toxins or by parasites that can cause pathological conditions or diseases. Chronic Granulomatous Disease occur
• Phagocytosis of microbes, molecules from damaged tissue, self-antigens, tumors
• Subsequent steps lead to maturation (expression of MHC II and costimulatory molecules, PRRs, with up-regulation Up-Regulation A positive regulatory effect on physiological processes at the molecular, cellular, or systemic level. At the molecular level, the major regulatory sites include membrane receptors, genes (gene expression regulation), mRNAs, and proteins. Pharmacokinetics and Pharmacodynamics of cytokine receptors Receptors Receptors are proteins located either on the surface of or within a cell that can bind to signaling molecules known as ligands (e.g., hormones) and cause some type of response within the cell. Receptors )
• Become more antigen-specific with maturity and participate in the adaptive immune response Adaptive immune response Immune responses against pathogens are divided into the innate and adaptive immune response systems. The adaptive immune response, also called the acquired immune system, consists of 2 main mechanisms: the humoral- and cellular-mediated immune responses. Adaptive Immune Response
• Once mature, dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions present antigens to T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions , which then proliferate.
• Positive feedback: effector T lymphocytes T lymphocytes Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions secrete IFN IFN Interferon (IFN) is a cytokine with antiviral properties (it interferes with viral infections) and various roles in immunoregulation. The different types are type I IFN (IFN-ɑ and IFN-β), type II IFN (IFN-ɣ), and type III IFN (IFN-ƛ). Interferons ‒ɣ → make the dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions produce ↑ IL-12  and ↑ microbicidal activity of macrophages

Dendritic cells release IL-12, which activates CD4 Th1 cells. These Th1 cells produce IL-2, stimulating production of more Th1 T-cell subsets. Th1 cells also release IFN-γ, which activates macrophages and activates fibroblasts to cause angiogenesis and fibrosis. If these macrophages are persistently stimulated by pathogens, such as Mycobacterium and Schistosoma , granulomas are formed.

Dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions versus follicular dendritic cells Follicular dendritic cells Non-hematopoietic cells, with extensive dendritic processes, found in the primary and secondary follicles of lymphoid tissue (the B cell zones). They are different from conventional dendritic cells associated with T-cells. They are derived from mesenchymal stem cells and are negative for class II mhc antigen and do not process or present antigen like the conventional dendritic cells do. Instead, follicular dendritic cells have fc receptors and C3b receptors that hold antigen in the form of antigen-antibody complexes on their surfaces for long periods for recognition by B-cells. MALT Lymphoma

It is important to note that follicular dendritic cells Follicular dendritic cells Non-hematopoietic cells, with extensive dendritic processes, found in the primary and secondary follicles of lymphoid tissue (the B cell zones). They are different from conventional dendritic cells associated with T-cells. They are derived from mesenchymal stem cells and are negative for class II mhc antigen and do not process or present antigen like the conventional dendritic cells do. Instead, follicular dendritic cells have fc receptors and C3b receptors that hold antigen in the form of antigen-antibody complexes on their surfaces for long periods for recognition by B-cells. MALT Lymphoma are completely unrelated to dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions in lineage and function. 

Follicular dendritic cells Follicular dendritic cells Non-hematopoietic cells, with extensive dendritic processes, found in the primary and secondary follicles of lymphoid tissue (the B cell zones). They are different from conventional dendritic cells associated with T-cells. They are derived from mesenchymal stem cells and are negative for class II mhc antigen and do not process or present antigen like the conventional dendritic cells do. Instead, follicular dendritic cells have fc receptors and C3b receptors that hold antigen in the form of antigen-antibody complexes on their surfaces for long periods for recognition by B-cells. MALT Lymphoma :

• Concentrated in the secondary lymphoid organs Lymphoid organs A system of organs and tissues that process and transport immune cells and lymph. Primary Lymphatic Organs where B-cell activation occurs
• Trap antigens on their surfaces that are then bound by the B-cell receptors Receptors Receptors are proteins located either on the surface of or within a cell that can bind to signaling molecules known as ligands (e.g., hormones) and cause some type of response within the cell. Receptors of B cells B cells Lymphoid cells concerned with humoral immunity. They are short-lived cells resembling bursa-derived lymphocytes of birds in their production of immunoglobulin upon appropriate stimulation. B cells: Types and Functions (B-cell activation)

Phagocytosis

• Engulfment of the pathogen in a vesicle Vesicle Primary Skin Lesions follows.                               
• The phagocyte forms a pseudopod that wraps around the pathogen, and this becomes a pinched-off membrane vesicle Vesicle Primary Skin Lesions called a phagosome. 
• A phagolysosome Phagolysosome Chédiak-Higashi Syndrome is formed as the phagosome fuses with a lysosome.
• In the compartment, the pathogen is eliminated by different microbial killing mechanisms.
• When the pathogen is destroyed, the phagocyte undergoes apoptosis Apoptosis A regulated cell death mechanism characterized by distinctive morphologic changes in the nucleus and cytoplasm, including the endonucleolytic cleavage of genomic DNA, at regularly spaced, internucleosomal sites, I.e., DNA fragmentation. It is genetically-programmed and serves as a balance to mitosis in regulating the size of animal tissues and in mediating pathologic processes associated with tumor growth. Ischemic Cell Damage (e.g., seen in pus) or the waste is eliminated by exocytosis Exocytosis Cellular release of material within membrane-limited vesicles by fusion of the vesicles with the cell membrane. The Cell: Cell Membrane .
• In antigen-presenting cells Antigen-presenting cells A heterogeneous group of immunocompetent cells that mediate the cellular immune response by processing and presenting antigens to the T-cells. Traditional antigen-presenting cells include macrophages; dendritic cells; langerhans cells; and B-lymphocytes. Follicular dendritic cells are not traditional antigen-presenting cells, but because they hold antigen on their cell surface in the form of immune complexes for b-cell recognition they are considered so by some authors. Adaptive Immune Response , parts of the pathogen material or peptides are transported to the cell surface for antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination presentation.

Eosinophils

• Recognized by their prominent eosinophilic cytoplasmic granules
• Located primarily in the lamina propria Lamina propria Whipple’s Disease of the GI tract
• Released extracellular traps contain eosinophil granules (that secrete their contents, including the cytotoxic Cytotoxic Parvovirus B19 major basic protein) when stimulated 
• Have cytotoxic Cytotoxic Parvovirus B19 effect against helminths Helminths Commonly known as parasitic worms, this group includes the acanthocephala; nematoda; and platyhelminths. Some authors consider certain species of leeches that can become temporarily parasitic as helminths. Anthelmintic Drugs and other parasites
• Also have extensive antibacterial Antibacterial Penicillins and antiviral Antiviral Antivirals for Hepatitis B activity
• Mediate eosinophilic GI diseases
• Circulating leukocytes Leukocytes White blood cells. These include granular leukocytes (basophils; eosinophils; and neutrophils) as well as non-granular leukocytes (lymphocytes and monocytes). White Myeloid Cells: Histology ; not found in tissues
• Express high-affinity receptors Receptors Receptors are proteins located either on the surface of or within a cell that can bind to signaling molecules known as ligands (e.g., hormones) and cause some type of response within the cell. Receptors for IgE IgE An immunoglobulin associated with mast cells. Overexpression has been associated with allergic hypersensitivity. Immunoglobulins: Types and Functions
• With IgE IgE An immunoglobulin associated with mast cells. Overexpression has been associated with allergic hypersensitivity. Immunoglobulins: Types and Functions receptors Receptors Receptors are proteins located either on the surface of or within a cell that can bind to signaling molecules known as ligands (e.g., hormones) and cause some type of response within the cell. Receptors , basophils participate in immediate hypersensitivity types of allergic immune response
• Provide resistance Resistance Physiologically, the opposition to flow of air caused by the forces of friction. As a part of pulmonary function testing, it is the ratio of driving pressure to the rate of air flow. Ventilation: Mechanics of Breathing against helminths Helminths Commonly known as parasitic worms, this group includes the acanthocephala; nematoda; and platyhelminths. Some authors consider certain species of leeches that can become temporarily parasitic as helminths. Anthelmintic Drugs   
• Activities are mediated by histamine, cathelicidin, and other mediators
• Produce IL-4 and IL-13, which promote Th2 Th2 A subset of helper-inducer T-lymphocytes which synthesize and secrete the interleukins il-4; il-5; il-6; and il-10. These cytokines influence b-cell development and antibody production as well as augmenting humoral responses. T cells: Types and Functions response

Eosinophil and basophil Both are granulocytes, with eosinophils possessing a bilobed nucleus and dark pink granules and basophils having a bilobed or trilobed nucleus, and dark blue granules.

• Morphologically similar to basophils
• Found in large numbers in interstitial tissues
• TLRs 1, 2, 4, and 6 (for the complement anaphylatoxin C5a)
• Receptors Receptors Receptors are proteins located either on the surface of or within a cell that can bind to signaling molecules known as ligands (e.g., hormones) and cause some type of response within the cell. Receptors for mannose-binding lectin (MBL)
• Participate in allergic responses and have antimicrobial and antiprotozoan functions
• TNF-ɑ 
• Inflammatory mediators (heparin, histamine, platelet-activating factor, leukotrienes Leukotrienes A family of biologically active compounds derived from arachidonic acid by oxidative metabolism through the 5-lipoxygenase pathway. They participate in host defense reactions and pathophysiological conditions such as immediate hypersensitivity and inflammation. They have potent actions on many essential organs and systems, including the cardiovascular, pulmonary, and central nervous system as well as the gastrointestinal tract and the immune system. Eicosanoids )
• Proteases Proteases Proteins and Peptides (e.g., tryptase Tryptase A family of neutral serine proteases with trypsin-like activity. Tryptases are primarily found in the secretory granules of mast cells and are released during mast cell degranulation. Exocrine Pancreatic Cancer , chymase)
• Antimicrobial peptides Antimicrobial peptides Innate Immunity: Barriers, Complement, and Cytokines such as cathelicidin and defensins Defensins Family of antimicrobial peptides that have been identified in humans, animals, and plants. They are thought to play a role in host defenses against infections, inflammation, wound repair, and acquired immunity. Innate Immunity: Barriers, Complement, and Cytokines

Natural killer cells Natural killer cells A specialized subset of T-lymphocytes that exhibit features of innate immunity similar to that of natural killer cells. They are reactive to glycolipids presented in the context of the major histocompatibility complex (MHC) class I-like molecule, CD1D antigen. Lymphocytes: Histology

• Lymphoid cells that do not express T- or B-cell receptors Receptors Receptors are proteins located either on the surface of or within a cell that can bind to signaling molecules known as ligands (e.g., hormones) and cause some type of response within the cell. Receptors
• Express a number of activating and inhibitory receptors Receptors Receptors are proteins located either on the surface of or within a cell that can bind to signaling molecules known as ligands (e.g., hormones) and cause some type of response within the cell. Receptors  
• Have granules with perforins and granzymes Granzymes A family of serine endopeptidases found in the secretory granules of leukocytes such as cytotoxic T-lymphocytes and natural killer cells. When secreted into the intercellular space granzymes act to eliminate transformed and virus-infected host cells. Lymphocytes: Histology
• Become senescent with age and obesity Obesity Obesity is a condition associated with excess body weight, specifically with the deposition of excessive adipose tissue. Obesity is considered a global epidemic. Major influences come from the western diet and sedentary lifestyles, but the exact mechanisms likely include a mixture of genetic and environmental factors. Obesity
• Fas– Fas ligand Fas ligand A transmembrane protein belonging to the tumor necrosis factor superfamily that was originally discovered on cells of the lymphoid-myeloid lineage, including activated T-lymphocytes and natural killer cells. It plays an important role in immune homeostasis and cell-mediated toxicity by binding to the fas receptor and triggering apoptosis. Tumor Necrosis Factor (TNF) caspase pathway
• Granzyme/ perforin Perforin A calcium-dependent pore-forming protein synthesized in cytolytic lymphocytes and sequestered in secretory granules. Upon immunological reaction between a cytolytic lymphocyte and a target cell, perforin is released at the plasma membrane and polymerizes into transmembrane tubules (forming pores) which lead to death of a target cell. Lymphocytes: Histology pathway
• Avoid attacking host cells through recognition of MHC I molecules expressed in all healthy host cells
• NK– T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions : have both T-cell and NK-cell surface markers and functions

Platelets Platelets Platelets are small cell fragments involved in hemostasis. Thrombopoiesis takes place primarily in the bone marrow through a series of cell differentiation and is influenced by several cytokines. Platelets are formed after fragmentation of the megakaryocyte cytoplasm. Platelets: Histology

• Description: circulating small cell fragments (bud off from megakaryocytes)
• Express PRRs
• Produce cytokines Cytokines Non-antibody proteins secreted by inflammatory leukocytes and some non-leukocytic cells, that act as intercellular mediators. They differ from classical hormones in that they are produced by a number of tissue or cell types rather than by specialized glands. They generally act locally in a paracrine or autocrine rather than endocrine manner. Adaptive Immune Response
• Recruit leukocytes Leukocytes White blood cells. These include granular leukocytes (basophils; eosinophils; and neutrophils) as well as non-granular leukocytes (lymphocytes and monocytes). White Myeloid Cells: Histology to sites of injury or inflammation Inflammation Inflammation is a complex set of responses to infection and injury involving leukocytes as the principal cellular mediators in the body’s defense against pathogenic organisms. Inflammation is also seen as a response to tissue injury in the process of wound healing. The 5 cardinal signs of inflammation are pain, heat, redness, swelling, and loss of function. Inflammation
• Megakaryocytes secrete IFN-α and IFN-β

Antigen-presenting cells Antigen-presenting cells A heterogeneous group of immunocompetent cells that mediate the cellular immune response by processing and presenting antigens to the T-cells. Traditional antigen-presenting cells include macrophages; dendritic cells; langerhans cells; and B-lymphocytes. Follicular dendritic cells are not traditional antigen-presenting cells, but because they hold antigen on their cell surface in the form of immune complexes for b-cell recognition they are considered so by some authors. Adaptive Immune Response (such as dendritic cells Dendritic cells Specialized cells of the hematopoietic system that have branch-like extensions. They are found throughout the lymphatic system, and in non-lymphoid tissues such as skin and the epithelia of the intestinal, respiratory, and reproductive tracts. They trap and process antigens, and present them to T-cells, thereby stimulating cell-mediated immunity. They are different from the non-hematopoietic follicular dendritic cells, which have a similar morphology and immune system function, but with respect to humoral immunity (antibody production). Skin: Structure and Functions and macrophages) detect, process, and present the antigens to T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions , allowing adaptive immunity to recognize and mount a response every time the pathogen is encountered (immunologic memory Memory Complex mental function having four distinct phases: (1) memorizing or learning, (2) retention, (3) recall, and (4) recognition. Clinically, it is usually subdivided into immediate, recent, and remote memory. Psychiatric Assessment ).

Major histocompatibility complex (MHC)

• Proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis found in antigen-presenting (and other) cells that are encoded by the HLA genes Genes A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms. DNA Types and Structure , located on chromosome Chromosome In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. Basic Terms of Genetics 6
• Principal function: present the antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination to adaptive immune system Immune system The body’s defense mechanism against foreign organisms or substances and deviant native cells. It includes the humoral immune response and the cell-mediated response and consists of a complex of interrelated cellular, molecular, and genetic components. Primary Lymphatic Organs ( T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions )
• Represents the interaction between the innate (e.g., antigen-presenting cells Antigen-presenting cells A heterogeneous group of immunocompetent cells that mediate the cellular immune response by processing and presenting antigens to the T-cells. Traditional antigen-presenting cells include macrophages; dendritic cells; langerhans cells; and B-lymphocytes. Follicular dendritic cells are not traditional antigen-presenting cells, but because they hold antigen on their cell surface in the form of immune complexes for b-cell recognition they are considered so by some authors. Adaptive Immune Response ) and adaptive immunity ( T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions )
• Found on all nucleated cells
• When a cell has an intracellular pathogen (e.g., virus Virus Viruses are infectious, obligate intracellular parasites composed of a nucleic acid core surrounded by a protein capsid. Viruses can be either naked (non-enveloped) or enveloped. The classification of viruses is complex and based on many factors, including type and structure of the nucleoid and capsid, the presence of an envelope, the replication cycle, and the host range. Virology ), MHC brings endogenous antigens to the surface, presenting them to CD8+ T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions . 
• Structure: 1 short and 1 long chain (ɑ chain with 3 domains: ɑ1,  ɑ2,  ɑ3), associated with the β₂-microglobulin
• Found only on certain immune cells (APCs)
• Present exogenous antigens (e.g., bacterial proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis ) to CD4+ T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions
• Structure: 2 ɑ and 2 β chains of equal length

Structures of MHC I and MHC II: MHC I has 1 short and 1 long chain (ɑ chain with 3 domains: ɑ 1 , ɑ 2 , and ɑ 3 ), associated with the β₂-microglobulin. MHC II has 2 ɑ and 2 β chains. The peptide antigen goes to the antigen-binding cleft. MHC: major histocompatibility complex

Routes of antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination presentation

• Proteasomes degrade proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis (within the cell) into peptides. 
• Peptide fragments are transported (via transporter associated with antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination processing) to the ER.
• In the ER, aminopeptidases Aminopeptidases A subclass of exopeptidases that act on the free n terminus end of a polypeptide liberating a single amino acid residue. Proteins and Peptides further trim the peptides.
• Antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination peptides are then loaded onto the MHC I molecules → to the Golgi apparatus for posttranslational modification
• Then the complexes are transported to the cell surface, where they are presented to CD8+ T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions . 
• Antigen-presenting cells Antigen-presenting cells A heterogeneous group of immunocompetent cells that mediate the cellular immune response by processing and presenting antigens to the T-cells. Traditional antigen-presenting cells include macrophages; dendritic cells; langerhans cells; and B-lymphocytes. Follicular dendritic cells are not traditional antigen-presenting cells, but because they hold antigen on their cell surface in the form of immune complexes for b-cell recognition they are considered so by some authors. Adaptive Immune Response take up extracellular antigens and are engulfed within phagosomes. 
• Phagosomes fuse with lysosomes Lysosomes A class of morphologically heterogeneous cytoplasmic particles in animal and plant tissues characterized by their content of hydrolytic enzymes and the structure-linked latency of these enzymes. The intracellular functions of lysosomes depend on their lytic potential. The single unit membrane of the lysosome acts as a barrier between the enzymes enclosed in the lysosome and the external substrate. The activity of the enzymes contained in lysosomes is limited or nil unless the vesicle in which they are enclosed is ruptured or undergoes membrane fusion. The Cell: Organelles (containing proteolytic enzymes Proteolytic enzymes Proteins and Peptides that cleave the phagocytosed proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis into small peptides).
• Newly synthesized MHC II molecules have the invariant chain, which binds the antigen-binding cleft.
• With the site occluded, other ER-resident peptides cannot bind BIND Hyperbilirubinemia of the Newborn the cleft. 
• From the ER, the invariant chain directs the MHC II complex to the acidified endosome Endosome Cytoplasmic vesicles formed when coated vesicles shed their clathrin coat. Endosomes internalize macromolecules bound by receptors on the cell surface. Hepatitis C Virus (where antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination peptides are). 
• In the endosome Endosome Cytoplasmic vesicles formed when coated vesicles shed their clathrin coat. Endosomes internalize macromolecules bound by receptors on the cell surface. Hepatitis C Virus , the invariant chain is released → peptides are loaded onto MHC II complexes (chaperoned by HLA-DM) 
• Peptide-loaded MHC II complexes are transported to the cell surface, allowing antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination presentation to CD4+ T cells T cells Lymphocytes responsible for cell-mediated immunity. Two types have been identified – cytotoxic (t-lymphocytes, cytotoxic) and helper T-lymphocytes (t-lymphocytes, helper-inducer). They are formed when lymphocytes circulate through the thymus gland and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen. T cells: Types and Functions .

Routes of antigen presentation by MHC class I and II molecules: In class I antigen presentation (left), proteasomes degrade endogenous antigens or proteins (within the cell) into peptides. Peptide fragments are transported (via transporter associated with antigen processing (TAP)) to the ER, where they are further trimmed by aminopeptidases and loaded onto the MHC class I molecule. MHC class I–loaded complexes go to the Golgi apparatus for posttranslational modification. Then the complexes are transported to the cell surface, where they are presented to CD8+ T cells. IN class II antigen presentation (right), extracellular/exogenous antigens are taken up within phagosomes by antigen-presenting cells. The phagosomes then fuse with proteolytic enzyme-filled lysosomes. This results in the breakdown of phagocytosed proteins into small peptides. Meanwhile, in the endoplasmic reticulum (ER), new MHC class II molecules are synthesized. These molecules have the invariant chain (pink structure in the right image, marked Ii), which binds the antigen-binding cleft. With the cleft occluded (by the invariant chain), ER-resident peptides cannot bind. The invariant chain directs the MHC II complex to the acidified endosome (where antigen peptides are) as it exits from the ER. When MHC II complexes are delivered to the endosome, the invariant chain is released, allowing loading of antigen peptides (chaperoned by a protein, HLA-DM) onto the MHC class II molecules. Once loaded, the formed antigen peptide-MHC class II complexes are brought to the cell surface, ready to present the antigen to CD4+ T cells. Ii: MHC class II–associated invariant chain MIIC: MHC class II compartment

Antigen-presenting–cell and T-cell interaction: Antigen-presenting cell interacts with T cell via signal 1 (T-cell receptor binding the cognate antigen presented by MHC molecule in the APC) and signal 2 (costimulatory molecule interaction between APC and T cell). With the proper antigen presentation, the mature T cell becomes activated.

MHC I versus MHC II

Related diseases.

The HLA region encodes several molecules that perform key functions in the immune system Immune system The body’s defense mechanism against foreign organisms or substances and deviant native cells. It includes the humoral immune response and the cell-mediated response and consists of a complex of interrelated cellular, molecular, and genetic components. Primary Lymphatic Organs . There is a robust association between the HLA region and several diseases.

• Severe congenital neutropenia Congenital neutropenia Severe Congenital Neutropenia ( SCN SCN Severe congenital neutropenia (SCN) affects myelopoiesis and has many different subtypes. Scn manifests in infancy with life-threatening bacterial infections. Severe Congenital Neutropenia ): condition with a deficiency of neutrophils. Severe congenital neutropenia Congenital neutropenia Severe Congenital Neutropenia manifests in infancy with life-threatening bacterial infections Infections Invasion of the host organism by microorganisms or their toxins or by parasites that can cause pathological conditions or diseases. Chronic Granulomatous Disease . Kostmann disease (SCN3) has an autosomal recessive inheritance Autosomal recessive inheritance Autosomal Recessive and Autosomal Dominant Inheritance pattern, whereas the most common subtype (SCN1) shows autosomal dominant inheritance Autosomal dominant inheritance Autosomal Recessive and Autosomal Dominant Inheritance . The most common cause is a mutation Mutation Genetic mutations are errors in DNA that can cause protein misfolding and dysfunction. There are various types of mutations, including chromosomal, point, frameshift, and expansion mutations. Types of Mutations in the ELANE gene Gene A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms. Basic Terms of Genetics . The treatment proven to be effective is the administration of granulocyte colony-stimulating factor Granulocyte colony-stimulating factor A glycoprotein of mw 25 kda containing internal disulfide bonds. It induces the survival, proliferation, and differentiation of neutrophilic granulocyte precursor cells and functionally activates mature blood neutrophils. Among the family of colony-stimulating factors, G-CSF is the most potent inducer of terminal differentiation to granulocytes and macrophages of leukemic myeloid cell lines. White Myeloid Cells: Histology , which elevates the decreased neutrophil count.
• Chediak-Higashi syndrome ( CHS CHS Cannabinoids ): autosomal recessive Autosomal recessive Autosomal inheritance, both dominant and recessive, refers to the transmission of genes from the 22 autosomal chromosomes. Autosomal recessive diseases are only expressed when 2 copies of the recessive allele are inherited. Autosomal Recessive and Autosomal Dominant Inheritance disorder that is caused by mutations affecting a lysosomal trafficking regulator Lysosomal trafficking regulator Chédiak-Higashi Syndrome protein. This mutation Mutation Genetic mutations are errors in DNA that can cause protein misfolding and dysfunction. There are various types of mutations, including chromosomal, point, frameshift, and expansion mutations. Types of Mutations plays a crucial role in the inability of neutrophils to kill phagocytosed microbes. NK-cell hyporesponsiveness is also noted in some cases. Individuals with CHS CHS Cannabinoids exhibit recurrent pyogenic infections Infections Invasion of the host organism by microorganisms or their toxins or by parasites that can cause pathological conditions or diseases. Chronic Granulomatous Disease , easy bleeding and bruising, and neurologic manifestations. 
• Chronic granulomatous disease Granulomatous disease A defect of leukocyte function in which phagocytic cells ingest but fail to digest bacteria, resulting in recurring bacterial infections with granuloma formation. When chronic granulomatous disease is caused by mutations in the cybb gene, the condition is inherited in an X-linked recessive pattern. When chronic granulomatous disease is caused by cyba, ncf1, ncf2, or ncf4 gene mutations, the condition is inherited in an autosomal recessive pattern. Common Variable Immunodeficiency (CVID) ( CGD CGD Chronic granulomatous disease (CGD), as the name implies, is a chronic disorder that is characterized by granuloma formation. This disorder is a consequence of defective phagocytic cells that are unable to produce bactericidal superoxide because of a defect in nicotinamide adenine dinucleotide phosphate (NADPH), the oxidase responsible for the respiratory burst in phagocytic leukocytes. Chronic Granulomatous Disease ): genetic condition characterized by recurrent severe bacterial and fungal infections Infections Invasion of the host organism by microorganisms or their toxins or by parasites that can cause pathological conditions or diseases. Chronic Granulomatous Disease , and granuloma formation. Defective nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide A coenzyme composed of ribosylnicotinamide 5′-diphosphate coupled to adenosine 5′-phosphate by pyrophosphate linkage. It is found widely in nature and is involved in numerous enzymatic reactions in which it serves as an electron carrier by being alternately oxidized (NAD+) and reduced (NADH). Pentose Phosphate Pathway phosphate Phosphate Inorganic salts of phosphoric acid. Electrolytes ( NADPH NADPH Nicotinamide adenine dinucleotide phosphate. A coenzyme composed of ribosylnicotinamide 5′-phosphate (nmn) coupled by pyrophosphate linkage to the 5′-phosphate adenosine 2. Pentose Phosphate Pathway ) oxidase Oxidase Neisseria (responsible for the respiratory burst Respiratory burst A large increase in oxygen uptake by neutrophils and most types of tissue macrophages through activation of an NADPH-cytochrome b-dependent oxidase that reduces oxygen to a superoxide. Individuals with an inherited defect in which the oxidase that reduces oxygen to superoxide is decreased or absent often die as a result of recurrent bacterial infections. Leukocyte Adhesion Deficiency Type 1 ) in neutrophils and macrophages leads to impaired phagocytosis. Infections Infections Invasion of the host organism by microorganisms or their toxins or by parasites that can cause pathological conditions or diseases. Chronic Granulomatous Disease commonly affect the lung, skin Skin The skin, also referred to as the integumentary system, is the largest organ of the body. The skin is primarily composed of the epidermis (outer layer) and dermis (deep layer). The epidermis is primarily composed of keratinocytes that undergo rapid turnover, while the dermis contains dense layers of connective tissue. Skin: Structure and Functions , lymph nodes Lymph Nodes They are oval or bean shaped bodies (1 – 30 mm in diameter) located along the lymphatic system. Lymphatic Drainage System: Anatomy , and liver Liver The liver is the largest gland in the human body. The liver is found in the superior right quadrant of the abdomen and weighs approximately 1.5 kilograms. Its main functions are detoxification, metabolism, nutrient storage (e.g., iron and vitamins), synthesis of coagulation factors, formation of bile, filtration, and storage of blood. Liver: Anatomy . A neutrophil function test, dihydrorhodamine (DHR) 123, is abnormal, and genotyping Genotyping Methods used to determine individuals’ specific alleles or snps (single nucleotide polymorphisms). Polymerase Chain Reaction (PCR) confirms the diagnosis.
• Castell-Rodríguez, A., Piñón-Zárate, G., Herrera- Enríquez, M., Jarquín-Yáñez, K., Medina-Solares, I. (2017). Dendritic cells: location, function, and clinical implications. In: Biology of Myelomonocytic Cells. https://www.intechopen.com/chapters/54824
• Chaplin, D.D. (2010). Overview of the immune response. J Allergy Clin Immunol 125(2 Suppl 2):S3–S23. https://pubmed.ncbi.nlm.nih.gov/20176265/
• Haynes, B.F., Soderberg, K.A., Fauci, A.S. (2018). Introduction to the immune system. Chapter 342 of Harrison’s Principles of Internal Medicine, 20th ed. McGraw-Hill. https://accessmedicine.mhmedical.com/content.aspx?bookid=2129&sectionid=192284326
• Johnston, R.B. (2021). An overview of the innate immune system. UptoDate. Retrieved July 2, 2021, from https://www.uptodate.com/contents/an-overview-of-the-innate-immune-system
• Levinson, W., Chin-Hong, P., Joyce, E.A., Nussbaum, J., Schwartz, B. (Eds.). (2020). Innate immunity. Chapter 58 of Review of Medical Microbiology & Immunology: A Guide to Clinical Infectious Diseases, 16th ed. McGraw-Hill. https://accessmedicine.mhmedical.com/content.aspx?bookid=2867&sectionid=242768129
• Maglione, P.J., Simchoni, N., Cunningham-Rundles, C. (2015). Toll-like receptor signaling in primary immune deficiencies. Ann NY Acad Sci 1356(1):1-21. https://pubmed.ncbi.nlm.nih.gov/25930993/
• Neerincx, A., Castro, W., Guarda, G., Kufer, T. (2013). NLRC5, at the heart of antigen presentation. Frontiers in Immunology. https://pubmed.ncbi.nlm.nih.gov/24319445/  
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The Role of Antigen Processing and Presentation in Cancer and the Efficacy of Immune Checkpoint Inhibitor Immunotherapy

Anastasia mpakali.

1 National Centre for Scientific Research Demokritos, Agia Paraskevi, 15341 Athens, Greece

Efstratios Stratikos

2 Laboratory of Biochemistry, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis Zographou, 15784 Athens, Greece

Associated Data

Not applicable.

Simple Summary

A new class of drugs, termed Immune Checkpoint Inhibitors, has revolutionized cancer therapy during the last few years. Unfortunately, these drugs are only effective for a subset of patients and cancer types. Recent work has suggested that how well cancer cells present some of their molecules to the immune system is critical for patient responses to immunotherapy with immune checkpoint inhibitors. Here, we review the role of the biochemical pathway of antigen presentation in cancer and discuss how it can be modulated to enhance the efficacy of cancer immunotherapy.

Recent clinical successes of cancer immunotherapy using immune checkpoint inhibitors (ICIs) are rapidly changing the landscape of cancer treatment. Regardless of initial impressive clinical results though, the therapeutic benefit of ICIs appears to be limited to a subset of patients and tumor types. Recent analyses have revealed that the potency of ICI therapies depends on the efficient presentation of tumor-specific antigens by cancer cells and professional antigen presenting cells. Here, we review current knowledge on the role of antigen presentation in cancer. We focus on intracellular antigen processing and presentation by Major Histocompatibility class I (MHCI) molecules and how it can affect cancer immune evasion. Finally, we discuss the pharmacological tractability of manipulating intracellular antigen processing as a complementary approach to enhance tumor immunogenicity and the effectiveness of ICI immunotherapy.

1. The Immune System and Cancer

The interplay between the immune system and cancer termed “cancer immunoediting” is a dynamic and continuously evolving process in which immune responses can eradicate tumor cells but also promote tumor progression through selective pressures [ 1 , 2 ]. The temporal evolution of the immune system–cancer interaction is usually considered to consist of at least three phases termed elimination, equilibrium, and escape [ 3 ]. During the elimination phase, often at the initial stages of carcinogenesis, the immune system aggressively destroys newly formed cancer cells. If this attack is successful in eliminating all pre-cancerous and cancerous cells, no clinically detectable tumors are formed. Failure to eliminate all cancer cells however can result in establishment of an equilibrium phase in which the immune system controls tumor growth but cannot fully eliminate it [ 4 ]. This phase is considered to include some degree of immune evasion and can lead to a strong selective pressure on cancer cells to mutate in ways to further avoid the immune surveillance either by becoming less immunogenic or by inducing a localized immunosuppressive state. Success in these processes leads to the escape phase that allows out-of-control cancer cell growth and the appearance of clinically visible tumors that are characterized by different mechanisms and magnitudes of immune evasion and suppression [ 5 ].

2. Mechanisms of Cancer Immune Evasion and the Role of Immune Checkpoints

Tumors can attempt to evade cellular immune responses either by excluding T cells from the tumor microenvironment (TME) or by establishing equilibrium with T cells that successfully migrate to the tumor [ 6 ]. The former mechanism, termed innate evasion, includes accumulation of defects in T cell priming and reduced intratumoral trafficking through aberrant cell-intrinsic signaling events. Such events include activation of the Wnt/β-catenin pathway [ 7 ], loss of function of PTEN [ 8 ], c-Myc signaling dependent activation [ 9 ], and loss of LKB1 signaling [ 10 ]. The latter mechanism, termed adaptive immune evasion can emerge from the selection of tumor cell clones that have progressively reduced their immunogenicity through the loss of expression of key tumor-specific antigens and/or the accumulation of mutations in genes involved in immune recognition [ 11 , 12 , 13 ]. Notably, loss of MHCI expression is a common mechanism utilized by tumors attempting to evade T cell cytotoxic responses [ 14 , 15 ]. Loss of immune signaling can also synergize with loss of antigenicity by interfering with interferon generation and function [ 16 , 17 ]. In general, synergism between innate and adaptive immune evasion can result to a major therapeutic challenge that may only be overcome by combining separate approaches that, in tandem, address problems in both the TME as well as tumor immunogenicity.

T-cell-mediated immunity is regulated by a balance between stimulatory and inhibitory signals [ 18 ]. After encountering their cognate antigen, T cells, via their CD28 receptor, are activated by stimulatory signals in the context of antigen presenting cells (APCs) in order to attack and eliminate cancerous cells. However, their inflammatory activity must then be diminished to preserve immune homeostasis. The inhibitory signals are provided by molecules called immune checkpoints (ICs) that, when activated, suppress T cell activity [ 19 ]. These molecules are receptors located on the surface of T lymphocytes that regulate the extent and duration of physiological immune responses and therefore limit tissue damage and maintain self-tolerance. Several inhibitory checkpoint molecules have been discovered to date, such as CTLA-4 (cytotoxic T lymphocyte-associated protein 4) , PD-1 (programmed cell death protein 1), LAG-3 (lymphocyte activation Gene-3), TIM-3 (T-cell immunoglobulin and mucin-domain containing 3), TIGIT (T cell immunoglobulin and ITIM domain), VISTA (V-Domain Ig Suppressor of T-Cell Activation), B7-H3, BTLA (B and T lymphocyte attenuator 4), and Siglec-15 [ 20 ]. CTLA-4 and PD-1 are the most well studied and play central roles in state-of-the-art immunotherapy strategies. CTLA-4 is upregulated immediately after TCR engagement and through its competition with the co-stimulatory molecule CD28 for the B7 ligands (CD80/B7.1 and CD86/B7.2) of the APCs, it limits autoreactive T cells early at their activation stage leading to immune tolerance and prevention of autoimmunity [ 21 , 22 ]. Besides its surface expression being upregulated, additional CTLA-4 is recruited to the immunologic synapse via intracellular vesicles to further dampen T cell receptor (TCR) signaling [ 23 ]. Through the recruitment of phosphatases, CTLA-4 interferes with the TCR-induced stop signal for stable immune conjugate formation, leading to fewer contact periods between T cells and APCs and finally to decreased T cell priming and proliferation. The CTLA-4 suppressive functions can be also mediated by regulatory T cells (T regs ), as it is expressed on their surface [ 24 , 25 ]. Recently, it was shown that CTLA-4 can deplete, through trans-endocytosis, available CD80 and CD86 ligands from the membranes of neighboring APCs to prevent their interaction with CD28 on T cells [ 26 ]. PD-1 is also expressed on activated T cells but acts at later stages of an immune response and interferes with previously activated T cells. If the stimulating antigen is cleared, PD-1 expression levels decrease on responding T cells whilst in the opposite case, its expression remains elevated. PD-1 has two tyrosine motifs in its cytoplasmic tail. Through its interaction with its ligands, PD-L1 and PD-L2, PD-1 is phosphorylated at these tyrosine residues, which leads to phosphatase recruitment. These phosphatases can then dephosphorylate downstream kinases and antagonize positive signals that take place through TCR and CD28, affecting TCR-mediated downstream signaling. The final outcome is impaired T cell activation, survival, cytokine production, and altered metabolism [ 27 ]. Sustained expression of PD-1 is considered to render T cells exhausted. PD-L1 expression is induced in response to inflammatory cytokines, such as IFNγ, and thus PD-1 regulation of T cell activity occurs in response to cytolytic and effector T cell function [ 28 ].

Signaling through PD-1 is a common mechanism that tumors utilize in order to put T cells in check and escape immunosurveillance. This can be achieved by upregulating PD-L1 expression on tumor cells themselves or on stromal and immune cells in the TME [ 25 , 29 ]. In mouse tumor cells, the upregulated expression of PD-L1 has been associated with impaired T cell mediated antitumor responses [ 30 , 31 , 32 ]. The combination of these findings with the recognition of ICs as negative regulators of T cell activation, gave rise to the idea that blocking the inhibitory action of ICs on T cells by using specific monoclonal antibodies, could improve T cell functions and enhance immune responses against cancer [ 33 ]. These pioneering cancer therapy approaches have now shifted the focus from attacking the tumor to assisting the host’s immune system to attack cancer cells. The presence of pre-existing cancer-specific T cells capable of recognizing tumor-specific antigens and neoantigens has been considered a necessary premise for this therapeutic approach [ 34 ]. However, although for many years the main mechanism of action of ICIs has been considered to be the re-activation of primed T cells, recently the role of novel T cells that are primed and recruited to the tumors after the initiation of immunotherapy has been emerging [ 35 , 36 ]. Regardless of the exact mechanism, the main advantage of ICI therapy is that it can induce durable responses representative of tumor-specific immunological memory formation [ 37 ]. Several antibody ICIs have already been FDA-approved since 2011 and have shown clinical efficacy in many cancer types ( Table 1 ) [ 38 ].

FDA-approved immune checkpoint inhibitors.

* RCC: Renal cell carcinoma, HCC: Hepatocellular carcinoma, NSCLC: Non-small cell lung cancer, SCLC: Small cell lung cancer, HNSCC: Head and neck squamous cell cancer, cHL: classical Hodgkin Lymphoma, TNBC: Triple negative breast cancer, MCC: Merkel cell carcinoma.

CTLA-4 blockade aims to induce robust activation of tumor reactive T cells. By sterically hindering the interaction of CTLA-4 receptor with B7 ligands, it leads to unrestrained CD28-mediated positive co-stimulation of T lymphocytes. The co-crystal structure of the first approved antibody against CTLA-4, ipilimumab, in complex with CTLA-4 revealed that the epitope recognized by ipilimumab overlaps with the B7 interaction domain [ 39 ]. Additionally, CTLA-4 blocking promotes antitumor responses through the deletion of T regs via antibody mediated cytotoxicity, as demonstrated in murine cancer models [ 40 , 41 ]. CTLA-4 inhibition is also able to broaden and remodel the peripheral TCR repertoire, as it was observed in cancer patients undergoing ipilimumab treatment [ 42 ]. Loss of CTLA-4 may lower the threshold for TCR ligation required for effective T cell activation since CTLA-4 normally acts to dampen TCR signal strength [ 25 ]. Blockade with either a-PD-1 or a-PD-L1 antibodies abrogates inhibition of TCR signaling and removes the brakes from T cells, unleashing their effector properties, while it also appears to be able to restore the function of exhausted T cells [ 43 , 44 ]. PD-1 blockade seems to be more effective in tumors infiltrated by tumor antigen-specific T cells that express PD-1 receptor but were kept in an inactive state due to the interaction of PD-1 with its ligands expressed by tumor cells and stromal cells within the TME [ 45 ].

3. ICI Therapy Failure and Tumor Immunogenicity

Although immunotherapy with ICIs holds much promise for durable outcomes in cancer regression and in some cases even cure, the majority of patients do not benefit by this course of treatment and either do not respond (innate/primary resistance) or relapse after an initial period of response (acquired/adaptive resistance) [ 46 ]. Emerging evidence from studies with patients treated with cancer immunotherapies indicates that the mechanisms of resistance broadly overlap with those normally used by cancers as they undergo immunoediting [ 47 ]. Several tumor cell-intrinsic and cell-extrinsic factors contribute to the resistance to therapy, leading to three different outcomes: i) insufficient generation of antitumor T cells, ii) inadequate function of tumor-specific T cells, and iii) impaired formation of T cell memory [ 46 , 48 ]. Amongst them, the immunogenicity of a tumor is considered to be a critical determinant of response to ICI therapy, as tumors devoid of tumor-specific antigens can never be recognized as foreign [ 49 ]. Anagnostou and colleagues examined the evolving landscape of tumor neoantigens during the acquisition of resistance to ICIs in non-small cell lung cancer (NSCLC) patients, and attributed this resistance to loss of mutations encoding cancer-specific neoantigens [ 50 ]. However, even a high mutational and neoantigen burden cannot lead to efficacious response if the patients’ cells lack a functional machinery for tumor antigen processing and presentation [ 46 , 51 ] since generation of reactive CD8+ T cells requires successful antigen processing and presentation of tumor-specific antigens [ 48 ].

4. Antigen Processing and Presentation in Cancer

For T cells to recognize malignant cells and attack them, two conditions are essential. First, the tumor cells have to report their intracellular changes on their surface and second, these changes must be sensed by T lymphocytes. The cellular mechanism that determines this direct interaction between the cancer and the adaptive immune system is the antigen processing and presentation pathway (APP) [ 52 ]. CD8+ T cells, via their TCR, can only detect aberrant cells in the context of peptide-MHC class I complexes. MHCI complexes are expressed on professional APCs that can activate naïve T cells, but also on all nucleated healthy and infected or transformed cells [ 53 ].

For a peptide to serve as an epitope and therefore be capable of inducing an effective adaptive response, it has to be first processed by the cellular antigen processing machinery (APM) and then loaded onto an MHCI molecule ( Figure 1 ). The processing and presentation pathway is a multi-step process in the context of the normal turnover of cellular proteins and often starts in the cytoplasm. There, intracellular proteins are ubiquitinated and fragmented into smaller pieces by the proteasome. An alternative pathway includes the proteasomal degradation of aberrant or misfolded proteins termed Defective Ribosomal Products (DRiPs) [ 54 ]. The constitutive proteasome is a barrel-shaped structure consisting of a catalytic 20S four-stacked ring core with chymotrypsin, trypsin, and caspase-like activities that is capped at each end by a regulatory 19S cap complex responsible for de-ubiquitination and unfolding of the trapped proteins that enter the main catalytic core. After exposure of the cells to inflammatory cytokines that generally enhance antigen presentation, new catalytic subunits named LMP2, LPM7, and LMP10 are produced and substitute these of the 20S proteasome to generate the immunoproteasome. This transition has been linked to changes in cleavage specificity, efficiency of MHCI ligand generation, and MHCI repertoire quantity [ 55 ]. Proteasomal cleavage generates peptides 2–26 residues long with a C-terminus anchor residue compatible with MHCI binding groove, but often extended at their N-terminus [ 55 ]. Peptides are then released in the cytosol and if they survive further degradation by cytosolic peptidases, are transferred into the endoplasmic reticulum (ER) by the Transporter associated with Antigen Processing, TAP. The TAP heterodimer, consisting from TAP1 and TAP2 subunits, forms a transmembrane pore in the ER membrane and preferentially transfers peptides 9–16 residues long, although longer peptides can be also transferred with much lower efficiency [ 56 ]. After a peptide enters the ER, its final assembly onto a nascent MHCI molecule is mainly orchestrated by a multi-subunit complex, called the Peptide Loading Complex (PLC) [ 57 ]. TAP constitutes an integral part of PLC, where it acts as a docking site for the MHC class I dedicated chaperone tapasin, and three other ER chaperones, the lectin calreticulin, calnexin, and the disulfide isomerase ERp57. Calnexin is important for early folding and oxidation of newly synthesized MHC heavy chain [ 56 ]. MHCI molecules are heterodimeric glycoproteins consisting of a polymorphic heavy chain (in humans encoded by the Human Leukocyte Antigen-HLA A, B, and C genes) and an invariable light chain, β2 microglobulin (β2m). MHCI molecules have a groove that can preferentially bind 8-11mer peptides. The exposed surface of this groove where the antigenic epitope is bound is the part of the MHCI complex that is recognized by the TCR [ 58 ]. Peptides that enter the ER and are too long to fit into MHCI are trimmed by the concerted action of two ER-resident aminopeptidases, ERAP1 and ERAP2 [ 59 ]. Calreticulin in combination with ERp57 assist with the folding and stabilization of the newly synthesized empty MHCI molecules. Tapasin mediates the recruitment of MHCI to the PLC and enables peptide loading and exchange, facilitating the formation of MHCI molecules with high affinity peptides. However, even after a peptide is loaded onto an MHCI molecule, an additional chaperone, TAP binding protein related protein, TAPBPR, assists with quality control to ensure peptide stable binding [ 60 ]. Having acquired a suitable peptide, the MHC class I molecule traffics to the cell surface through the Golgi network for presentation to T cells [ 56 ].

Overview of the Major Histocompatibility Class (MHC) class I pathway of antigen processing and presentation and the alternative pathway of cross-presentation.

Tumors are particularly immunogenic and presentation of their specific antigens to T cells in an MHCI-restricted manner would lead to their eradication. Defects and alterations in the components of the APM are often found in tumors as cancer progression requires tumor cells to acquire the ability to avoid immune recognition [ 61 ]. Alterations in tumor APM can result not only in the downregulation of cell-surface expression MHCI molecules but can also alter the repertoire of antigenic peptides presented to the T lymphocytes. Since successful treatment with ICIs relies on re-activation of T cells, alterations in antigen processing and presentation of antigens can result to impaired antitumor responses and therapy resistance [ 16 , 62 , 63 ].

Alterations in antigen processing and presentation pathway may occur at any step of synthesis, assembly, transport, and surface expression of MHCI molecules or at any step of antigen editing ( Figure 2 ). Truncating alterations, loss of heterozygosity, frameshift, and loss-of-function mutations affecting the β2m protein in human tumor cells that lead to instability of MHCI complexes, impaired folding, and diminished transport to the cell surface, have been associated with resistance to ICI therapy. In lung cancer patients, disruption of MHCI-mediated antigen presentation due to β2m loss of heterozygosity conferred resistance to PD-1 blockade therapy [ 64 ]. In melanoma metastatic patients treated with checkpoint inhibitors, point mutations, deletions, truncations, and loss of heterozygosity in β2m have been associated with resistance to ICI immunotherapy [ 16 , 62 ]. Furthermore, loss of expression of thiol reductase ERp57 has been demonstrated in several tumor types to correlate with poor prognosis [ 65 , 66 , 67 ]. Downregulation of calreticulin expression has been observed in colorectal and bladder cancers as well as in myeloproliferative neoplasms and has been associated with impaired antigen processing and presentation [ 68 , 69 , 70 ]. Defects have also been found in the IFNγ-inducible proteasome components [ 66 , 71 , 72 ]. Loss or downregulation of the transporter TAP have also been recorded in many cancer cell lines and primary tumors [ 72 , 73 , 74 ]. In all these cases, patients had a poor disease prognosis and diminished MHCI surface expression on tumor cells that correlated with changes in their antigenic peptide repertoire. In melanoma cells, micro-RNA downregulation of TAP expression led to reduction of MHCI surface expression and decreased T cell recognition [ 75 ]. In addition, the expression of Tapasin, another important APM component, has been found altered in several types of cancer [ 76 , 77 , 78 ]. MHCI surface expression was significantly decreased in all these patients and correlated with tumor progression. Impaired tapasin function led to MHCI molecules loaded with low-affinity, suboptimal antigenic epitopes. Furthermore, it reduced antigen presentation of tumor-specific antigens and blocked the presentation of certain immunodominant epitopes [ 77 , 78 , 79 ]. Mutations in tapasin and structural defects in IFNγ-related genes were found in recurrent metastatic melanoma with disease progression after active immunotherapy [ 80 ]. Furthermore, endoplasmic reticulum aminopeptidases, ERAP1 and ERAP2, exhibit variable expression levels in different cancer types [ 81 ]. Although mutations in these enzymes are rare, their expression in cancer is often either downregulated or upregulated while SNPs affecting their enzymatic activity can influence the immunopeptidome presented by MHCI molecules [ 82 , 83 , 84 , 85 , 86 , 87 ]. As these enzymes can both trim and destroy epitopes destined for binding onto MHCI molecules, their expression levels and activity strongly influence the peptide pool available for loading onto MHCI and can thus affect the immunogenicity of tumors [ 88 , 89 ]. In some cancers, ERAP1 overexpression leads to destruction of tumor-specific immunodominant epitopes and induction of anti-tumor CD8+ responses, linking antigen destruction with tumor escape [ 90 , 91 , 92 ]. In other cases, ERAP1 downregulation can lead to cancer rejection through Natural Killer cell mediated cytotoxicity [ 93 , 94 ]. ERAP2 overexpression in patients with oral cavity squamous cell carcinoma has been associated with metastasis from the primary tumor and poor prognosis [ 95 ], while the absence of ERAP2 in choriocarcinoma cells reduced their ability to activate T lymphocytes [ 96 ]. Downregulation of the mouse homologue, ERAAP, in mouse tumors increased the efficacy of a-PD1 blockade therapy [ 97 ]. In bladder cancer patients receiving a-PD1 therapy, expression quantitative trait loci affecting the expression of both ERAP1 and ERAP2 were found to associate with favored response to therapy and prolonged survival, probably due to alterations in the repertoire of peptides available for presentation to T cells [ 98 ]. Recently, functional ERAP1 allotypes have been correlated with tumor-infiltration by CD8+ T cells in cervical and oropharyngeal squamous cell carcinomas due to changes in processing of particular antigenic epitopes [ 99 ]. Overall, intracellular antigen processing is emerging as a master regulator of the immunogenicity of cancer [ 100 , 101 ].

Defects in different components of the antigen processing and presentation machinery that can underlie immune evasion by cancer.

As most components of the APP machinery are IFNγ inducible, defects in IFNγ signaling cascade can limit MHCI surface expression. The main proteins that interfere with this pathway are the transcription factors IFN-regulatory factor 1 (IRF-1) and STAT-1, and the kinases Janus-associated kinase JAK-1 and JAK-2. Tumor cells with activated IFNγ pathways can respond to cytokine secretion by immune cells located into the TME and become visible to T cells. Multiple studies have linked defects in IFNγ signaling with resistance to ICI therapy [ 16 , 17 , 102 , 103 ]. Genetic analysis of tumors from patients with melanoma and colon cancer who did not respond to PD1 blockade therapy despite their high mutation burden and high percentage of pre-existing tumor specific T cells, were identified to acquire loss of function mutations in JAK1/2 kinases and decreased MHCI surface expression [ 102 , 104 ]. Although indirectly associated to the IFNγ pathway, loss of the protein tyrosine phosphatase Ptpn2 was correlated to enhanced levels of antigen-loaded MHCI molecules on the surface of tumors and to sensitivity of tumor cells to immunotherapy [ 97 ].

Epigenetic events in cancer cells can regulate the expression of immune-related genes, resulting in changes in antigen processing and presentation that impair tumor recognition [ 105 ]. DNA methylation and histone modifications of MHCI heavy chain gene promoters leads to transcriptional silencing and decreased MHCI surface expression, causing impaired antigen presentation and immune evasion [ 106 , 107 ]. Additional components of the APM machinery have been found to be epigenetically regulated in many cancer types [ 107 , 108 ]. In melanoma, increased histone methyltransferase Ezh2 expression during a-CTLA-4 immunotherapy, decreased the antigen presentation ability of cancer cells while its inactivation reversed the resistance to therapy and synergistically suppressed tumor growth [ 109 ]. In prostate cancer, epigenetic silencing of the crucial component JAK1 kinase of the IFNγ signaling pathway led to IFNγ-insensitivity-mediated tumor evasion and resistance to immunotherapy [ 110 ]. Moreover, methylation of the NLRC5 MHCI trans-activator caused suppression of MHCI molecules and other components of the APM machinery in mice and an impaired ability to induce CD8+ T cell activation in cells [ 111 ]. In addition, Merkel cell carcinoma patients with low expression of APM components that was mediated by histone deacetylation, were resistant to a-PD1 therapy [ 112 , 113 ]. Antigen presentation efficiency is also diminished in human tumors characterized by large chromosomal instability and structural alterations. Although these tumors initially show induction of MHCI-restricted antigen presentation due to activation of cGAS/STING cytosolic DNA sensing pathway that detects tumor derived DNA and other pro-inflammatory signaling pathways, as they evolve under immune pressure, they suppress their antigen presentation machinery and adopt an immunologically poor phenotype. An experimental model of such tumor aneuploidy revealed that the suppression of antigen processing and presentation genes can be at least partly attributed to DNA hypermethylation of the corresponding genes, while the expression level of DNA methylotransferases was found significantly elevated [ 114 ].

5. MHCI Expression in Cancers

Downregulation of MHCI favors escape of tumor cells from immune surveillance [ 115 ]. Many studies in tumor cell lines and biopsies from patients reported total or partial loss of MHCI surface expression as a frequent event in cancer [ 72 , 116 , 117 , 118 ]. According to Garrido and colleagues, the loss or downregulation of surface MHCI expression is an active process that takes place gradually as tumors develop [ 119 ]. As such, at the early phase of tumor development, cancer cells are mostly MHCI positive. This induces T cell infiltration at the tumor microenvironment that recognize and kill cancer cells capable of presenting tumor-specific antigens on their MHCI molecules. Gradually, a vast diversity of tumor clones with variable MHCI surface expression levels is generated. A Darwinian type T cell-mediated selective pressure leads to tumors characterized by the presence of only MHCI negative cancer cells. This phase is accompanied by dramatic changes of the tumor tissue architecture that prevents T cells from entering the cancer niche as they are retained in the surrounding stroma [ 119 , 120 ]. This immune selection of MHCI-negative tumor cells has been demonstrated after immunotherapy in cancer patients and in experimental cancer models as the therapeutic application of checkpoint blockade increases the selective pressure towards tumor cells [ 121 ].

Apart from mutations, epigenetic modifications and structural alterations, tumors can adopt additional mechanisms to decrease their MHCI surface expression. A phenomenon often observed in tumors is the surface expression of non-classical MHCI molecules, such as HLA-G, HLA-E, and HLA-F. Although these molecules can present antigenic peptides in the context of antiviral defense, it is not clear whether their role in cancer is related to antigen presentation or they function as inhibitory ligands through their interaction with receptors on effector cells [ 122 , 123 ]. MHCI molecules can also be downregulated by other regulatory mechanisms, that involve signal transduction cascades, oncogenes, and tumor suppressor genes. Mutations in the BRAF oncogene (such as the V600E) lead to internalization of MHCI molecules from the cell surface of melanoma tumor cells and its sequestration within endocytic compartments, resulting in impaired recognition by the adaptive immune system [ 124 ]. In addition, autophagy, a conserved nutrient sensing system that induces the degradation of cytoplasmic proteins and damaged organelles by lysosomes can interfere with MHCI surface expression. In pancreatic ductal adenocarcinoma, Yamamoto and colleagues showed that MHCI molecules are selectively targeted for lysosomal degradation by an autophagy-dependent manner leading to alterations of immunogenicity of the tumor and impaired antigen presentation while its inhibition acts in synergism with ICI therapy and results in enhanced antitumor responses [ 125 ]. The SND1 oncoprotein, highly expressed in various cancers, prevents normal assembly of MHCI molecules by leading nascent synthesized MHCI heavy chain to ER-associated degradation (ERAD). Deletion of SND1 in tumor mouse models restores tumor antigen presentation to T cells both in vitro and in vivo and enhances T cell infiltration into the tumors [ 126 ]. Moreover, additional tumor suppressor genes (such as Fhit and p53) and oncogenes (such as Her2), interfere with MHCI expression in cancer cells [ 112 , 127 , 128 ]. In many human cancers, MHCI downregulation also associates with impaired signaling by transcription factors, such as NFkB and IRF2 that regulate activation of transcription of the MHCI heavy chain [ 112 ]. Additionally, IRF2 loss is associated with impaired peptide transport from the cytosol to the ER and peptide trimming [ 129 ]. Recently, it was demonstrated that the RNA binding protein MEX3B is linked with resistance to cancer immunotherapy in melanoma patients, by binding and destabilizing the HLA-A mRNA resulting in decreased HLA-A expression on the surface of tumor cells and thereby protecting the tumor cells by T cell-mediated recognition and elimination [ 130 ]. Finally, several long non-coding RNAs and miRNAs have been shown to modulate MHCI expression levels in several cancers [ 131 , 132 , 133 , 134 ].

In a recently published study, Chowell and colleagues analyzed the impact of individual’s specific MHC class I germline alleles on the clinical outcome of ICI therapy. By carrying out high-resolution MHCI genotyping of two patient cohorts with advanced melanoma and NSCLC that had received treatment with IC molecules, the authors observed that homozygosity in at least one human MHCI locus was linked to reduced survival periods, independently of mutational load, age, tumor stage, or type of therapy. Antigen presenting MHCI molecules are highly polymorphic, especially at their peptide binding grooves, and therefore each allele can bind and present a restricted set of antigenic epitopes. As result/Consequently, individuals homozygous in at least one MHCI locus may present a smaller, less diverse repertoire of tumor antigens to CD8+ T cells and thus may be less likely to present potent epitopes that induce highly effective antitumor responses that can be enhanced by ICI therapy [ 135 , 136 ]. Given that only a small percentage of presented tumor antigens in cancer patients are immunogenic, it seems that even small differences in MHCI molecules can significantly affect the adaptive responses and the efficacy of immunotherapy. This may also explain why MHCI homozygous patients with tumors bearing low neoantigen load show decreased survival and fail to respond to ICI therapeutic strategies compared to heterozygous patients and why specific HLA supertypes are associated with increased immune responsiveness. Moreover, Chowell and colleagues provided an additional link between MHCI heterozygosity and the presentation of a greater variety of tumor-specific antigens. By deep-sequencing of TCRs from tumor samples collected on-therapy, the authors observed enhanced clonality in heterozygous patients, concluding that the diversity of MHCI molecules modulates the selection and the resulting clonal expansion of T cells reactive against neoantigens and tumor-specific antigens after treatment with ICIs [ 137 ]. Accordingly, Marty and coworkers demonstrated that an individual’s MHCI genotype can predict cancer susceptibility as oncogenic mutations found in a tumor were linked to this genotype. Their study suggests that MHCI genotypes can act as a barrier that constrains the possible mutations that a developing tumor can accumulate [ 138 ].

6. Dendritic Cells and Cross-Presentation in Cancer

Although tumor cells often express MHCI molecules on their cell surface they tend to be poor antigen presenters and immune stimulators since they often lack costimulatory molecules and thus cannot effectively stimulate naïve T lymphocytes [ 139 ]. De novo generation and initiation of adaptive immune responses specific to tumor antigens, requires the cross-presentation capability of professional APCs that capture exogenous derived antigens, process and present them in order to prime naïve T cells [ 140 ]. The most potent known APCs are the dendritic cells (DCs), that constitute a heterogeneous cell population subdivided to several different subtypes [ 141 ]. DCs differentiate from bone marrow progenitors and reside in lymphoid and peripheral tissues where they act as sentinels of the immune system. Under steady-state conditions, differentiated DCs are found in their immature form. Immature DCs show a high endocytic potential and capture antigens but express low levels of MHCI and costimulatory molecules and as a result they do not prime T cells, but rather induce immune tolerance [ 142 ]. In order to be able to prime naïve T cells, DCs must first be activated and shift to their mature form. Their maturation is characterized by movement of MHCI to the cell surface, upregulation of the costimulatory molecules CD80 and CD86, higher expression of the C-C chemokine receptor 7 (CCR7), enhanced migration to lymph nodes (LNs), and increased cytokine production that drive T cell stimulation and clonal expansion [ 143 , 144 , 145 ]. DC activation is normally considered to result from detection of pathogen or damage associated molecular patterns (PAMPs/DAMPs) recognized by specific receptors. Within tumors, several of these receptors recognize endogenous DAMPs released or expressed on the surface of dead or dying cells. Immunogenic death of cancer cells, either spontaneously or due to therapeutic interventions, is an active process that releases alarmins and chemotactic factors that attract and activate DCs [ 146 ].

The inflammatory environment of a tumor, which includes cytokines, chemokines, and growth factors, fosters infiltration by DCs [ 147 ]. In tumors, DCs have access to large amounts of tumor antigens. After capturing and processing them through either the cytosolic or the vacuolar pathway, DCs migrate to the draining LNs to present these antigens and prime tumor-specific T cells. Memory and effector T cells return to the tumor site to perform surveillance and killing activities [ 148 ]. Studies with DCs isolated from tumor-bearing mice confirm their ability to cross-present tumor antigens and induce adaptive immune responses [ 149 , 150 ]. Apart from migratory DCs, non-migratory DCs that remain in the tumor may interact with T cells and prime them [ 147 ]. Additionally, by secreting IL-12 and other cytokines, non-migratory DCs can maintain and regulate antitumor responses. Antigen experienced T cells require cognate interactions with tissue DCs presenting antigens at a sufficient dose and duration to expand in situ and achieve their full effector activity [ 151 ]. Additionally, DCs in tumors can also be involved in priming of T cells, when found in ectopic or tertiary lymphoid structures in the immediate proximity of the tumor mass [ 152 , 153 ]. This phenomenon is especially important for the response against neoantigens that develop as tumor progresses [ 145 ]. Infiltration of DCs into tumor sites is associated with prolonged survival and reduced incidence of metastasis in patients with various types of solid tumors [ 154 ].

Cancer often develops evasion mechanisms that interfere with proper DC function [ 155 , 156 , 157 ]. In cancer patients, defective DC function is highly associated with impaired immune responses against antigens expressed by tumors [ 158 ]. The inherent plasticity of DCs and the balance between stimulatory and suppressive signals within the TME dictate whether DCs can induce and maintain a T cell response or not. In many cases, their number, distribution, phenotype, and function can change as the tumor progresses. Studies have shown that the number of DCs in peripheral blood of patients with head and neck squamous carcinoma is different from that of healthy individuals [ 159 ]. In a model of spontaneous ovarian cancer, Scarlett and colleagues observed a functional switch in DCs from an immunostimulatory to an immunosuppressive phenotype. Moreover, the depletion of DCs at early stages correlates to tumor growth while the depletion in later stages results in tumor regression. Finally, tumor DCs progressively upregulate PD-1 and PD-L1 and this phenomenon has been associated with T cell suppression and loss of Tumor infiltrating Lymphocytes (TILs) [ 160 ]. In the TME, DCs have either inefficient or totally absent antigen presenting capability or are polarized into immunosuppressive/tolerogenic regulatory DCs that suppress T cell activity [ 147 , 161 , 162 ]. The TME constitutes a challenging environment with limited availability of/for? oxygen due to poor vascularization and nutrients as well as increased concentration of metabolic products, which interfere with DC function, attenuating DC efficiency for cross priming [ 163 ]. A prerequisite for DC activation is their metabolic reprogramming to meet increased demands for protein synthesis and secretion of chemokines and cytokines that is accompanied by an increase in glucose uptake and enhanced levels of glycolysis. Competition for glucose uptake with other cells in the TME can render DCs unable to function properly [ 147 ]. The unique nature of the TME has also been highlighted by two separate recent studies that associated the impaired antigen presentation capacity of DCs with defects in trafficking of MHCI to the cell surface due to incorporation of tumor-derived oxidized lipids into DC lipid bodies. In this case, MHCI rather accumulate inside late endosomes [ 157 , 164 ]. Activation of the β-catenin pathway is another mechanism that cancer utilizes in order to inhibit cross-priming as activation of this pathway induces a tolerogenic state in DCs. Wnt ligands and other molecules, both in tumor cells and inside DCs, mediate DC exclusion from TME and inhibition of their antitumor activity, respectively. The DC intrinsic signaling route is also active in tumor infiltrating DCs in order to disrupt cross-presentation and reprogram DCs to induce tolerance [ 161 ]. In addition, many other factors (VEGF, IL-10, IL-6, colony stimulating factor CSF-1) inhibit maturation of bone marrow progenitors or monocytes into DCs, and instead drive monocytes toward a suppressive phenotype as they promote development of MDSCs and TAMs [ 165 ].

The clinical success of immunotherapy with checkpoint inhibitors relies significantly on effective processing and cross-presentation of tumor-specific antigens captured by DCs [ 158 ]. The blockade of inhibitory receptors on the cell surface of T cells by monoclonal antibodies, can intensify antitumor responses initially primed by DCs [ 166 ]. Tumor-bearing mice with impaired cross-presentation pathways showed resistance to therapy with antibodies targeting ICs [ 167 , 168 ]. During the last few years, new strategies have been emerging that aim to strengthen the therapeutic efficacy of checkpoint blockade treatment with DC-based vaccination, i.e., DCs loaded with tumor (neo)antigens for presentation to the immune system, as available preclinical and clinical data have demonstrated that DC-vaccination synergizes with ICIs for improved therapeutic outcomes [ 169 , 170 ]. In intracranial glioma tumor-bearing mice, the combined administration of an a-PD1 monoclonal antibody with a DC vaccine, led to long-term survival that was dependent on CD8+ T cells that infiltrated the tumor [ 170 ], while in a murine lung cancer model the combination of DC vaccination with ICIs led to 80% tumor eradication. The treated mice developed immunological memory that fostered cancer recurrence-free survival. In the same mice, monotherapy using either agent did not result in eradication of the tumor [ 171 , 172 ]. In human patients with active myeloma, the synergistic effect of the two therapies led to enhanced T cell responses against myeloma targets [ 173 ]. Ge and colleagues demonstrated that blocking the PD1/PD-L1 pathway with monoclonal antibodies, induces DC maturation and proliferation and that suppressing IC molecules during DC vaccination prolonged survival in a breast tumor-bearing mouse model [ 174 ]. Okada and colleagues used different MHCI-restricted tumor-associated neoantigens simultaneously with mature DCs and proposed that using this type of therapy at early stages of cancer can lead to generation of clinically useful neoantigen-specific T cells [ 175 ]. According to Linette and colleagues, vaccination using DCs appears to be necessary as an adjuvant to ICI therapy since most T cell clones specific for tumor neoantigens have been demonstrated to be naïve and below the limit of detection in patients with melanoma. In other words, a combinational therapy enhances both direct and cross-presentation and has the potential to boost the frequency and diversity of tumor-specific T cells and thus strengthen immune responses [ 176 ]. Finally, numerous studies have explored whether modulation of intratumoral APCs could increase the response to ICI therapies. These studies demonstrated that intratumoral DCs that sustain the potential to re-stimulate immune cells in the context of tumor microenvironment, are required for efficacious therapy outcomes, while their paucity limits the efficacy of ICIs [ 177 , 178 , 179 ].

7. The Immunopeptidome and Cancer

The sum of peptides bound and presented by MHCI on the surface of cells is increasingly referred to as the cellular immunopeptidome [ 180 ]. Under malignant conditions, the iummunopeptidome has been found altered both quantitatively and qualitatively. These altered tumor antigenic peptides may be recognized by the adaptive immune system as foreign and therefore induce immune responses. Indeed, in 2014, Gubin and colleagues first demonstrated that cancer immunotherapy treatments that boost T cell activity, overcoming tumor suppression induced by the tumor themselves, depend on T cell recognition of tumor-specific antigens [ 49 ]. Therefore, a detailed knowledge of the immunopeptidome constitution and deeper understanding of the characteristics of suitable tumor-associated rejection antigens can improve the current therapeutic immunotherapy interventions and offer new opportunities towards the development of personalized treatments. Several research efforts have already aimed at the identification of naturally presented antigens in different types of hematological and solid tumors while new tools and techniques (i.e., mass spectrometry-based and in silico, proteogenomic techniques etc.) are being developed and integrated, aiming for a more precise characterization and validation of the cancer-specific immunopeptidomes [ 181 , 182 , 183 , 184 , 185 , 186 , 187 , 188 ].

8. Tumor Antigens and Tumor-Associated Antigenic Peptides

A significant challenge in the field of immunotherapy is the identification of MHCI-presented peptides that are able to mediate T cell-based tumor rejection. Long-term clinical benefits of cancer immunotherapy treatments rely on T lymphocytes that recognize tumor antigens [ 189 ]. The major factors that determine whether an antigen is a good immunotherapy target are: (i) its immunogenicity, i.e., its ability to provoke an immune response after (re)-activation of T cells induced by ICIs, (ii) its tumor specificity, (iii) its prevalence and expression level on tumor cells, and (iv) its role in the oncogenic process [ 190 ].

Tumor antigens are classified into antigens of high tumoral and low tumoral specificity [ 189 ]. The first category includes antigens that are strictly tumor-specific, such as viral antigens generated in cancers of viral etiology and antigens derived from mutations or rearrangements in coding sequences and chromosome translocation events. The second group encompasses differentiation antigens, i.e., antigens expressed in both tumor and the corresponding healthy tissue but over-expressed in tumors [ 189 ]. A special category of tumor-associated antigens are RNA-editing derived epitopes. RNA editing is a posttranscriptional mechanism that generates sequence variations in proteins by enzymatic modification of nucleotides in mRNA sequences. This mechanism has been found dysregulated in cancers and peptides generated this way are presented to the immune system and elicit immune responses. In a recent study, Zhang and collaborators identified over-edited peptides from tumor tissues and provided evidence that effector CD8+ T cells specific for these peptides can be found in human tumors [ 191 ]. Moreover, potential tumor antigens can emerge after posttranslational modifications that occur on the antigens themselves and influence their binding affinity for MHCI [ 192 , 193 ]. Another class of antigenic peptides demonstrated to provoke immune responses and may constitute tumor rejection antigens, are the proteasome-generated spliced peptides [ 194 ]. In this case, distinct peptidic fragments (from the same protein or from different proteins) produced by the proteasome, are ligated in situ , producing sequences that are non-contiguous in the genome and are not found in proteins in the cell. In a recent study, Liepe and coworkers provided evidence that up to 30% of peptides bound to MHCI molecules can be spliced peptides [ 195 ]. However, this high prevalence of spliced peptides has been controversial and reanalysis of the original results by Mylonas and colleagues using multiple computational and verification tools estimated spliced peptides percentage to be much lower, in the range of 2–6% [ 196 ]. Finally, tumor antigens, named cryptic antigens, may also be derived from non-canonical translation of protein-coding genes or from translation of non-coding sequences. It has been proposed that up to 10% of the MHCI bound peptides can originate from non-coding genomic regions, untranslated regions and exonic out-of-frame translation. Additional sources of cryptic antigens can be long non-coding RNAs, altered mRNA splicing events, small nucleolar RNAs, and proteins encoded in ribosomal DNA [ 197 ]. If one takes into account that 99% of tumor-specific mutations are located in non-coding regions, these cryptic MHCI antigens can be a very rich source of tumor-specific antigens [ 197 , 198 ].

9. Epigenetic Control of Tumor Antigen Expression and Presentation

Tumor cells frequently exhibit epigenetic aberrations that significantly impact the repertoire of expressed proteins and therefore presented peptides, affecting recognition by immune cells. A class of tumor antigens that is epigenetically regulated and re-expressed in tumors is cancer testis antigens (CTAs). In healthy adults, CTAs are expressed only in male germ and trophoblastic cells [ 199 ]. However, ectopic expression has been observed in tumor cells of different histology—possibly indicating a role in oncogenesis and tumor growth—and is associated with global and promoter-specific DNA demethylation and histone modifications [ 115 , 199 ]. CTAs expressed by cancer cells are considered as tumor-specific antigens due to the fact that germ cells do not normally express MHCI molecules on their surface and additionally, due to the highly immunogenic capacity of CTAs. Indeed, potent cellular and humoral responses against these antigens, especially melanoma-associated MAGE and PRAME families and NY-ESO-1, have been observed in patients, while the use of demethylating agents in tumor cell lines increased their expression leading to recognition and destruction of the cancer cells by antigen-specific T cells [ 200 , 201 ].

Combination of ICI treatment with CTA vaccines has been demonstrated to have a synergistic positive effect. In melanoma patients, utilization of such a combinational treatment led to higher treatment response rates [ 202 , 203 ]. Immunological analysis showed that treatment with CTLA-4 immune-checkpoint antibody ipilimumab in metastatic melanoma patients enhanced NY-ESO-1 specific T cell responses and provided durable clinical benefits [ 204 ]. However, tumors can still find mechanisms to evade CTA-specific immune recognition and CTAs have been found downregulated in many cancers [ 202 ]. Moreover, dedifferentiated liposarcoma, leiomyosarcoma, and synovial sarcoma tumors with positive expression of PRAME cancer testis antigens have been demonstrated to reduce the expression levels of many components of the APP (such as MHC molecules, β2m, TAP2 and LMPs) in order to avoid immune recognition [ 205 ].

Apart from cancer testis antigens, other categories of antigens that could serve as tumor rejection antigens also appear to be under epigenetic regulation. Studies using DNA methyltransferase inhibitors demonstrated that these agents can induce the expression of transposable elements including mainly endogenous retroviruses (ERVs) [ 206 ]. ERVs are the most abundant viral elements in the human virome that are silenced due to DNA methylation in somatic cells [ 207 ]. Their activation in tumors results in a state of viral mimicry that can lead to generation of neoantigens in treated cancer cells. Their induced expression mimics exogenous retroviral infection and turns on viral defense genes resulting in innate immune responses, attraction of cytotoxic T cells in the TME, and IFNγ release that in turn induces transcription of APM components [ 207 ]. Indeed, a-PD-1 responsiveness has been positively correlated with ERVs expression in cancers [ 208 ]. Moreover, high molecular weight melanoma-associated antigens (HMW-MAAs) have been demonstrated to undergo demethylation at their gene promoter in melanoma lesions and cell lines, resulting in their re-expression, but whether these antigens can provoke immune responses remains elusive [ 209 ]. In a recent study, neoantigen expression levels were affected by promoter hypermethylation of genes harboring neoantigenic mutations in 23% of cases studied [ 11 ]. Qamra and colleagues analyzed chromatin profiles and the epigenomic promoter landscape in gastric adenocarcinoma and observed that epigenetically activated alternative tumor-specific promoters can favor immune evasion through depletion of immunogenic peptides and reduction of tumor antigenicity [ 210 ].

10. Neoantigens

Tumorigenesis and cancer outgrowth are closely related to genetic diversity and accumulation of non-synonymous somatic alterations. These alterations can be missense mutations, silent mutations, insertions, and deletions as well as copy number gains and losses that result in new peptide sequences which are strictly tumor-specific. A single alteration in amino acid sequence can interfere with T cell recognition in three different ways: (i) by creating an anchor residue that changes the binding affinity of the new peptide with the MHCI molecule; (ii) by changing the TCR binding properties resulting in a conformationally altered peptide-MHCI complex that can be recognized by different T cell populations and (iii) by altering processing of the protein by the cellular APM that could result to presentation of an epitope that normally would be degraded [ 211 , 212 ]. The number of mutations within a tumor genome is defined as tumor mutation burden (TMB). A high level of TMB raises the possibilities of generation of neoantigens and the emergence of neoantigens diverges cancer cells from normal, healthy cells. Cancer cells can now be recognized as foreign by the immune system as high levels of mutational load is believed to enhance antigen presentation to T cells and increase the chances of tumors being identified by widening the T cell killing repertoire [ 213 , 214 ]. The success of ICI therapy relies on reinvigoration of pre-existing T cells that although are kept under tight control by modulatory mechanisms, have the ability to recognize cancer cells and attack them when this control is unleashed [ 215 ]. Indeed, studies have demonstrated that neoantigens can elicit responses after immunotherapy treatment and T cells recognizing tumor-related neoepitopes have been identified in different cancers [ 216 , 217 , 218 ]. There is extensive published literature that correlates high mutation burden and neoantigen frequency with durable survival and regression benefit from/after ICI therapy in several types of tumors. Neoantigens have been proposed to be good predictive and prognostic markers of better clinical outcomes, although tumors with low mutational load can still respond to checkpoint blockade, indicating a non-linear correlation and the involvement of additional factors [ 219 , 220 , 221 , 222 , 223 , 224 , 225 , 226 , 227 , 228 , 229 , 230 , 231 , 232 , 233 , 234 , 235 , 236 ].

An important source of neoantigens comes from the accumulation of mutations that occur in the genome when the DNA repair mechanisms of the cell are deregulated. During the cell cycle, cells progress through a series of checkpoints before mitotic division to ensure replication fidelity. Cells are well equipped with mechanisms that recognize and correct DNA damages, such as proofreading polymerases, mismatch repair pathways, base and nucleotide excision pathways, and homologous repair mechanisms. However, tumors often develop defects in these mechanisms and, as a result, DNA replication errors accumulate leading to a large number of mutations that induce genomic instability, which in turn promote cancer growth. The major causes that drive repair deficiencies in cancer correlate with inherited and de novo germline and somatic alterations, at the DNA sequence level, in genes that constitute components of the repair machinery, as well as epigenetic alterations (DNA methylation, histone modifications, nucleosome remodeling, and RNA-mediated targeting) that lead to transcriptional silencing of the associated genes or changes in chromatin dynamics required for DNA repair [ 237 ].

Many studies have demonstrated the strong correlation between inactivation of DNA repair pathways and genomic instability with significant higher mutational burden, tumor neoantigen load, and immune cell infiltration [ 238 , 239 , 240 , 241 , 242 ]. Rospo and colleagues used a colorectal cancer model system and found that alterations in DNA repair genes facilitate the acquisition of dynamic neoantigen profiles that fluctuate over time [ 243 ] while similar results were also observed in lung squamous cell carcinoma [ 240 ]. A CRISPR/Cas9-mediated targeting of the mismatch repair (MMR) component Mlh1 in murine breast, colon, and pancreatic ductal adenocarcinomas, revealed that MMR deficiency is associated with high mutational burden, TCR diversity, and significantly elevated neoantigen production. Furthermore, neoantigen production had continuous renewal potential compared to MMR-proficient cells that exhibited stable mutational load and neoantigen profiles [ 244 ]. The hyper-mutated phenotype that characterizes these types of tumors has been demonstrated to associate with higher rates of response to ICI therapy and durable clinical benefit [ 245 , 246 , 247 , 248 ]. In a study evaluating clinical data in patients with 12 different types of MMR-deficient tumors treated with an a-PD-1 agent, Le and colleagues observed rapid in vivo expansion of neoantigen-specific T cell clones reactive to mutant neoantigenic peptides found in the tumor. Such peptides may constitute a cohort of neoantigens useful for evaluating responses to IC treatment [ 247 ]. Collectively, it appears that there is growing evidence that the MMR deficient phenotype can serve as a good predictive biomarker of clinical response to ICI therapy.

11. T Cell Epitopes Associated with Impaired Peptide Processing

T cell epitopes associated with impaired peptide processing (TEIPP) constitute a unique, alternative repertoire of CD8+ T cell epitopes. TEIPP peptides are non-mutated self-antigens arising from housekeeping genes and emerge only in immune-edited tumors with low MHCI expression and defects in the APM as functional TAP seems to prevent their presentation. Their processing can also be conducted by alternative routes, such as the signal peptide route or the convertase family. TEIPP peptides are thought to be present within the ER of cells carrying intact TAP but cannot be presented due to their competition with the large flow of TAP-pumped peptides that are normally loaded onto MHCI molecules. A CD8+ T cell subset was discovered that selectively recognizes and targets tumor cells with defects in their APM and not cells with proficient APM. This T cell subset is positively selected in the thymus but remains in a naïve state in the periphery so it is not affected by tolerance [ 249 , 250 , 251 ]. The ppCT 16–25 peptide derived from the signal peptide of pre-procalcitonin was the first human tumor epitope identified whose surface expression is associated with impaired TAP transporter function [ 252 ]. Moreover, in a recent study, 16 different HLA-A*02:01 presented TEIPP peptides were identified in mouse tumor models with defects in TAP transporter [ 253 ]. In addition, successful targeting of immune-escaped tumour variants by TEIPP-specific T cells was demonstrated [ 253 ]. TEIPP could be considered as tumor-specific neoantigens since their surface presentation is favored only under conditions of TAP dysfunction.

Recent work has highlighted that dysfunction of another APM component, ERAP1 (or ERAAP in mouse) can also lead to up-regulation of non-classical MHC class Ib molecules that normally present peptides from the signal sequence of MHCI [ 254 ]. Presentation by these non-classical MHC led to robust CD8+ responses [ 254 ]. Interestingly, ERAP1 downregulation affected the immunopeptidome of both classical and non-classical MHCI [ 255 ]. It was thus proposed that MHC class Ib presentation of signal sequence peptides may constitute a mechanism for immune surveillance for the dysfunction of the aminopeptidase trimming component of the APM [ 256 ].

12. Strategies for Enhancing ICI Therapy Effectiveness: The Role of Antigen Presentation

Despite impressive clinical results, resistance to ICI immunotherapy is commonly a bottleneck in the successful treatment of several cancer types. Many approaches are currently under investigation aiming to surmount resistance to ICIs and improve clinical outcomes, often focusing on combining various therapeutic modalities (traditional therapies, other immunotherapy regiments as well as molecularly targeted therapies) on a checkpoint inhibitor backbone [ 257 ]. From a mechanism point of view, ongoing approaches aim to promote antigen processing and presentation, improve tumor antigen release and neoantigen supply, enhance T cell priming, expansion, survival, and effector functions, make the TME more friendly for immune cells, attenuate tumor-induced immunosuppressive factors, and promote proinflammatory/immunogenic pathways [ 258 , 259 , 260 ]. Moreover, there is significant evidence that gut microbiota diversity and composition can affect ICI responses and resistance in many cancers, by educating local and systemic immune responses, enhancing beneficial effects of metabolites, and dampening immune-related side-effects [ 261 ]. A summary of ongoing pre-clinical and clinical efforts focusing on overcoming resistance to ICI therapies is shown in Figure 3 .

Combinatorial strategies under investigation that aim to enhance efficacy of immune checkpoint inhibitor (ICI) immunotherapy (PRC: Polycomb repressive complex).

It is becoming increasingly clear that antigen processing and presentation is both central to cancer immune evasion and also a key puzzle piece in cancer immunotherapy. In order, however, to be able to manipulate APP to enhance cancer immunotherapy regiments, it is first necessary to understand the exact mechanisms by which APP is altered in cancer. Tumor cells can manipulate antigen presentation either by altering the cellular proteome or any of the components of the APP machinery. Therapeutically, several of the components of the APP machinery could be targeted in order to enhance the immunogenicity of cancer: the ubiquitin-proteasome degradation pathway, cytosolic peptidases, the TAP transporter, the peptide loading complex, peptide editing chaperones such as Tapasin or TAPBPR, ER aminopeptidases, and the MHCI themselves. In addition, induction of changes in the cellular proteome can regulate antigen presentation. In one study, researchers demonstrated a correlation between protein homeostasis and tumor antigen presentation by showing IFNγ-independent changes the MHCI peptide repertoire by low-level inhibition of the Heat Shock Protein Hsp90 [ 262 ]. Recently, Ilca and colleagues used a soluble form of the peptide editor TAPBPR and found an efficient way to bypass the peptides that are naturally presented and load onto tumour cells immunogenic peptides that resulted in robust immune responses [ 263 ].

One component of the APP that appears amenable to pharmacological targeting are the ER aminopeptidases ERAP1 and ERAP2 and the cross-presentation related aminopeptidase Insulin-regulated aminopeptidase IRAP. ERAP1 and ERAP2 appear to have a significant amount of specialization for antigen processing, whereas IRAP participates in additional biological processes including T cell receptor signaling [ 264 ]. Both ERAP1 and ERAP2 have been shown to be downregulated in some cancers [ 265 ], play key roles in the shaping of the cellular immunopeptidome [ 85 ], and their activity has been associated with changes in anti-cancer immune responses [ 101 ]. Furthermore, ERAP1 inhibitors have been shown to regulate the immunopeptidome [ 86 ] and elicit antitumor cytotoxic responses [ 90 , 91 , 94 ]. IRAP has been shown to be important in cross-presentation [ 266 ] and an IRAP inhibitor to be able to enhance cytotoxic responses ex vivo [ 267 ]. Furthermore, the development of inhibitors for these enzymes has reached significant maturity [ 268 , 269 , 270 ]. However, the synergism between inhibition of intracellular antigen processing by aminopeptidases and enhancement of antitumor immunity by ICI has not been explored yet.

A potential synergism between aminopeptidase inhibition and ICI is depicted in Figure 4 . As shown in panel A, an immune-evading tumor can be using ERAP1/ERAP2 to destroy tumor-associated antigenic peptides and over-expresses immune checkpoints such as PD-L1 to avoid T cell responses. Therapeutic interventions using ICIs, such as a-PD-1, can help promote T cell re-engagement but lack of presentation of appropriate tumor-associated antigenic peptides represents a bottleneck on antitumor cytotoxic responses (Panel B). Inhibition of ERAP1 or ERAP2, can help reactivate such responses by protecting tumor-associated antigenic peptides from degradation (Panel C). While this combinatorial approach is promising, it has not been experimentally evaluated and can suffer from a number of serious caveats since it cannot address exclusion of T cells from the TME or other means of T cell inactivation and antigen presentation silencing. Thus, it may be limited to specific cases or require combination with additional immunotherapy approaches. Given however the important role of APP in antitumor responses and the multitude of combinatorial cancer immunotherapy approaches currently under investigation, the modulation of intracellular antigen processing by aminopeptidase inhibitors is highly likely to find an application in enhancing tumor antigenicity.

Schematic representation of antigen processing and presentation in cancer immune evasion and immune re-activation by ICIs and manipulation of intracellular antigen processing. ( Panel A ) tumor antigens are processed by the proteasome but then destroyed by ER aminopeptidases ERAP1 or ERAP2 resulting in lack of presentation on the cell surface. Overexpression of PD-L1 on the cancer cell surface downregulates cytotoxic T-lymphocyte responses. Synergism between these two mechanisms results in efficient immune evasion by the tumor. ( Panel B ) Immune-checkpoint inhibitors can help activate T cells but lack of tumor antigen presentation limits cytotoxic responses. ( Panel C ) inhibition of ERAP1 and ERAP2 can help rescue tumor-associated antigenic peptides from destruction and promote their presentation, which, in combination with ICI treatment, can help re-activate T cell cytotoxic responses against the tumor.

13. Concluding Remarks

Expanding the benefits of cancer immunotherapy with ICIs to more patients and cancer types is probably one of the most urgent challenges in modern cancer therapy. The initial enthusiasm with ICI clinical successes gradually gave way to the realization that the interplay between the immune system and cancer is extremely complex and poorly understood. Many facets of this interplay have to synergize to circumvent the established evolutionary immune evasion of cancer. Not surprisingly, many immunotherapy approaches under investigation aim to combine multiple modulations of the immune system, including T cells and the TME, to achieve synergistic therapeutic effects. Antigen processing and presentation is undoubtably a key component in the immune evasion by cancer and thus its modulation constitutes a highly promising avenue for therapy. Still, being only one part of a larger puzzle, time will tell if manipulation of antigen presentation will be an effective monotherapy or it will find its place as a component of combination immunotherapy.

Acknowledgments

The authors thank Angeliki Chroni for critical advice and editing of the manuscript.

Abbreviations

A.M acknowledges financial support from the Hellenic Foundation for Research and Innovation (H.F.R.I) post-doctoral grant no 303.

Institutional Review Board Statement

Informed consent statement, data availability statement, conflicts of interest.

The authors declare no conflict of interest.

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ORIGINAL RESEARCH article

The purinergic receptor p2x7 as a modulator of viral vector-mediated antigen cross-presentation.

• 1 Institute of Virology, Universitätsklinikum Düsseldorf, Düsselorf, Germany
• 2 Institute of Molecular Medicine II, Universitätsklinikum Düsseldorf, Düsseldorf, Germany
• 3 Department of Medical Sciences, University of Ferrara, Ferrara, Italy
• 4 Institute of Infection Immunity, University of Utrecht, Utrecht, Netherlands
• 5 Biological and Medical Research Center (BMFZ), Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8 + T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing.

Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation.

Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection.

Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Introduction

The P2X7 receptor (P2RX7) belongs to the ionotropic purinergic P2X subfamily and is mostly expressed in immune, endothelial and epithelial cells ( 1 , 2 ). High concentrations of adenosine triphosphate (ATP) are known to activate the ion channel as a danger-associated molecular pattern (DAMP), leading to the intracellular increase of Na + and Ca 2+ and the efflux of K + . P2RX7 has been shown to be crucial for the regulation of various signaling pathways, such as the inflammasome pathway or those that lead to the release of cytokines, cell death or mitochondrial activation ( 3 – 6 ). Next to its contribution to various pathological diseases, P2RX7 activation is also associated with the release of extracellular vesicles (EVs) or particles (EPs) ( 7 – 9 ). They contain various immunostimulatory molecules known to be pivotal for the activation of antigen presentation processes ( 10 , 11 ). Recently, functional P2RX7 expression has been linked to increased viral loads of human herpes virus 6A ( 12 ). Furthermore, data from our lab ( 13 , 14 ) indicate increased expression of the P2X7 receptor during infection with Modified Vaccinia Virus Ankara (MVA).

MVA is a highly attenuated double-stranded DNA virus belonging to the family of the Poxviridiae and the genus Orthopoxvirus ( 15 – 17 ). For the generation of MVA, the parental strain Chorioallantois Vaccinia Virus (CVA) was passaged over 570 times in chicken embryo fibroblasts, leading to six large deletions in the MVA genome and the inability to replicate in most mammalian cells ( 17 – 20 ). Since MVA fails to generate infectious particles in humans, it has been developed as a suitable vector for vaccine design ( 21 – 23 ). It is able to express a large amount of recombinant DNA, and it induces strong humoral and cellular immune responses upon vaccination ( 22 , 24 ).

Interestingly, robust and long-lived cytotoxic T cell (CTL) immunity is dependent on cross-presentation during MVA infection ( 25 , 26 ). Upon infection with MVA, cells undergo apoptosis, containing and releasing antigens to be phagocytosed by professional antigen-presenting cells (APCs) ( 27 , 28 ). Upon internalization of the antigen, two distinct pathways can lead to the loading of MHC class I molecules (MHCI): the vacuolar and the cytosolic antigen-processing pathway ( 29 ). A peptide-MHCI-complex can either be generated by TAP interacting with internalized phagosomes containing the peptide to be processed or by processing already internalized peptides via the endoplasmic reticulum ( 30 ). In the vacuolar pathway, the processing and loading, both will occur in the vacuoles themselves ( 31 ). The preformed MHCI-peptide complex is then released and exported to the cell surface where CTL can be activated and release inflammatory cytokines, such as IFNү and TNFα ( 32 ). Since cross-presentation is essential for optimal CD8 + T cell priming for various pathogens as well as vector delivery systems, its molecular regulation has encouraged intense investigations. More evidence suggests that the stimulus for successful cross-presentation does not originate from the non-infected antigen-presenting cell, but rather from the bystanding initially infected cell, which we term feeder cell. We have recently shown that STING in feeder cells is involved in regulating CD8 + T cell responses via type I interferon production acting on the cross-presenting APC ( 33 ).

In this study, we aim to analyze the role of other innate triggers in feeder cells for MVA-induced antigen cross-presentation. The innate immune system serves as the first line of defense once a pathogen is encountered and P2RX7 as a member of the innate system has been shown to be potently activated by extracellular ATP, which is released by different stimuli. ATP is essential during vaccinia virus infection and therefore for the regulation of immune responses ( 34 , 35 ). P2RX7 has been described to be involved in antigen presentation ( 36 ). It alters the secretome in cells bearing the active P2RX7, such as the production of extracellular vesicles that might contain antigens or the production and release of varying inflammatory cytokines and chemokines ( 10 , 37 , 38 ). Additionally, P2RX7 activity has been associated with the expression of Nfatc1 , belonging to the group of primary response genes modulated by immune signals ( 39 – 41 ). Therefore, we investigated the involvement of the P2X7 receptor, as a member of the innate immune system, in infected feeder cells during cross-presentation of MVA-derived antigens.

The regulation of cross-presentation has been intensively studied for years, however, detailed knowledge about the molecular mechanisms that underlie the relevant pathways as well as about the innate triggers to initiate the process in the cross-presenting APC is lacking. In the present study, we aimed to investigate the potential role of P2RX7 as an innate stimulus in infected feeder cells for the initiation and modulation of cross-presentation in the non-infected bystander APC. We show that the ATP-sensitive P2RX7 from the BALB/c strain in feeder cells is essential for enhancing CD8 + T cell responses via cross-presentation in vitro . We show that various P2RX7-dependent pathways that we analyzed and which are crucial for the initiation of immune responses, such as the release of inflammatory cytokines, mRNA and the presence of apoptotic stimuli, were modulated in the presence of functional P2RX7 in infected feeder cells. Our findings suggest that the improved cross-presentation capacity of antigen-presenting cells co-cultured with infected feeder cells bearing active P2RX7 might be due to the activation of several pathways in feeder cells that may act together to orchestrate the immune response. To our knowledge, this is the first report instigating the function of the P2RX7 for regulation of MVA-mediated T cell immunity.

The plasma membrane P2X7 receptor is not functional in Cloudman (CM) cells and reconstitution with active P2RX7 from BALB/c mice enhances the release of extracellular particles after MVA infection

In line with the literature ( 42 ) and our sequencing data ( Supplementary Figure 1A ), the fluorometric analysis of intracellular Ca 2+ influx failed to demonstrate activation of the plasma membrane-located P2RX7 in Cloudman (CM) mock-or MVA-PK1L-Ova infected cells upon Bz-ATP specific stimulus ( Figure 1A ). Although recent New Generation Sequencing analysis ( 14 ) has shown that the expression of the P2X7 receptor was upregulated after MVA infection, qRT-PCR analysis of infected CM cells failed to demonstrate an upregulation of expression but indicated a stable constitutive expression after infection ( Supplementary Figure 1B ). For the matter of simplification, we distinguish between active and inactive P2RX7, referring to the BALB/c P2RX7 or DBA/C57BL/6 P2RX7 (less sensitive to ATP stimuli ( 42 )), respectively. To exclude that the lack of intracellular Ca 2+ increase was due to faulty loading of the fluorescent indicator FURA-2-AM, we additionally stimulated the cells with ionomycin, which is a receptor-independent trigger for a maximal increase of intracellular Ca 2+ . Even higher Bz-ATP stimuli could not increase intracellular Ca 2+ concentrations ( Supplementary Figure 1C ). Interestingly, when stimulating the CM cells with ATP, known to activate additional purinergic receptors besides P2RX7, we observed a gain in intracellular Ca 2+ amounts, suggesting the activity of other receptors of this or the P2Y receptor family ( 43 ) ( Supplementary Figure 1D ). Next, we were interested in studying the role of active P2RX7 and transfected our CM cells with the fully functional P2RX7 expressed by BALB/c mice ( 42 ). We were able to demonstrate its activity after transfection, by an increase in the concentration of intracellular Ca 2+ upon Bz-ATP stimulus ( Figure 1B ). This response was reversed when the reconstituted cells were treated with the P2RX7-specific competitive inhibitor A740003 at 20µM. Transfection with the empty vector control, similar to CM WT cells, did not alter the amount of intracellular Ca 2+ upon Bz-ATP treatment. Toxicity of A740003 was excluded ( Supplementary Figure 1E ). Furthermore, the receptor activity represented by an increased Ca 2+ influx in the P2RX7-transfected cells appeared to be significantly higher at earlier time points during MVA infection after 4 h.p.i. ( Supplementary Figure 1F ), suggesting that the infection itself modulates the activity of the receptor. However, calcium levels did not change after 20h MVA infection when compared to uninfected cells indicating that the receptor activity is only transiently increased after infection ( Figure 1C ). Interestingly, we did not observe P2RX7-specific pore function in P2RX7 transfected cells ( Supplementary Figure 1G ) ( 44 ). The release of extracellular particles, which is reported to be partly P2RX7 dependent ( 7 ), was enhanced when P2RX7 transfected cells were infected with MVA, supporting the regulation of P2RX7-specific functions during MVA infection ( Figures 1D, E ). Overall, we confirmed the reconstitution and functionality of P2RX7 in CM cells after transfection.

Figure 1 The P2X7 receptor is inactive in Cloudman (CM) cells (DBA background) and transfection with functional P2RX7 from BALB/c mice restores P2RX7-specific functions. (A) Fluorometric analysis of intracellular Ca 2+ (iCa 2+ ) influx as a known marker of P2RX7 activity to demonstrate the function of the plasma membrane-located P2RX7 in CM cells infected with MVA or mock. Activity of P2RX7 in CM wildtype (WT) cells infected either with MVA-PK1l-Ova (20hpi/MOI1) expressing ovalbumin under the control of the vaccinia virus early promoter PK1L or mock-infected was investigated by measurement of intracellular Ca 2+ concentrations of FURA-2-AM loaded cells upon stimulus with 200µM Bz-ATP. (B) CM cells were transfected with empty vector (CM pcDNA3) or P2RX7 containing plasmid DNA. Activity was assessed at least one week post transfection by fluorometric assay. CM P2RX7 cells were additionally pre-treated for 5min with 20µM A740003 (CM P2RX7 A740003), a P2RX7 specific inhibitor. (C) Intracellular calcium concentrations were further tested in CM P2RX7-transfected cells 20h post-MVA infection (MOI1) (CM P2RX7 MVA) or mock-infected cells (CM P2RX7). (D) Quantification of extracellular particles per total number of cells was started after addition of 200µM Bz-ATP for a time frame of approximately 30sec (three subsequent frames) from (E) MVA- (MOI1) or mock-infected CM WT or transfected cells (CM P2RX7 or CM pcDAN3) cells. Cells were stained with quinacrine nucleic acid stain and PKH26 membrane stain and then stimulated with 200µM Bz-ATP to visualize the release of particles using confocal image analysis. Data shown, represent one from at least n=3 (A–C) or n=2 independent experiments (D, E) . Statistical significance (P) ***P ≤ 0.001.

Active P2RX7 in feeder cells promotes MVA antigen cross-presentation

Recent studies have shown that innate triggers derived from infected feeder cells are relevant for the activation of T cells by antigen-presenting cells ( 33 ). We were interested in investigating whether the presence of a functional P2X7 receptor in feeder cells may have an impact on the antigen uptake and presentation capacity of bone marrow-derived dendritic cells (BMDCs) for activation of CD8 + T cells. We demonstrate that using MVA-OVA-infected feeder cells bearing the active P2X7 receptor led to a significantly higher CD8 + T cell activation as determined by IFNү production in B8R- specific T cells or by TNFα production in either B8R- or OVA-specific T cell lines when co-cultured with uninfected dendritic cells as cross-presenting APC ( Figure 2A ). These data are in line with the P2RX7-dependent release of these cytokines in mice ( 45 ). The frequency of cross-presenting BMDCs with SIINFEKL/H2-K b complexes on the cell surface, as well as the amount of these peptide/MHCI complexes per cell, was significantly increased ( Figure 2B left, middle). Interestingly, the presence of the active P2RX7 in CM cells led to the increase of MHCII surface expression in co-cultured BMDCs ( Figure 2B right). The expression of other maturation markers, such as CD40 or CD86 on co-cultured BMDCs was not affected (data not shown). Furthermore, pre-treatment of CM P2RX7 cells with A740003 before co-culture with BMDCs led to a significantly reduced CD8 + T cell activation ( Supplementary Figure 2A ) and SIINFEKL/H2-K b expression ( Supplementary Figure 2B ) which was comparable to CM WT or CM pcDNA3 cells. To corroborate the specific function of P2RX7, we used HEK293 as feeder cells expressing active human P2RX7 ( Figure 2D ). Indeed, the co-incubation of BMDCs with MVA-infected HEK293 hP2RX7 feeder cells resulted in increased SIINFEKL/H2-K b expression ( Figure 2C ). In sum, we demonstrated that the presence of functional P2RX7 in feeder cells aids in improving antigen cross-presentation upon MVA infection.

Figure 2 Antigen cross-presentation is enhanced in the presence of active P2RX7 in CM feeder cells. GM-CSF bone marrow derived-dendritic cells (BMDCs) were co-cultured with CM WT (CM), control plasmid (CM pcDNA3) P2RX7 transfected (CM P2RX7) feeder cells infected with MVA-PK1L-Ova expressing ovalbumin under control of the viral early promoter PK1L (MVA) at MOI1 or mock-infected (Ø). At 20hpi, B8R- or Ova-specific CD8 + T cells were added to the co-culture for 4h. Presence of intracellular activation markers was determined by flow cytometric analysis (FACS) of (A) IFNү (left) or TNFα (right) production. (B) Frequency (left) and mean fluorescent intensity (MFI) (middle) of SIINFEKL/H2-K b surface expression (FACS). MHCII (%) expression in BMDCs (right). (C) SIINFEKL/Kb expression was also assessed in BMDC that had been co-cultured with HEK293 cells (WT or hP2RX7 transfected) infected with either mock (Ø) or MVA-PK1L-Ova (MVA) at MOI1 for 20h. (D) Fluorometric analysis of intracellular calcium concentrations in HEK293 WT (HEK293) and hP2RX7 transfected (HEK293 hP2RX7) cells with or without MVA-PK1L-OVA (MOI1, 20h) infection and 200µM Bz-ATP stimulus. Data are pooled from at least n=3 independent experiments (n=3-5) and shown as means ± SD. P values indicate statistical significance (P) with *P ≤ 0.05 **P ≤ 0.01; ***P ≤ 0.001.

Functional P2RX7 does not alter antigen availability or replication capacity of MVA but impacts the gene expression of viral antigens

We first hypothesized that the increased SIINFEKL/H2-K b surface expression on antigen-presenting cells and the improved CD8 + T cell activation that we found when BMDCs were co-cultured with P2RX7 feeder cells, might be due to an increased amount of viral antigens. We analyzed the expression of early antigens, such as B8R , a native MVA antigen, or Ova , expressed under the control of the early MVA promoter PK1L, or the late viral antigen A19L in CM P2RX7 or CM pcDNA3 transfected cells and compared it to CM WT cells. Expression of these antigens was initially lower in CM P2RX7 cells compared to CM WT or the empty vector control CM pcDNA3. However, at 24hpi B8R , Ova and A19L mRNA fold change in CM P2RX7 cells was significantly higher when compared to CM WT or CM pcDNA3 cells ( Figure 3A ). We concluded that the expression kinetics of viral genes in CM P2RX7 was delayed, although mRNA expression at later time points in these cells was significantly higher. This was further confirmed when analyzing the replication capacity of MVA in the different CM feeder cell lines. We also observed a higher residual viral titer at 0hpi in CM P2RX7 ( Figure 3B ), suggesting a possible role of the P2X7 receptor for viral entry, as previously stated ( 12 ). Since MVA has lost its ability to replicate in most mammalian cells ( 19 ), we wanted to exclude the possibility that the presence of this receptor was affecting viral replication behavior and, as a consequence, promote antigen cross-presentation. However, at 24hpi viral particle amounts in CM P2RX7 cells were comparable to CM WT or CM pcDNA3 cells ( Figure 3B right). Similarly, the synthesis of OVA protein in CM P2RX7 cells was initially reduced at 8hpi but comparable at 20hpi ( Figure 3C ), indicating that expression of viral genes and synthesis of corresponding proteins might be delayed in CM P2RX7 transfected cells.

Figure 3 Expression of active P2RX7 in feeder cells alters several signaling pathways that may be correlated with enhanced antigen availability and cross-presentation by BMDCs. (A) CM WT (CM) or pcDNA3 or P2RX7 transfected cells (CM pcDNA3 or CM P2RX7) were infected with MVA-PK1L-Ova at MOI1 for 0h to 24h to assess the expression of viral antigens B8R and A19L or recombinant antigen Ova, respectively. mRNA fold change is shown as expression of the respective gene at each time point compared to the 0h time point of CM WT cells. (B) Viral growth kinetics of MVA-p7.5-GFP expressing green fluorescent protein (GFP) under control of the viral early/late promoter p7.5. (Left) Wildtype or transfected CM cells were infected for 0h to 24hpi and viral titers were determined by back titration on DF1 cells at the indicated hpi as tissue culture infectious dose (TCID 50 ). (Right) Final viral loads after 24hpi were measured by subtracting viral output at 24hpi with viral input at 0hpi. (C) Western Blot analysis of WT or transfected CM cells upon MVA-PK1L-Ova infection at 8hpi or 20hpi (left). Quantification of OVA (middle) or cleaved CASPASE-8 (right) protein amounts in cellular extracts. (D) Phosphatidylserine residues on the cell surface of mock- (Ø) or MVA-infected (MOI1) WT or transfected CM cells at either 6hpi or 20hpi. Quantification of either APC-negative (APC-) (live/dye non-permissive) (left) or APC-positive (APC+) (dead/dye permissive) (right) within phosphatidylserine positive cells, depicting either early or late apoptotic cells, respectively. Experiments are shown as means with SEM (A) or SD of n=3 independent biological replicates with statistical significance (P) *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

The above data exclude that altered protein amounts in feeder cells as the source of antigenic uptake by antigen-presenting cells contributed to the improved antigen presentation by BMDCs co-cultured with infected CM P2RX7 cells. It has been postulated that the antigen has to be released but needs to be still cell-associated for the antigen-presenting cells to phagocytose and process it for cross-presentation to CD8 + T cells ( 46 – 48 ). We confirmed the activation of the extrinsic apoptosis pathway by cleavage of Caspase 8 ( Figure 3C ). The exposure of phosphatidylserine residues on the cell surface is important for the activation of phagocytosis by APCs and has been linked to P2RX7 activation ( 49 – 51 ). We demonstrate that the presence of the functional P2X7 receptor led to the increased surface expression of phosphatidylserine residues in cells having a permeable cell membrane ( Figure 3D right) when mock-infected or infected with MVA for 6h. In contrast, MVA-infected CM P2RX7 with intact cell membrane, hence alive cells, displayed reduced phosphatidylserine levels as compared to CM WT or CM pcDNA3 cells after 20h MVA infection. Overall, we speculate that expression of the functional P2X7 receptor in CM feeder cells does not modulate the antigen-presentation capacity by APCs by increasing the total amount of antigenic protein in feeder cells, but rather allows for increased viral mRNA levels in infected cells at later time points as well as by altering apoptotic pathways. This depicts the importance of further analyses on the RNA level and the possible altered localization of P2RX7 affecting other cellular pathways.

Extracellular particles as well as supernatant released from feeder cells with active P2RX7 promote MVA antigen cross-presentation

Extracellular particles are known to contain crucial regulatory molecules for cell-to-cell communication ( 11 ). Similarly, supernatant from P2RX7 transfected cells differs from control cells ( 37 ). Here we hypothesized that both the extracellular particle fraction (EP-fraction), as well as the supernatant fraction (sup-fraction) from CM P2RX7 cells, are responsible for the enhanced antigen cross-presentation we observed. We first isolated the EP-fraction and the supernatant fraction as depicted in Figure 4A and determined both, mRNA and protein content. OVA protein amounts were comparable in wildtype and transfected CM cells after overnight infection in both, the EP- and the sup-fraction ( Figure 4B ). Interestingly, mRNA levels of the MVA early antigen B8R , but not Ova or A19L expression, were significantly higher in the EP-fraction at 0 hours post-infection leading to the hypothesis that the lower mRNA expression we observed in the cell extracts ( Figure 3A ) might be due to the release of RNA in extracellular particles ( Figure 4D ). This finding highlights the importance of focusing on the role of RNA in the regulation of MVA antigen presentation. We also showed that CM P2RX7 transfected cells allowed the secretion of significantly higher amounts of inflammatory cytokines pre- as well as post-infection ( Figure 4C ), demonstrating that the presence of functional P2RX7 modulates other signaling pathways as well. A detailed overview of the secreted cytokines can be seen in Supplementary Figure 3A . To further investigate the function of these culture sub-fractions from P2RX7 transfected cells, we delivered EP- and sup-fractions to the co-culture of uninfected CM cells and BMDCs to monitor subsequent CD8 + T cell activation and expression of SIINFEKL/H2-K b complexes on dendritic cells. The EP-fraction from CM P2RX7 was not able to induce stronger CD8 + T cell IFNү or TNFα production as compared to the EP-fraction derived from pcDNA3 transfected CM cells. However, the sup-fraction led to increased IFNү and TNFα production when added to the co-culture of CM and BMDCs ( Figure 4F ). Interestingly, both fractions from CM P2RX7 cells were able to induce a significantly higher SIINFEKL/H2-K b expression as compared to the empty vector control-derived fractions ( Figure 4G ). To establish whether these fractions could initiate similar CD8 + T cell activation as the infection of CM P2RX7 cells, these were added to MVA-infected CM WT cells ( Supplementary Figures 3B–E ). Even though both fractions seem to contribute to the increased expression of SIINFEKL/H2-K b on BMDCs, the addition of EPs or supernatants from infected P2RX7 CM cells could not further increase CD8 + T cell activation or SIINFEKL/H2-K b expression on BMDCs significantly. In addition, we filtered supernatants from infected feeder cells to remove any cell components larger than 0.2µM, such as apoptotic bodies (but leaving exosomes and microvesicles in the fraction) and added this filtered supernatant (fil sup) to either uninfected CM or MVA-infected cells ( Figure 4H left, middle). We found a significant contribution of the filtered supernatant from CM P2RX7 cells for the activation of dendritic cells and subsequent presentation of the SIINFEKL peptide on its MHCI. The total amount of cross-presenting BMDCs (frequency) with SIINFEKL/H2-Kb complexes at the cell surface as well as the amount of SIINFEKL/H2-Kb complexes per BMDC (MFI) was significantly increased ( Figure 4H left, middle). Notably, MHC I expression (total H2-K b ) of the antigen-presenting cells was not altered upon the addition of this filtered supernatant ( Figure 4H right). To sum this up, we demonstrate that the cellular fraction of P2RX7 transfected cells as well as other subcellular fractions are critically involved in enhancing antigen cross-presentation.

Figure 4 Subcellular fractions of P2RX7 transfected CM feeder cells enhance MVA-mediated antigen cross-presentation upon co-cultivation with cross-presenting BMDCs. (A) Experimental setup to extract extracellular particle (EP) fraction and supernatant fraction (sup) from infected cells. Figure created in Biorender with permission. (B) Both fractions were analyzed for OVA protein content at 20 hours post MVA-PK1L-Ova infection (MVA) at MOI1.5 (EP) or MOI1 (sup). (C) Expression and release of inflammatory chemokines was assessed in the supernatant fraction of CM, CM pcDNA3 or CM P2RX7 cells that were either mock- or MVA-infected (MOI1 for 20h) by Legendplex assay. (D) mRNA expression. Quantification of Ova (upper) or B8R mRNA (lower) at 0hpi and 20hpi in EP-fractions of MVA-PK1L-OVA infected cells (MOI1.5). (E, F) EP- or sup-fractions from MVA-PK1L-Ova infected CM pcDNA3 or CM P2RX7 cells that were added to the co-culture of uninfected CM WT feeder cells with uninfected BMDCs which were then co-cultured with B8R- or Ova-specific CD8 + T cells for 4h to assess IFNү or TNFα production upon antigen-specific activation. (G) SIINFEKL/H2-K b surface expression of BMDCs was determined after adding either the EP- or the sup-fraction from control (pcDNA3) or P2RX7 transfected (P2RX7) cells to uninfected BMDCs co-cultured with uninfected CM WT (CM) cells. (H) SIINFEKL/H2-K b surface expression of BMDCs by frequency (left) or mean fluorescence intensity (middle) after the addition of filtered supernatants from MVA-infected CM pcDNA3 or CM P2RX7 cells to either mock-infected (Ø) or MVA-PK1L-OVA (MVA) at MOI1 infected CM cells. (Right) MHCI expression (total H2-K b ) of BMDCs was assessed as a control. Plots show the mean of n=3 independent experiments with SD and statistical significance (P) *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. ns, not significant.

The presence of active P2RX7 modulates mitochondrial function

As a plasma membrane receptor, P2RX7 is associated with the activation of the canonical inflammasome pathway ( 52 ). Recent studies, however, show that P2RX7 can also localize to and function intracellularly on mitochondrial structures ( 53 ). We confirmed the presence of intracellular P2X7 receptors in our feeder cells. These were significantly increased in P2RX7 transfected CM cells ( Figure 5A ). Mitochondria and energy metabolism are impacted during viral infection and since the presence of P2RX7 on mitochondrial surfaces of HEK293 hP2RX7 and N13 microglial cells has been shown recently by others ( 53 , 54 ), we determined the mitochondrial activity in our feeder cells. As expected, maximal respiration and spare capacity were significantly increased in CM cells bearing the active P2X7 receptor as compared to CM WT or empty vector-transfected cells. Basal respiration was only increased in CM P2RX7 cells when compared to CM pcDNA3 cells. Also after infection, the maximal respiration and spare capacity were significantly upregulated in CM P2RX7 cells ( Figure 5B ). In addition, the extracellular acidification rate (ECAR), an indicator for glycolysis processes ( 55 ), was significantly higher in both, mock-infected and MVA-infected CM P2RX7 cells, as compared to CM WT or CM pcDNA3 cells ( Figure 5C ). Interestingly, intracellular ATP concentrations in the uninfected CM P2RX7 cells were comparable to CM WT cells but slightly lower than in CM pcDNA3 cells. After infection, however, intracellular ATP was significantly increased in CM P2RX7 cells only ( Figure 5D ), while extracellular ATP inversely correlated after infection showing a significant decrease for CM P2RX7 cells only ( Figure 5E ). In summary, we report that presence of P2RX7 significantly alters the energy metabolism of the cells by increasing the availability of mitochondrial ATP after MVA infection.

Figure 5 Presence of functional P2RX7 affects mitochondrial functions. (A) Intracellular expression of P2RX7. Mean fluorescent intensities (MFI) in mock- (Ø) or MVA-infected (MOI1, 20hpi) (MVA) CM WT (CM), empty vector control (CM pcDNA3) or P2RX7 (CM P2RX7) transfected cells. (B) Mitochondrial activity was assessed by measurement of the oxygen consumption rate (OCR) after treatment of CM WT, CM pcDNA3 or CM P2RX7 transfected cells with modulators of the electron transport chain such as Oligomycin, FCCP, and Rotenone/Antimycin A) (upper graphs) with (right) or without MVA infection (left) according to the Mitostress test kit (Seahorse). (lower graph) Mock-infected or MVA-infected (MOI5 for 6h) CM WT, CM pcDNA3, or CM P2RX7 cells were used to determine basal and maximal respiration as well as spare capacity upon addition of above mentioned modulators. (C) Comparable conditions were used to assess the extracellular acidification rate (ECAR) as an indicator for glycolysis processes in the indicated mock or MVA-infected (MOI5 for 6h) cells. (D) Quantification of intracellular or (E) extracellular ATP in CM WT, CM pcDNA3, or CM P2RX7 transfected cells either mock or MVA-infected (MOI5 for 6h). Data shown are of at least n=3 independent experiments, depicted as means with SD or SEM (D, E) and statistical significance (P) *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. ns, not significant.

Studies have demonstrated the significant role of the P2X7 receptor in response to viral infections, exhibiting both protective and pathological functions ( 56 ). However, the function of P2RX7 during MVA infection and its relevance for antigen cross-presentation has not been investigated yet. Our findings suggest that the presence of an active P2X7 receptor in MVA-infected feeder cells can lead to a strongly increased antigen-presentation capacity by cross-presenting dendritic cells. This highlights a new function of purinergic receptor signaling in feeder cells serving as antigenic source for cross-presentation by dendritic cells.

Before delving into the specific role of the P2X7 receptor, we ensured its lack of function in the CM feeder cells ( Figure 1A , Supplementary Figure 1C ). As expected and as described in the literature, there was no discernible increase in intracellular Ca 2+ in these cells, despite the stimulus with high concentrations of Bz-ATP, an agonist of P2X7 receptors ( 42 , 43 ). Interestingly, when CM cells were treated with ATP, we could observe a desensitizing peak, which was ATP-dependent and strongly indicated the activation of other P2 receptors ( 43 ). However, when we reconstituted CM cells with the wild type, fully ATP-sensitive P2RX7 derived from BALB/c mice, we observed an increase in intracellular Ca 2+ , which was reversible upon treatment with A740003, a P2RX7-specific inhibitor ( 57 ). It also appeared that P2RX7 may play an important role during the initial phase of MVA infection, as infection for 4h resulted in a slight increase in intracellular Ca 2+ values ( Supplementary Figure 1G ), while infection for 20h left intracellular Ca 2+ levels unaffected ( Figure 1C ).

Based on our studies, MVA infection regulates P2RX7-specific functions, since the release of extracellular particless, which has been reported to be at least partly P2RX7-dependent ( 7 , 8 ), was enhanced after MVA infection in the presence of an active P2RX7 ( Figures 1D, E ). We anticipate, however, that wildtype or empty vector-transfected CM cells with inactive P2RX7 would release apoptotic bodies instead of EPs, as MVA has been shown to trigger the initiation of apoptosis pathways in infected cells ( 11 , 27 ). Further CM cell-specific studies are underway to understand the molecular pathways by which P2RX7 signaling modulates EP release mechanisms.

The presence of active P2RX7 in feeder cells during MVA infection led to a dramatic increase in CD8 + T cell activation and enhanced expression of SIINFEKL/H2-K b complexes on murine antigen-presenting cells via cross-presentation ( Figures 2A, B ). This finding was corroborated in human cells by using a human feeder cell line, namely HEK293 cells, expressing a functional human P2RX7, while pre-treatment of CM P2RX7 with the P2RX7-specific inhibitor A740003 abrogated it ( Supplementary Figures 2A, B ). These results provide substantial evidence for the involvement of P2RX7 in feeder cells during MVA infection for antigen cross-presentation by dendritic cells. Since the presence of active P2RX7 in CM cells led to increased MHC II expression in dendritic cells ( Figure 2B right) ( 58 , 59 ), we hypothesize that P2RX7 alters the microenvironment e.g. by secretion of cytokines which stimulate non-infected bystander dendritic cells and has been previously suggested to be relevant for antigen presentation ( 60 ). Expression of P2RX7 has been associated with improved antigen presentation, especially due to the release of P2RX7-dependent extracellular particles containing inflammatory molecules and antigens ( 61 , 62 ). Our data indicates a substantial P2RX7-dependent modulation of the production of MVA-derived antigen in feeder cells and its release or presentation to DCs enhancing MVA-mediated antigen cross-presentation.

Recent studies imply that feeder cells play a crucial role for antigen cross-presentation ( 33 ). In order to better understand this process, we decided to investigate the expression of viral antigens in feeder cells. Interestingly, viral particles were attached to the cell surface, but not internalized in CM P2RX7 cells as demonstrated by the increased viral particle load at 0hpi ( Figure 3B left). In line with this finding, mRNA levels of all antigens tested (viral B8R and A19L as well as recombinant Ova ) were initially lower in CM P2RX7 compared to CM WT cells, but were significantly increased at 20 hpi ( Figure 3A ) indicating that viral antigen expression kinetics is delayed in P2RX7 cells. This altered kinetics was corroborated by western blot analysis of OVA antigen synthesis in infected CM P2RX7 cells compared to the controls ( Figure 3C left and middle). This implies that P2RX7 might play a role for viral entry, as previously described for other viruses such as HHV-6A and HBV/HDV ( 12 , 63 ). Importantly, the MVA replication capacity in feeder cells was not altered in the absence or presence of P2RX7 resulting in comparable viral titers/multiplication rates ( Figure 3B right) ( 19 ), thereby excluding that increased amounts of antigen due to increased viral replication in CM P2RX7 cells altered the cross-presentation capacities of APCs.

APCs require cell-associated antigens to be phagocytosed and processed for antigen cross-presentation ( 28 , 47 ). It has been shown that expression of P2RX7 activates Caspase-8-mediated apoptosis and leads to the exposure of phosphatidylserine at the cell surface ( 64 , 65 ). In fact, our studies demonstrate high expression of active Caspase-8 ( Figure 3C left/right) and an increase of late apoptosis in CM P2RX7 cells at early time (8hpi) after mock or MVA infection ( Figure 3D right) in the presence of P2RX7 thereby enhancing the decoration of the cell with the early apoptotic marker phosphatidylserine. Interestingly, at later time (20hpi), only cells without functional P2RX7 showed expression of phosphatidylserine on the cell surface (early apoptotic) ( Figure 3D ). Phosphatidylserine at the cell surface is required for vaccinia virus including MVA to enter cells ( 66 ). Vaccinia virus entry is facilitated by ‘apoptotic mimicry’, hence by flagging phosphatidylserine on mature virions and identifying it as apoptotic debris for uptake ( 67 ). Additionally, vaccinia virus is able to transfer phosphatidylserine molecules from the lipid bilayer of cell membranes to increase its infectivity ( 68 ), likely because the exposure of phosphatidylserine on the cell surface may act as an ‘eat-me’ signal for phagocytes ( 69 ) which potentially explains the increased expression of the BMDC maturation marker CD40 when co-cultured with uninfected CM P2RX7 cells. EPs, which emerge from the cell membrane, incorporate membrane-specific molecules, including phosphatidylserine ( 11 , 70 ). Rausch and colleagues have proposed that vesicles bearing phosphatidylserine can trigger CD8 + T cell activation ( 70 ). Interestingly, in an experimental setting where either EP- or sup-fractions serve as the only source of antigen for cross-presentation ( Figures 4E, F ), we observed an increased CD8 + T cell activation which was accompanied by enhanced antigen-specific peptide/MHC class I surface expression in cross-presenting DC ( Figure 4G ). The above findings suggest that expression of a functional P2X7 receptor in feeder cells not only modulates antigen cross-presentation in APCs at the protein level, as previously suggested ( 71 ), but also influences viral gene expression and viral entry in feeder cells as well as phagocytosis by BMDCs.

P2RX7-expressing cells feature an altered secretome e.g. released extracellular particles might contain antigens, cytokines and regulatory RNAs that are important for antigen-presentation ( 8 , 37 , 39 , 61 ). Although the amount of OVA protein in isolated extracellular particles or in the supernatant of MVA-infected cells was comparable in the absence or presence of P2RX7 ( Figure 4B ), the composition and expression level of (pro)inflammatory cytokines and chemokines in the supernatant of P2RX7 expressing cells was significantly altered ( Figure 4C , Supplementary Figure 3A ) in the presence of P2RX7. These results support the importance of P2RX7 during the initial phase of MVA infection as well as at the later stage, when infected feeder cells are co-cultured with antigen-presenting cells and continuously supply the microenvironment with stimulatory molecules and enhance cross-presentation ( 7 , 72 ). The EP fraction and, significantly stronger the supernatant fraction of CM P2RX7 cells increased CD8 + T cell activation and SIINFEKL/H2-K b expression by cross-presenting BMDCs when added to mock-infected feeder cells ( Figures 4F, G ). This effect was less pronounced when these fractions were added to MVA-infected feeder cells ( Supplementary Figure 3B–E ). The feeder cells produce apoptotic bodies upon MVA infection, which may contain antigens for phagocytosis. Since these were not eliminated by our isolation method ( 46 ), we filtered the supernatant (0.2µM) to exclude apoptotic bodies in this fraction. We confirmed that the secretome of CM P2RX7 cells significantly contributed to improved antigen cross-presentation by BMDCs ( Figure 4H left/middle). This effect could be attributed to both, the secreted pro-inflammatory cytokines as well as small vesicles such as exosomes or micro vesicles released due to the presence of P2RX7 ( 8 , 73 ). Additional soluble as well as cell-associated factors from infected feeder cells may be needed to fully license DCs for enhanced cross-presentation, as the total MHCI expression of BMDCs remained unchanged upon the addition of filtered supernatant fractions ( Figure 4D right).

The P2RX7 protein is known to be expressed on the plasma membrane as well as on intracellular membrane structures, suggesting that it may have multiple functions depending on the compartment within the cell. Sarti and colleagues have previously described the enhancement of mitochondrial metabolism by P2RX7 ( 53 ). We confirmed the intracellular presence of P2RX7 in our feeder cells ( Figure 5A ) as well as an increase in mitochondrial activity in the presence of P2RX7 in our MVA-infection model ( Figures 5B, C ). P2RX7 expression correlated with maximal respiration rate and spare capacity in MVA- and mock-infected cells, demonstrating enhanced ability of the cells to respond to stress ( 74 ). Furthermore, the extracellular acidification rate (ECAR) was significantly enhanced in the presence of active P2RX7, delineating the altered glycolysis pathway in these cells ( Figure 5C ), in line with previously reported glycolytic activity attributed to P2RX7 ( 75 ). These results suggest that P2RX7 is able to change the bioenergetics state of cells ( 76 ), with or without MVA infection. ATP is required for efficient vaccinia virus production ( 77 ). Importantly, we observed higher ATP levels within P2RX7 competent feeder cells which were significantly increased after MVA infection ( Figure 5D ). In contrast, basal secretion of ATP by these cells (extracellular ATP level) was less or comparable when infected with MVA, while cells with inactive P2RX7 released significantly higher amounts of ATP into the supernatant after MVA infection ( Figure 5E ). As shown before, cells infected with MVA undergo apoptosis. Since ATP is released during cell death processes ( 27 , 78 ), we suggest in line with others that ATP regulation is P2RX7-dependent in BALB/c P2RX7-bearing feeder cells, but it is apoptosis-dependent in feeder cells lacking the fully functional P2RX7 ( 79 ). Further studies are required to analyze if the available ATP can act in an autocrine manner and reactivate P2X7 receptors of the feeder cell, as previously reported ( 80 ). It has been described that infection of viral pathogens may drive metabolic reprogramming to allow for adaptation of the cell to biosynthetic and energetic needs required for viral replication ( 81 , 82 ). We demonstrate that cells expressing a functional P2RX7 seem to handle MVA infections better due to prolonged active cell metabolism and increased energy levels resulting in increased overall cell fitness and delayed apoptotic cell death. The lack of ethidium bromide pore opening ( Supplementary Figure 1G ), another potential characteristic of P2RX7 plasma membrane expression ( 44 ), indicates a rather protective role of P2RX7 in feeder cells. In this respect, active Caspase-8 found in cells with functional P2RX7 seems to be activated in the absence of cell death, leading to the release of inflammatory cytokines and the restriction of pathogen growth ( 83 ). A signaling pathway associated with these processes and affected by P2RX7-dependent modulation may involve NF-κB ( 84 ).

In this study, we have uncovered significant factors influenced by the ATP-sensitive P2X7 receptor in MVA-infected feeder cells that promote antigen cross-presentation by dendritic cells. These factors include (i) the secretion of pro-inflammatory cytokines, (ii) delayed viral entry during MVA infection of CM P2RX7 cells associated with delayed viral gene expression and subsequent viral antigen synthesis. We further identified (iii) the release of small vesicles (<0.2µM) such as exosomes and microvesicles as well as viral mRNA containing EPs, as a potential source of viral antigens challenging the fact that only cell-associated antigens may be involved in the activation of CD8 + T cells by antigen cross-presentation. Additionally, we found that (iv) the presence of late apoptotic markers in feeder cells as well as (v) improved mitochondrial functions in feeder cells contribute to a favorable microenvironment for enhanced cross-presentation. Based on our results, we suggest that various signaling pathways triggered by active P2RX7 in infected CM feeder cells interplay and significantly contribute to the increased antigen cross-presentation capability of BMDCs leading to enhanced SIINFEKL//H2-K b expression in BMDCs and subsequent CD8 + T cell activation.

Limitations of study

Since the ECAR measured in this assay is only a quantitative measurement of the total amount of acid (H + ) produced during both the tricarboxylic acid cycle and glycolysis, further assays may help to determine more specific alterations in the glycolytic metabolism ( 55 ). This study did not include an in-depth analysis of different extracellular particle fractions such as apoptotic bodies, microvesicles and exosomes. Further fractionation and subsequent characterization will allow to determine the exact content of these particles. Further work should address the role of DBA P2RX7 in CM cells. Even though the plasma membrane receptor is not functional in cells with a DBA background due to the P451L mutation ( 42 ), further studies may try to characterize other functions of this receptor during MVA infection in vitro and in vivo . Up to now, animal models such as appropriate P2RX7 knockout mice are lacking to investigate the role of the receptor in vivo . Since ATP may play a role during the co-culture of immune cells ( 85 ), future studies should assess its role in feeder cells, APCs and during co-culture. In addition, understanding how the altered mitochondrial metabolism affects EP release and composition as well as cross-presentation on the molecular level would be important for future studies. Due to the limitations of our in vitro murine model for the cross-presentation of MVA antigens, future research should include human cells.

Materials and methods

The identifiers of all reagents and resources used are listed in Supplementary Table 1 ( Supplementary Material ).

For isolation of bone marrow female 12-to 16-week adult C57BL/6N mice were purchased from Janvier and were allowed to acclimate for a minimum of one week in the in-house animal facility. For weekly T cell stimulation, the spleen of adult C57BL/6N mice was used. Animals were maintained at the Zentrale Einrichtung für Tierversuchsanstalt (ZETT) at the University of Düsseldorf under specific pathogen-free conditions. Experimental procedures have been approved by the regional authorities (North Rhine-Westphalia State Environment Agency - LUA NRW, Germany) and the animal use committee at the University of Düsseldorf (Reg. No O119/11).

Recombinant MVA were generated by homologous recombination as previously described ( 33 , 86 ). All stock preparations of MVA used in this study were diluted to a concentration of 1x10 9 viral particles/mL and maintained at -80°C. Viral aliquots were thawed in a water bath, sonicated for one minute, briefly vortexed and spun down for usage. Freeze/thawed aliquots were not used more than three times.

Infection of cells

Unless differently stated, cells were harvested, pelleted in a falcon and infected with MVA (MOI1, unless otherwise specified) for one to two hours at 37°C, 5% CO 2 with intermittent shaking every 15min. Cells were washed twice before incubation with other cells for cross-presentation experiments. For the remaining experiments cells were seeded and incubated immediately. Since harvesting at different time points was required for expression kinetics analyses and titration experiments, cells were seeded and allowed to adhere before infection. Infection was then performed directly on the plate with intermittent shaking and washing after one hour of incubation at 37°C and 5% CO 2 . For infection in 96-well plates (Mitochondrial function assay and intracellular ATP determination assay) virus was added to each well of the plate, shaken every 15min for 1h and subsequently incubated for the remaining time frame at 37°C and 5% CO 2.

Fluorometric analysis of intracellular Ca 2+ levels or EtBr-guided pore opening

1x10 6 cells were either mock- or MVA (MVA-PK1L-OVA) infected at an MOI of 1 for either 4h or 20h. For Ca 2+ measurements infected cells were placed in a falcon for loading with 4µM FURA-2 AM (Sigma-Aldrich) in saline solution (12.5mM NaCl, 0.5mM KCl, 0.1mM MgSO 4 , 2mM HEPES, 0.55mM D-glucose, 0.5mM NaHCO 3 (all Sigma-Aldrich)) supplemented with 0.5mM CaCl 2 (pH 7.4, Merck) and 250µM sulfinpyrazone (Sigma-Aldrich) at 37°C for 20min. Cells were then washed, resuspended in saline solution and stimulated with the indicated concentrations of Bz-ATP (Sigma-Aldrich) and 1µM ionomycin (Invitrogen) for the recording of intracellular Ca 2+ release. For detection of pore opening at the cell membrane, cells were loaded with 2µL ethidium bromide (EtBr (Sigma-Aldrich)) and stimulated with 200µM Bz-ATP and 100µM digitonin (Sigma-Aldrich). Measurements were done in a thermostat quartz cuvette using a Perkin-Elmer KS50 rotating and heating system at a wavelength of 340/380nm (excitation) and 505nm (emission) for intracellular Ca 2+ release and at a wavelength of 360nm (excitation) and 580nm (emission) for EtBr- pore opening assay.

Generation of stably transfected cell lines

Cloudman S91 cells (ATCC CCL-53.1) were seeded at a density of 1.5x10 5 cells per well in a 6-well plate and transfected with either 3µG pcDNA3 control or P2RX7 encoding plasmid DNA using Lipofectamine reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, plasmid DNA was dissolved in medium, incubated with 1µL PlusReagent for 5min and then 3µL Lipofectamine was added and further incubated for 30min at room temperature. The Lipofectamine-DNA mixture was then added dropwise to the cells and cells were selected for geneticin (0.2mg/mL) resistance two days post-transfection. Transfection efficacy was confirmed by Western Blot analysis of P2RX7 synthesis and by fluorometric analysis measuring the P2RX7-dependent intracellular Ca 2+ increase.

Live cell confocal imaging

Cells were grown on a round cover dish placed in a 6-well plate at a density of 5x10 5 cells per well. Cells were stained with 2µM PKH-26 (Sigma-Aldrich) and 2µM Quinacrine (Sigma-Aldrich) for 10min at 37°C and 5% CO 2 in saline saccharose solution (30mM saccharose, 0.1mM K 2 HPO 4 , 0.1mM MgSO 4 , 0.5mM D-glucose, 0.2mM HEPES (all Sigma-Aldrich)) supplemented with CaCl 2 (pH7.4). Cells were placed in a holder device for round cover glasses and stimulated with 200µM Bz-ATP. Images were acquired in 6-second intervals for approximately 15min. Images were taken at 60x magnification of the Olympus Fluoview FV3000 (Olympus). Data visualization was achieved using OMERO software (Open microscopy imaging).

Generation of bone marrow-derived dendritic cells

Bone marrow was obtained from 12-to 16-week-old C57BL/6N and 5x10 6 bone marrow cells were seeded with 10% GM-CSF (obtained from supernatant of B16 cells expressing GM-CSF, originally kindly provided by Georg Häcker, Freiburg) in RPMI-medium (Gibco) containing 10% heat-inactivated FBS and 50µM 2-mercaptoethanol (M2 Medium) in 10cm Petri-dishes. On day three fresh M2 Medium and GM-CSF was added to the primary culture and on day six 10mL medium was replaced with fresh M2 Medium containing GM-CSF. BMDCs were used on day seven for all experiments.

T cell restimulation

CD8 + T cell lines were generated as described recently ( 33 ). For weekly T cell stimulation, both EL4 cells (ATCC TIB-39) and naïve splenocytes from C57BL/6N were irradiated with 100Gy or 30Gy, respectively. EL4 cells were loaded with 1µg/mL B8R-peptide (TSYKFESV; immunodominant peptide derived from the B8 protein from vaccinia virus) or Ova-peptide (SIINFEKL; derived from ovalbumin) and then co-incubated with splenocytes, CD8 + specific T cells and M2 Medium containing 5% TCGF (T-cell growth factor). Both peptides are H2-K b -restricted.

Cross-presentation assay

Cloudman S91 murine melanoma (CM) cells (MHC I haplotype H2-d) were used as feeder cells for antigen cross-presentation assays. A total of 2x10 6 cells were either mock- or MVA-PK1L-OVA (MOI1) infected for 20h, washed and subsequently incubated with psoralen (1µg/mL) (Sigma-Aldrich) for 15min at 37°C and 5% CO 2 and treated with UV-A light (PUVA) for further 15min. Cells were harvested, transferred to a falcon and washed with medium. CM feeder cells were co-incubated with uninfected BMDCs, which were previously generated from bone marrow of C57BL/6N mice (MHC I haplotype H2-K b ) at a ratio of 1:1 in a 6 cm dish for 18h. The next day, the co-culture of CM and BMDCs was harvested, washed in M2 Medium and resuspended in M2 medium in a final volume of 1mL. One part of the co-culture suspension (200µL) was immediately stained for the surface expression of peptide/MHCI complexes (SIINFEKL peptide within H2-K b ) on BMDCs, while 100µL of the CM-BMDCs co-culture (containing 2x10 5 BMDCs as antigen-presenting cells) was further incubated with 2x10 5 B8R- or Ova- specific CD8 + T cells in the presence of 1µg/mL Brefeldin A (Sigma Aldrich) for 4h at 37°C and 5% CO 2 . Further analysis of CD8 + T-cell activation is described below.

Intracellular cytokine staining (ICS)

To determine the antigen presentation capacity of dendritic cells in the cross-presentation setting, peptide-specific T cell lines were used as a read out system. After 4h incubation (see above cross-presentation assay), cells were washed with PBS and dead cells were excluded by staining with Fixable viability dye eFluor 506 (Invitrogen) (1:600) for 20min on ice. Cells were washed with FACS buffer (PBS supplemented with 1% BSA and 0.02% sodium azide) and then stained using anti-mouse CD8α eFluor 450 (eBioscience) (1:300) for 20min on ice. Subsequently, cells were permeabilized with BD Cytofix (BD Biosciences) for 15min on ice and then stained with Anti-mouse IFNy APC (Invitrogen) (1:400) and Anti-mouse TNFα PE-Cyanine7 (Invitrogen) (1:300) in 1:10 diluted BD Perm/Wash for 30min on ice. Cells were washed twice and resuspended in 1% PFA for subsequent analysis using the FACS Canto II device (BD Biosciences).

SIINFEKL/H2-K b surface staining/MHC II maturation staining

Antigen processing and presentation capacity was also assessed by measuring the MHCI/peptide complex formation as SIINFEKL/H2-K b expression on the surface of the dendritic cells. For this co-cultured BMDCs (see above cross-presentation assay) were washed with PBS and dead cells were stained with Fixable viability dye eFluor 660 (Invitrogen) (1:2000) for 20min on ice. Fc-receptors were blocked using anti-mouse CD16/CD32 (eBioscience) (1:200). After Fc-blocking, surface staining was performed for 30min on ice using anti-mouse CD11c PE (Invitrogen), anti-mouse H2-Kb FITC (Biolegend) and anti-mouse SIINFEKL/H2-K b PE-Cyanine 7 (eBioscience) (all 1:300 in FACS buffer). Cells were washed twice and resuspended in 1% PFA for subsequent analysis using FACS Canto II. Alternatively, cells were stained with Fixable viability dye eFluor 506 (Invitrogen) (1:600) for 20min on ice, followed by the Fc-blocking step and surface staining with CD11c APC-Cyanine 7 (BD Pharmingen) and MHCII PE (all 1:300 in FACS buffer).

Viral or cellular gene expression

For kinetic analysis of viral gene expression (0h to 24hpi), 2x10 6 cells were infected with MVA-PK1L-Ova (MOI1) for 1h at 4°C, resulting in the virus attachment to the cell surface. After washing cells were harvested at the indicated time points, spun down and the pellet was resuspended for total RNA isolation as described in the manufacturer´s protocol (RNeasy Mini Kit (Qiagen)). Briefly, cells were lysed using RLT buffer containing 10µL 2-ß-mercaptoethanol and mixed with one volume of 70% ethanol for subsequent isolation using the RNeasy Mini spin column. cDNA was then transcribed using the Revert Aid H minus first strand cDNA synthesis (Thermo Fisher Scientific) according to the manufacturer’s instructions and used as a template for subsequent quantitative PCR reaction with PowerUp SYBR Green Master Mix (Applied Biosciences). Expression of viral B8R , Ova , A19L and cellular P2rx7 genes was normalized to expression of 18S-rRNA housekeeping gene and ΔΔCT was calculated by further comparison of ΔCT values with the 0h time point of CM wildtype cells. Primer sequences are listed in Supplementary Table 1 .

Viral replication

In order to determine the replication capacity of MVA, 1x10 6 CM cells (WT, pcDNA3- or P2RX7- transfected) were infected with MVA-p7.5-GFP at MOI5 for 0h (1h at RT), washed and further incubated until harvested at 4h, 8h or 24hpi. Collected samples were vortexed and subjected to three rounds of freeze-thaw-sonication cycles to release viral particles. Viral suspensions were then used to prepare serial dilutions that were plated on 96-well plates containing MVA-permissive DF-1 cells (ATCC CRL-12203) (80% confluent). Fluorescent signal and cytopathic effect was monitored for seven days post-infection to determine the 50% endpoint titer of viral particles per milliliter by using the Spearman-Karber method to calculate the tissue culture infectious dose 50 (TCID 50 ).

Western Blot analysis

For extraction of proteins, 2x10 6 cells were infected (see above “infection of cells”) and harvested at the indicated time points. Collected cells were spun down by centrifugation, washed with PBS and resuspended in RIPA buffer (Thermo Fisher Scientific) containing HALT Protease & Phosphatase Inhibitor cocktail (Thermo Fisher Scientific) (1:100). After three rounds of freeze-thaw-sonication cycles, supernatants were harvested after a single centrifugation step at full speed for five minutes at 4°C. Protein content was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). SDS-PAGE and blotting on nitrocellulose membranes was performed as described elsewhere ( 87 ). Membranes were incubated with Anti-Ovalbumin (Rockland) (1:20 000); Anti-Cleaved caspase-8 (Cell signaling) (1:1000); Anti-P2RX7 (Sigma-Aldrich) (1:200) and Anti-ß- Actin (Sigma-Aldrich) (1:50 000). Relative quantification of specific proteins was done by calculating ratio of the protein of interest with the ß-Actin loading control using the ImageJ analysis tool (US National Institutes of Health, Bethesda, USA).

Phosphatidylserine exposure analysis

Surface staining of phosphatidylserine residues on MVA-infected cells was done according to Apotracker-Green protocol (Biolegend) at either 6hpi or 20hpi. Briefly, 2x10 5 cells were washed with FACS buffer and incubated in 400nM Apotracker-Green staining solution for 20min at room temperature. Cells were subsequently stained with fixable viability dye eFluor 660 (1:2000) for 20min on ice, washed and immediately analyzed by FACS. Cells were either gated for APC-negative (non-permissive for viability dye) and FITC-positive (Apotracker Green-positive) populations, designating early apoptotic cells or gated for APC- and FITC-double-positive populations, indicating late apoptotic cells.

Isolation of extracellular particles and supernatant fractions

For extracellular particle isolation the protocol was adapted according to Pegoraro and colleagues ( 8 ). Four T75 flasks (approximately 8x10 6 cells/flask) were seeded with CM cells one day before infection to obtain 90% confluency. Before infection, one flask per cell line was counted in order to calculate the respective MOI. Cells were allowed to rest for 30min at room temperature and after washing 3mL medium was added in each flask. MVA-PK1L-Ova (MOI 1.5) was added and flasks were shaken every 15min for one hour (at 4°C for RNA isolation). After one hour cells were washed and harvested (0h value) or further incubated for a total of 20h at 37°C and 5% CO 2 . For harvesting, medium was discarded, cells were washed with PBS and 3mL saline solution supplemented with 0.05mM CaCl 2 was added. Cells were stimulated with 200µM Bz-ATP for 30min at 37°C. Thereafter, the supernatant was aspirated and centrifuged at 300g for five minutes at 4°C to remove cell debris. The cleared supernatant was harvested, aliquoted in Eppendorf tubes and centrifuged at 20.000 g for one hour at 4°C. The supernatant was discarded and the remaining extracellular particle fraction (EP-fraction) was either used for quantitative RNA analyses (resuspended in RLT buffer with 2-ß-mercaptoethanol), western blot analyses (resuspended in RIPA buffer with HALT Protease & Phosphatase Inhibitor cocktail) or for cross-presentation assays (resuspended in PBS). For cross-presentation assays, EP fractions were additionally PUVA treated prior to the last centrifugation step, as described above.

For isolation of supernatants, 2x10 6 cells per condition tested were used. Cells were either MVA-PK1L-Ova (MOI1) or mock-infected for the indicated time (8h or 20h for western blot analysis; 20h for Legendplex and cross-presentation assays), harvested and supernatants (sup-fraction) were collected after centrifugation at 300g for five minutes. For indicated experiments, supernatant fractions were further passed through a 0.2µM size pore filter (fil sup-fraction) to be used for cross-presentation experiments. All supernatant fractions (sup- or fil sup-fractions) used for cross-presentation assays were additionally PUVA treated as described above.

Cytokine and chemokine analysis in supernatants

The release of cytokines/chemokines was analyzed using the Legendplex MU anti-virus response panel (Biolegend). Briefly, 2x10 6 cells were either MVA-PK1L-Ova (MOI1) or mock-infected for 20h. After harvesting the cell suspensions, supernatants were collected after centrifugation at 300g for 5min and processed according to the manufacturer’s instructions. Data was analyzed using the Biolegend LEGENDplex Data Analysis Software (Biolegend).

Cross-presentation assays using EP- or supernatant-fractions

Infection of CM feeder cells was performed as described above for cross-presentation assays. On day two, CM WT feeder cells (either mock- or MVA-PK1L-OVA infected, MOI1) were co-incubated with BMDCs and, additionally, pulsed with either EP-, sup- or fil sup-fractions. These fractions were isolated from either infected CM pcDNA3 (transfected cells with inactive P2RX7) or infected CM P2RX7 cells (transfected cells with active P2RX7) after 20hpi as described above. On day 3, cross-presentation assays were continued as described above.

Intracellular quantification of P2RX7

To assess the expression of P2RX7, 2x10 5 CM cells (WT, pcDNA3- or P2RX7-transfected) were mock- or MVA-PK1L-OVA (MOI1) infected. After 20hpi, cells were stained with fixable viability dye eFluor 660 (1:2000) for 20min on ice, permeabilized with BD Cytofix for 15min on ice and then stained with anti-P2RX7 (1:200) for one hour on ice to quantify the intracellular presence of P2RX7. Cells were further incubated with anti-mouse-IgG-PE (Jackson laboratories) (1:200) secondary antibody for 30min on ice, washed and immediately used for FACS analysis by FACS Canto II.

Mitochondrial metabolism analysis

The day prior to infection, 2x10 4 cells were seeded in a Seahorse XF96 Cell culture Microplate (Agilent Technologies). Cells were allowed to adhere for 1h at room temperature and were further incubated at 37°C at 5% CO 2 overnight. The next day cells were infected with MVA-PK1L-Ova (MOI5, 6h). Mitochondrial function was assessed using the Seahorse XF Cell Mito Stress test (Agilent technologies) according to the manufacturer’s instructions. Compounds have been used at the concentration of 15µM for Oligomycin, 5µM for FCCP and 5µM for Rot/AA.

Intracellular ATP measurements

The day prior to infection, 5x10 4 cells were seeded in a 96 flat well chimney base plate and incubated overnight at 37°C and 5% CO 2 . Cells were infected with MVA-PK1L-Ova for 6h (MOI5) before intracellular ATP concentrations were determined using the Luminescent ATP detection assay kit (Abcam) as described in the supplier´s protocol. Briefly, cells were lysed and ATP was stabilized by a detergent during a shaking step. After the addition of the substrate solution, prompted luminescence was measured and compared to ATP standard samples using a Spark plate reader (Tecan).

Extracellular ATP measurement

1x10 6 CM cells (WT, pcDNA3- or P2RX7-transfected either MVA-PK1L-Ova or mock-infected, MOI5) were seeded in 1mL in a 6-well plate. The supernatant was harvested after 6hpi. For each condition, 50µL supernatant was incubated with 50µL of FirezymeB Diluent buffer (Firezyme). Samples as well as an ATP standard (Sigma- Aldrich) were compared in a standard curve at serial dilutions run by the Luminometer Victor 3 1420 Multiwell counter (Perkin Elmer) with automated addition of 100µL Enliten Luciferase/Luciferin reagent (Promega) to detect emitted luminescence.

Quantification and statistical analysis

Details on statistical analyses are integrated in figure legends. When indicated, data was normalized to untreated or WT control cells. Unpaired two-tailed student’s t-test was used to calculate statistical significances using Prism 8 (GraphPad Software). Extracellular particles ( Figure 1C ) were quantified by counting three adjacent frames of each replicate upon Bz-ATP stimulus and normalized to cell numbers (determined by quinacrine staining) per frame. Graphical data represent mean values with error bars indicating SD or SEM with P-values of ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***) and ≤ 0.0001 (****) indicating significant differences between groups.

Data availability statement

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Ethics statement

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. The animal study was approved by North Rhine-Westphalia State Environment Agency - LUA NRW, Germany) and the animal use committee at the University of Düsseldorf (Reg. No O119/11). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

YL: Writing – review & editing, Data curation, Formal analysis, Investigation, Validation, Writing – original draft. SM: Investigation, Writing – review & editing. GA: Investigation, Writing – review & editing. JW: Investigation, Methodology, Writing – review & editing. IK: Investigation, Methodology, Writing – review & editing. EDM: Investigation, Methodology, Writing – review & editing. AP: Investigation, Methodology, Writing – review & editing. RL: Methodology, Writing – review & editing. KK: Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. PP: Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. RT: Investigation, Methodology, Writing – review & editing. FDV: Methodology, Writing – review & editing. EA: Methodology, Supervision, Validation, Writing – review & editing. ID: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Grants GK1949/2 and DR632/2-1 project No 452147069 to ID and by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 812915 to ID. EA and FDV were supported by grants from the Italian association for cancer research (AIRC grant numbers: IG 22837, IG 13025 and IG 18581).

Acknowledgments

We thank the Laboratory of Clinical Pathology, especially Luigia Ruo (Department of Medical Sciences, University of Ferrara) for support in fluorometric assays and Sha Tao and Cornelia Barnowski (Institute of Virology, Universitätsklinikum Düsseldorf) for answering scientific and technical questions. We acknowledge Professor Massimo Bonora (Department of Medical Sciences, University of Ferrara) for support with Live Confocal Imaging. Computational infrastructure and support were provided by the Centre for Information and Media Technology at Heinrich-Heine-University Düsseldorf.

Conflict of interest

FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a biotech Company involved in the development of anti-P2X7 antibodies, and a Consultant with Breye Therapeutics.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2024.1360140/full#supplementary-material

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Keywords: Modified Vaccinia Virus Ankara, cross-presentation, P2RX7, extracellular vesicles, cytokines

Citation: Longo Y, Mascaraque SM, Andreacchio G, Werner J, Katahira I, De Marchi E, Pegoraro A, Lebbink RJ, Köhrer K, Petzsch P, Tao R, Di Virgilio F, Adinolfi E and Drexler I (2024) The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation. Front. Immunol. 15:1360140. doi: 10.3389/fimmu.2024.1360140

Received: 22 December 2023; Accepted: 05 April 2024; Published: 22 April 2024.

Reviewed by:

Copyright © 2024 Longo, Mascaraque, Andreacchio, Werner, Katahira, De Marchi, Pegoraro, Lebbink, Köhrer, Petzsch, Tao, Di Virgilio, Adinolfi and Drexler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Ingo Drexler, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

• Open access
• Published: 22 April 2024

Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine

• Suyang Wang 1 ,
• Chao-Cheng Chen 1 ,
• Ming-Hung Hu 1 ,
• Michelle Cheng 1 ,
• Hsin-Fang Tu 1 ,
• Ya-Chea Tsai 1 ,
• Jr-Ming Yang 1 ,
• T. C. Wu 1 , 2 , 3 , 4 ,
• Chuan-Hsiang Huang 1 &
• Chien-Fu Hung   ORCID: orcid.org/0000-0001-9170-7797 1 , 2  

Journal of Translational Medicine volume  22 , Article number:  378 ( 2024 ) Cite this article

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Bacteria-based cancer therapy have demonstrated innovative strategies to combat tumors. Recent studies have focused on gram-negative bacterial outer membrane vesicles (OMVs) as a novel cancer immunotherapy strategy due to its intrinsic properties as a versatile carrier.

Here, we developed an Human Papillomavirus (HPV)-associated E7 antigen displaying Salmonella -derived OMV vaccine, utilizing a Poly(L-arginine) cell penetrating peptide (CPP) to enhance HPV16 E7 (aa49-67) H-2 Db and OMV affinity, termed SOMV-9RE7.

Due to OMV’s intrinsic immunogenic properties, SOMV-9RE7 effectively activates adaptive immunity through antigen-presenting cell uptake and antigen cross-presentation. Vaccination of engineered OMVs shows immediate tumor suppression and recruitment of infiltrating tumor-reactive immune cells.

The simplicity of the arginine coating strategy boasts the versatility of immuno-stimulating OMVs that can be broadly implemented to personalized bacterial immunotherapeutic applications.

Current advanced cancer immunotherapy treatments boost humoral and cellular immunity without the non-specific targets and toxic effects on normal cells as conventional cancer treatment [ 1 , 2 ]. However, despite these advances, the inability to predict treatment efficacy, the need for additional biomarkers, the development of resistance to cancer immunotherapies, and the high treatment costs continue to serve as a limitation in immunotherapeutic treatments [ 3 , 4 ]. Therefore, therapy with live tumor-targeting bacteria has received attention as a unique option to overcome these challenges [ 5 ]. Studies have shown that bacteria-based therapies can serve as a monotherapy or complement to other anticancer therapies [ 5 , 6 , 7 ]. Previously, we showed that live Salmonella and cytokine combination therapy induced potent T-cell immunity and long-term tumor control in mice [ 8 ]. We also demonstrated that heat-inactivated Salmonella (S. typhimurium) could display tumor antigens to achieve tumor-specific immune responses [ 9 ]. Nevertheless, a significant limitation of live bacteria lies in its off-target toxicity and lowered efficacy in inactivated bacteria [ 10 ]. Therefore, further investigation is needed to develop an approach with bacteria-based therapies that is more potent than heat-inactivated bacteria therapies, while also addressing the safety concerns of using live attenuated bacteria.

In recent years, studies have shown that gram-negative bacteria can naturally release outer membrane vesicles (OMVs), which comprises of lipopolysaccharides (LPS), outer membrane proteins, periplasmic proteins, and phospholipids and can serve as carriers for various substances such as toxins, metabolites, enzymes, virulence factors, and genetic material (DNA and RNA) [ 11 , 12 , 13 ]. Unlike attenuated bacteria, OMVs are considered safer and can effectively stimulate the immune system by delivering key immunogens from their parent bacteria [ 13 , 14 , 15 ]. Genetic engineering techniques have shown that the construction of recombinant OMVs improves target precision through surface protein and carries exogenous proteins for improved immunogenicity [ 16 , 17 ]. As a neoantigen vaccine, OMVs can fuse multiple surface proteins and therefore simultaneously display various distinct tumor antigens to elicit a synergistic antitumor immune response in metastatic lung melanoma and subcutaneous colorectal cancer models [ 18 ]. These studies have further underscored the impact of bacteria OMVs as a versatile immunotherapeutic approach in developing cancer vaccines, as it presents a balance between immunogenicity and safety [ 15 , 19 , 20 , 21 ].

Previously, we optimized a bacteria antigen-display strategy through modification of the Human Papillomavirus (HPV)-associated E7 antigen, incorporating nine arginine residues (9RE7) for enhanced E7 coating [ 8 ]. Poly-l-arginine is a cell penetrating peptide (CPP), often used for mammalian cell uptake and delivery of drugs or macromolecules such as proteins and enzymes [ 22 , 23 ]. Here, we coated 9RE7 on Salmonella OMV (SOMV), a naturally released OMV derived from Salmonella SL7207, and synthesized SOMV-9RE7 which will be investigated to serve as a safer and more effective method to delivery HPV E7 antigen. By generating systemic E7-specific CD8+ T cells and recruiting them to the tumor microenvironment (TME), SOMV-9RE7 exhibited promising antitumor effects. These results demonstrate a broad application of 9RE7 peptide and an alternative to traditional bacteria immunotherapy.

Material and methods

Cell preparation.

E7-expressing TC-1 tumor cells and dendritic cells were grown in vitro in RPMI 1640 media containing 10% (v/v) fetal bovine serum, 50 units/mL of penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM non-essential amino acids, and 0.1% (v/v) 2-mercaptoethanol under 37 °C with 5% CO2. E7-specific CD8+ splenocytes were isolated from mice vaccinated with E7 DNA and incubated with E7 expressing cells and maintained in culture.

Bacteria-derived outer membrane vesicle

Salmonella SL7207 was grown overnight in LB broth at 37 °C. On the next day, 1 mL of the overnight Salmonella culture was added to 9 mL of fresh LB medium and incubated until O.D. 600 reading of 0.5. This freshly cultured Salmonella was added to fresh LB medium at 1:100 dilution in 250 mL culture at 37 °C for 16 h. The supernatant was collected after centrifugation and filtered through a 0.45 µm MCE Membrane Filter (Millipore Sigma). Filtered medium was transferred to ultracentrifuge tubes and at 150,000 RPM for 3 h (Beckman). The supernatant was removed and particles at the bottom of the tubes were collected and suspended in 1 mL PBS and stored at − 80 °C. OMV yield is calculated using Bio-Rad protein assay with a concentration of approximately 1.5 mg/mL.

Peptide synthesis

Peptides used in this study include RRRRRRRRR-RAHYNIVTF (E7 protein amino acids 49–57), termed 9RE7, and was synthesized by GenScript (Piscataway, NJ, USA) at a purity of over 90%.

SOMV-9RE7 generation and characterization

SOMV-9RE7 is synthesized by combining SOMV and 9RE7 in PBS buffer to be vortexed for 30 min. Subsequent dialysis is performed using a 50kD Amicron Ultra Centrifugal Filter (Millipore Sigma) to remove unbound peptides. For characterization of SOMV-9RE7, 10 µg of SOMV was mixed with 1 µg of FITC conjugated peptides E7 or 9RE7 in the PBS buffer. Bacteria/peptide mixture was vortexed at room temperature for 30 min, followed by dialysis with the 50kD Amicron Ultra Centrifugal Filter (Millipore Sigma) to remove unbound peptides. FITC signals were measured by 13-color B-Y-R-V CytoFLEX S (Beckman Coulter). Particle size and charge was determined by Malvern Zetasizer (Worcestershire, UK). 40% of 9RE7 remained coated on SOMV after dialysis. This was determined by interpolating the standard curve of FITC-labeled SOMV-9RE7 at 500 nm with Nanodrop One (Thermo Fisher Scientific).

In vitro T cell activation

10 µg of SOMV and 1 µg 9RE7 are used to synthesize SOMV-9RE7 as described above. E7-specific CD8+ T cell activation follows previously established protocol [ 24 , 25 ]. SOMV-9RE7 is incubated with 1 × 10 5 dendritic cell line in 96 well plate cultured with complete RPMI media at 37 °C, 5% CO2 overnight. After aspirating culture medium and washing with PBS, 5 × 10 5 E7-specific CD8+ T cells were added to the dendritic cell line and blocked with Brefeldin A + Monensin Golgi Plug (Thermo Fisher Scientific) overnight. Cells were collected and stained with APC-A750-conjugated anti-mouse CD8α antibody (Biolegend) before permeabilization with eBioscience Fixation (Invitrogen) and intracellular staining FITC-conjugated IFNγ antibodies.

Mice vaccination

For tumor inoculation, 1 × 10 5 TC-1 cells in 50 µL of PBS were subcutaneously injected into 6–8 weeks old female C57BL/6 mice at the lower right abdomen. Largest length and width were measured by digital calipers twice per week. Tumor volumes were calculated by the formula: V = (Length × Width 2 )/2. At the indicated time points, TC-1 tumor-bearing mice were vaccinated subcutaneously in the tumor graft region with 10 µg of 9RE7 peptides, 10 µg SOMV, or SOMV-9RE7 (10 µg of SOMV and 10 µg of 9RE7).

Flow cytometry analyses

Blood samples were collected from vaccinated mice after final treatment. Red blood cell (RBC) lysis using RBC lysis buffer (eBioscience) collected peripheral blood mononuclear cells (PBMC). For tumor tissue sample preparation, tissue was collected from mice and transferred to FACS buffer in gentleMACS C tubes (Miltenyi Biotec). Tissue digestion enzymes including Collagenase I, Collagenase IV, and DNase I were added to samples. Samples were dissociated with gentleMACS Dissociator (Miltenyi Biotec) before incubating for 20 min. After centrifugation and buffer exchange, tumor samples were purified by loading onto Ficoll-Paque Plus (GE Healthcare Life Sciences, Marlborough, MA). Tubes were centrifuged for 20 min and Ficoll-RPMI interface was collected. Samples were then counted, plated at equal cell numbers, and prepared for flow cytometry. Spleen grinded through Corning® 70 μm Cell Strainer (Millipore Sigma) with syringe stopper and suspended in RPMI medium. Next, RBC lysis was performed and splenocytes were counted and plated at appropriate cell numbers.

For FACS analysis, live cells were identified with Zombie Aqua live/dead (BioLegend) and Fc Block to reduce nonspecific antibody binding. Peripheral antigen-reactive CD8+ T cell population in PBMC was identified with PE-conjugated HPV16 E7aa49–57 peptide loaded H-2 Db E7 tetramer and APC-A750-conjugated anti-mouse CD8α antibodies. For tumor infiltrating lymphocyte and splenocyte analyses, we used APC-A700-conjugated anti-mouse CD45 antibodies, BV421-conjugated anti-mouse CD3 antibodies, PE-Cy5-conjugated anti-mouse CD8 antibodies, PE-conjugated HPV16 E7 tetramer, and BV-650-conjugated anti-mouse IFNγ antibodies. FACS analysis was performed using CytoFLEX S (Beckman Coulter Life Sciences) and fluorescent compensation was generated using single-antibody controls. All flow cytometry data and gating strategies were performed by FlowJo software.

Statistical analysis

GraphPad Prism V.10 software was used to perform data statistical analysis. Data is represented as means and standard error of the mean. Kaplan–Meier survival plots were used to estimate the survival percentage and tumor-free rate. Long rank tests were used to compare the survival time between treatment groups. Comparison between individual data points were analyzed for variance with one-way ANOVA and the Tukey–Kramer multiple comparison test, *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns = not significant.

Poly-arginine anchored E7 peptide can be coated on Salmonella -derived outer membrane vesicles as an immunogenic antigen carrier

First, we wanted to demonstrate that SOMV can bind to 9RE7 peptide efficiently, so we characterized the binding affinity of 9RE7 to SOMV using FITC-conjugated peptides. We were able to visualize the 9RE7-FITC and E7-FITC distribution on SOMV with nanoscale flow cytometry. In the flow cytometry histogram, SOMV coated with 9RE7-FITC had a more homogeneous population than coating with E7-FITC (Fig.  1 A). By calculating the mean fluorescence intensity, SOMV-9RE7-FITC had a significantly higher FITC signal than SOMV-E7-FITC, increasing gMFI by three folds and showing the critical role of the 9R moiety in achieving high affinity peptide coating (Fig.  1 B). Next, we measured the particle size distributions and surface charges of SOMV and SOMV-9RE7 to elucidate on particle properties. Using dynamic light analysis, we observed a similar size distribution of SOMV as SOMV-9RE7, average zeta size of 132.6 nm and 153.2 nm respectively, with no significant changes in particle size after modification with 9RE7 (Fig.  1 C, D ). However, there was a significant alteration to the vesicle surface charge, where the negative charge of SOMV had a positive shift by attaching 9RE7 that increased its Zeta-potential from -6.7 mV to -1.9 mV (Fig.  1 E). Therefore, we have demonstrated the successful creation of a stable particle, SOMV-9RE7, using the arginine-anchored E7 peptide platform.

Characterization of 9RE7 peptide presenting Salmonella- derived outer membrane vesicle. Nanoscale flow cytometry analysis of binding efficiency between Salmonella -derived outer membrane vesicle (SOMV) and nona-arginine extended HPV E7 peptide (9RE7). 10 µg of SOMV is incubated with 1 µg FITC-conjugated E7 peptide (E7-FITC), termed SOMV-E7-FITC, or FITC-conjugated 9RE7 (9RE7-FITC) peptide, termed SOMV-9RE7-FITC, with A representative flow cytometry histogram of FITC channel and B bar graph of gMFI on peptide coated SOMV. C  Dynamic light scattering particle size frequency of SOMV and 9RE7-coated SOMV (SOMV-9RE7). D Z-average size comparison of SOMV and SOMV-9RE7. E Zeta potential measurements of the surface electric charge on SOMV and SOMV-9RE7. F Flow cytometry analysis of E7-specific T cell activation by SOMV-9RE7 in vitro. SOMV, 9RE7, and SOMV-9RE7 are prepared and dialyzed with 50kD MWCO Amicon centrifugal filter before incubated with dendritic cell line overnight. Upon replacing culture medium, E7-specific T cells are added to dendritic cells for another 24 h and blocked golgi protein transport inhibitor. After collecting the cell mixture, E7-specific T cells are stained with APC-A750-conjugated anti-mouse CD8α antibody stainings before cell membrane permeation and FITC anti-mouse IFNγ antibody for flow cytometry analyses. Representative flow cytometric images show CD8α and IFNγ gating. G Bar graph summary of T cells with positive IFNγ population. *p < 0.05, ****p < 0.0001

Next, we sought to show the immunogenic properties of SOMV-9RE7 as an antigen delivery particle. We prepared SOMV, 9RE7, and SOMV-9RE7 as described in methods prior to co-culturing with dendritic cells in RPMI medium overnight. Then, E7-specific CD8+ splenocytes were added to each group for stimulation overnight. From flow cytometry analysis gating for CD8 and intracellular IFNγ, SOMV-9RE7 treated group showed the highest splenocyte activation, and SOMV alone could not induce IFNγ expression (Fig.  1 F, G ). Importantly, 9RE7 after centrifugal filtration had no IFNγ expression, meaning that the dialysis protocol effectively removed free peptides. Our results indicated that SOMV-9RE7 can activate antigen presenting cells (APCs) and cross-present Major Histocompatibility Complex (MHC) class I restricted antigen to engage E7-specific CD8+ T cells in vitro.

Local treatment of SOMV-9RE7 show significant anti-tumor efficacy by inducing adaptive antigen-specific immunity

We investigated the response of the HPV positive TC-1 model to SOMV-9RE7 treatment. At 7 and 14 days after TC-1 inoculation, we delivered SOMV-9RE7 subcutaneously on the tumor side, while comparing it to 9RE7 and SOMV treatments, as shown in the schema in Fig.  2 A. Initial vaccination of SOMV and SOMV-9RE7 caused mild infection responses in the form of weight loss after 1 day, but the vaccinated mice quickly recovered within 3 days after treatment (Additional file 1 : Fig. S1). SOMV-9RE7 therapy had a visible impact on tumor growth after the first dose by significantly reducing tumor size compared to the control group, whereas 9RE7 or SOMV treatments had no long-term impact on tumor growth (Fig.  2 B). Accordingly, SOMV-9RE7 treated mice had significantly longer survival than the control group by doubling the last survival day (Fig.  2 C). PBMC was collected from each group after the second dose to test for development of antigen-reactive lymphocytes. In parallel to the antitumor results, flow cytometry analysis showed a significantly higher population of E7+ cytotoxic T lymphocytes (CTLs) under SOMV-9RE7 treatment (Fig.  2 D, E ). Tumor antigen 9RE7 was insufficient to induce E7-specific immunity without presence of an adjuvant. These results have shown that SOMV-9RE7 can suppress TC-1 tumor growth through mediating potent E7-specific CD8+ T cell mediated response in vivo.

Antitumor efficacy and antigen-specific immune response under SOMV-9RE7 treatment in TC1 tumor-bearing mouse model. A Schematic illustration of experiment schedule. C57BL/6 mice are inoculated with 1 × 10 5 TC-1 cells subcutaneously (s.c.) on day 0. They are given weekly treatments s.c. of either 10 µg 9RE7, 10 µg SOMV, or SOMV-9RE7 (10 µg SOMV incubated with 10 µg of 9RE7) for 2 weeks. PBMC is collected from the mice 5 days after the final treatment to measure. B Tumor growth curve of TC-1 tumor-bearing mice. C Kaplan–Meier survival curve of TC-1 tumor-bearing mice. D Quantification of E7-specific T cell population. PBMC was collected from each group and processed by staining with PE-conjugated HPV16 E7aa49-57 peptide-loaded H-2Db E7 tetramer and APC-A750-conjugated anti-mouse CD8α antibodies. Representative flow images show gating strategy for identifying CD8+ and PE+ T cells for all 4 groups and E bar graph representation are shown. **p < 0.01, ***p < 0.001, ****p < 0.0001

SOMV-9RE7 induces effector T cell proliferation and activity in the tumor microenvironment and spleen

To analyze the effect on SOMV-9RE7 on the organ systems, we adjusted the treatment schedule. 14 days after TC-1 inoculation, we initiated treatment for three doses before harvesting tumor and spleens from each group (Fig.  3 A). The SOMV-9RE7 treated mice had significantly smaller tumors in weight compared to the other groups (Fig.  3 B). Tumors were processed into single mononuclear cells for FACS analysis where we saw the highest CD45+ CD3+ lymphocyte accumulation in SOMV-9RE7 treated TME, whereas SOMV treatment did not induce lymphocyte trafficking in the TME (Fig.  3 C, D ). Within the total tumor infiltrating-lymphocytes (TILs), we examined the CD8+ CTL population (Fig.  3 E). The 9RE7 peptide treatment had a higher CD8+ population than the control or SOMV group, but peptide-coated vesicle SOMV-9RE7 had significantly more CTLs than the other groups (Fig.  3 F). More importantly, upon examining antigen-specific CTLs staining with HPV16 E7 tetramer (Fig.  3 G), there was only a small E7 positive populations in control, 9RE7, and SOMV groups compared to SOMV-9RE7, which had a significant E7-specific CTL population (Fig.  3 H). Finally, we examined E7-specific effector functional activity and saw significantly more IFNγ expression in SOMV-9RE7 treated E7+ CD8+ TILs (Fig.  3 I, J).

Analysis of tumor infiltrating lymphocytes in SOMV-9RE7 treated TC-1 tumor-bearing mouse model. A Treatment schedule of C57BL/6 mice inoculated with 1 × 10 5 TC-1 cells s.c. and treatments are given on day 14 for three doses. Tumor and spleen from each group are harvested on day 30. B Tumor weight of each treatment group after harvestation. Treated tumors are processed and isolated for mononuclear cells. Total TIL populations are identified by staining for APC-A700-conjugated anti-mouse CD45 and BV421-conjugated anti-mouse CD3 antibodies. Representative flow cytometry images show C CD45+ and CD3+ positive lymphocyte population and D bar graph analysis of CD45+ and CD3+ TIL percentages of each treatment. E CD8+ T cell subpopulation in CD3 + /CD45 + cells are identified with PE-Cy5-conjugated anti-mouse CD8 antibody and F bar graph analysis of CD8+ TIL population. Then, PE-conjugated HPV16 E7 tetramer are used to quantify E7-specific TIL with G gating strategy for E7 positive cytotoxic T cells and H bar graph analysis of E7 percentage. G E7-specific effector T cell functionality is determined by intracellular staining with BV650-conjugated IFNγ antibody, and J calculation of IFNγ expression in each group

At the same time, we analyzed splenocyte populations to compare TIL profile of the TME. Flow cytometry analysis of splenocytes shows a slight increase in CD45+ CD3+ splenic lymphocytes in the SOMV-9RE7 group compared to control mice (Fig.  4 A, B ). Interestingly, SOMV treated mice spleens showed a significant decrease in total CD45+ CD3+ lymphocytes than SOMV-9RE7 treatment (Fig.  4 B). Both SOMV and SOMV-9RE7 had increased CD8+ T cell populations (Fig.  4 C, D ), with only SOMV-9RE7 treated mice developing a significant E7-specific splenocyte population (Fig.  4 E, F ). Upon analyzing effector cell activation, SOMV-9RE7 treated E7-specific splenocytes had significantly more IFNγ activity than other groups (Fig.  4 G, H ). Combining both TIL and splenocyte analysis, SOMV-9RE7 has shown it effectively develops adaptive immunity in the lymphoid system, while actively recruiting E7 antigen-reactive T cells to the TME for tumor-targeting immunity.

Characterization of splenocyte populations in SOMV-9RE7 vaccinated TC-1 tumor model. A Flow cytometry analysis of splenic lymphocyte population from previously described treatments by staining with APC-A700-conjugated anti-mouse CD45 and BV421-conjugated anti-mouse CD3 antibodies. B Percentage of CD45+ and CD3+ splenocyte population in each treatment group. C Cytotoxic lymphocyte population within splenic lymphocytes is identified by staining with PE-Cy5-conjugated anti-mouse CD8 antibody and D bar graph shows CD8+ T cells from each group. E Within CD8+ T cells, E7 antigen-specific T cells are gated with PE-conjugated HPV16 E7 tetramer and F E7+ populations are analyzed from each group. ( G ) E7-specific CTL effector function is presented by intracellular staining with BV650-conjugated IFNγ antibody and H percentage of IFNγ expression with bar graph

In this study, we wanted to demonstrate the wide applicability of our efficient arginine peptide-enhanced antigen coating platform that is not only restricted to bacteria as carriers [ 8 ], but also bacteria-derived OMVs. Here, we saw the importance of arginine residues in enhancing affinity between 9RE7 peptide and SOMV. In contrast, FITC-labeled E7 coating of SOMV resulted in heterogeneous populations of SOMV-E7-FITC, indicating non-uniform distribution of E7-FITC on SOMV (Fig.  1 A). Further investigation of synthesized SOMV-9RE7 confirmed its 9RE7 presentation due to a positive surface charge increase from SOMV (Fig.  1 E). These results suggest that the 9RE7 peptide coating strategy can also be applied to OMVs, demonstrating its diverse utility as an immunotherapeutic method.

By retaining the similar inflammatory components to parental gram-negative bacteria, OMVs can engage APCs through TLR4 recognition and cross-present antigens to T cells [ 26 ]. Previously we saw that 9RE7 peptide could stimulate E7-specific through peptide loading on dendritic cells [ 8 ]. This creates a potential confound of false positive activation from unbound 9RE7 in SOMV-9RE7 synthesis that wasn’t removed during the filtration procedure. To eliminate this artifact, the 9RE7 control group was dialyzed with a 50kD centrifugal filter to examine peptide removal efficiency. From the low T cell activation treated with filtrated 9RE7 (Fig.  1 G), it can be confirmed that the increased IFNγ expression in SOMV-9RE7 treated group was not an artifact from unremoved free peptide.

Delivering SOMV-9RE7 subcutaneously in TC-1 tumor-bearing mice led to significant anti-tumor effects after the first dose. Development of E7 antigen-specific T cells was confirmed in PBMC after two dose treatments, further confirming the effectiveness of the 9RE7 antigen coating strategy. SOMV-9RE7’s therapeutic effect is potentially due to direct activation of APCs in the tumor-draining lymph-node for immediate onset of adaptive immunity [ 27 ]. Due to the native inflammatory agents present on SOMV, it induced mild inflammatory responses including symptoms of redness at the injection site and weight loss of around 5%. These symptoms cleared quickly within one or two days after injection. They became much less pronounced following the second dose, meaning the mice have begun to tolerate the treatment. In future experiments, a reduced overall dose or a gradual dose escalation of SOMV can be implemented to prevent septic shock and overactive immune response.

Analyzing the immunologic profile in the TME, we observed a significant infiltration and activation of E7-specific CTLs in SOMV-9RE7 treated mice. The same trends in T cell proliferation were reflected in splenocytes. On the other hand, control treatments 9RE7 and SOMV yielded no significant amounts of E7-specific TILs (Fig.  3 H) or splenocytes (Fig.  3 F). Therefore, we can confidently conclude that 9RE7 antigen-display contributed to the differential E7+ T cell expansion difference between SOMV and SOMV-9RE7, which led to tumor-specific control. Interestingly, 9RE7 treated tumors showed a slight increase in CD8+ TILs (Fig.  3 F), while splenocytes of SOMV treatment display an increase in CD8+ subpopulation (Fig.  4 D) despite overall CD45+ CD3+ splenocyte decreased (Fig.  4 B). These discrepancies in the observed local and systemic immunological profiles, however, cannot convey a comprehensive understanding of tumor-associated immune responses that correlate to tumor control. This further highlights the consistent cytotoxic lymphocyte profiles in SOMV-9RE7 immunotherapy that drive tumor-associated immunity.

Thus, we have demonstrated the successful synthesis of an MHC class I tumor-associated epitope displaying OMV vaccine with the polyarginine CPP method. Furthermore, SOMV-9RE7 exhibited immunogenic properties that activate E7-specific T cells via APC cross-presentation in vitro. Finally, administration of SOMV-9RE7 showed superior anti-tumor effects on TC-1 tumors by increasing E7-specific T cell infiltration and boosting systemic adaptive immunity. Future investigation will focus on the differences in therapeutic efficacy of 9RE7-coated Salmonella and SOMV to determine the more efficacious and safer platform for bacterial immunotherapy in HPV-associated and other cancer models. Previous bacteria-based combination therapy results showed highly synergistic benefits of pairing cytokine or immune checkpoint inhibitors to enhance the efficacy of bacteria therapy [ 8 , 9 , 28 ]. Our future experiments will dive into augmenting the CD8+ T cell-mediated tumor-specific immune cascade to determine synergistic combination therapy targets. In a phase II clincal trial of treating metastatic melanoma with modified HLA-A2*0201 bound gp100:209–217(210 M) peptide vaccine in combination with cytokine interlekin-2 (IL-2), the glycoprotein carrier can be replaced with our SOMV platform to introduce bacterial immunotherapy as a combination treatment of melanoma [ 29 ]. Due to the simplicity and efficiency of polyarginine coating strategy, it can be broadly applied to personalized neoantigen targeted therapy that utilizes next-generation sequencing to identify highly immunogenic tumor-specific neoantigen [ 30 ]. Our peptide-loaded SOMV can be seamlessly and effectively implemented in other neoantigen vaccines, such as the iNeo-Vac-P01 for pancreatic cancer[ 31 ], and the HER2-derived MHC I peptide E75 vaccine used in clinical trial for ductal carcinoma in situ [ 32 ]. Finally, small exogenous proteins and antibodies can also be displayed on OMVs using polyarginine CPPs as anchors. Engineered HPV L2 minor capsid targeting monoclonal antibodies can potentially be delivered through OMV surface presentation, which may lead to more efficient virus neutralization and offer new solutions to antibody penetration and targeting challenges [ 33 , 34 ].

By implementing our innovative 9RE7 antigen coating strategy to OMV and introducing the pioneering SOMV-9RE7, we have provided a feasible and economical approach for developing bacteria-based antigen-displaying vaccines. This strategy employs a straightforward production technique, eliminating the need for designing recombinant bacterial constructs, which allows the control over the peptide to OMV ratio. Furthermore, recent studies involving OMV vaccines in phase II clinical trials have demonstrated promising results [ 35 ], indicating the translational potential of SOMV vaccines. While our SOMV-9RE7 work represents a groundbreaking advancement in bacteria-immunotherapy and vaccines for HPV-associated cancer, future preclinical and clinical research endeavors will continue to expand upon this strategy.

Conclusions

This study is innovative in introducing the novel SOMV-9RE7 vaccine as a bacteria immunotherapy tailored to target HPV-associated cancers. We demonstrated that the combination of SOMV with 9RE7 enhances antitumor effects as well as effector T cell proliferation and activity in the tumor microenvironment and in the spleen. Our findings strongly suggest that SOMV-9RE7 vaccines represents a promising, cost-effective, and viable strategy. More clinical evidence will be needed to confirm these findings and provide a more comprehensive potential of this approach.

Availability of data and materials

All data relevant to the study are included in the article or uploaded as Additional information. Data and materials are available on reasonable request.

Abbreviations

Outer membrane vesicles

Lipopolysaccharides

Human Papillomavirus

Nine arginine residues linked to HPV E7 (aa49-57) short peptide

Cell penetrating peptide

Salmonella -Derived outer membrane vesicles

Tumor microenvironment

Antigen presenting cells

Major Histocompatibility Complex

Cytotoxic T lymphocytes

Tumor infiltrating-lymphocytes

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Acknowledgements

We thank the laboratory animal center at the Johns Hopkins School of Medicine for animal care.

This study was supported by the National Institutes of Health, National Cancer Institute Specialized Program of Research Excellence (SPORE) in Cervical Cancer grant (NIH/NCI P50CA098252) and NCI awards (R01CA237067, R21DE029910-01, R21CA256020, and 1R21CA234516-01A1).

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Suyang Wang, Chao-Cheng Chen, Ming-Hung Hu, Michelle Cheng, Hsin-Fang Tu, Ya-Chea Tsai, Jr-Ming Yang, T. C. Wu, Chuan-Hsiang Huang & Chien-Fu Hung

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Conception and design: S.W., CF.H. Conducting experiments: S.W., CC.C., YC.T. Analysis and interpretation of data: S.W., MH.H., HF.T., YC.T., JM.Y. Writing and review of manuscript: S.W., M.C., CF.H. Study supervision: T.C. W, CH.H., CF.H.

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Additional file 1..

TC-1 tumor-bearing mice weight under SOMV-9RE7 treatment. Body mass of mice from each treatment group is measured twice a week. Vaccinations are given on days 7 and 14.

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Wang, S., Chen, CC., Hu, MH. et al. Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine. J Transl Med 22 , 378 (2024). https://doi.org/10.1186/s12967-024-05195-7

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Inab: initiating coverage – enhancing the insurgents of the immune system.

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We are initiating coverage of IN8bio, Inc. (NASDAQ:INAB) with a valuation of $5.00 per share. This value is based on our estimates for a successful development and commercialization of INB-100 in patients with leukemia, specifically acute myeloid leukemia (AML) following haploidentical hematopoietic stem cell transplant (HSCT) and INB-400 in recurrent and newly diagnosed glioblastoma multiforme (GBM) patients. INB-100 is the subject of a Phase I study and is enrolling patients in an expansion cohort. It is slated to report long-term follow-up results at scientific meetings in 2024. INB-200 has completed Phase I enrollment and will report GBM data at scientific conferences in 2024 including the American Society of Clinical Oncology (ASCO) meeting in June. This program has passed the baton to INB-400, which may add an allogeneic arm to the GBM program in 2025. The Phase II INB-400 study initiated enrollment in 1Q:24.

The leukemia program uses haploidentical allogeneic cells while the GBM program employs autologous ones. Subsequent programs may explore the use of fully off-the-shelf (OTS) allogeneic cells in order to benefit from the quality, quantity, cost and immediate availability of such a product. Orphan designation has been granted to the GBM program and we expect AML will also be allowed this expedited pathway. IN8bio expects to pursue the Regenerative Medicine Advanced Therapy (RMAT) designation which allows a gene and cell therapy certain regulatory advantages such as priority review, if it can show evidence that it can benefit a serious or life-threatening disease.

We anticipate that both the INB-100 and INB-400 programs will evolve into adaptive studies in 2025 that will eventually support FDA approval. Based on our estimates of enrollment duration, time to achieve desired endpoints and regulatory submission, we could see a BLA submitted in 2028 for INB-400 and in 2029 for INB-100. We anticipate that pivotal studies and commercialization will benefit from a partner in the United States and around the world.

IN8bio has demonstrated clinical activity and initial safety for both its leukemia and GBM programs. As of the latest update in December 2023, 100% of patients enrolled in the INB-100 leukemia study have achieved and maintained a complete response (CR). 70% of the patients produced a CR lasting more than six months and 60% enjoy a CR greater than one year. As of the latest update in November 2023, the INB-200 GBM study has enrolled 22 patients with the majority of treated patients exceeding expected progression free survival (PFS) based on their profile. No dose limiting toxicities, cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome (ICAN) have been observed. INB-400 is in the early stages of enrollment and has not yet produced public data.

γδ T cells are a type of immune cell that present characteristics of both the innate and adaptive immune system. They comprise a subset of T cells that differ from the more common αβ T cells in that they can recognize a tumor directly without reliance on antigen presentation. γδ T cells are relatively uncommon in the immune system repertoire, comprising roughly 5% of the total T lymphocyte population and residing in many of the peripheral tissues that interface with the outside world. Since the γδ T cells are not antigen specific, they may be effective in allogeneic therapies which could potentially lead to an off the shelf (OTS) γδ T cell product.

IN8bio’s program is differentiated from other immunotherapies by its DeltEx platform, genetically modified Drug Resistant Immunotherapy (DRI) and manufacturing prowess which is applied to its γδ T cells. The platform is able to collect, activate, genetically modify and expand γδ T cells for use in cell therapy. The DRI feature in particular is key in combination therapy with chemotherapy which allows γδ T cells to overexpress DNA damage repair proteins which shield them from chemotherapy damage. Additionally, the chemotherapy upregulates stress antigens on cancer cells, which act as a beacon to attract the γδ T cells. These features have shown success in early-stage trials and support advancement into later stage studies.

IN8bio is pursuing initial indications in leukemia and GBM. In the broader leukemia setting there are over 60,000 cases per year in the US and just under 500,000 worldwide. We expect IN8bio to first pursue AML, which comprises about a third of the total cases of leukemia. It is considered a rare disease as it falls under the 200,000 cases per year threshold for orphan indications. GBM is also a rare disease with about 14,000 new cases per year in the US and 300,000 around the globe. If the therapies are successful in these initial indications, we expect to see further expansion into a broader leukemia setting and in other solid tumors.

Both leukemia and GBM present substantial unmet needs. Most leukemia patients relapse after HSCT while their immune system reconstitutes and patients require supportive therapy to eradicate any remaining leukemic cells. GBM patients have a very short expected survival of 12 to 18 months and ~5% five-year survival rate. These difficult cancers are particularly amenable to γδ T cell therapy which can leverage this flexible and durable immune cell to improve survival. Based on the strong data generated, IN8bio recently raised over $14 million in a securities purchase agreement bringing its end of year 2023 cash pile to over $21 million. In addition to the cash on hand, IN8bio has access to an at-the-market (ATM) facility that has historically contributed to the company’s coffers. Company management has stated that it has sufficient capital to fund operations into the first quarter of 2025.

We expect further updates on the leukemia and GBM programs throughout 2024 and expect that if data continues to be favorable, additional financial support will follow. Assuming sufficient capital is raised, the company plans to accelerate clinical trials in 2025 that may be adaptive into pivotal trials that can support a biologic license application (BLA) filing. Based on these assumptions, we anticipate a regulatory filing for the GBM program in 2028 and the leukemia program in 2029 followed by approval in the following year for each.

Key reasons to own IN8bio shares:

➢ The γδ T cell therapy platform has produced remarkable overall survival (OS)

o 100% of INB-100 leukemia patients achieved median CR >6 months and 60% >1 year

▪ Allogeneic γδ T cells persist and expand beyond 1 year after administration

o INB-200 GBM patients exceed standard of care median PFS and median OS

▪ Four of 12 subjects have achieved OS of > than 1 year

➢ DeltEx Platform

o Ability to produce expanded and modified γδ T cells ex vivo at high rates

o Can apply gene therapy to shield γδ T cells from chemotherapy

o Cell therapy employs small batch production using cell processing systems

➢ Offers preclinical programs that address shortcomings in other cell therapies

o Non-signaling chimeric antigen receptor (nsCAR) γδ T cell (INB-300)

o Induced pluripotent stem cell (iPSC) derived γδ T cells (INB-500)

➢ Presence of γδ T cells highly correlated with improved cancer prognosis

o Supported by multiple studies (Gentles, Godder, Meraviglia)

➢ γδ T cells offer features from both the innate and adaptive immune system

o Detect cellular stress

o Can kill cancer cells without priming

o Can differentiate between stressed and healthy cells

➢ Robust intellectual property

o Patent portfolio addressing multiple disease states

o Internally developed know-how and trade secrets

In the following sections we describe immuno-oncology (I-O), elaborate on cell therapy and introduce γδ T cells. We look at how γδ T cells differ from other types of immune cells and describe how they present features of both the innate and adaptive immune responses. We follow with a description of IN8bio’s DeltEx platform and DRI modifications. The next section examines the company’s clinical programs including INB-100, INB-200 and INB-400. We also include a brief description of IN8bio’s preclinical assets. A discussion of IN8bio’s leukemia and GBM is given along with why we expect to see a refinement in focus to AML as well as newly diagnosed and recurrent GBM. This section is followed by a short review of IN8bio’s intellectual property (IP) and anticipated regulatory pathway. The epidemiology, etiology and other details of IN8bio’s primary indications in leukemia and GBM are included. We then discuss other competitors in the immuno-oncology space, including several peers advancing γδ T cell programs. The subsequent segment reviews recent financial and operational history for the company followed by an introduction to the management team and a summary of company-specific risks. Our valuation discussion details the assumptions behind our target price. Our work generates a valuation of $5.00 per share for IN8bio, Inc.

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• Published: 19 April 2024

Synthetic cationic helical polypeptides for the stimulation of antitumour innate immune pathways in antigen-presenting cells

• DaeYong Lee 1 , 2   na1 ,
• Kristin Huntoon   ORCID: orcid.org/0000-0002-7211-8986 1 , 2   na1 ,
• Yifan Wang   ORCID: orcid.org/0000-0001-7387-0654 3   na1 ,
• Minjeong Kang 3 ,
• Yifei Lu   ORCID: orcid.org/0000-0003-2268-2738 1 , 2 ,
• Seong Dong Jeong 1 , 2 ,
• Todd M. Link 4 ,
• Thomas D. Gallup   ORCID: orcid.org/0000-0003-2471-4342 1 , 2 ,
• Yaqing Qie 1 , 2 ,
• Xuefeng Li 3 ,
• Shiyan Dong   ORCID: orcid.org/0000-0003-2912-8895 3 ,
• Benjamin R. Schrank   ORCID: orcid.org/0000-0001-8138-4250 3 ,
• Adam J. Grippin 3 ,
• Abin Antony   ORCID: orcid.org/0000-0001-8907-1056 3 ,
• JongHoon Ha 3 ,
• Mengyu Chang 3 ,
• Liang Wang   ORCID: orcid.org/0000-0001-5038-694X 3 ,
• Dadi Jiang   ORCID: orcid.org/0000-0002-0935-3908 3 ,
• Jing Li 3 ,
• Albert C. Koong 3 ,
• John A. Tainer 4 ,
• Wen Jiang   ORCID: orcid.org/0000-0001-9154-633X 3 &
• Betty Y. S. Kim   ORCID: orcid.org/0000-0001-6890-8355 1 , 2  

Nature Biomedical Engineering ( 2024 ) Cite this article

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• Biomedical engineering
• Biomedical materials
• Immunotherapy
• Tumour immunology

Intracellular DNA sensors regulate innate immunity and can provide a bridge to adaptive immunogenicity. However, the activation of the sensors in antigen-presenting cells (APCs) by natural agonists such as double-stranded DNAs or cyclic nucleotides is impeded by poor intracellular delivery, serum stability, enzymatic degradation and rapid systemic clearance. Here we show that the hydrophobicity, electrostatic charge and secondary conformation of helical polypeptides can be optimized to stimulate innate immune pathways via endoplasmic reticulum stress in APCs. One of the three polypeptides that we engineered activated two major intracellular DNA-sensing pathways (cGAS–STING (for cyclic guanosine monophosphate–adenosine monophosphate synthase–stimulator of interferon genes) and Toll-like receptor 9) preferentially in APCs by promoting the release of mitochondrial DNA, which led to the efficient priming of effector T cells. In syngeneic mouse models of locally advanced and metastatic breast cancers, the polypeptides led to potent DNA-sensor-mediated antitumour responses when intravenously given as monotherapy or with immune checkpoint inhibitors. The activation of multiple innate immune pathways via engineered cationic polypeptides may offer therapeutic advantages in the generation of antitumour immune responses.

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Acknowledgements

We thank C. Wogan of the Division of Radiation Oncology, MD Anderson Cancer Center, for editorial assistance. This work is supported in part by the National Cancer Institute grant (1K08 CA241070) to W.J., US Department of Defense grant (W81XWH-19-1-0325) to B.Y.S.K. and the Cancer Center Support (Core) grant (P30 CA016672) from the National Cancer Institute, National Institutes of Health, to The University of Texas MD Anderson Cancer Center (principal investigator P.W. Pisters).

Author information

These authors contributed equally: DaeYong Lee, Kristin Huntoon, Yifan Wang.

Authors and Affiliations

Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

DaeYong Lee, Kristin Huntoon, Yifei Lu, Seong Dong Jeong, Thomas D. Gallup, Yaqing Qie & Betty Y. S. Kim

Brain Tumour Center, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Yifan Wang, Minjeong Kang, Xuefeng Li, Shiyan Dong, Benjamin R. Schrank, Adam J. Grippin, Abin Antony, JongHoon Ha, Mengyu Chang, Liang Wang, Dadi Jiang, Jing Li, Albert C. Koong & Wen Jiang

Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Todd M. Link & John A. Tainer

Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT, USA

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Contributions

D.Y.L., B.Y.S.K. and W.J. conceived the project and designed experiments. B.Y.S.K. and W.J. supervised the project. D.Y.L. carried out all the experiments and analysed all the data. Y.W., K.H., Y.L., M.K., S.D.J. and Y.Q. performed the in vivo studies. T.M.L. and J.A.T. performed size exclusion chromatography for molecular weight determination. Y.W., X.L., S.D., Y.A. and J.L. helped to interpret the data. D.Y.L., W.J. and B.Y.S.K. wrote the paper with help from all authors.

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Correspondence to Wen Jiang or Betty Y. S. Kim .

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Extended data

Extended data fig. 1 varying hydrophobicity in the engineered polypeptide affects immunogenicity via er stress-mediated mtdna release and effector functions in macrophages..

a , Chemical structure of cationic polypeptides with different amine-containing analogues. Cationic polypeptides including hydrophilic analogues and cyclic structures more favorably induced. ( b ) ER stress and ( c ) mtDNA release in BMDMs ( n  = 3). The representative western blot images were shown from at least twice independent results. Cationic polypeptide tethered with a hydrophilic building block and cyclic structure increased (d) phagocytosis of EO771 breast cancer cells and (e) cross-presentation of the model antigen SIINFEKL-H2Kb ( n  = 3). (f) Gene expression of pro-inflammatory cytokines was affected by hydrophobicity of polypeptides and the chemical structure of amine-including analogues ( n  = 3). One-way ANOVAs with Bonferroni post hoc correction were used in c , d , e , f . All data are expressed as means±s.d.

Source data

Extended data fig. 2 varying the electrostatic charge of the polypeptide affects immunogenicity via er stress-mediated mtdna release and effector functions in macrophages..

a , Chemical structure of polypeptides with different electrolytes. P1 more favorably induced ( b ) ER stress and ( c ) mtDNA release in BMDMs greater than did PTMA (strongly cationic) and PS (anionic) (n = 3). The representative western blot images were shown from at least twice independent results. P1 improved ( d ) phagocytosis of EO771 breast cancer cells and ( e ) cross-presentation of the model antigen SIINFEKL-H2Kb to a greater extent than did PTMA or PS ( n  = 3). f , Expression of genes for pro-inflammatory cytokines was regulated by types of electrolytes and strength of cationic charges ( n  = 3). One-way ANOVAs with Bonferroni post hoc correction were used in c , d , e , f . All data are expressed as means±s.d.

Extended Data Fig. 3 Varying the length of the side chain of the polypeptide regulates immunogenicity via ER stress-mediated mtDNA release and effector functions in macrophages.

a , Chemical structures of polypeptides with side chains of different lengths. P1 induced ( b ) ER stress and ( c ) mtDNA release in BMDMs more strongly than did P3 (mid-length) and PS (short length) ( n  = 3). The representative western blot images were shown from at least twice independent results. P1 improved ( d ) phagocytosis of EO771 breast cancer cells and (e) cross-presentation of the model antigen SIINFEKL-H2Kb to a greater extent than did P3 and P1 ( n  = 3), ordinary one-way ANOVA. f , Expression of genes for pro-inflammatory cytokines was affected by the length of side chains in the polypeptides ( n  = 3). One-way ANOVAs with Bonferroni post hoc correction were used in c , d , e , f . All data are expressed as means±s.d.

Extended Data Fig. 4 P1 promotes innate and adaptive immune activation within the tumour microenvironment.

a , P1 increased the population of tumour-infiltrating CD8 + T cells and IFNγ-producing CD8 + T cells, but decreased that of T reg cells (CD3 + CD4 + CD25 + FoxP3 + ), relative to P2 and P3 ( n  = 3 biological replicates). Tumour-infiltrating lymphocytes were obtained on day 16 (2 days after the last treatment). b , P1 promoted M1 macrophage polarization (M1 macrophage: CD80 + CD86 + CD206 – ; M2 macrophage: CD80 – CD206 + ; macrophages: CD11b + CD11c – F4/80 + ) and maturation of dendritic cells (DCs) (Mature DCs: CD80 + CD86 + DC; DC: CD11c + MHC-II + F4/80 – ) but did not affect the number of MDSCs (CD11b + CD11c – MHC-II – F4/80 – Gr-1 + ) within the tumour microenvironment relative to P2 and P3 ( n  = 3), unpaired two-tailed Student’s t test in comparison with Cont and the indicated conditions. All data are expressed as means±s.d.

Extended Data Fig. 5 P1 combined with αPD1 evoked strong antitumour immunity in mice bearing metastatic tumours.

a , Timeline for tumour establishment and administration of P1 and αPD1. b , Growth curves for 4T1 tumours in mice after the indicated treatments ( n  = 8 biologically independent mice for HEPES; n  = 9 biologically independent mice for P1; n  = 10 biologically independent mice for P1 + αPD1 and cGAMP+αPD1), unpaired two-tailed Student’s t test in comparison with HEPES or the indicated conditions on day 30 after tumour inoculation. c , Photographs of excised 4T1 tumour tissues on day 17. d , Kaplan-Meier survival curves of 4T1 tumour-bearing mice; log-rank (Mantel-Cox) test in comparison with HEPES or the indicated conditions. e , Flow cytometry of tumour-infiltrating T lymphocytes (T reg cells: CD3 + CD4 + CD25 + FoxP3 + ) and their subsets ( n  = 4 biological replicates), which were harvested on day 17. f , Immunofluorescence stains for CD4, CD8, and Iba1 in macrophages. Representative images from random fields of view in one of the three biologically independent mice. Scale bar, 30 μm. g . Profiles of tumour-infiltrating myeloid cells (M1 macrophage: CD80 + CD86 + CD206 − ; M2 macrophage: CD80 − CD206 + macrophage: CD11b + CD11c − F4/80 + ; mature DC: CD80 + CD86 + DC; DC: CD11c + MHC-II + F4/80 − ; MDSC: CD11b + CD11c − MHC-II − F4/80 − Gr-1 + ) as evaluated by flow cytometry ( n  = 4 biological replicates); unpaired two-tailed Student’s t test in comparison with Cont or the indicated condition. All data are expressed as means±s.d.

Extended Data Fig. 6 P1 combined with αPD1 generates robust systemic antitumour immunity.

a , P1 + αPD1 treatment increased the production of IFNγ from CD8 + T cells and decreased the population of T reg cells (CD3 + CD4 + CD25 + FoxP3 + ) in tumour-draining lymph nodes and spleen as compared with the other treatment conditions ( n  = 4 biological replicates), unpaired two-tailed Student’s t test in comparison with HEPES or the indicated conditions. b , P1 + αPD1 treatment increased the population of mature DCs (CD11c + F4/80 − MHC-II + CD80 + CD86 + ) and pro-inflammatory macrophages polarization (CD80 + CD86 + CD11b + CD11c − F4/80 + for migratory macrophages in spleen, CD80 + CD86 + CD11b + CD11c – F4/80 + for macrophages in lymph nodes), but maintained the numbers of MDSCs (CD11b + CD11c - F4/80 - MHC-II − Gr-1 + ) in tumour-draining lymph nodes and spleen, relative to the other treatments (n = 4 biological replicates); unpaired two-tailed Student’s t test in comparison with HEPES or the indicated conditions. All data are expressed as means±s.d.

Extended Data Fig. 7 Activation of MyD88 and STING is required to stimulate the p-IRF3 axis in tumour-homing DCs treated with P1 + αPD1.

Immunofluorescence images show that DCs treated with P1 + αPD1 promoted p-IRF3 nuclear translocation (white arrows) in WT but not in STING –/– or MyD88 –/– cells. Scale bar, 30 μm. One-way ANOVA with Bonferroni post hoc correction was used to determine statistical significance. Numbers of nucleus-translocating p-IRF3 + DCs in the immunofluorescence images were counted with ImageJ software ( n  = 6 biological replicates). All data are expressed as means±s.d.

Extended Data Fig. 8 TLR9 activation is required to recruit tumour-infiltrating T cells by stimulating IRF7 signaling in tumour-homing APCs treated with P1 + αPD1.

a , P1 + αPD1 treatment promoted p-IRF7 nuclear translocation in macrophages and DCs in WT but not in TLR9 –/– mice. Scale bar, 30 μm; unpaired two-tailed Student’s t test in comparison to the indicated conditions ( n  = 8 biological replicates). b , P1 + αPD1 treatment of TLR9 –/– mice blocked recruitment of both CD4 + and CD8 + T cells into tumours. Scale bar, 20 μm; unpaired two-tailed Student’s t test in comparison to the indicated conditions ( n  = 6 biological replicates). Numbers of cells in the immunofluorescence images were counted by ImageJ software. All data are expressed as means±s.d.

Extended Data Fig. 9 Activation of STING and MyD88 in cancer cells is not required for P1 + αPD1’s antitumour effect.

a , Immunoblots verifying that EO771 STING –/– cells did not express STING, and EO771 MyD88 –/– cells did not express MyD88. The representative western blot images are shown from at least two independent experiments. b , Tumour growth curves for mice with EO771 WT, EO771 STING –/– , or EO771 MyD88 –/– tumours after intravenous injection of HEPES or P1 + αPD1 ( n  = 5 biologically independent mice for all treatment groups). c , Kaplan-Meier survival curves for mice with EO771 WT, EO771 STING –/– , or EO771 MyD88 –/– tumours; log-rank (Mantel-Cox) test compared with the indicated conditions. All data are expressed as means±s.d.

Extended Data Fig. 10 CD8 + T-cell depletion abolished adaptive immunity conferred by P1 + αPD1.

a , CD8 + T cell populations in splenocytes, evaluated by flow cytometry on day 17 after tumour cell inoculation ( n  = 3 biological replicates). ( b , c ) CD8 depletion ( b ) abrogated the antitumour effect of P1 + αPD1 and ( c ) reduced the survival of EO771 tumour-bearing mice ( n  = 6 biologically independent mice for all treatment groups); unpaired two-tailed Student’s t test in comparison to P1 + αPD1 at day 24 after tumour inoculation for tumour growth curve; log-rank (Mantel-Cox) test for Kaplan-Meier survival curve. d , Excised tumours from each experimental group show that CD8 depletion eliminated the antitumour effect of P1 + αPD1. e , Immunofluorescence stains of tumour-infiltrating CD8 + T cells show that CD8 depletion inhibited the recruitment of CD8 + T cells within tumours. Scale bar, 30 μm. f , CD8 depletion reduced the proportions of tumour-infiltrating CD8 + T cells and IFNγ + CD8 + T cells, but did not change the proportion of Tregs ( n  = 3 biological replicates); unpaired two-tailed Student’s t test; n.s., not significant compared with the indicated conditions. All data are expressed as means±s.d.

Supplementary information

Supplementary information.

Supplementary methods and figures, and lists of reagents.

Reporting Summary

Peer review file, supplementary data 1.

Statistical data for Supplementary Figs. 4, 5, 7–19, 22–25 and 27–36.

Supplementary Data 2

Uncropped western blots for Supplementary Figs. 12, 22 and 24–29.

Source Data Figs. 1–8 and Extended Data Figs. 1–10

Statistical source data for Figs. 1–8 and Extended Data Figs. 1–10.

Source Data Fig. 3 and Extended Data Figs. 1–3 and 9

Uncropped western blots for Fig. 3 and Extended Data Figs. 1–3 and 9.

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Lee, D., Huntoon, K., Wang, Y. et al. Synthetic cationic helical polypeptides for the stimulation of antitumour innate immune pathways in antigen-presenting cells. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01194-7

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Received : 15 March 2023

Accepted : 01 March 2024

Published : 19 April 2024

DOI : https://doi.org/10.1038/s41551-024-01194-7

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