MHC, Antigen Presentation, HLA, and Rheumatic Diseases - Detailed Notes

Part 1: Major Histocompatibility Complex (MHC) - Structure and Organization

Overview

"The HLA locus consists of more than 200 genes that encode cell surface proteins involved in presenting intracellular antigens to the immune system." - Tietz Textbook of Laboratory Medicine, 7th Edition

Molecular Structure

"Unlike class I, the class II groove is open at both ends and therefore can bind longer peptides, typically about 15-20 amino acids long. However, just as for class I, it is a stretch of about 9 amino acids that are directly involved in the interaction." - Roitt's Essential Immunology

HLA Polymorphism

• Protects species from microbial molecular mimicry
• Maintains diversity of antigenic recognition in the lymphoid system
• Ensures heterozygosity (hybrid vigor)

Part 2: Antigen Processing and Presentation Pathways

Figure: (a) Endogenous pathway - cytoplasmic proteins are proteasomally degraded, peptides transported via TAP into the ER, loaded onto MHC class I, and presented to CD8+ T cells. (b) Exogenous pathway - extracellular proteins are endocytosed, degraded in lysosomes, loaded onto MHC class II (after CLIP displacement), and presented to CD4+ T cells. (Source: Roitt's Essential Immunology)

A. Endogenous Antigen Processing Pathway (MHC Class I)

1. Protein ubiquitination: Intracytoplasmic proteins (viral, tumor, normal self-proteins) are tagged with multiple ubiquitin molecules in an ATP-dependent reaction. Misfolded/disassembled proteins from the ER (via ER-associated protein degradation, ERAD) and mitochondrial-derived proteins can also enter this pathway.
2. Proteasomal degradation: Polyubiquitinated proteins are directed to the 26S proteasome, which cleaves them into peptide fragments. The standard (housekeeping) proteasome generates mostly fragments that are too short or too long for MHC class I binding.
3. Immunoproteasome: IFN-gamma induces replacement of three standard catalytic subunits (beta-1, beta-2, beta-5) with immunoproteasome-specific subunits (beta-1i/LMP2, beta-2i/MECL-1, beta-5i/LMP7), encoded within the MHC. This "immunoproteasome" has modified cleavage specificity that greatly increases the proportion of 8-10 amino acid peptides optimal for MHC class I binding.
4. TAP transport: Peptide fragments are translocated from the cytosol into the endoplasmic reticulum (ER) lumen by the TAP1/TAP2 heterodimer (Transporters associated with Antigen Processing), ABC transporter proteins also encoded within the MHC. Peptides slightly too long undergo further trimming by ER-resident aminopeptidases ERAP-1 and ERAP-2 in humans (ERAAP in mice). Notably, ERAP1 variants are associated with ankylosing spondylitis, highlighting the role of peptide trimming in disease pathogenesis.
5. MHC class I assembly: In the ER, newly synthesized MHC class I heavy chains associate with beta-2 microglobulin and the chaperone calnexin (prior to beta-2m association) and then calreticulin + ERp57 (after). The peptide loading complex (calreticulin, ERp57, tapasin) stabilizes the empty groove and facilitates optimal peptide loading. Tapasin bridges TAP and the MHC class I molecule.
6. Surface transport: The stable MHC class I-peptide complex is transported via the trans-Golgi network to the cell surface.
7. T-cell recognition: CD8+ cytotoxic T lymphocytes recognize the MHC class I-peptide complex via their TCR. The peptide occupies the groove with anchor residues pointing into the groove pockets and a minority of side-chains (17-27% solvent accessibility) pointing toward the TCR.

B. Exogenous Antigen Processing Pathway (MHC Class II)

1. Endocytosis/Phagocytosis: Extracellular antigens (bacteria, soluble proteins, immune complexes) are internalized into endosomes/phagosomes.
2. Endosomal/lysosomal degradation: The endocytic vesicles progressively acidify and acquire proteases (cathepsins B, D, S, L). These degrade the ingested proteins into peptide fragments within protease-containing endosomes/lysosomes.
3. MHC class II synthesis with invariant chain (Ii): Newly synthesized MHC class II alpha-beta heterodimers in the ER associate with the invariant chain (Ii). The Ii serves two functions:
   • Its CLIP (Class II-associated Invariant chain Peptide) segment acts as a "molecular stopper," sitting in the peptide-binding groove and preventing premature peptide loading by ER-resident peptides (which would otherwise compete with class I).
   • The Ii contains targeting signals that direct the MHC class II-Ii complex away from the secretory pathway toward the endocytic compartments.
4. MIIC formation and CLIP exchange: The MHC class II-Ii complex is transported to specialized late endosomal compartments called MIIC (MHC class II compartments). Here, Ii is progressively degraded by lysosomal proteases, leaving only the CLIP fragment in the groove. The non-classical MHC class II molecule HLA-DM (encoded within the MHC) then catalyzes the exchange of CLIP for high-affinity antigenic peptides. HLA-DO modulates HLA-DM activity in B cells.
5. Peptide loading and surface expression: Antigenic peptides generated in endosomes compete for binding to MHC class II. The class II-peptide complex is then transported to the cell surface.
6. T-cell recognition: CD4+ helper T lymphocytes recognize MHC class II-peptide complexes. Class II-bound peptides extend from both ends of the open groove with greater solvent accessibility (35%) and more side-chains pointing toward the TCR.

C. Cross-Presentation Pathway

D. CD1 Pathway

Part 3: Role of HLA in Susceptibility and Pathogenesis of Rheumatic Diseases

General Principles

1. Arthritogenic peptide hypothesis: Specific HLA alleles present self-peptides (autoantigens) to autoreactive T cells, driving pathological autoimmune responses.
2. Thymic selection hypothesis: HLA alleles shape the TCR repertoire during thymic development, selecting for (or failing to delete) autoreactive T cell clones.
3. Molecular mimicry: Structural similarities between the HLA-peptide complex and microbial antigens may trigger cross-reactive immune responses.

3A. Rheumatoid Arthritis (RA)

• DRB1*0401, *0404, *0405 (DR4 subtypes) - strongest risk in White/North European populations
• DRB1*0101 (DR1) - risk in some ethnic groups; lower relative risk than DR4
• DRB1*1001 (DR10) - risk in Southern European and some Middle Eastern populations
• DRB1*0901 - risk allele in Asians

• Influences the shape of the antigen-binding groove (positions 70-74)
• May determine which peptides are bound and presented to T cells
• Is positioned where it can interact with both incoming peptides and TCR

"Most of these risk alleles share a common sequence: Q or K-R-R-A-A, which has been termed the shared epitope (SE). This structural feature is located on the α-helical portion of the DR β chain in a position where it may influence both peptide binding and T cell receptor interactions with the DRB1 molecule." - Firestein & Kelley's Textbook of Rheumatology

• PTPN22 (R620W variant) - disrupts Csk-mediated inhibition of Lck, leading to TCR signaling threshold lowering
• STAT4, CTLA4, PADI4, TRAF1-C5, PTPRC, CD40, and many others
• Heritability of RA ~65%; HLA-DRB1 contributes approximately 30-50% of genetic risk

3B. Ankylosing Spondylitis (AS) and Spondyloarthropathies

• 80-95% of AS patients are HLA-B27 positive (White Europeans); odds ratio ~90-100
• In White Europeans, HLA-B27 frequency is ~8% in the general population vs ~90% in AS patients
• Only ~5% of HLA-B27-positive individuals develop AS, indicating that HLA-B27 is necessary but not sufficient
• Heritability of AS is >90%; HLA-B27 contributes <50% of this genetic risk
• First-degree relatives of AS patients have 5-16x greater risk of AS than HLA-B27+ general population individuals - implying HLA-B27-modifying genes

"Of patients who have ankylosing spondylitis, 88% to 96% are HLA-B27 positive, but only 5% of the HLA-B27 population develops ankylosing spondylitis." - Campbell's Operative Orthopaedics

1. Arthritogenic peptide hypothesis: HLA-B27 presents specific self or bacterial (Klebsiella, Chlamydia) peptides to CD8+ cytotoxic T cells, triggering joint inflammation.
2. Misfolding/UPR hypothesis: HLA-B27 heavy chains have a propensity to misfold in the ER and form homodimers. Misfolded B27 activates the unfolded protein response (UPR), leading to IL-23 production and downstream Th17 pathway activation.
3. Free heavy chain hypothesis: Cell surface HLA-B27 homodimers (B27-HC) engage NK and T cell receptors (e.g., KIR3DL2), activating Th17 responses.
4. ERAP1 interaction: ERAP1 (aminopeptidase involved in trimming peptides for class I loading) variants strongly modify AS risk in an HLA-B27-dependent manner - loss-of-function ERAP1 variants protect against AS only in HLA-B27-positive individuals, directly implicating peptide processing in pathogenesis.

3C. Systemic Lupus Erythematosus (SLE)

• HLA-DR2 (DRB1*1501) and HLA-DR3 (DRB1*0301) are associated with increased SLE susceptibility (OR ~2-3)
• DR2/DR3 heterozygotes have higher risk than either homozygote
• HLA-DR2 associates particularly with anti-dsDNA and anti-Sm antibodies
• HLA-DR3 associates with anti-Ro/SSA and anti-La/SSB antibodies
• C4A null allele (class III MHC) - complement deficiency impairs immune complex clearance and is a strong SLE risk factor
• Non-MHC genes: IRF5, TREX1, STAT4, BLK, PTPN22, FCGR2A/3A, ITGAM

3D. Primary Sjogren's Syndrome (pSS)

• HLA-DR3 (DRB1*0301) and HLA-DQ2 (DQB1*0201) are the main risk alleles
• HLA-DQ associations mediated through linkage disequilibrium with DR3
• Anti-Ro/SSA antibodies associate strongly with DR3
• HLA-DRB1*1501 (DR2) also associated in some populations

3E. Juvenile Idiopathic Arthritis (JIA)

• RF-positive polyarticular JIA: Shares HLA-DRB1 shared epitope alleles with adult RA (DRB1*01/*04)
• Oligoarticular/RF-negative: HLA-DRB1*0801, *11, *1301; DRB1*1301-DQA1*01-DQB1*06 haplotype associated with oligoarticular disease
• Enthesitis-related arthritis (ERA): HLA-B27 associated (as in adult AS)
• Systemic JIA: Less clear HLA association; different pathogenic mechanism (autoinflammatory)

"The shared epitope (consisting of selected HLA-DRB1*01/*04 alleles) is associated only with RF-positive polyarticular JIA, a finding supporting the fact that this JIA category is the childhood equivalent of adult RA." - Rheumatology, 2-Volume Set (Elsevier)

3F. Other Rheumatic Diseases and HLA Associations (Summary Table)

Part 4: Note on HLA Typing

Why HLA Typing?

1. Transplantation - organ and hematopoietic stem cell transplantation (HSCT)
2. Disease susceptibility/diagnosis - ankylosing spondylitis, celiac disease, narcolepsy
3. Pharmacogenomics - predicting drug hypersensitivity (abacavir/HLA-B*57:01; carbamazepine/HLA-B*15:02)
4. Research - population genetics, autoimmune disease studies

Methods of HLA Typing

1. Serological (Complement-Dependent Cytotoxicity - CDC) - Historical

• Lymphocytes incubated with panels of anti-HLA antibodies + complement
• Dead cells indicate HLA match
• Disadvantages: Can only distinguish major allele groups (low resolution); requires viable lymphocytes; laborious; cannot distinguish many alleles
• Now largely replaced by molecular methods

2. Cellular Typing - Historical

• Mixed Lymphocyte Reaction (MLR) - measures T cell proliferative response to allogeneic cells
• Primed Lymphocyte Test (PLT)
• Also replaced by molecular methods

3. Molecular/DNA-Based Methods - Current Standard

"HLA alleles are defined by their gene sequences and individuals can be typed by the polymerase chain reaction (PCR) using discriminating pairs of primers. Molecules encoded by the class II HLA-DP, -DQ, and -DR loci provoke CD4 T-cell responses, whereas HLA-A, -B, and -C gene products are targets for alloreactive CD8 T-cells." - Roitt's Essential Immunology

• Multiple parallel PCR reactions each using primers specific for one allele
• Presence/absence of PCR product identifies the allele
• Medium resolution; fast; suited for routine typing

• PCR amplification of HLA loci, then hybridization with labeled allele-specific probes
• Can be done in reverse (reverse SSO/Luminex bead array) - highly multiplexed
• Medium to high resolution; now widely automated

• Sanger sequencing of amplified HLA exons (exons 2 and 3 for class I; exon 2 for class II)
• High-resolution (allele-level) typing
• Gold standard but laborious; subject to phase ambiguities

• Full-length HLA gene sequencing at ultra-high resolution
• Resolves phase ambiguities
• Becoming the new gold standard, especially for HSCT
• Cost-effective at scale; can type all HLA loci simultaneously

• Fluorescent-labeled heteroduplex analysis
• Automated, high-throughput

• Single SNP variants in LD with HLA alleles (e.g., HCP5 rs2395029 as surrogate for HLA-B*57:01 with 99.9% predictive value)
• Useful in population screening; risk of false negatives in copy number variation regions
• Supplemented by serological phenotyping (B57/B58 detection) or skin patch testing

Resolution Levels in HLA Typing

"The level of HLA typing resolution required for hematopoietic stem cell transplantation differs from that of renal transplantation. Higher-resolution HLA typing is required to match the donor and recipient for stem cell transplantation because stem cell transplantation involves the transfer of an entire immune system to the patient." - Henry's Clinical Diagnosis and Management by Laboratory Methods

HLA Typing for HSCT

HLA-B*57:01 Testing in Pharmacogenomics

• HLA-B*15:02 before carbamazepine (Stevens-Johnson syndrome - particularly in Asian populations)
• HLA-B*58:01 before allopurinol (SJS/TEN in Asians)
• HLA-A*31:01 before carbamazepine in Europeans

Key Summary

• Roitt's Essential Immunology (Essentials)
• Firestein & Kelley's Textbook of Rheumatology, 2-Volume Set
• Rheumatology 2-Volume Set (Elsevier, 2022)
• Tietz Textbook of Laboratory Medicine, 7th Edition
• Henry's Clinical Diagnosis and Management by Laboratory Methods
• Janeway's Immunobiology, 10th Edition
• Cellular and Molecular Immunology (Abbas)
• Campbell's Operative Orthopaedics, 15th Ed.