Introduction to immunopeptidomics
The major histocompatibility complex (MHC) plays a critical role in adaptive immunity through antigen presentation. MHC associated antigens derived from either endogenous or exogenous proteins are recognized by T cells and trigger an immune response. Mass spectrometry-based immunopeptidomics is a rapidly growing proteomics application that enables the identification of MHC associated antigens extracted from biological samples. Targeting MHC antigens isolated using mass spectrometry-based immunopeptidomics provides scientists with novel ways to predict antigens for applications such as cancer treatment, vaccine development, immunotherapy, and drug discovery.
The basics of immunopeptidomics
The major histocompatibility complex locus regulates a highly dense and structurally diverse region that encodes cell surface proteins for adaptive immunity in vertebrates. Once antigens are detected by MHC molecules and bound to the complex, they are known as immunopeptides. These MHC bound antigens are presented to the immune system, whereby T cells can identify the peptides on cell surfaces to initiate an immune response.
Recognition of antigens as small peptide fragments bound to an MHC molecule and displayed at the cell surface is a distinctive feature of T cells. Understanding how MHC molecules present antigens to T cells is central to adaptive immunity and is essential to manipulating the immune system. T cells are critical for adaptive immunity since they protect the body from infections and can target cancer cells. These cell surface immunopeptides can be isolated, identified and quantified through mass spectrometry. Immunopeptidomics combines proteomics and immunology to study the antigen repertoire, or the immunopeptidome, by assessing MHC bound peptides isolated from biological samples using mass spectrometry for quantitative analysis and characterization.
MHC associated peptides (MAPs)
While the origin of MHC associated peptides, or MAPs, are preferentially driven by recycled proteins such as defective proteins and ribosomal products, their presence and diversity are impacted by many factors. MAPs are highly diverse (10,000+ peptides per cell) and are impacted by processes such as translation, phosphorylation, protein degradation via ubiquitin proteasome pathways, and the enzymatic proteases responsible for structural breakdowns. Neoantigens, due to variations at the amino acid level that often arise in cancerous tissue, can occur within the immunopeptidome to influence cellular activity and health. With MAPs being small in sequence length at 8–25 amino acids and highly diverse among individuals, precise isolation and quantification of the immunopeptidome can only be provided with the accuracy of mass spectrometry. Whether you’re looking to validate a sequence, transcribe a complex peptide mixture, identify genomic location or transcript expression, or take personalized medicine further with vaccine design, mass spectrometry is vital for analyzing MAP expression.
MHC antigen processing pathways
The MHC system, known as the human leukocyte antigen (HLA) complex in humans, encodes proteins that make up MHC Class I, MHC Class II, or MHC Class III proteins. The HLA region, located on the short arm of chromosome 6 on band 6p21.3, is responsible for generating an immune response and is homologous to the MHC system. The MHC antigen presentation pathway is a critical process for the adaptive immune system that regulates T lymphocyte (T cell) immune responses toward pathogen-infected and cancerous cells. Antigen presentation occurs through MHC molecules, which present MAPs on the cell surface for recognition by T cells via T cell receptors. Through the recognition response, MHC molecules presenting MAPs bind to the T cell receptors to activate the T cell and initiate an immune response. The two major classes of MHC molecules are MHC Class I and II. Both MHC-I and -II are transmembrane cell surface molecules in the glycoprotein family.
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Figure 1. Depiction of antigen presenting cells and the MHC Class I and II pathways. The pathways show: 1) Antigen uptake 2) Antigen processing 3) MHC associated peptide (MAP) formation and 4) MHC cell surface expression for T cell recognition.
MHC-I molecules consist of a transmembrane alpha domain where peptide binding occurs and a smaller beta 2 microglobulin subunit, while the MHC-II molecule structure includes an alpha and beta domain that together make up the peptide binding region. MHC Class I molecules present peptides derived from endogenous pathogens or proteins (known as intracellular antigens) to CD8+ cytotoxic T cells. In contrast, MHC Class II molecules are responsible for presenting peptides derived from exogenous proteins or pathogens (known as extracellular antigens) to CD4+ helper T cells. MHC Class I are restricted to nucleated cells, whereas specialized antigen presenting cells such as dendritic cells, macrophages, and B cells express MHC Class II molecules. Both Class I and II present MAPs to T cell receptors, initiating an immune response as described below.
MHC Class I antigen presentation
MHC-I molecules present peptide fragments derived from intracellular proteins onto the cell surface of nucleated cells. In the MHC-I antigen presentation pathway, endogenous proteins from either pathogens or the cell itself are degraded through the ubiquitin-proteasome pathway (UPP) into small peptides that are 8–11 amino acids in length. The peptide antigens are transported from the cytoplasm to the rough endoplasmic reticulum by the transporter associated with antigen processing (TAP) protein, where a peptide loading complex consisting of tapasin, Erp57, and calreticulin (Crt) loads them onto the MHC-I molecule. The MHC-I peptide complex moves through the ER and Golgi apparatus to the cell surface for recognition by CD8+ cytotoxic T cells. If the peptides presented are pathogenic, mutated, or different from normal peptides, the CD8+ cytotoxic T cells will initiate apoptosis of the infected or malignant cell and release cytokines to trigger an immune response.
MHC Class II antigen presentation
MHC-II molecules provide the immune system with information about what is going on outside of the cell. MHC Class II antigen presentation occurs when exogenous proteins are internalized by antigen presenting cells through phagocytosis and endocytosis. These exogenous proteins are degraded by the endosomal-lysosomal pathway as they proceed through early endosomes, then to endosomes, and finally to lysosomes through an environment of increasing acidity. Acid hydrolase enzymes in the endosomes and lysosomes digest the proteins into peptides consisting of 12–25 amino acids, which are loaded onto the MHC Class II molecules.
MHC-II molecules are synthesized in the ER and contain an invariant chain that blocks the peptide binding site, preventing endogenous peptides from loading onto the MHC-II. The MHC Class II molecules move from the ER through the Golgi to the endosome, guided by the HLA-DM protein. The invariant chain is processed into a shorter peptide called CLIP, and when the HLA-DM releases the CLIP, the MHC-II can then be loaded with exogenous antigens and transported to the cell surface. These cell surface MHC-II antigens are recognized by CD4+ helper T cells, initiating an immune response with the release of cytokines, recruitment of other immune cells, and antibody production.
T cells in MHC signaling
While innate immunity activates a rapid, non-specific inflammatory response, adaptive immunity generates memory T and B cells to allow for a long-lasting, effective response to reinfection over the course of a lifetime. Adaptive immunity can occur after exposure to an antigen from either a pathogen or a vaccination. Antigens are described by where they originate; thus, autoantigens (or self-antigens) are derived from proteins produced within the body’s own cells, while non-self-antigens originate from outside the body and can be derived from external sources such as pathogens, toxins, or transplanted tissues. The ability of the immune system to differentiate between self and non-self-antigens is crucial for maintaining immune homeostasis and preventing autoimmune responses, where the immune system mistakenly targets self-antigens.
Helper T cells, killer T cells, and macrophages are the three main kinds of lymphocytes involved in cell-mediated immunity. When T cells recognize a foreign fragment attached to the MHC molecule, they bind to the MHC-peptide complex and activate an immune response. The maturation and differentiation of naïve T cells into helper or killer T cells are dependent on the binding specificity of MHC proteins to external antigens. The binding of T cell receptors (TCR) to either Class I or Class II MHC molecules directly influences differentiation of the selected cells into either CD4+ (helper) or CD8+ cytotoxic effector and memory cells (killer) T cells, to mediate a direct immune response. The ligand for the TCR is always a peptide bound to an MHC molecule; the CD8 receptor on a cytotoxic T cell can only bind to MHC-I, and the CD4 receptor on a helper T cell can only bind to MHC-II.
Peptides that bind to Class I MHC molecules are usually derived from intracellular proteins (i.e., cytoplasmic) and are generated by the proteasome, transported to the endoplasmic reticulum, loaded onto MHC molecules, and displayed on the cell surface to circulating T lymphocytes. The peptide/MHC-I complex binding to a TCR triggers a cytotoxic response. On Class II MHC molecules, the bound peptides are usually derived from extracellular antigens generated by endosomal proteolysis of proteins after they are endocytosed by specialized antigen presenting cells such as B cells. TCR recognition of such peptide/MHC-II complexes triggers the release of cytokines, creating an inflammatory response among the cells, such as antibody secretion.
While the central role of T cells is to stimulate both humoral and cellular immune responses, it is crucially important that T cells do not react with self-proteins. In healthy cells, MHC molecules present self-peptides, and no reaction occurs from T cells. B cells usually require help from T cells to secrete antibodies. Any given B cell whose receptor mutates to become self-reactive would, under normal circumstances, fail to make antibodies due to lack of self-reactive T cells, which would provide the helper cells to fight the immunity.
Autoimmune disease is caused by autoantibodies and/or autoreactive T cells targeting self-antigens. Polymorphisms, epigenetic modifications, and post translation modifications (PTMs) lead to altered gene expression and function, which in turn can change peptides, leading to abnormal T cell responses that underlie autoimmune disease. Common autoimmune disorders such as celiac disease, type-1 diabetes, inflammatory bowel disease (IBD), multiple sclerosis, autoimmune thyroid disease, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) have been linked to the immune system mistakenly attacking its own tissues. Mass spectrometry-based immunopeptidomics provides a valuable mechanism for identifying MHC ligands that contain alterations such as PTMs, providing insights into both the pathogenesis and therapeutic options of autoimmune diseases.
Isolating MHC antigens by mass spectrometry
The immunopeptidome consists of a complex mixture of peptides at varying concentrations but of similar length, complicating immunopeptidomic analysis. Primary cells and tissue, cell lines, and disease state tissue can be lysed to release MHC-associated peptide complexes into solution for purification. Detecting and identifying MHC-bound peptides on cell surfaces can be challenging due to their low abundance, making it difficult to effectively measure and identify these peptides using traditional working concentrations for mass spectrometry analysis. Thus, immunopeptidomic analysis requires a large amount of peptides from starting tissue material in addition to specialized software to identify the bound peptides that can analyze up to thousands of peptide sequences simultaneously.
To combat these barriers to sample prep, immunopeptidomic samples are generally prepared by isolating desired peptides bound to MHCs using an antibody (allele-specific or pan) or an engineered affinity tag system from lysed cells or tissues to concentrate the sample peptides. In addition to using an immunoaffinity column equipped with MHC or HLA antibodies to capture the molecules and associated peptides or a specific subtype, magnetic and agarose or Sepharose beads with Protein A, G, and A/G can be used to support immunoaffinity purification.
Targeted and non-targeted data acquisition mass spectrometry can be employed in immunopeptidomics. Scientists may require discovery (or non-targeted data acquisition) using label-free quantitation, TMT isobaric labeling or metabolic SILAC labeling techniques. Scientists may also opt to use targeted data validation methods by mass spectrometry to evaluate peptides for applications such as drug discovery. Once MAPs are identified in discovery, targeted quantitation using mass spectrometry for validation of the peptide antigens using products such as AQUA custom peptides or SureQuant assay kits can be performed to complete the analysis.
Immunopeptidomic applications
Immunopeptidomics aids in understanding disease pathogenesis, particularly autoimmune disease and cancers, by using mass spectrometry to quantitatively analyze MHC associated peptides in the immunopeptidome. This approach enables the identification of peptide antigens presented during immune responses or disease states by comparing them against protein sequence databases. Immunopeptidomics has made significant contributions to immunotherapy and vaccine development for cancer by aiding in the development of personalized treatment approaches through the identification of patient tumor specific antigens. Identifying peptide antigens that stimulate a robust immune response also contributes to the generation of effective vaccines against infectious disease. Analysis of the immunopeptidome offers valuable insights for precision medicine, immunotherapy, and advancing the understanding of immune-related diseases.
Characterization for cancer immunotherapy
Identifying and characterizing the immunopeptidome of cancerous tissue enables the discovery of novel tumor specific antigens (TSAs) which are used as targets for cancer immunotherapy. To develop immunotherapies against specific peptides, relevant targets of a tumor are identified using T cell epitopes. Studying the MHC-peptide complexes presented by tumor cells can provide insight into the immune responses against cancer and aid in cancer biomarker identification.
Immunopeptidomics allows for the identification of neoantigens, which are novel peptide antigens derived from mutated proteins that are specifically expressed by cancer cells but not found in normal (control) cells. Since the expression of neoantigens can vary between different cancer types and individuals, their identification and characterization are important for developing personalized immunotherapies, helping lead to improved treatment outcomes. Analysis of the peptide repertoire over time can assess the effectiveness of immunotherapies for tumors, monitor treatment response, discover biomarkers for early cancer detection, and help predict patient outcomes.
Immunopeptidomic studies have been used to identify HLA Class I peptides that are more abundant in triple negative breast cancer samples than normal breast tissue. These peptides are being further validated as targets for cancer vaccines and T cell immunotherapy. Immunopeptidomic analysis is also being used to profile epithelial cells biopsied from ovarian tumors to identify MAPs specifically expressed in cancerous tissue to aid in the development of immunotherapies. Immunopeptidomics brings significant improvements to immunotherapy applications by providing the specificity for minute peptide changes required for detecting de novo mutations and creating personalized medicine that CAR-T therapy affords for better patient outcomes, improving the lives of cancer patients.
Immunopeptidomic analysis aids in vaccine development
Immunopeptidomics has played a significant role in combating infectious disease. The identification of peptides presented by MHC molecules on pathogen infected cells can reveal bacterial and viral antigens as candidates for vaccine development. Immunopeptidomic analysis of pathogen infected cells has identified T cell epitopes for the bacteria Mycobacterium tuberculosis and Chlamydia trachomatis, and the parasite Leishmania major. More recently, this method helped identify viral T cell epitopes and was used to develop potential HIV and SARS-CoV-2 vaccines.
To develop effective cancer vaccines, the identification of epitopes recognized by T cells is crucial. Immunopeptidomics has been a key application in cancer vaccine development, providing an avenue where tumor specific antigens are extracted from biopsied tissues and analyzed. To develop a vaccine targeting cancer cells, several steps are involved.
- Affinity purification and peptide identification: Immunoprecipitation and tandem mass spectrometry are used to isolate MHC-restricted tumor peptides, then quantify the peptides to help identify cross-reactive T cells for functional characterization of candidate peptides, which should elicit a strong immune response.
- Validate candidate peptides: Using RNAseq and mass spectrometry analysis, scientists can characterize these key MHC-restricted peptides, then validate and quantify those candidate peptides.
- Validate candidate peptides for carrier binding (i.e., characterize the binding affinity of the peptides to the oncolytic adenovirus).
- Assess tumor reactivity to peptides: Determine if tumor tissue growth is impeded.
By analyzing the tumor peptide repertoire, specific peptide signatures or patterns can be identified that correlate with disease progression or treatment response. Immunopeptidomics is used to help improve vaccine development to help cover a wide population of associated peptides with specific antigen expression. Recent research has propelled immunopeptidomics to the forefront for personalized oncolytic cancer vaccines.
Role of PROTACs in MHC peptide presentation
Peptides presented by MHC Class I molecules are derived from endogenous proteins that are degraded through the ubiquitin-proteasome pathway. Targeted protein degradation through proteolysis targeting chimeras (PROTACs®) has been shown to induce changes in the expression of MHC Class I peptides. Some of these peptides originate from the specific protein targeted for degradation, potentially identifying new antigen targets for T cell immunotherapy and increasing the ability of PROTACs to elicit a biological response toward undruggable targets. PROTACs can also be used to generate MHC peptide antigens from mutated proteins that are not easily degraded by the ubiquitin proteasome system, enabling the discovery of additional neoantigens for cancer immunotherapy.
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