# Lecture 5: Antigen Processing and Presentation
## Unit 2.7: Immunology
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## Learning Objectives
By the end of this lecture, students will be able to:
1. Describe the structure and function of MHC class I and II molecules
2. Explain the pathways of antigen processing and presentation
3. Describe the genetics of the major histocompatibility complex
4. Explain the role of professional antigen-presenting cells
5. Describe cross-presentation and its significance
6. Explain clinical correlates of MHC and antigen presentation
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## Lecture Outline
### I. Major Histocompatibility Complex Overview
The major histocompatibility complex (MHC) comprises a cluster of highly polymorphic genes encoding cell surface glycoproteins essential for adaptive immunity, originally discovered through their role in determining tissue transplant compatibility between individuals. MHC molecules bind peptide fragments derived from proteins and display them on the cell surface for surveillance by T lymphocytes, thereby enabling the immune system to detect the presence of foreign antigens, including those derived from intracellular pathogens and tumors. This antigen presentation function makes MHC molecules central to virtually all adaptive immune responses and explains their critical importance in transplantation, autoimmunity, and infectious disease susceptibility.
The MHC serves several fundamental functions in immunity that extend beyond simple antigen display. First, MHC molecules enable T cells to distinguish self from non-self by presenting peptide fragments that reflect the proteome of each cell, allowing detection of virus-infected or transformed cells. Second, the MHC determines transplant compatibility, as differences in MHC molecules between donor and recipient constitute the primary barrier to organ and tissue transplantation. Third, specific MHC alleles are strongly associated with susceptibility or resistance to autoimmune diseases, infections, and even certain malignancies, making MHC genetics clinically relevant for disease risk assessment. Fourth, MHC molecules shape the T cell repertoire during thymic development through positive and negative selection, determining which T cell specificities survive to populate the periphery.
MHC molecules are classified into two main types that differ in structure, tissue distribution, and function. MHC class I molecules are expressed on virtually all nucleated cells and present peptides derived primarily from cytoplasmic proteins (endogenous pathway) to CD8+ T cells. MHC class II molecules have restricted expression, being found constitutively only on professional antigen-presenting cells (dendritic cells, macrophages, and B cells), and present peptides derived from extracellular proteins captured by endocytosis (exogenous pathway) to CD4+ T cells. A third category, MHC class III genes, encodes various immune-related proteins including complement components and cytokines but does not encode antigen-presenting molecules.
In humans, the MHC is termed the human leukocyte antigen (HLA) complex and spans approximately 4 megabases on the short arm of chromosome 6 (6p21.3). The classical MHC class I genes include HLA-A, HLA-B, and HLA-C, while class II genes include HLA-DR, HLA-DP, and HLA-DQ. Each individual inherits one haplotype (set of MHC genes) from each parent, and because MHC genes are codominantly expressed, each cell displays both maternal and paternal MHC molecules. The extreme polymorphism of HLA genes, with thousands of alleles identified at each locus, generates enormous diversity within populations, ensuring that at least some individuals will mount effective immune responses against any given pathogen, thereby promoting species survival.
Panel A: Chromosome 6 map showing the organization of the HLA complex with class I region (telomeric, containing HLA-A, HLA-B, HLA-C genes), class III region (central, containing complement genes C2, C4, factor B, and TNF genes), and class II region (centromeric, containing HLA-DP, HLA-DQ, HLA-DR genes), with approximate genetic distances indicated
Panel B: Comparison of MHC class I and class II structures showing class I as heavy chain (alpha1, alpha2, alpha3 domains) plus beta2-microglobulin, and class II as alpha chain (alpha1, alpha2) plus beta chain (beta1, beta2), with peptide-binding grooves highlighted and CD8/CD4 binding sites indicated
Panel C: Tissue distribution diagram showing MHC class I expression on all nucleated cells (liver hepatocyte, neuron, epithelial cell, muscle cell) with absence on red blood cells, and MHC class II restricted expression on professional APCs (dendritic cells, macrophages, B cells) with inducible expression on other cells by IFN-gamma
Panel D: Codominant expression illustration showing diploid cell expressing both maternal (HLA-A1, B8, DR3) and paternal (HLA-A2, B44, DR4) MHC molecules simultaneously on the cell surface, doubling the peptide repertoire that can be presented
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### II. MHC Class I Structure
MHC class I molecules are heterodimeric glycoproteins consisting of a polymorphic heavy chain (alpha chain) non-covalently associated with the invariant light chain beta2-microglobulin, together forming a stable complex capable of binding and presenting peptide antigens to CD8+ T cells. The heavy chain is encoded within the MHC on chromosome 6, while beta2-microglobulin is encoded on chromosome 15, representing a unique arrangement where components of a functional receptor are encoded on different chromosomes. The mature class I molecule displays remarkable structural conservation across all classical HLA-A, HLA-B, and HLA-C alleles, reflecting the fundamental importance of this architecture for immune function.
The heavy chain comprises three extracellular domains (alpha1, alpha2, and alpha3), a transmembrane region, and a short cytoplasmic tail. The membrane-distal alpha1 and alpha2 domains form the peptide-binding groove, a structure resembling a trough with a floor of eight antiparallel beta strands topped by two alpha helices that form the walls. This groove accommodates peptides of 8-10 amino acids, with the ends of the groove closed by conserved residues that constrain peptide length. The floor and walls of the groove contain pockets (designated A through F) that accommodate specific amino acid side chains of the bound peptide, with pockets B and F being particularly important for anchoring the peptide through its amino- and carboxy-terminal residues.
Peptide binding to MHC class I exhibits allele-specific preferences determined by the polymorphic residues lining the binding groove, particularly those forming the B and F anchor pockets. Each HLA allele binds peptides sharing specific anchor residues at positions 2 and 9 (or the C-terminus), defining the peptide-binding motif for that allele. For example, HLA-A2 preferentially binds peptides with leucine at position 2 and valine or leucine at the C-terminus, while HLA-B27 favors arginine at position 2. This allele-specific binding means that different individuals present different subsets of peptides from the same protein, contributing to variation in immune responses among individuals. The bound peptide is an integral component of the stable MHC-peptide complex, and class I molecules without bound peptide are unstable and rapidly degraded.
Beta2-microglobulin associates non-covalently with the alpha3 domain and is essential for proper folding, stability, and cell surface expression of MHC class I molecules. The alpha3 domain and beta2-microglobulin together form an immunoglobulin-like structure that positions the peptide-binding groove away from the cell surface. Importantly, the alpha3 domain contains the binding site for the CD8 coreceptor, which interacts with a conserved loop on the alpha3 domain during T cell recognition. This interaction brings the CD8-associated Lck kinase into proximity with the TCR-CD3 complex, enhancing signal transduction. The extreme polymorphism of class I molecules, with over 7,000 HLA-A alleles, over 8,000 HLA-B alleles, and over 6,000 HLA-C alleles documented, is concentrated in the alpha1 and alpha2 domains that form the peptide-binding groove, directly affecting the peptide repertoire that can be presented.
Panel A: Three-dimensional ribbon diagram of MHC class I structure showing the heavy chain alpha1, alpha2, and alpha3 domains colored distinctly, beta2-microglobulin in a contrasting color, a peptide seated in the binding groove, and the cell membrane with transmembrane region, with domains and functional regions labeled
Panel B: Top-down view of the peptide-binding groove showing the beta-sheet floor, alpha-helix walls, closed ends, and peptide accommodated within, with anchor residue pockets (A through F) highlighted and a typical 9-mer peptide with key anchor positions (P2, P9) indicated
Panel C: Illustration of allele-specific peptide binding showing two different HLA alleles (HLA-A2 and HLA-B27) with their distinct pocket structures and the different peptide motifs they accommodate, demonstrating how polymorphism affects peptide repertoire
Panel D: Assembly and stability diagram showing that peptide binding is required for stable class I expression, with empty class I molecules being retained in ER and unstable, while peptide-loaded class I achieves stable conformation and traffics to the cell surface
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### III. MHC Class II Structure
MHC class II molecules are heterodimeric glycoproteins composed of two transmembrane chains, designated alpha and beta, both encoded within the MHC region and both contributing to formation of the peptide-binding groove. Unlike class I molecules where only the heavy chain is polymorphic, both class II chains exhibit polymorphism, although the beta chain is typically more polymorphic than the alpha chain. This structural arrangement enables class II molecules to present longer peptides than class I and to accommodate peptides with more variable binding registers, reflecting their distinct role in presenting exogenous antigens to CD4+ T helper cells.
Each chain of the class II molecule contains two extracellular domains: alpha1 and alpha2 for the alpha chain, and beta1 and beta2 for the beta chain. The peptide-binding groove is formed by the alpha1 domain of the alpha chain and the beta1 domain of the beta chain coming together to create a structure superficially similar to the class I groove but with critical differences. Most notably, the class II groove has open ends rather than the closed ends of class I, allowing bound peptides to extend beyond the groove at both termini. This architectural difference explains why class II-bound peptides are typically 13-25 amino acids or longer, although only a core of approximately 9 amino acids actually sits within the groove.
Peptide binding to class II molecules involves anchor residues that occupy pockets within the groove, similar to class I, but the open-ended nature of the groove provides greater flexibility in peptide binding. Class II molecules exhibit less stringent length requirements, and the same peptide can bind in slightly different registers (positions within the groove), although one register typically predominates. The polymorphic residues lining the class II groove determine which peptides bind efficiently, and as with class I, different HLA-DR, HLA-DP, and HLA-DQ alleles present different peptide repertoires. The beta2 domain of the beta chain contains the binding site for the CD4 coreceptor, which recognizes a conserved region and enhances T cell activation similarly to the CD8-class I interaction.
Expression of class II molecules is tightly regulated and normally restricted to professional antigen-presenting cells, but can be induced on many other cell types by interferon-gamma. This inducibility is controlled by the class II transactivator (CIITA), a master regulator that does not bind DNA directly but rather coordinates assembly of the transcriptional machinery on class II gene promoters. CIITA expression is itself controlled by different promoters in different cell types: a constitutive promoter drives expression in dendritic cells and macrophages, while an IFN-gamma-inducible promoter enables expression in non-professional APCs during inflammation. Deficiency of CIITA or other components of the class II transcription machinery causes bare lymphocyte syndrome type II, a severe combined immunodeficiency characterized by absence of MHC class II expression.
Panel A: Three-dimensional structure of MHC class II showing alpha chain (alpha1, alpha2 domains) and beta chain (beta1, beta2 domains) forming the heterodimer, with the peptide-binding groove at the top, CD4 binding site on beta2, and both transmembrane regions anchoring the molecule to the membrane
Panel B: Comparison of class I and class II peptide-binding grooves viewed from above, highlighting closed ends of class I accommodating 8-10mer peptides versus open ends of class II allowing peptide extensions, with a 15-mer peptide shown in class II with core binding region and flanking extensions
Panel C: CIITA regulation of class II expression showing different promoters (pI for dendritic cells, pIII for B cells, pIV for IFN-gamma induction), CIITA protein coordinating transcription factors at class II gene promoters, and resulting cell-type-specific and inducible expression patterns
Panel D: Flow diagram of class II gene organization showing HLA-DR, HLA-DQ, and HLA-DP regions on chromosome 6, with alpha and beta genes for each, and the resulting heterodimers, noting that DR can form multiple isotypes due to multiple DRB genes
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### IV. MHC Class I Antigen Processing
The MHC class I antigen processing pathway, also called the endogenous or cytosolic pathway, generates peptides from proteins synthesized within the cell and loads them onto MHC class I molecules for presentation to CD8+ T cells. This pathway enables surveillance of the intracellular proteome, allowing the immune system to detect cells harboring intracellular pathogens or expressing abnormal proteins due to malignant transformation. The pathway involves protein degradation in the cytoplasm, peptide transport into the endoplasmic reticulum, and assembly of peptide-MHC complexes before transport to the cell surface.
The proteasome is the primary proteolytic machinery responsible for generating peptides for class I presentation. This large barrel-shaped complex comprises a 20S catalytic core containing multiple proteolytic active sites, capped by 19S regulatory particles that recognize ubiquitinated proteins and unfold them for entry into the catalytic chamber. The standard proteasome contains constitutive catalytic subunits, but during inflammation or infection, interferon-gamma induces expression of alternative subunits (LMP2, LMP7, MECL-1) that replace the constitutive subunits to form the immunoproteasome. The immunoproteasome exhibits altered cleavage preferences that favor production of peptides with hydrophobic or basic C-termini, which are preferred anchors for many class I alleles, thereby enhancing antigen presentation during immune responses.
Peptides generated by the proteasome are transported from the cytoplasm into the endoplasmic reticulum by the transporter associated with antigen processing (TAP), a heterodimeric ABC transporter composed of TAP1 and TAP2 subunits. TAP preferentially transports peptides of 8-16 amino acids with hydrophobic or basic C-terminal residues, matching the substrate preferences of class I molecules. The transported peptides are often longer than the optimal 8-10 residues for class I binding and may be further trimmed by ER-resident aminopeptidases, particularly ERAP1 and ERAP2, which remove N-terminal residues to generate peptides of optimal length. TAP deficiency results in bare lymphocyte syndrome type I, characterized by reduced class I surface expression and susceptibility to recurrent respiratory infections, though less severe than complete class I deficiency.
Assembly of peptide-MHC class I complexes in the ER involves a dedicated loading complex that ensures only properly folded, peptide-loaded molecules reach the cell surface. Newly synthesized class I heavy chains first associate with the chaperone calnexin, which promotes proper folding. Following beta2-microglobulin association, the complex transfers to a loading complex comprising calreticulin, ERp57 (a thiol oxidoreductase), and tapasin. Tapasin physically bridges the class I molecule to TAP, positioning the peptide-binding groove near the peptide supply while also acting as a peptide editor that favors loading of high-affinity peptides. Only class I molecules loaded with stably-binding peptides pass quality control and exit the ER; empty or suboptimally loaded molecules are retained and eventually degraded. This stringent quality control ensures that cell surface class I molecules present a representative sample of the cytoplasmic proteome.
Panel A: Overview of the endogenous pathway showing cytoplasmic protein being ubiquitinated and degraded by the proteasome to peptide fragments, TAP transporting peptides into the ER, peptide loading onto class I molecules in the ER, and transport through Golgi to the plasma membrane for CD8+ T cell surveillance
Panel B: Detailed proteasome structure showing 20S core particle with catalytic chamber, 19S regulatory caps, and comparison of constitutive versus immunoproteasome subunits (LMP2, LMP7, MECL-1 replacing constitutive subunits), with indication of altered cleavage preferences favoring class I-compatible peptides
Panel C: TAP transporter structure and function showing TAP1 and TAP2 subunits forming the channel, ATP hydrolysis driving transport, peptide substrate preferences (8-16 aa, C-terminal hydrophobic/basic), and position in ER membrane connecting cytoplasm to ER lumen
Panel D: Peptide loading complex showing class I heavy chain, beta2-microglobulin, calreticulin, ERp57, and tapasin bridging to TAP, with peptide editing function illustrated by preferential release of low-affinity peptides and stable loading of high-affinity peptides
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### V. MHC Class II Antigen Processing
The MHC class II antigen processing pathway, also called the exogenous or endocytic pathway, captures extracellular proteins through phagocytosis, endocytosis, or macropinocytosis, processes them in endosomal compartments, and loads the resulting peptides onto class II molecules for presentation to CD4+ T helper cells. This pathway is particularly important for responses to extracellular pathogens, including most bacteria, parasites, and toxins, as well as for presentation of antigens captured from apoptotic cells and immune complexes. Professional antigen-presenting cells are specialized for this pathway, constitutively expressing class II and possessing enhanced endocytic and phagocytic capabilities.
Newly synthesized class II alpha and beta chains assemble in the endoplasmic reticulum, where they associate with the invariant chain (Ii, CD74), a specialized chaperone that serves multiple essential functions. First, the invariant chain occupies the peptide-binding groove of nascent class II molecules, preventing premature binding of ER-resident peptides that would otherwise be loaded through the default class I pathway. The portion of Ii that occupies the groove is called CLIP (class II-associated invariant chain peptide). Second, Ii trimerizes, with each Ii molecule associating with one alphabeta class II heterodimer, creating a nonameric complex. Third, Ii contains targeting signals in its cytoplasmic tail that direct the class II-Ii complex from the ER through the Golgi to the endosomal pathway rather than directly to the cell surface.
Within the endosomal pathway, progressively acidic compartments contain proteases (cathepsins) that degrade the invariant chain, leaving only the CLIP fragment in the class II groove. Simultaneously, exogenous proteins captured by endocytosis are delivered to these same compartments and degraded to peptides by cathepsins B, D, L, S, and other endosomal proteases. The specialized MHC class II compartment (MIIC) is where peptide loading occurs, characterized by high concentrations of class II molecules, HLA-DM, and processed peptides. HLA-DM is a non-classical class II molecule that catalyzes CLIP release and peptide exchange, binding to class II and inducing conformational changes that facilitate release of CLIP or weakly-bound peptides and loading of higher-affinity peptides from the endosomal environment.
The process of peptide editing by HLA-DM ensures that class II molecules are loaded with the most stably-binding peptides available, optimizing the presentation of immunodominant epitopes. HLA-DM remains associated with class II until a peptide with sufficient affinity stabilizes the class II molecule, at which point DM releases and the peptide-class II complex can traffic to the cell surface. HLA-DO, another non-classical class II molecule expressed primarily in B cells and thymic epithelium, modulates DM function by forming stable DM-DO complexes that have reduced CLIP-exchange activity, potentially altering the repertoire of presented peptides in these cell types. Cathepsin S is particularly important for Ii degradation in dendritic cells and B cells, while cathepsin L predominates in thymic epithelium, representing tissue-specific regulation of the processing machinery.
Panel A: Overview of the exogenous pathway showing extracellular antigen being captured by endocytosis/phagocytosis, delivery to progressively acidic endosomal compartments with cathepsin-mediated degradation, class II-Ii complex trafficking from ER to endosomes, CLIP removal and peptide loading in MIIC, and transport of peptide-class II to the cell surface for CD4+ T cell recognition
Panel B: Invariant chain structure and functions showing the trimerized Ii with three class II heterodimers attached, CLIP region occupying each groove, ER retention signal, and endosomal targeting signals in the cytoplasmic tail, with annotation of each functional domain
Panel C: Sequential diagram of Ii processing showing intact Ii-class II complex in early endosome, progressive cathepsin-mediated Ii degradation leaving CLIP, HLA-DM catalyzing CLIP release, and peptide loading with stable peptide-class II complex formation
Panel D: HLA-DM function illustration showing DM binding to class II-CLIP complex, inducing conformational change releasing CLIP, facilitating peptide sampling and exchange, with comparison of weak-binding peptide release versus stable-binding peptide retention, and role of HLA-DO in modulating DM activity in B cells
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### VI. Professional Antigen-Presenting Cells
Professional antigen-presenting cells (APCs) are specialized immune cells that constitutively express MHC class II molecules and are optimized for antigen capture, processing, and presentation to T lymphocytes. The three main professional APCs are dendritic cells, macrophages, and B cells, each with distinct properties that suit them for different roles in initiating and sustaining adaptive immune responses. The defining feature of professional APCs is their ability to provide not only the peptide-MHC signal (signal 1) but also the costimulatory signals (signal 2) and cytokine signals (signal 3) required for full T cell activation.
Dendritic cells (DCs) are the most potent APCs and are uniquely capable of activating naive T cells, making them essential for initiating primary immune responses. DCs exist in two functional states: immature DCs residing in peripheral tissues actively capture antigens through macropinocytosis, receptor-mediated endocytosis, and phagocytosis but express low levels of costimulatory molecules. Upon encountering pathogens and receiving activation signals through pattern recognition receptors, DCs undergo maturation characterized by decreased endocytic capacity, upregulation of MHC class II and costimulatory molecules (CD80, CD86, CD40), increased expression of the chemokine receptor CCR7 that directs migration to draining lymph nodes, and acquisition of cytokine-producing capacity. Mature DCs in lymph node T cell zones present antigen to naive T cells and provide the signals necessary for their activation and differentiation.
Macrophages serve dual roles as both innate immune effector cells and antigen-presenting cells, though they primarily present antigen to previously activated (effector and memory) T cells rather than naive cells. Resident macrophages in tissues continuously sample their environment through phagocytosis and present antigens constitutively, while activated macrophages upregulate MHC class II and costimulatory molecules. The primary function of antigen presentation by macrophages is to receive help from effector CD4+ T cells: Th1 cells recognizing antigen on macrophages produce IFN-gamma that enhances macrophage microbicidal activity, creating a positive feedback loop that optimizes pathogen killing. Different tissue macrophage populations (Kupffer cells in liver, alveolar macrophages in lung, microglia in brain) perform tissue-specific homeostatic and immune functions.
B cells are unique among professional APCs in that they capture antigen through their surface immunoglobulin (BCR), providing exquisite specificity for antigen uptake. B cells are relatively inefficient at presenting antigens they capture through nonspecific mechanisms, but are highly efficient at presenting their cognate antigen, concentrating it 1000-10,000-fold compared to non-specific uptake. This antigen-specific capture and presentation is critical for T-B collaboration: B cells that have bound antigen through their BCR internalize, process, and present peptides from that antigen to T follicular helper cells, receiving survival and differentiation signals in return. This interaction occurs primarily at the T-B border and in germinal centers, where Tfh cells select high-affinity B cells based on their ability to efficiently capture and present antigen.
Panel A: Dendritic cell maturation process showing immature DC in tissue with high endocytic capacity, pathogen encounter triggering maturation through PRR activation, migration through lymphatics with CCR7 upregulation, and mature DC in lymph node T zone with high MHC II, CD80/86, and CD40 presenting to naive T cell
Panel B: Comparison of the three professional APCs showing dendritic cell (initiates primary responses, most potent), macrophage (amplifies responses, receives T cell help for enhanced killing), and B cell (cognate antigen capture via BCR, presents to Tfh for B cell help), with key characteristics and primary functions
Panel C: Macrophage activation cycle showing macrophage presenting antigen to Th1 cell, receiving IFN-gamma, upregulating MHC II and microbicidal mechanisms, enhanced pathogen killing, with indication that this is particularly important for intracellular pathogens like Mycobacteria
Panel D: B cell antigen presentation showing BCR-mediated specific antigen capture, internalization to endosomal compartment, processing and class II loading, and presentation to Tfh cell at T-B border, with Tfh-derived signals (CD40L, IL-21) promoting B cell activation, survival, and differentiation
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### VII. Cross-Presentation
Cross-presentation is a specialized pathway by which certain dendritic cells can present exogenous antigens on MHC class I molecules, thereby activating CD8+ cytotoxic T cell responses against pathogens and tumors that do not directly infect antigen-presenting cells. This pathway represents an important exception to the general rule that class I presents endogenous antigens and class II presents exogenous antigens. Cross-presentation is essential for generating CTL responses against viruses that preferentially infect non-hematopoietic cells (such as epithelial cells) and against tumors that lack the costimulatory capacity to directly prime naive CD8+ T cells.
The biological importance of cross-presentation is best understood in the context of antiviral and anti-tumor immunity. Many viruses infect tissue parenchymal cells but not dendritic cells, and since naive CD8+ T cells require activation by professional APCs in lymph nodes, there must be a mechanism for DCs to acquire and present viral antigens without being infected. Cross-presentation solves this problem: DCs can phagocytose virus-infected cells that have died, process viral proteins, and present them on class I for CTL priming. Similarly, tumor cells generally cannot activate naive T cells due to insufficient costimulation, but DCs can acquire tumor antigens through phagocytosis of dying tumor cells and cross-present them to prime anti-tumor CTL responses. The clinical success of checkpoint inhibitors demonstrates the relevance of these naturally-primed anti-tumor responses.
The mechanisms of cross-presentation are incompletely understood, but two major pathways have been proposed. In the vacuolar pathway, internalized antigens are processed within endosomal compartments and loaded onto recycling class I molecules that traffic through these compartments. In the cytosolic pathway, internalized antigens escape from endosomes into the cytoplasm, where they are processed by the proteasome and loaded onto class I through the conventional ER-based machinery. Evidence supports contributions from both pathways, possibly depending on the nature of the antigen and the DC subset involved. Some internalized antigens may be recognized by the ER-associated degradation (ERAD) machinery, which exports them to the cytoplasm for proteasomal degradation.
Not all dendritic cells cross-present with equal efficiency. In mice, the CD8alpha+ conventional DC subset (cDC1) is specialized for cross-presentation, and genetic ablation of this population severely impairs cross-priming of CTL responses. The human equivalent is CD141+ (BDCA3+) DCs, characterized by expression of the chemokine receptor XCR1 and the C-type lectin CLEC9A, which recognizes dead cell debris and facilitates cross-presentation. These DCs express specialized machinery that favors cross-presentation, including reduced phagolysosomal acidification that preserves antigens from complete degradation, and efficient mechanisms for antigen export to the cytoplasm. Understanding cross-presentation has practical implications for vaccine design, as strategies that target antigen to cross-presenting DCs or enhance antigen escape to the cytoplasm can improve generation of CTL responses.
Panel A: Cross-presentation concept diagram showing conventional pathways (endogenous to class I, exogenous to class II) and cross-presentation as exception (exogenous antigen to class I), with annotation of why this is important for viruses not infecting DCs and for tumor immunity
Panel B: Two proposed mechanisms of cross-presentation showing vacuolar pathway (antigen processed in endosome, loads onto recycling class I in endosome) and cytosolic pathway (antigen escapes to cytoplasm, conventional proteasome-TAP-ER loading), with indication that both may contribute depending on context
Panel C: Cross-presenting DC subset illustration showing cDC1 cells (CD8alpha+ in mouse, CD141+/XCR1+/CLEC9A+ in human) with specialized features including CLEC9A receptor recognizing dead cell debris, reduced phagosomal acidification preserving antigens, and efficient cytosolic antigen export
Panel D: Clinical relevance of cross-presentation showing cross-priming in viral infection (DC phagocytoses infected cell, cross-presents viral antigens to CTL), anti-tumor immunity (DC acquires tumor antigens from dying tumor cell, cross-presents to prime tumor-specific CTL), and vaccine strategies targeting cross-presenting DCs
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### VIII. MHC Genetics
The genetics of the MHC exhibit several unusual features that distinguish this region from most other parts of the genome, including extreme polymorphism, linkage disequilibrium, and codominant expression, all of which have important implications for transplantation, disease susceptibility, and population immunity. These genetic characteristics have been shaped by evolutionary pressure from pathogens, which has selected for diversity in antigen presentation capacity at both the individual and population levels. Understanding MHC genetics is essential for transplant matching, interpretation of disease associations, and appreciation of individual variation in immune responses.
Codominant expression means that both maternal and paternal MHC alleles are expressed simultaneously on cell surfaces, effectively doubling the number of distinct MHC molecules available for antigen presentation. An individual heterozygous at HLA-A, HLA-B, and HLA-C expresses six different class I molecules (two from each locus), while homozygotes express only three distinct molecules. Similarly, class II expression includes products from both chromosomes, with additional complexity arising from the potential for alpha-beta chain pairing between products of different loci or different parental alleles (though trans-complementation is limited by structural constraints). This codominant expression maximizes the peptide repertoire that can be presented and likely provides heterozygote advantage in pathogen resistance.
The extreme polymorphism of HLA genes is the highest of any coding genes in the human genome, with thousands of alleles documented at each classical locus. This polymorphism is concentrated in the residues that form the peptide-binding groove, directly affecting which peptides can be presented. Population-level diversity ensures that any given pathogen is unlikely to evade presentation by all HLA types, providing species-level protection against pathogens that might otherwise evolve to evade a monomorphic MHC. The polymorphism is maintained by balancing selection, including both heterozygote advantage and frequency-dependent selection (where rare alleles may provide advantage against pathogens adapted to common alleles). Certain HLA alleles have been associated with protection against specific pathogens, most notably HLA-B27, HLA-B57, and HLA-B58 with slower HIV progression.
Linkage disequilibrium refers to the non-random association of alleles at different loci, occurring when certain allele combinations (haplotypes) are more common in the population than expected by chance. In the MHC, strong linkage disequilibrium extends across the entire region, meaning that knowing one HLA allele provides information about others on the same chromosome. Common haplotypes include HLA-A1-B8-DR3 in European populations and HLA-A33-B58-DR3 in Asian populations. Linkage disequilibrium has practical implications for transplant matching, as matching at one locus often implies matching at linked loci. It also complicates disease association studies, as associations with one HLA allele might reflect linkage to the causative allele rather than direct causation. Modern high-resolution HLA typing by sequencing has become the standard for transplantation and is increasingly used to define precise disease associations.
Panel A: Codominant expression illustration showing a cell from a heterozygous individual expressing both maternal alleles (HLA-A1, B8, Cw7, DR3, DQ2) and paternal alleles (HLA-A3, B7, Cw7, DR15, DQ6) simultaneously, with annotation showing how this doubles the potential peptide repertoire
Panel B: Graphical representation of HLA polymorphism showing the number of alleles at each locus (HLA-A >7000, HLA-B >8000, HLA-C >6000, HLA-DRB1 >3000, HLA-DQB1 >1800, HLA-DPB1 >1500), with indication that polymorphism is concentrated in peptide-binding groove residues
Panel C: Linkage disequilibrium diagram showing chromosome 6 with HLA region, demonstrating that certain allele combinations (A1-B8-DR3 haplotype) are inherited together more often than expected by chance, with population frequency data and explanation of why this occurs (selection and population history)
Panel D: HLA typing methods evolution showing serological typing (historical, antibody-based, low resolution), PCR-based methods (SSP, SSO, intermediate resolution), and next-generation sequencing (current standard, high resolution, identifies novel alleles), with applications for each (transplant matching, disease association)
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### IX. MHC and Disease Associations
Associations between specific HLA alleles and disease susceptibility represent some of the strongest and most reproducible genetic risk factors in medicine, with certain HLA-disease associations conferring relative risks exceeding 100-fold. These associations predominantly involve autoimmune diseases, reflecting the central role of MHC molecules in determining which self-antigens are presented to T cells and in shaping the T cell repertoire during thymic selection. Understanding HLA-disease associations has provided insights into disease mechanisms and has practical applications in disease risk prediction and diagnosis.
The strongest HLA-disease association in medicine is between HLA-B27 and ankylosing spondylitis, with over 90% of patients carrying this allele compared to approximately 8% of the general population, yielding a relative risk of approximately 90-fold. HLA-B27 is also associated with other spondyloarthropathies including reactive arthritis, psoriatic arthritis, and inflammatory bowel disease-associated arthritis. Despite decades of research, the mechanism underlying this association remains incompletely understood. Proposed hypotheses include the arthritogenic peptide hypothesis (B27 presents a unique peptide that cross-reacts with a microbial antigen), the B27 misfolding hypothesis (B27 heavy chains misfold in the ER, triggering stress responses and inflammation), and the free heavy chain hypothesis (B27 heavy chains dissociate from beta2-microglobulin and form homodimers that stimulate aberrant immune responses). Importantly, most HLA-B27-positive individuals never develop spondyloarthropathy, indicating that additional genetic and environmental factors are required.
Type 1 diabetes mellitus provides a paradigm for understanding how class II alleles influence autoimmune susceptibility. The strongest genetic risk factors for T1DM are HLA-DR3 and HLA-DR4, particularly when present together in DR3/DR4 heterozygotes. However, the actual risk is conferred by specific DQ alleles in linkage disequilibrium with DR3 and DR4, specifically DQ2 (DQA1*05:01-DQB1*02:01) and DQ8 (DQA1*03:01-DQB1*03:02). Notably, certain DQ alleles are protective, particularly DQ6 (DQB1*06:02), which is rarely found in T1DM patients even when present with risk-conferring DR alleles. The structural basis for these effects involves the ability of risk versus protective alleles to present insulin-derived peptides to autoreactive T cells, with crystallographic studies revealing how specific residues in the DQ groove determine peptide binding and T cell recognition.
Celiac disease provides perhaps the clearest mechanistic understanding of an HLA-disease association. Virtually all celiac disease patients carry HLA-DQ2 or HLA-DQ8, which present gluten-derived peptides to CD4+ T cells in the intestinal lamina propria. The key insight is that gluten peptides are poor binders of DQ2/DQ8 in their native form but become high-affinity binders after deamidation by tissue transglutaminase, which converts glutamine residues to negatively charged glutamate that fits into positively charged pockets in the DQ2 binding groove. This explains both the HLA requirement (specific binding groove) and the role of tissue transglutaminase (generates the immunodominant epitopes). Similar molecular insights are emerging for other HLA-associated diseases, enabling development of targeted therapies and antigen-specific tolerance strategies.
Panel A: Overview of HLA-disease associations showing major autoimmune diseases with their associated HLA alleles and relative risks: ankylosing spondylitis (B27, RR~90), type 1 diabetes (DR3/DR4, DQ2/DQ8, RR~15), celiac disease (DQ2/DQ8, RR~7), rheumatoid arthritis (DR4 shared epitope, RR~5), multiple sclerosis (DR2/DRB1*15:01, RR~3)
Panel B: HLA-B27 and spondyloarthropathy showing proposed mechanisms including arthritogenic peptide presentation, B27 heavy chain misfolding causing ER stress, and free B27 heavy chain homodimers engaging aberrant receptors, with note that mechanism remains debated
Panel C: Type 1 diabetes HLA associations showing DR-DQ linkage, specific risk alleles (DQ2, DQ8) and protective alleles (DQ6), and molecular model of DQ8 presenting insulin B chain peptide to autoreactive T cell with key groove-peptide contacts highlighted
Panel D: Celiac disease mechanism showing gluten peptide being deamidated by tissue transglutaminase (converting glutamine to glutamate), deamidated peptide fitting into DQ2 groove with negative charges matching positive pockets, presentation to gluten-specific T cell, and resulting intestinal inflammation and villous atrophy
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### X. Clinical Correlates
The clinical significance of MHC and antigen presentation extends beyond disease associations to include immunodeficiency disorders affecting presentation machinery, immune evasion mechanisms exploited by pathogens and tumors, transplantation matching, and vaccine design strategies. These applications demonstrate how fundamental understanding of antigen presentation translates directly into clinical practice and therapeutic development.
Bare lymphocyte syndrome encompasses rare immunodeficiencies caused by defects in antigen presentation machinery. Type I bare lymphocyte syndrome results from TAP1 or TAP2 deficiency, causing reduced MHC class I surface expression. Affected individuals develop recurrent bacterial infections of the respiratory tract and bronchiectasis, but generally survive to adulthood, likely because class I-independent immune mechanisms provide partial protection. Type II bare lymphocyte syndrome is more severe, resulting from defects in transcription factors required for class II expression (CIITA, RFX5, RFXAP, or RFXANK). Complete absence of class II expression causes profound CD4+ T cell deficiency with severe infections in infancy and death without hematopoietic stem cell transplantation. These natural experiments demonstrate the essential but distinct roles of class I and class II in human immunity.
Many pathogens and tumors have evolved mechanisms to evade T cell recognition by interfering with antigen presentation. Viruses employ diverse strategies to reduce class I surface expression: herpes simplex virus produces ICP47, which blocks TAP; human cytomegalovirus encodes US6 (inhibits TAP), US2 and US11 (target class I for degradation), and US3 (retains class I in ER); adenovirus E3/19K retains class I in ER; and HIV Nef downregulates class I from the cell surface. Tumors frequently lose class I expression through genetic mechanisms (beta2-microglobulin mutations, HLA gene deletions) or epigenetic silencing. While class I loss enables escape from CTL recognition, it renders cells susceptible to NK cell killing through missing-self recognition, illustrating how the innate and adaptive immune systems provide complementary surveillance.
Transplantation requires consideration of HLA matching, particularly for hematopoietic stem cell transplantation (HSCT), where stringent matching at HLA-A, HLA-B, HLA-C, and HLA-DRB1 (8/8 match) significantly improves outcomes. Solid organ transplantation has less stringent matching requirements, though zero-mismatch kidney transplants have superior long-term graft survival. Virtual crossmatching, which predicts compatibility based on recipient HLA antibody specificities and donor HLA typing, has largely replaced traditional cell-based crossmatching. Highly sensitized patients with antibodies against many HLA types pose significant challenges for finding compatible donors, and desensitization protocols to reduce HLA antibody levels are sometimes employed to enable transplantation across positive crossmatches.
Panel A: Bare lymphocyte syndromes showing Type I (TAP deficiency causing reduced class I expression, recurrent sinopulmonary infections, generally compatible with survival) versus Type II (CIITA or RFX deficiency causing absent class II expression, severe combined immunodeficiency, fatal without HSCT), with the different clinical presentations reflecting class I versus class II functions
Panel B: Viral immune evasion mechanisms showing multiple strategies to reduce class I expression: ICP47 blocking TAP (HSV), US6/US2/US11 targeting class I pathway (CMV), E3/19K retaining class I in ER (adenovirus), and Nef removing class I from surface (HIV), with tumor class I loss through mutation or epigenetic silencing also depicted
Panel C: Transplant matching diagram showing HLA loci matched for HSCT (HLA-A, -B, -C, -DRB1 for 8/8 match), impact of mismatch on graft-versus-host disease and graft failure, and solid organ considerations with virtual crossmatch algorithm using donor HLA type and recipient antibody profile to predict compatibility
Panel D: MHC-based vaccine strategies showing peptide prediction algorithms identifying HLA-binding epitopes from pathogen or tumor proteins, considerations of population HLA diversity requiring multi-epitope vaccines or pan-HLA-binding epitopes, and personalized cancer vaccines based on individual patient HLA type and tumor mutations
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## Summary
- MHC class I molecules are expressed on all nucleated cells, present endogenous peptides (8-10 aa) to CD8+ T cells, and consist of polymorphic heavy chain plus beta2-microglobulin
- MHC class II molecules are restricted to APCs, present exogenous peptides (13-25 aa) to CD4+ T cells, and consist of polymorphic alpha and beta chains with open-ended groove
- The class I pathway involves proteasome degradation of cytoplasmic proteins, TAP transport into ER, and loading complex assembly with tapasin-mediated peptide editing
- The class II pathway involves endocytic antigen capture, invariant chain blocking and targeting, cathepsin processing, HLA-DM-mediated CLIP removal and peptide exchange
- Professional APCs include dendritic cells (most potent, activate naive T cells), macrophages (receive T cell help), and B cells (BCR-mediated specific capture)
- Cross-presentation by cDC1 dendritic cells loads exogenous antigens onto class I for CTL priming against non-infecting pathogens and tumors
- MHC genetics feature extreme polymorphism, codominant expression, and linkage disequilibrium, with practical implications for transplant matching
- HLA-disease associations include B27-ankylosing spondylitis (strongest), DR3/DR4/DQ2/DQ8-type 1 diabetes, and DQ2/DQ8-celiac disease
- Bare lymphocyte syndrome Type I (TAP deficiency) and Type II (class II transcription defects) demonstrate essential but distinct roles of each pathway
- Viral immune evasion strategies targeting class I presentation are countered by NK cell missing-self recognition
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## Key Terms
| Term | Definition |
|------|------------|
| MHC class I | Molecule presenting endogenous peptides to CD8+ T cells, expressed on all nucleated cells |
| MHC class II | Molecule presenting exogenous peptides to CD4+ T cells, restricted to APCs |
| TAP | Transporter associated with antigen processing; delivers peptides to ER for class I loading |
| Proteasome | Cytoplasmic complex degrading ubiquitinated proteins to peptides for class I presentation |
| Invariant chain | Chaperone blocking class II groove and targeting to endosomes |
| HLA-DM | Non-classical class II that catalyzes CLIP removal and peptide exchange |
| Cross-presentation | Pathway loading exogenous antigens onto class I for CD8+ T cell activation |
| HLA polymorphism | Extreme genetic diversity of MHC genes affecting peptide binding repertoire |
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