17.10.03 · mol-cell-bio / immunology

MHC and antigen presentation: class I and II pathways, and T cell activation

stub3 tiersLean: nonepending prereqs

Anchor (Master): Janeway's Immunobiology, 10th ed. (2022), Ch. 5-6

Intuition Beginner

Every cell in your body puts fragments of the proteins it is making on its outer surface, like samples displayed in a shop window. These fragments are small pieces of protein called peptides, and they are held in specialised display molecules called MHC molecules (major histocompatibility complex). Patroling T cells walk past and inspect these displays. If a cell is healthy, the displayed peptides are normal "self" fragments, and T cells ignore them. If a cell has been infected by a virus, some of the displayed peptides come from viral proteins — and T cells recognise these foreign fragments as a sign of danger.

There are two main types of MHC molecule, and they show different kinds of peptides to different kinds of T cells. MHC class I molecules are found on nearly every cell in the body. They display peptides from proteins that were synthesised inside the cell itself — so if a virus hijacks the cell's protein-making machinery, the resulting viral peptides appear on MHC class I. These are inspected by CD8+ T cells (also called cytotoxic T cells or killer T cells), which destroy any cell displaying foreign peptides.

MHC class II molecules are found only on specialised immune cells — dendritic cells, macrophages, and B cells. These cells engulf pathogens from outside, break them down in internal compartments, and load the resulting peptides onto MHC class II. The displayed peptides are then inspected by CD4+ T cells (helper T cells), which do not kill directly but coordinate the broader immune response by releasing signalling molecules called cytokines and activating B cells.

The distinction matters: MHC class I reports on what is happening inside the cell, while MHC class II reports on what the immune cell has swallowed from outside. Together, they give T cells a complete picture of both intracellular and extracellular threats.

Visual Beginner

The diagram shows the two MHC pathways side by side. On the left, the MHC class I pathway: a virus-infected cell degrades viral proteins in the proteasome, transports the resulting peptides into the endoplasmic reticulum (ER) via TAP, loads them onto MHC class I molecules, and displays the peptide-MHC complex on the cell surface for inspection by a CD8+ cytotoxic T cell.

On the right, the MHC class II pathway: a dendritic cell engulfs a bacterium, degrades it in an endosomal compartment, loads the resulting peptides onto MHC class II molecules (which were held in the ER by the invariant chain until reaching the endosome), and displays them for inspection by a CD4+ helper T cell. The key structural difference is visible in the cross-section: MHC class I has a closed binding groove that accommodates shorter peptides (8-10 amino acids), while MHC class II has an open groove that accommodates longer peptides (13-25 amino acids).

Worked example Beginner

Trace what happens when an influenza virus infects an epithelial cell in your airway, from viral protein synthesis to T cell detection.

Step 1: Viral protein production. The virus enters the epithelial cell and hijacks its ribosomes to make viral proteins. Some of these newly made viral proteins are tagged for destruction by the cell's waste-disposal system — a large protein complex called the proteasome.

Step 2: Proteasomal degradation. The proteasome chops the viral proteins into short peptide fragments, typically 8-10 amino acids long. These fragments are small enough to fit into the binding groove of an MHC class I molecule.

Step 3: Transport into the ER. The peptides are moved from the cytosol into the endoplasmic reticulum (ER) by a transport protein called TAP (transporter associated with antigen processing). TAP acts like a ticket gate: it only lets through peptides of the right size range.

Step 4: Loading onto MHC class I. Inside the ER, newly assembled MHC class I molecules wait for a peptide. A chaperone protein called tapasin holds the empty MHC I near TAP, testing peptides as they come through. When a peptide fits snugly into the binding groove, the MHC I molecule changes shape, stabilises, and is released for transport to the cell surface.

Step 5: Surface display. The peptide-loaded MHC class I travels through the Golgi apparatus to the cell surface. Every infected cell now displays viral peptides in its MHC class I "shop windows."

Step 6: T cell recognition. A passing CD8+ cytotoxic T cell whose receptor happens to fit this particular viral peptide recognises it. The T cell binds tightly, becomes activated, and releases toxic proteins (perforin and granzymes) that kill the infected cell before it can produce more virus.

Check your understanding Beginner

Exercise (easy, multiple choice).

Which type of MHC molecule is found on nearly all nucleated cells and displays peptides from proteins synthesised inside the cell?

A. MHC class II B. MHC class I C. Both MHC class I and II equally D. Neither — MHC molecules are only on immune cells

Hint

Think about which MHC pathway reports on intracellular infections such as viruses. Every infected cell needs to be able to signal that it has been compromised.

Answer

B. MHC class I. Nearly all nucleated cells express MHC class I and use it to display peptides derived from intracellular proteins. This allows CD8+ cytotoxic T cells to detect any cell that has been infected by a virus or has become cancerous (producing abnormal internal proteins). MHC class II is restricted to professional antigen-presenting cells (dendritic cells, macrophages, B cells).

Exercise (easy, multiple choice).

What is the role of TAP (transporter associated with antigen processing) in the MHC class I pathway?

A. It transports MHC class I molecules to the cell surface. B. It transports peptides from the cytosol into the endoplasmic reticulum. C. It degrades viral proteins into peptide fragments. D. It helps CD8+ T cells recognise peptide-MHC complexes.

Hint

Peptides are generated in the cytosol by the proteasome, but MHC class I molecules are assembled inside the ER. How do the peptides get from one compartment to the other?

Answer

B. TAP transports peptides from the cytosol into the endoplasmic reticulum, where they can be loaded onto MHC class I molecules. TAP is a member of the ABC transporter family and uses ATP hydrolysis to shuttle peptides of 8-16 amino acids across the ER membrane. Without functional TAP (as in TAP deficiency syndrome), MHC class I molecules remain empty, unstable, and are not displayed on the cell surface — causing susceptibility to chronic viral infections.

Formal definition Intermediate+

The major histocompatibility complex (MHC) is a cluster of highly polymorphic genes encoding cell-surface glycoproteins that bind peptide fragments and present them to T lymphocytes. In humans, the MHC is called HLA (human leukocyte antigen); in mice, H-2. MHC molecules are divided into two classes with distinct structures, expression patterns, peptide sources, and T cell partners.

MHC class I structure and genetics

MHC class I molecules are heterodimers of a polymorphic heavy chain (approximately 45 kDa, encoded by HLA-A, HLA-B, or HLA-C in humans) and the invariant beta-2-microglobulin ( $β_{2}$ m, 12 kDa, encoded on chromosome 15, outside the MHC). The heavy chain has three extracellular domains ( $α_{1}$ , $α_{2}$ , $α_{3}$ ), a transmembrane helix, and a short cytoplasmic tail. The $α_{1}$ and $α_{2}$ domains form the peptide-binding groove — a cleft bounded by two alpha helices resting on a floor of eight antiparallel beta strands. The groove is closed at both ends, constraining bound peptides to 8-10 amino acids (occasionally up to 11). The $α_{3}$ domain and $β_{2}$ m have immunoglobulin-like folds; $α_{3}$ is the binding site for the CD8 co-receptor.

MHC class I genes are the most polymorphic in the human genome. HLA-B alone has over 6,000 alleles. This polymorphism is concentrated in the peptide-binding groove: different alleles bind different sets of peptides with distinct sequence motifs. For example, HLA-A*02:01 prefers peptides with leucine at position 2 and valine at the C-terminus; HLA-B*27:05 prefers arginine at position 2. The peptide-binding specificity arises from pockets (designated A through F) in the groove that accommodate specific peptide side chains.

MHC class I pathway: endogenous antigen processing

The MHC class I pathway monitors the intracellular protein pool and presents peptides derived from cytosolic and nuclear proteins to CD8+ cytotoxic T cells.

Proteasomal degradation. The 26S proteasome is a barrel-shaped protease complex that degrades ubiquitin-tagged proteins into peptides of 3-25 amino acids. In cells exposed to interferon-gamma, three constitutive catalytic subunits ( $β_{1}$ , $β_{2}$ , $β_{5}$ ) are replaced by the immuno-subunits LMP2 ( $β_{1}$ i), MECL-1 ( $β_{2}$ i), and LMP7 ( $β_{5}$ i), forming the immunoproteasome. The immunoproteasome generates peptides with hydrophobic or basic C-termini — the preferred anchor residues for most MHC class I alleles — and degrades them less rapidly, favouring antigen production. The proteasome activator PA28 (11S regulator) further modulates cleavage specificity.

TAP transport. Peptides generated by the proteasome are translocated from the cytosol into the ER lumen by TAP (transporter associated with antigen processing), a heterodimer of TAP1 and TAP2. TAP is an ABC (ATP-binding cassette) transporter that preferentially translocates peptides of 8-16 amino acids with hydrophobic or basic C-terminal residues. Peptides longer than the optimal 8-10 residues can be trimmed in the ER by ERAP1 (endoplasmic reticulum aminopeptidase 1) and ERAP2, which sequentially remove N-terminal residues to generate the correct length for MHC class I binding.

MHC class I assembly and peptide loading. The MHC class I heavy chain is synthesised in the ER and folds with the assistance of the chaperone calnexin and the thiol oxidoreductase ERp57. After $β_{2}$ m association, calnexin is replaced by calreticulin, and the partial MHC I complex enters the peptide-loading complex (PLC), a multi-protein assembly comprising TAP, tapasin, calreticulin, and ERp57. Tapasin bridges the MHC I molecule to TAP, positioning the empty binding groove near the peptide translocation channel. Tapasin edits the peptide repertoire: it retains MHC I molecules that have bound low-affinity peptides, giving them the opportunity to exchange for higher-affinity peptides. This peptide editing function ensures that only stably loaded MHC I molecules are released from the ER.

Surface expression. Stably loaded MHC I-peptide complexes exit the ER, transit the Golgi, and are delivered to the plasma membrane. The half-life of a peptide-loaded MHC I molecule on the cell surface is several hours; empty MHC I molecules are unstable and are retained in the ER for degradation.

MHC class II structure and genetics

MHC class II molecules are heterodimers of an alpha chain (approximately 33 kDa) and a beta chain (approximately 28 kDa), both encoded within the MHC (HLA-DR, HLA-DP, HLA-DQ in humans). Each chain has two extracellular domains: the membrane-distal $α_{1}$ / $β_{1}$ domains form the peptide-binding groove, while the membrane-proximal $α_{2}$ / $β_{2}$ domains have immunoglobulin-like folds. The $β_{2}$ domain is the binding site for the CD4 co-receptor. Unlike MHC class I, the MHC class II groove is open at both ends, allowing bound peptides to extend beyond the groove. This accommodates longer peptides of 13-25 amino acids, with the core 9-mer anchored in the groove and flanking residues protruding from both ends.

MHC class II pathway: exogenous antigen processing

The MHC class II pathway monitors extracellular material taken up by professional antigen-presenting cells (dendritic cells, macrophages, B cells) and presents peptides to CD4+ helper T cells.

Invariant chain and ER trafficking. Newly synthesised MHC class II alpha-beta dimers in the ER are occupied by the invariant chain (Ii, CD74). The invariant chain has three functions: (1) it blocks the peptide-binding groove, preventing premature loading of ER-resident peptides; (2) it directs MHC II transport from the ER through the Golgi to MHC class II compartments (MIICs), late endosomal compartments specialised for antigen processing, via targeting signals in its cytoplasmic tail; and (3) it facilitates MHC II folding and assembly.

Endosomal degradation and CLIP. In the acidic MIIC, proteases (cathepsins S, L, and D) progressively degrade the invariant chain, leaving a short fragment called CLIP (class II-associated invariant chain peptide) occupying the groove. The antigen-presenting cell has simultaneously internalised extracellular material (pathogens, proteins, immune complexes) into endosomes, where the same cathepsins degrade these exogenous proteins into peptide fragments.

HLA-DM and peptide editing. HLA-DM (a non-classical MHC II molecule that does not present peptide to T cells) catalyses the removal of CLIP from the MHC II groove and facilitates loading of antigenic peptides. HLA-DM acts as a peptide editor: it accelerates the dissociation of low-stability peptides (including CLIP) from the MHC II groove, allowing higher-affinity antigenic peptides to bind. This kinetic proofreading mechanism is analogous to the tapasin-mediated editing in the MHC I pathway. HLA-DO (expressed in B cells and thymic epithelial cells) modulates HLA-DM activity, broadening the peptide repertoire presented.

Surface expression. Peptide-loaded MHC class II molecules are transported to the plasma membrane. On the surface, the MHC II-peptide complex is available for inspection by CD4+ T cells. Mature dendritic cells upregulate both MHC class II and co-stimulatory molecules (B7-1/B7-2) upon activation by innate immune signals, providing both signal 1 (peptide-MHC) and signal 2 (co-stimulation) required for naive CD4+ T cell activation.

MHC polymorphism and transplant rejection

The extreme polymorphism of MHC genes has profound consequences for transplantation. Each individual expresses up to six classical MHC class I molecules (two each from HLA-A, HLA-B, HLA-C, one from each parental chromosome) and multiple MHC class II molecules. Allogeneic MHC molecules (from a different individual) are recognised as foreign by 1-10% of the host's T cells — an anomalously high frequency compared to the 1-in-a-million frequency for any given foreign peptide presented by self-MHC. This strong alloreactive response is the molecular basis of transplant rejection.

The high alloreactive precursor frequency arises because allogeneic MHC-peptide complexes can engage T cell receptors through two mechanisms: (1) the TCR recognises the foreign MHC molecule itself (which differs from self-MHC at multiple polymorphic residues in the peptide-binding groove), and (2) the set of peptides bound by the foreign MHC is different from those bound by self-MHC, creating many novel peptide-MHC surfaces for TCR recognition.

Cross-presentation

Cross-presentation is the ability of certain dendritic cells to load exogenous antigens onto MHC class I molecules, allowing CD8+ T cell priming against pathogens that do not directly infect dendritic cells (e.g., tumors, viruses that infect non-immune cell types). Two mechanistic pathways have been proposed:

Cytosolic pathway. Internalised antigens are exported from endosomes into the cytosol, where they enter the standard MHC class I pathway (proteasomal degradation, TAP transport, ER loading).

Vacuolar pathway. Antigens are degraded within endosomal compartments by lysosomal proteases, and the resulting peptides load onto recycling MHC class I molecules within the endosome itself, independently of TAP and the proteasome.

Cross-presenting dendritic cells (often identified by the marker XCR1 in mice, or as CD141+ BDCA-3+ dendritic cells in humans) are specialised for activating CD8+ T cell responses against viruses and tumors.

Counterexamples to common slips

MHC class I only presents viral peptides. MHC class I displays a random sampling of all intracellular proteins being degraded at any given time — the vast majority are normal self-peptides. Only a small fraction of the presented peptides are pathogen-derived during infection. T cells are trained to ignore the self-peptides and respond only to the foreign ones.
MHC class II is only on dendritic cells and macrophages. Under inflammatory conditions (particularly interferon-gamma exposure), many non-immune cell types can be induced to express MHC class II — including endothelial cells, fibroblasts, and epithelial cells. However, these cells generally lack co-stimulatory molecules and may induce T cell anergy rather than activation.
MHC molecules bind whole proteins. MHC molecules bind only peptide fragments of 8-10 amino acids (class I) or 13-25 amino acids (class II). The protein must be proteolytically processed before loading. This is the key difference between T cell recognition (which requires processed peptide fragments) and B cell/antibody recognition (which can bind intact, folded proteins).

Key mechanism Intermediate+

Mechanism: The immunological synapse and T cell activation.

T cell activation requires sustained contact between a T cell and an antigen-presenting cell, organised into a specialised cell-cell junction called the immunological synapse. The synapse forms over 5-30 minutes and has a characteristic concentric structure visible by total internal reflection fluorescence (TIRF) microscopy.

Supramolecular activation clusters (SMACs). The mature synapse consists of three concentric zones:

cSMAC (central SMAC). The innermost zone, enriched in TCR-peptide complexes and the signalling protein PKC-theta. The cSMAC functions as a site for sustained TCR signalling and TCR endocytosis (downregulation after activation). Only a small fraction of engaged TCRs are in the cSMAC at any given time; the majority are in the periphery.

pSMAC (peripheral SMAC). A ring surrounding the cSMAC, enriched in the adhesion molecules LFA-1 (lymphocyte function-associated antigen 1, also called CD11a/CD18) on the T cell binding ICAM-1 (intercellular adhesion molecule 1) on the APC. The pSMAC provides the mechanical stability that maintains cell-cell contact for hours. LFA-1 undergoes a conformational change from low-affinity to high-affinity binding upon TCR signalling (inside-out signalling), strengthening adhesion specifically when antigen is present.

dSMAC (distal SMAC). The outermost zone, enriched in the large glycoprotein CD43 and CD45 (the tyrosine phosphatase required for Lck activation). CD43 is actively excluded from the synapse centre by the actin cytoskeleton, creating a physical barrier that prevents non-specific interactions.

Kinetic proofreading and serial triggering. The synapse implements two quantitative features of T cell activation:

Kinetic proofreading. A T cell receptor must be engaged by peptide for a minimum dwell time (estimated at 2-5 seconds) to trigger downstream signalling. Short-lived interactions dissociate before the signalling cascade is fully initiated. This time threshold acts as a kinetic proofreading mechanism that discriminates between high-affinity (agonist) and low-affinity (self) peptide complexes based on their dissociation rates ( $k_{off}$ ).

Serial triggering. A single peptide complex can engage and trigger multiple TCRs sequentially. One pMHC binds a TCR, triggers signalling, the TCR is internalised, and the same pMHC is free to engage another TCR. Estimates suggest that a single pMHC can serially trigger 10-200 TCRs, amplifying the signal from a small number of antigenic pMHC complexes into a robust activation response.

Signal integration. Full T cell activation requires 4-6 hours of sustained signalling. The number of triggered TCRs, the duration of contact, and the strength of co-stimulation (CD28-B7) are integrated to determine the outcome: activation and proliferation, anergy (functional inactivation), or deletion (apoptosis). This is why naive T cells are activated only by mature dendritic cells in lymph nodes — the dendritic cell provides the stable, prolonged contact and co-stimulatory signals necessary for the signal integration threshold to be reached.

Exercises Intermediate+

Exercise 1 (easy, multiple choice).

Which of the following correctly pairs the MHC class with its T cell partner?

A. MHC class I — CD4+ helper T cells B. MHC class II — CD8+ cytotoxic T cells C. MHC class I — CD8+ cytotoxic T cells D. Both classes activate both T cell types equally

Hint

The CD8 co-receptor binds the $α_{3}$ domain of MHC class I. The CD4 co-receptor binds the $β_{2}$ domain of MHC class II. Which co-receptor goes with which T cell type?

Answer

C. MHC class I presents peptides to CD8+ cytotoxic T cells; MHC class II presents peptides to CD4+ helper T cells. The co-receptors reinforce this specificity: CD8 binds a conserved site on MHC class I, and CD4 binds a conserved site on MHC class II. During T cell development in the thymus, positive selection ensures that T cells whose TCRs recognise MHC class I become CD8+, while those recognising MHC class II become CD4+.

Exercise 2 (easy, multiple choice).

What is the function of the invariant chain (Ii) in the MHC class II pathway?

A. It degrades antigens in the endosome. B. It blocks the MHC II peptide-binding groove in the ER and directs MHC II to endosomal compartments. C. It transports peptides from the cytosol into the ER. D. It acts as a co-stimulatory molecule on the cell surface.

Hint

MHC class II molecules are assembled in the ER, but they must load peptides derived from extracellular proteins in endosomal compartments. How does the cell prevent premature peptide loading in the ER?

Answer

B. The invariant chain (Ii) serves three roles: it blocks the MHC II peptide-binding groove to prevent loading of ER-resident peptides, it contains targeting signals that direct MHC II from the ER through the Golgi to the MIIC (MHC class II compartment), and it assists in MHC II folding. In the MIIC, proteases degrade Ii to leave CLIP in the groove, and HLA-DM then exchanges CLIP for antigenic peptides.

Exercise 3 (medium, symbolic).

Compare the MHC class I and MHC class II pathways in terms of: (a) source of peptides, (b) site of peptide loading, (c) peptide transport mechanism, (d) peptide length, (e) T cell type activated. Present your answer as a side-by-side comparison.

Hint

MHC class I monitors the inside of the cell; MHC class II monitors what has been taken in from outside. Consider where in the cell each pathway loads peptides and how the peptides reach that location.

Answer

Feature	MHC Class I	MHC Class II
(a) Peptide source	Cytosolic and nuclear proteins (endogenous)	Extracellular proteins taken up by endocytosis (exogenous)
(b) Site of loading	Endoplasmic reticulum	MIIC (late endosomal compartment)
(c) Transport	TAP (ABC transporter) moves peptides from cytosol to ER	No specialised transporter needed; antigens are degraded in the same compartment where loading occurs
(d) Peptide length	8-10 amino acids (closed groove)	13-25 amino acids (open groove)
(e) T cell type	CD8+ cytotoxic T cells	CD4+ helper T cells

Exercise 4 (medium, symbolic).

Explain the role of tapasin in the MHC class I pathway. What is peptide editing, and why does it matter for immune surveillance?

Hint

Not all peptides that enter the ER have equal affinity for MHC class I. Tapasin acts as a quality control checkpoint. Consider what would happen if MHC I molecules were released with suboptimal peptides.

Answer

Tapasin is a chaperone that bridges MHC class I molecules to TAP in the peptide-loading complex. It performs peptide editing by retaining MHC I molecules that have bound low-affinity peptides, giving them the opportunity to exchange these for higher-affinity peptides. MHC I molecules that have loaded optimal peptides undergo a stabilising conformational change and are released from tapasin for transport to the cell surface.

Peptide editing matters because the stability of the peptide-MHC complex on the cell surface determines how long it is available for T cell inspection. A suboptimal peptide that dissociates quickly leaves an empty, unstable MHC I that is internalised and degraded — the antigenic signal is lost. By selecting for high-affinity peptides, tapasin maximises the density and duration of antigenic peptide display, improving the sensitivity of CD8+ T cell surveillance. Cells lacking tapasin show reduced surface MHC class I expression and impaired antigen presentation.

Exercise 5 (medium, symbolic).

What is cross-presentation, and why is it necessary for CD8+ T cell responses against tumors? Why cannot standard MHC class I presentation by dendritic cells alone prime these responses?

Hint

Tumor cells are not professional antigen-presenting cells. They express MHC class I with tumor peptides, but they lack co-stimulatory molecules. Consider what happens when a CD8+ T cell encounters a tumor cell without co-stimulation.

Answer

Cross-presentation is the process by which dendritic cells take up exogenous antigens (from tumor cells, dying infected cells, or immune complexes) and load them onto MHC class I molecules for presentation to CD8+ T cells.

It is necessary because naive CD8+ T cell activation requires both signal 1 (peptide-MHC I) and signal 2 (co-stimulation, specifically B7 molecules). Tumor cells express MHC class I displaying tumor peptides, but they do not express B7 co-stimulatory molecules. A naive CD8+ T cell that encounters a tumor cell directly receives signal 1 without signal 2, leading to anergy (functional inactivation) rather than activation.

Professional dendritic cells that have cross-presented tumor antigens provide both signals: MHC class I loaded with tumor peptides (signal 1) and B7 molecules upregulated by innate immune activation (signal 2). This allows proper priming and expansion of tumor-specific CD8+ T cells, which can then migrate to the tumor and kill tumor cells without requiring co-stimulation.

Exercise 6 (hard, symbolic).

Describe the immunological synapse structure (cSMAC, pSMAC, dSMAC) and explain how kinetic proofreading and serial triggering work together to achieve sensitive yet specific T cell activation. Why is sustained signalling (4-6 hours) required?

Hint

A single peptide-MHC complex on an APC surface must activate a T cell that has thousands of TCRs. The synapse concentrates signalling molecules and uses time as a discrimination mechanism. Consider how the half-life of TCR-pMHC binding determines whether downstream signalling cascades have enough time to complete.

Answer

The immunological synapse is a concentrically organised cell-cell contact:

cSMAC (central): Accumulates TCR-peptide complexes. Serves as the primary signalling zone and a site for TCR endocytosis and degradation (signal attenuation).

pSMAC (peripheral): Ring of LFA-1/ICAM-1 adhesion. LFA-1 undergoes affinity maturation (inside-out signalling triggered by TCR engagement), strengthening adhesion specifically when antigen is present. This mechanical stability maintains contact for the hours needed for full activation.

dSMAC (distal): Enriched in CD43 and CD45. Actin-dependent exclusion of CD43 creates a barrier that prevents non-specific adhesion molecules from interfering with synapse formation.

Kinetic proofreading requires that a TCR remain engaged by pMHC for a minimum dwell time (approximately 2-5 seconds) before downstream signalling proceeds. Self-peptides have fast $k_{off}$ rates (short dwell times) that fall below this threshold, while foreign agonist peptides have slower $k_{off}$ rates that exceed it. This provides specificity: only peptides with sufficiently slow off-rates trigger signalling.

Serial triggering amplifies sensitivity: one pMHC sequentially engages and triggers 10-200 TCRs. Even a dendritic cell displaying as few as 10-100 copies of a specific pMHC can trigger thousands of TCRs on a single T cell over several hours.

Together, kinetic proofreading ensures specificity (fast-off-rate self-peptides are rejected) while serial triggering ensures sensitivity (few antigenic pMHC complexes suffice for activation). The 4-6 hour requirement for sustained signalling provides an additional integration threshold: transient encounters (which might result from low-level cross-reactivity with self-peptides) do not reach the cumulative number of triggered TCRs needed for full activation. Only genuine, sustained antigen presentation by a mature dendritic cell passes all three thresholds.

MHC peptide-binding motifs, immunoproteasome, and immunopeptidomics Master

MHC peptide-binding motifs

Each MHC allele has a characteristic peptide-binding motif — a pattern of preferred amino acids at specific positions within the bound peptide. These motifs arise from the architecture of the peptide-binding groove, which contains a series of pockets (designated A through F) that accommodate the side chains of the peptide at positions P1, P2, P3, and so on.

For MHC class I, the binding motif is defined by two or three anchor residues whose side chains fit into deep pockets in the groove. The B pocket accommodates the side chain at P2; the F pocket accommodates the C-terminal residue. For example:

HLA-A*02:01. Prefers leucine or methionine at P2 (B pocket) and valine, leucine, or alanine at the C-terminus (F pocket). Typical 9-mer motif: x-L/M-x-x-x-x-x-x-V/L/A.

HLA-B*27:05. Requires arginine at P2 (deep, negatively charged B pocket) and prefers hydrophobic or basic residues at the C-terminus. Typical 9-mer motif: x-R-x-x-x-x-x-x-x (K/R/H/Y).

HLA-B*08:01. Prefers basic residues at P2 and P3, with a hydrophobic anchor at the C-terminus.

For MHC class II, the binding motif is less strict because the open groove accommodates longer peptides. The core 9-mer binding register has anchor positions at P1, P4, P6, and P9, with pockets in the groove that accommodate specific side chains. The binding motif for a given MHC II allele is often described as a pseudosequence or a matrix of position-specific scoring values rather than simple anchor residues.

The practical consequence: the set of peptides presented by any individual is determined by their MHC genotype. Two individuals with different HLA alleles present different peptide sets from the same pathogen, leading to different T cell responses. This HLA heterozygosity at the population level is maintained by balancing selection — individuals heterozygous at HLA loci can present a broader repertoire of pathogen-derived peptides and have a fitness advantage, as demonstrated by studies of HIV progression (HLA-B heterozygosity correlates with delayed AIDS onset).

Immunoproteasome

Cells exposed to interferon-gamma (produced by NK cells and Th1 cells during infection) replace the three constitutive catalytic subunits of the proteasome with immuno-subunits: LMP2 ( $β_{1}$ i, encoded by PSMB9 in the MHC), MECL-1 ( $β_{2}$ i, encoded by PSMB10), and LMP7 ( $β_{5}$ i, encoded by PSMB8 in the MHC). The resulting immunoproteasome has altered cleavage specificity:

Increased chymotrypsin-like activity (cleavage after hydrophobic residues), producing peptides with hydrophobic C-termini preferred by most MHC class I alleles.
Decreased caspase-like activity (reduced cleavage after acidic residues), reducing production of peptides that are poor fits for MHC I.
Faster processing of ubiquitinated substrates, increasing the rate of antigen production.

The immunoproteasome also has distinct roles beyond antigen processing: it is constitutively expressed in professional antigen-presenting cells, and immunoproteasome deficiency in mice causes defects in T cell responses and increased susceptibility to infections. The immunoproteasome is a therapeutic target: selective inhibitors (such as ONX-0914, which preferentially inhibits LMP7) can modulate autoimmune and inflammatory responses by reducing the generation of autoantigenic peptides.

ERAP trimming

ERAP1 (endoplasmic reticulum aminopeptidase 1) and ERAP2 trim N-extended peptides in the ER to the optimal length for MHC class I binding. ERAP1 removes N-terminal residues from peptides of 9-16 amino acids, but has minimal activity on peptides of 8 residues or shorter — this length selectivity ensures that peptides are trimmed to, but not beyond, the optimal MHC I binding length. ERAP1 also trims peptides that have already been loaded onto MHC I if the N-terminus extends beyond the groove.

ERAP1 polymorphisms are associated with autoimmune diseases. The K528 variant (rs30187, associated with ankylosing spondylitis, psoriasis, and Birdshot chorioretinopathy) has reduced trimming activity, potentially leading to altered peptide repertoires that include more N-extended peptides. The resulting changes in the MHC I peptidome may alter CD8+ T cell selection in the thymus or peripheral T cell responses, predisposing to autoimmunity.

HLA-E, HLA-G, and NK cell checkpoints

Non-classical MHC molecules (HLA-E, HLA-F, HLA-G) have limited polymorphism and serve specialised regulatory functions.

HLA-E binds peptides derived from the signal sequences of classical MHC class I molecules (predominantly VMAPRTLLL from HLA-A, -B, and -C leader sequences). Peptide-loaded HLA-E is recognised by the inhibitory receptor NKG2A/CD94 on NK cells and some CD8+ T cells. When a cell has normal MHC class I expression, HLA-E displays the leader peptide and engages NKG2A, delivering an inhibitory signal that prevents NK cell killing. When MHC class I is downregulated (as in viral infection or cancer), HLA-E lacks its canonical peptide and is destabilised, removing the NKG2A inhibitory signal and allowing NK cell activation.

This mechanism makes HLA-E/NKG2A an immune checkpoint analogous to PD-1/PD-L1. Monalizumab (anti-NKG2A antibody) blocks this checkpoint and is in clinical trials for cancer immunotherapy, particularly in combination with anti-PD-1 antibodies. Tumors that downregulate classical MHC I to escape CD8+ T cells become vulnerable to NK cells when NKG2A is blocked.

HLA-G is expressed predominantly at the maternal-fetal interface (extravillous trophoblast cells) and binds the inhibitory receptors ILT2 (LILRB1) and ILT4 (LILRB2) on NK cells, T cells, and macrophages. HLA-G mediates immune tolerance at the maternal-fetal interface, protecting the semi-allogeneic fetus from maternal immune attack. Tumors can exploit this pathway by upregulating HLA-G expression.

MHC tetramer technology

MHC tetramers are reagents used to detect and quantify antigen-specific T cells by flow cytometry. A tetramer consists of four recombinant MHC molecules, each loaded with the same peptide, multimerised on a streptavidin backbone conjugated to a fluorochrome. The principle: a single pMHC monomer binds TCR with low affinity (too weak for reliable staining), but four pMHC molecules binding simultaneously to multiple TCRs on the T cell surface provide sufficient avidity for stable, detectable binding.

MHC tetramers have enabled direct enumeration of antigen-specific T cells during infections, vaccinations, and autoimmune diseases. Key findings include: HIV-specific CD8+ T cells can reach 1-10% of all CD8+ T cells during acute infection; tumor-infiltrating lymphocytes are enriched for tumor-specific T cells that are often functionally exhausted; and the naive precursor frequency for a given antigen-specific T cell is approximately 1 in $1 0^{5}$ to $1 0^{6}$ CD8+ T cells.

Transplant immunology

Hyperacute rejection (minutes to hours) is mediated by pre-existing anti-HLA antibodies in the recipient (from prior transfusion, pregnancy, or transplantation) that bind donor endothelial cells, activate complement, and cause immediate graft thrombosis. Prevented by crossmatch testing (recipient serum vs. donor lymphocytes) before transplantation.

Acute cellular rejection (days to weeks) is mediated by alloreactive CD8+ T cells that recognise donor MHC class I (direct allorecognition — recipient T cells respond to intact donor MHC on donor APCs) and CD4+ T cells that respond to donor MHC class II. Treatment: high-dose corticosteroids, anti-thymocyte globulin, or calcineurin inhibitors (cyclosporine, tacrolimus — which block calcineurin-dependent NFAT activation and IL-2 production, suppressing T cell activation).

Chronic rejection (months to years) involves both immune and non-immune mechanisms: chronic T cell and antibody-mediated injury to graft vasculature leads to transplant vasculopathy — progressive intimal thickening of graft arteries that eventually occludes the lumen. This is the leading cause of long-term graft loss and is poorly controlled by current immunosuppression.

HLA matching between donor and recipient reduces the strength of the alloreactive response. The most important loci for kidney transplantation are HLA-A, HLA-B, and HLA-DR (the "six-antigen match"). Each mismatched antigen increases the risk of acute rejection and reduces long-term graft survival. For haematopoietic stem cell transplantation, high-resolution HLA matching (4-digit allele level) at HLA-A, -B, -C, -DRB1, and -DQB1 is required to minimise graft-versus-host disease.

Autoimmune disease associations

Certain HLA alleles are strongly associated with autoimmune diseases, indicating that the MHC peptide-binding repertoire influences self-tolerance:

HLA-B*27 and ankylosing spondylitis. The relative risk for HLA-B*27-positive individuals is approximately 80-fold (the strongest HLA-disease association). Proposed mechanisms include: (a) presentation of arthritogenic peptides from joint bacteria (Klebsiella, Salmonella) that cross-react with self-peptides; (b) misfolding of HLA-B*27 heavy chains in the ER, triggering the unfolded protein response and inflammatory signalling; (c) surface expression of HLA-B*27 free heavy chains (without $β_{2}$ m) that activate NK cells via KIR receptors.

HLA-DR4 (DRB1*04:01) and rheumatoid arthritis. The "shared epitope" (a specific amino acid sequence QKRAA at positions 70-74 of the DR $β$ 1 chain) is present in multiple RA-associated HLA-DR alleles. The shared epitope may influence peptide selection during thymic development, allowing escape of autoreactive CD4+ T cells that target joint antigens (citrullinated vimentin, fibrinogen, and collagen).

HLA-DQ2/DQ8 and type 1 diabetes. HLA-DQ2 (DQA1*05:01/DQB1*02:01) and HLA-DQ8 (DQA1*03:01/DQB1*03:02) are present in over 90% of type 1 diabetes patients. These alleles present peptides from pancreatic beta-cell proteins (insulin B chain, GAD65, IA-2) to CD4+ T cells, initiating the autoimmune destruction of insulin-producing cells.

Cancer immune evasion via MHC downregulation

Tumors employ multiple strategies to evade MHC-restricted T cell recognition:

MHC class I downregulation. Loss of MHC class I surface expression is observed in 15-90% of human tumors (depending on tumor type) and occurs through multiple mechanisms: loss of $β_{2}$ m expression (preventing MHC I assembly, observed in melanoma and Hodgkin lymphoma), epigenetic silencing of HLA genes, downregulation of TAP1/TAP2, and loss of the immunoproteasome subunits. Tumors with complete MHC I loss are invisible to CD8+ T cells but become vulnerable to NK cells (which detect "missing self").

Selective HLA loss. Some tumors lose only specific HLA alleles (partial loss), maintaining enough MHC I expression to inhibit NK cells while eliminating the specific alleles that present immunodominant tumor antigens. This is particularly common in tumors with known immunodominant epitopes restricted to a single HLA allele.

PD-L1 upregulation. Tumors frequently upregulate PD-L1 (binds PD-1 on T cells, delivering an inhibitory signal that dampens TCR signalling). PD-L1 expression is induced by interferon-gamma produced by tumour-infiltrating T cells (adaptive immune resistance) and by oncogenic signalling pathways (constitutive expression). Anti-PD-1/PD-L1 antibodies restore T cell function but are effective only when tumor antigens are presented — tumors with complete MHC I loss do not respond to checkpoint immunotherapy.

Immunopeptidomics

Immunopeptidomics is the comprehensive identification of peptides bound to MHC molecules using mass spectrometry. The approach involves immunoaffinity purification of MHC molecules (using pan-HLA class I antibodies such as W6/32 or allele-specific antibodies), acid elution of bound peptides, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) sequencing of the eluted peptides.

Key findings from immunopeptidomics:

A single cell displays approximately $1 0^{4}$ to $1 0^{5}$ distinct peptides on MHC class I at any given time, drawn from a subset of the proteome enriched for rapidly turned-over proteins and defective ribosomal products (DRiPs).
The MHC I peptidome is dominated by peptides from abundant cytosolic proteins (ribosomal proteins, chaperones, metabolic enzymes), with pathogen-derived peptides representing a small minority even during active infection.
Tumor immunopeptidomics has identified neoantigens — peptides derived from tumour-specific mutations that are presented on MHC I and recognised by T cells. Neoantigen burden (the number of predicted neoantigens based on tumour exome sequencing and HLA binding predictions) correlates with response to checkpoint immunotherapy across multiple tumour types.
Post-translational modifications (phosphorylation, citrullination, glycosylation) generate modified peptides that can be presented on MHC and trigger autoimmune responses: citrullinated peptides on HLA-DR4 in rheumatoid arthritis, phosphorylated peptides on HLA-B in some cancers.

Connections Master

Innate immunity 17.10.01. MHC class II expression and co-stimulatory molecule upregulation on dendritic cells are driven by innate immune signals (TLR engagement, inflammatory cytokines). Without innate immune activation, dendritic cells present antigen without co-stimulation, inducing T cell anergy rather than activation. Interferon-gamma (produced by NK cells and Th1 cells) induces immunoproteasome formation and upregulates MHC class I expression, linking innate immune detection to enhanced antigen presentation.
Adaptive immunity 17.10.02 pending. MHC-restricted antigen presentation is the mechanism by which T cells are activated during adaptive immunity. The two-signal model of T cell activation (signal 1: peptide-MHC; signal 2: co-stimulation) connects antigen processing to clonal selection — only T cells whose TCRs match the presented peptide are activated and undergo clonal expansion.
Protein structure 17.01.02 pending. The MHC peptide-binding groove is a structural feature whose shape (closed vs. open ends, pocket geometry) determines peptide length and sequence specificity. The polymorphic residues that line the groove affect peptide binding through direct molecular interactions (hydrogen bonds, van der Waals contacts, electrostatic complementarity). Understanding MHC specificity requires the same structural reasoning introduced in the protein structure unit.
Membrane transport 17.02.02. TAP is an ABC transporter that uses ATP hydrolysis to move peptides across the ER membrane against a concentration gradient. The mechanism (alternating access model, ATP-driven conformational change) is the same as for other ABC transporters discussed in the membrane transport unit.
ER and Golgi trafficking 17.03.04 pending. Both MHC class I and class II molecules are assembled in the ER and transit the secretory pathway to the cell surface. MHC class II trafficking to the MIIC involves targeting signals in the invariant chain cytoplasmic tail and diversion from the default secretory pathway. The quality control mechanisms in the ER (chaperone-assisted folding, retention of incompletely assembled complexes) regulate MHC assembly and surface expression.
Cell signaling 17.07.01. The TCR signalling cascade downstream of peptide-MHC recognition (Lck, ZAP-70, LAT, PLC-gamma1, NFAT) uses the same kinase cascade architecture as RTK and GPCR signalling. The immunological synapse is a spatially organised signalling platform analogous to signalling microdomains in other receptor systems.
DNA repair and V(D)J recombination 17.06.02 pending. The MHC locus is the most polymorphic region in the human genome, maintained by balancing selection. The HLA genes include the immunoproteasome subunits LMP2 and LMP7 and the TAP genes within the MHC itself, reflecting co-evolution of antigen processing and presentation machinery.

Historical notes Master

The discovery of MHC restriction by Rolf Zinkernagel and Peter Doherty in 1974 is one of the most consequential experiments in immunology. Working at the John Curtin School of Medical Research in Canberra, they studied cytotoxic T cell responses to lymphocytic choriomeningitis virus (LCMV) in mice. They found that cytotoxic T cells from an LCMV-infected mouse of strain A would kill LCMV-infected cells from the same strain (A) but not LCMV-infected cells from a different strain (B), even though both were infected with the same virus. The T cells were "restricted" to recognising viral antigen only when presented by the host's own MHC molecules. This result, published in Nature 248 (1974) 701-702, established that T cells recognise a composite of peptide and MHC — not antigen alone — and earned Zinkernagel and Doherty the 1996 Nobel Prize in Physiology or Medicine.

The MHC itself was discovered decades earlier through tumour transplantation experiments in mice. George Snell at the Jackson Laboratory in the 1940s-1950s identified the H-2 locus as the major determinant of graft acceptance or rejection between mouse strains. Jean Dausset discovered the human equivalent (HLA system) in 1958 by identifying leukocyte-agglutinating antibodies in multiply-transfused patients. Baruj Benacerraf demonstrated that immune response genes controlling antibody production mapped to the MHC in guinea pigs. Snell, Dausset, and Benacerraf shared the 1980 Nobel Prize for their work on the MHC and immunogenetics.

The structure of MHC class I was solved by Pamela Bjorkman and Don Wiley in 1987 (Nature 329, 506-512), revealing the peptide-binding groove as a cleft between two alpha helices on a beta-sheet floor. The groove contained electron density corresponding to an unknown mixture of bound peptides — the first direct structural evidence that MHC molecules are peptide receptors. The MHC class II structure followed in 1993 (Brown et al., Nature 364, 33-39), showing the open-ended groove that accommodates longer peptides.

The MHC class I antigen processing pathway was elucidated in the late 1980s and 1990s through a combination of genetic and biochemical approaches. The TAP transporter was identified in 1990 by sequencing the genes between LMP2 and LMP7 in the MHC; mutations in TAP caused MHC class I deficiency. Tapasin was discovered in 1997 by Peter Cresswell's laboratory. The immunoproteasome was characterised in the mid-1990s, with the demonstration that interferon-gamma induces replacement of constitutive proteasome subunits with immuno-subunits.

The immunological synapse was visualised for the first time in 1998 by Grakoui et al. (Science 285, 221-227) using supported lipid bilayers containing ICAM-1 and peptide-MHC to mimic an APC surface. Time-lapse imaging revealed the centripetal reorganisation of TCR and adhesion molecules into the mature cSMAC-pSMAC pattern. The kinetic proofreading model was proposed by McKeithan (1995, PNAS 92, 5042-5046), and serial triggering was demonstrated by Valitutti et al. (1995, Nature 375, 148-151), who showed that a small number of pMHC complexes could trigger downregulation of a large number of TCRs.

Cross-presentation was first demonstrated by Michael Bevan in 1976 (J. Exp. Med. 143, 1283-1288), who showed that CD8+ T cells could be primed by antigens from cells that did not share MHC type with the T cell — indicating that the antigen had been processed and re-presented by host antigen-presenting cells. The molecular mechanisms (cytosolic vs. vacuolar pathways) remain actively investigated.

Immunopeptidomics emerged in the 2000s with the application of high-sensitivity mass spectrometry to MHC-bound peptides. Donald Hunt's laboratory at the University of Virginia pioneered the sequencing of MHC-bound peptides by LC-MS/MS, and the field has expanded with improvements in mass spectrometry sensitivity and computational peptide identification. Tumor neoantigen identification by immunopeptidomics has become a cornerstone of personalised cancer immunotherapy, with neoantigen vaccines entering clinical trials.

Bibliography Master

Zinkernagel, R. M. & Doherty, P. C. — Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiaillogeneic system, Nature 248 (1974) 701-702.
Bjorkman, P. J. et al. — Structure of the human class I histocompatibility antigen, HLA-A2, Nature 329 (1987) 506-512.
Brown, J. H. et al. — Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1, Nature 364 (1993) 33-39.
Grakoui, A. et al. — The immunological synapse: a molecular machine controlling T cell activation, Science 285 (1999) 221-227.
McKeithan, T. W. — Kinetic proofreading in T-cell receptor signal transduction, Proc. Natl. Acad. Sci. USA 92 (1995) 5042-5046.
Valitutti, S. et al. — Serial triggering of many T-cell receptors by a few peptide-MHC complexes, Nature 375 (1995) 148-151.
Bevan, M. J. — Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay, J. Exp. Med. 143 (1976) 1283-1288.
York, I. A., Chang, S. C., Saric, T. et al. — The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8-9 residues, Nat. Immunol. 3 (2002) 1177-1184.
Alberts, B. et al. — Molecular Biology of the Cell, 7th ed. (Garland Science, 2022), Ch. 24 The Adaptive Immune System.
Murphy, K. & Weaver, C. — Janeway's Immunobiology, 10th ed. (Garland Science, 2022), Ch. 5-6.
Lodish, H. et al. — Molecular Cell Biology, 9th ed. (W. H. Freeman, 2021), Ch. 24.
Snell, G. D. — The H-2 locus of the mouse: observations and theory concerning its role in transplantation and the immune response, Transplant. Proc. 3 (1971) 1131-1142.
Dausset, J. — Iso-leuko-anticorps, Acta Haematol. 20 (1958) 156-166.
Bassani-Sternberg, M. et al. — Deciphering HLA-I motifs across HLA peptidomes improves neo-antigen predictions and identifies alkylation residues underlying HLA loss, Mol. Cell. Proteomics 16 (2017) 1818-1832.

Prerequisites

17.10.01 pending
17.10.02 pending

Used in

17.10.04

Tier anchors

beginner: Alberts et al., Molecular Biology of the Cell, 7th ed. (2022), Ch. 24
intermediate: Lodish et al., Molecular Cell Biology, 9th ed. (2021), Ch. 24
master: Janeway's Immunobiology, 10th ed. (2022), Ch. 5-6

References

Alberts et al. — Molecular Biology of the Cell, 7th ed. (Garland Science, 2022) · Ch. 24 The Adaptive Immune System — MHC and Antigen Presentation
Lodish et al. — Molecular Cell Biology, 9th ed. (W. H. Freeman, 2021) · Ch. 24 MHC Molecules and Antigen Presentation
Janeway's Immunobiology, 10th ed. (Garland Science, 2022) · Ch. 5-6 Antigen Processing and Presentation
Zinkernagel, R. M. & Doherty, P. C. — Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis · Nature 248 (1974) 701-702; MHC restriction discovery

Estimated time

beginner: 15m
intermediate: 30m
master: 45m