Lymphocyte receptors and their role in the immunopathogenesis of ankylosing spondylitis

Abstract

The functional plasticity of the human immune system is enabled by the diversity of immune-response genes encoded within polymorphic genomic regions. Throughout evolutionary history, heterogeneity yielded by expansion and diversification of immune-related loci has maximised the potential for productive defences against invasive pathogens, while ensuring infection risk is stratified across populations. Concurrently, self-tolerance mechanisms have emerged to prevent immune retaliation against host constituents. Disrupted self-tolerance results in autoimmunity, which can manifest at diverse body sites and drive pathology through the destruction of tissues and organs. The aetiology of immune-mediated diseases (IMD) typically involves interacting environment and inherited genetic factors, making the molecular processes that culminate in these conditions exceedingly difficult to define.This thesis addresses the immunological mechanisms driving ankylosing spondylitis (AS), a chronic IMD affecting 0.1-0.5% of individuals of European decent. AS is characterised by inflammation targeting the axial skeleton, contributing to joint erosion, consequential reactive bone deposition, and, in severe instances, bony fusion (ankylosis) of the pelvis and spine. AS is the prototypic spondyloarthropathy (SpA), sharing symptomatology with related SpAs including reactive arthritis (ReA), a self-limiting arthritis triggered by gastrointestinal and urogenital infection. Up to 70% of AS patients show evidence of bowel inflammation and 5-10% are diagnosed with inflammatory bowel disease (IBD), sparking the hypothesis that AS may stem from cross-recognition of gut-derived microbial antigens and autoreactive self-proteins at the joints.Over 100 genetic loci are associated with AS risk. The primary determinant of disease susceptibility is inheritance of HLA-B*27, a class I human leukocyte antigen (HLA) allele found in >85% of patients and contributing ~20% to disease heritability. Endogenous peptides displayed by HLA class I molecules provide an interface of communication between host cells and lymphocytes, which engage surface HLA through hypervariable receptors. Polymorphisms in HLA class I peptide processing machinery show strong associations with AS and have been demonstrated to alter the HLA-B*27 presented peptidome. Irrespective of this, the link between genetic associations and lymphocyte responses remains unresolved. This thesis explores the dynamics of HLA interactions with T-cells and natural killer (NK) cells of the immune system, specifically in the context of the profound HLA-B*27 association with AS.Chapter 2 addresses the hypothesis that CD8+ T-cells retaliate against HLA-B*27-presented arthritogenic peptide/s to drive autoinflammation in AS. Sequencing of the hypervariable peptide-contact region of the T-cell receptor (TCR) s-chain was used to profile CD4+ and CD8+ T-cells from AS patients and HLA-B*27 matched controls in search of disease-associated public clonotypes indicative of common antigen exposure. The peripheral CD4+ and CD8+ TCR repertoire of AS patients exhibited significantly increased diversity relative to controls, attributed to a reduction in large clonal expansions, suggesting global perturbation in adaptive immunity. Additional to broad repertoire diversity changes, public CD8+ clonotypes were detected at significantly increased incidence in the blood of HLA-B*27+ve AS patients. One such clonotype utilised a TCR hypervariable region identical to that previously detected in the inflamed synovium of individuals with bacterial-induced ReA. Two disease-associated clonotypes were also negatively associated with HLA-B*27 in controls, implying an inherent degree of autoreactivity warranting T-cell deletion in healthy HLA-B*27 carriers.Chapter 3 and 4 dissect the consequences of heterogeneity at the killer immunoglobulin-like receptor (KIR) locus on AS risk. KIRs expressed on NK and T-cells exhibit specificity for structural motifs on HLA ligands and are inherited in copy-number variable haplotypes that exhibit immense allelic diversity, translating to functional diversification of lymphocyte responses across individuals. Chapter 3 describes the largest genetic association study to assess KIR-HLA epistasis in any phenotypic context. Imputed KIR and HLA dosages for 8,107 Immunochip genotyped AS cases and 12,214 controls allowed identification of statistical interactions between co-inherited receptors and their ligands that alter disease risk. Chapter 4 further interrogates functional diversity at the KIR3DL1/S1 locus, encoding as alternate alleles an inhibitory and activating receptor known to engage HLA-B and govern NK cell cytotoxicity. Six subgroups of KIR3DL1 alleles differing in their surface expression and signalling characteristics were typed by PCR in 415 AS patients and 292 controls, and the consequences of KIR3DL1/S1 allelic variation on the NK cell proinflammatory cytokine profile was assessed in patient serum.In summary, this thesis details experimental and statistical approaches to assess interactions between HLA ligands and lymphocyte receptors in the context of AS. It discloses an apparent degree of immunodeficiency in AS patient T-cell responses that is yet to be addressed in the literature, supporting a model whereby perturbed clearance of infectious stimuli may proceed chronic inflammation and the emergence of cross-reactive CD8+ T-cells. It also presents an interrogation of KIR associations with disease, emphasising how independently inherited genetic factors can collectively contribute to disease risk by altering the dynamics of cellular interactions. The implications of these findings in understanding the immunopathogenesis of AS and shaping the future of therapeutic development are discussed in depth in the final chapter of this thesis.