Premed · Premed · Immunology
Lecture 13: Antigen Processing and Presentation
Immunology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the MHC class I antigen processing and presentation pathway (endogenous/cytosolic pathway)
- Describe the MHC class II antigen processing and presentation pathway (exogenous/endosomal pathway)
- Explain the concept of cross-presentation and its immunological importance
- Identify the roles of key molecules in antigen processing (proteasome, TAP, invariant chain, HLA-DM)
- Describe alternative antigen presentation pathways (CD1, MR1)
Lecture Content
I. Overview of Antigen Processing and Presentation
T cells cannot recognize free, native antigens the way antibodies can. Instead, they require that protein antigens be degraded into short peptide fragments and displayed on MHC molecules at the cell surface. This fundamental requirement means that two main pathways exist to funnel antigens into the appropriate MHC loading compartments. The MHC class I pathway (also called the endogenous or cytosolic pathway) presents peptides derived from intracellular sources -- such as viral proteins synthesized within an infected cell, tumor antigens, and normal self-proteins -- to CD8+ T cells. The MHC class II pathway (the exogenous or endosomal pathway) presents peptides derived from extracellular proteins that have been phagocytosed or endocytosed, delivering them to CD4+ T cells. A third pathway, cross-presentation, allows exogenous antigens to be loaded onto MHC class I molecules, enabling CD8+ T cell responses against pathogens that do not directly infect antigen-presenting cells.
II. MHC Class I Pathway (Endogenous/Cytosolic Pathway)
The MHC class I pathway presents peptides derived from proteins synthesized within the cell. Because all nucleated cells express MHC class I, every cell in the body has the capacity to display intracellular antigens and alert CD8+ T cells to infection or transformation.
The pathway proceeds through several coordinated steps. First, cytosolic and nuclear proteins are tagged with ubiquitin and degraded by the 26S proteasome. Upon stimulation with IFN-gamma, the constitutive proteasome subunits are replaced by inducible subunits (LMP2, LMP7, and MECL-1), forming the immunoproteasome, which preferentially generates peptides with hydrophobic or basic C-terminal residues that are well suited for binding in the MHC class I groove. A major source of MHC I peptides is defective ribosomal products (DRiPs) -- newly synthesized, misfolded proteins that are rapidly degraded, enabling the immune system to survey what is being translated in real time.
Next, the generated peptides must be transported from the cytosol into the endoplasmic reticulum. This is accomplished by TAP (transporter associated with antigen processing), a TAP1/TAP2 heterodimer embedded in the ER membrane. TAP is an ATP-dependent transporter that preferentially translocates peptides of 8 to 16 amino acids with hydrophobic or basic C-terminal residues. Deficiency of TAP results in bare lymphocyte syndrome type I, characterized by markedly reduced MHC class I surface expression.
Within the ER, the MHC class I alpha chain first folds with assistance from the chaperone calnexin, then associates with beta-2-microglobulin to form a partially folded complex. This complex is then loaded into the peptide-loading complex (PLC), which consists of TAP (providing the peptide source), tapasin (which bridges MHC I to TAP and stabilizes empty MHC I molecules), ERp57 (a thiol oxidoreductase that assists in disulfide bond formation), and calreticulin (a chaperone that stabilizes MHC I). Tapasin functions as a "peptide editor," ensuring that only high-affinity peptides are stably loaded. Meanwhile, ER aminopeptidases ERAP1 and ERAP2 trim the N-terminal extensions of peptides to the optimal length of 8 to 10 amino acids.
Once a stable peptide-MHC I complex forms, it is released from the PLC and transported through the Golgi apparatus to the cell surface, where it is presented to CD8+ T cells. The TCR recognizes the peptide-MHC complex, while the CD8 co-receptor binds the alpha-3 domain to stabilize the interaction.
<image>A step-by-step diagram of the MHC class I antigen processing pathway inside a cell. Panel A: In the cytoplasm, a virus-infected cell produces viral proteins, which are tagged with ubiquitin and fed into the proteasome (barrel-shaped structure). The proteasome degrades proteins into short peptides (8-16 aa). Panel B: Peptides are transported through the TAP1/TAP2 transporter in the ER membrane into the ER lumen. Panel C: Inside the ER, the peptide-loading complex is shown: the MHC class I alpha chain (associated with beta-2-microglobulin) is held by calreticulin and connected to TAP via tapasin, with ERp57 assisting. ERAP1 trims peptide N-termini to optimal length. A high-affinity peptide is loaded into the MHC I groove. Panel D: The stable peptide-MHC I complex travels through the Golgi to the cell surface, where it is displayed to a CD8+ T cell. The TCR contacts the peptide-MHC I complex, and CD8 binds the alpha-3 domain.</image>
III. MHC Class II Pathway (Exogenous/Endosomal Pathway)
The MHC class II pathway presents peptides derived from extracellular proteins that have been internalized by professional antigen-presenting cells. Only professional APCs -- dendritic cells, macrophages, and B cells -- constitutively express MHC class II, reflecting their specialized role in activating CD4+ T helper cells.
The process begins with antigen uptake. Macrophages and DCs internalize antigens through phagocytosis, while B cells use receptor-mediated endocytosis via their BCR, and DCs can also capture antigen through the mannose receptor, DEC-205, or nonspecific macropinocytosis. Once internalized, antigens are delivered through a progressively acidifying endosomal pathway -- from early endosomes (pH 6.0) to late endosomes to lysosomes (pH 4.5). This acidification activates acid-dependent proteases, particularly the cathepsins (B, D, L, and S), which are cysteine and aspartyl proteases that degrade proteins into peptide fragments of 13 to 25 amino acids suitable for MHC class II binding.
Meanwhile, MHC class II alpha and beta chains are synthesized in the ER, where they associate with the invariant chain (Ii, CD74). The invariant chain serves three critical functions: it blocks the peptide-binding groove to prevent premature loading of ER-resident peptides (which are reserved for MHC class I), acts as a chaperone for proper MHC II folding and assembly, and contains targeting signals that direct the MHC II complex from the Golgi to the endosomal and lysosomal compartments. As the invariant chain traverses the endosomal system, cathepsins progressively degrade it, leaving behind a small remnant called CLIP (class II-associated invariant chain peptide) that remains lodged in the groove.
The exchange of CLIP for an antigenic peptide is catalyzed by HLA-DM, a non-classical MHC class II molecule found in the MHC class II compartment (MIIC). HLA-DM acts as a "peptide editor," promoting the binding of high-affinity peptides and the release of low-affinity ones, thereby ensuring that the most stable peptide-MHC II complexes reach the cell surface. In B cells and thymic epithelial cells, HLA-DO modulates this process by inhibiting HLA-DM activity. The stable peptide-MHC II complex is then transported to the cell surface, where it is presented to CD4+ T helper cells, with the CD4 co-receptor binding the beta-2 domain of MHC II.
<image>A step-by-step diagram of the MHC class II antigen processing pathway in an antigen-presenting cell (dendritic cell). Panel A: Extracellular bacteria are phagocytosed or endocytosed into the cell, forming an endosome that acidifies progressively (pH indicated). Cathepsin enzymes degrade the bacterial proteins into peptide fragments. Panel B: In the ER, MHC class II alpha and beta chains assemble with the invariant chain (Ii), which occupies the peptide-binding groove and is shown as a trimer of alpha-beta-Ii complexes. The complex exits the ER via Golgi and is directed to the endosomal compartment (MIIC) by Ii targeting signals. Panel C: In the MIIC, Ii is progressively degraded by cathepsins, leaving only the CLIP fragment in the groove. HLA-DM catalyzes the removal of CLIP and loading of an antigenic peptide (from the degraded bacterium). Panel D: The stable peptide-MHC II complex is transported to the cell surface, where it is recognized by a CD4+ T helper cell. The TCR contacts the peptide-MHC II complex, and CD4 binds the beta-2 domain.</image>
IV. Cross-Presentation
Cross-presentation refers to the presentation of exogenous antigens on MHC class I molecules, a pathway that is essential for generating CD8+ CTL responses against pathogens that do not directly infect antigen-presenting cells -- such as tumors and viruses that infect non-APCs. This pathway is performed primarily by conventional dendritic cells type 1 (cDC1s), identified as CD8alpha+ DCs in mice and BDCA3+/CD141+ DCs in humans.
Two mechanisms have been proposed. In the cytosolic pathway, antigen escapes from the endosome or phagosome into the cytosol, where it is degraded by the proteasome and transported into the ER via TAP, following the standard MHC class I loading pathway. In the vacuolar pathway, antigen is processed by cathepsins within endosomal compartments, and MHC class I molecules are loaded directly in those compartments without TAP involvement. Cross-presentation is critical for antiviral and antitumor immunity, for the cross-priming of naive CD8+ T cells by dendritic cells, and for the effectiveness of many vaccines, where DCs must cross-present vaccine antigens to activate CTL responses.
V. Alternative Antigen Presentation Pathways
Not all immune surveillance depends on classical peptide-MHC interactions. CD1 molecules are non-classical MHC class I-like molecules that present lipid and glycolipid antigens. Five isoforms exist: CD1a, CD1b, and CD1c present microbial lipids -- such as mycobacterial mycolic acids and lipoarabinomannan -- to lipid-specific T cells. CD1d presents lipid antigens to NKT cells (invariant NKT or iNKT cells), with alpha-galactosylceramide (alpha-GalCer) serving as a potent CD1d-restricted NKT cell agonist. Lipid antigens are loaded in endosomal compartments in a manner analogous to the MHC class II pathway.
MR1 (MHC-related protein 1) presents vitamin B metabolites, specifically riboflavin derivatives produced by bacteria and yeast, to MAIT cells (mucosal-associated invariant T cells). MAIT cells are abundant in the blood and mucosal tissues and mount rapid, innate-like responses to bacterial infections.
VI. Immune Evasion of Antigen Presentation
Many pathogens have evolved sophisticated strategies to evade MHC-mediated antigen presentation. Viral evasion of the MHC class I pathway is particularly well characterized. Herpes simplex virus produces ICP47, which blocks TAP and prevents peptide transport into the ER. Cytomegalovirus employs multiple mechanisms: US6 blocks TAP, US2 and US11 redirect MHC class I molecules for proteasomal degradation through a process called dislocation, and US3 retains MHC I in the ER. Adenovirus E3-19K likewise retains MHC I in the ER. HIV Nef downregulates MHC I from the cell surface by redirecting it to lysosomes, and Kaposi's sarcoma herpesvirus proteins K3 and K5 ubiquitinate MHC I, triggering its endocytosis and degradation.
Evasion of the MHC class II pathway also occurs. Mycobacterium tuberculosis inhibits phagosome-lysosome fusion, thereby preventing antigen processing. HIV Nef also downregulates MHC class II, and some viruses produce IL-10 homologs that suppress MHC II expression. Importantly, these evasion strategies create an evolutionary arms race: cells that downregulate MHC class I to escape CTL detection become susceptible to NK cell killing through the missing-self recognition mechanism, maintaining selective pressure against complete MHC loss.
