Abstract
Granulomatosis with polyangiitis (GPA) is a systemic autoimmune disease. The underlying pathology is primarily caused by the formation of anti-neutrophil cytoplasmic antibodies (ANCA), leading to ANCA associated vasculitis (AAV). While ANCA produced by B cells are a key mediator of the underlying pathogenic process, there is increasing evidence from human and murine studies to strongly support a role for CD4+ T cells in GPA pathogenesis. Previous studies using peripheral blood mononuclear cells (PBMCs) and whole blood from GPA patients suggest that CD4+ T cell subsets, particularly regulatory T cells (Treg) and circulating follicular helper T (cTfh), may be altered in frequencies and/or function in GPA patients.The aim of this thesis was to identify and compare Treg and cTfh frequencies in GPA patients and healthy controls (HC) and to investigate potential cell intrinsic dysregulation and/or extracellular factor(s) responsible for the functional imbalance in these CD4+ T cell subsets in GPA.
Phenotypic profiling of CD4 T cells by FACS, revealed the aberrant presence of CD25 expression on GPA naive CD4+ T cells. The data reveal a significant increase in Tfh-like phenotypes in GPA, with GPA CD4+ T cells exhibiting elevated expression of BCL6 (a key transcription factor for Tfh differentiation) and IL-21 (a Tfh signature cytokine).
Microarray analysis was performed on naïve CD4+ T cells, from GPA and HC donors, with and without TCR stimulation. These data revealed that antimicrobial genes were the main differentially expressed genes irrespective of stimulation, a finding consistent with the previously accepted notion that chronic infection is an important underlying driver in GPA pathogenesis. Additionally, TCR activation induced the expression of key genes associated with Tfh lineage, such as BCL6 and TRIM8, consistent with the observation of increased Tfh-like phenotypes in GPA.
Significantly, the microarray data suggested the down-regulation of the IL-2/STAT5 pathway in GPA naïve CD4+ T cells. Furthermore, IL2-induced STAT5 activation was confirmed to be decreased in GPA naïve CD4 T cells, by western blot analysis. As the IL2/STAT5 signalling module is a major negative regulator of Tfh differentiation, these findings provide a molecular mechanism for the aberrant enhanced development of Tfh cells in GPA for the first time.
In contrast, frequencies of Tregs and their capacity to suppress effector T cell proliferation were comparable between CD4+ T cells from GPA patients and HC. However, cytokine analysis of supernatants from the suppression assays, reveal significantly that GPA Tregs failed to specifically inhibit the expression of the pro-inflammatory cytokine GM-CSF, which has previously been shown to be a critical mediator of neutrophil activation during the development of GPA pathogenesis.
Finally, the contribution of extracellular factors in plasma to CD4+ T cell dysregulation was assessed by co-culture assays and mass spectrometry. Factors present in plasma could induce expression of CD25 on CD4 T cells from healthy donors. Pro-inflammatory cytokine IL-18 was elevated in plasma from GPA patients. Intriguingly, a comparative mass-spectrometry analysis of plasma samples suggested a potential dysregulation of lipid metabolism in GPA.
In conclusion, this study presents key novel evidence for the involvement of both cell intrinsic dysregulation and extracellular factors in the imbalance of CD4+ T cell differentiation in GPA.
| Date of Award | 1 May 2020 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Susan John (Supervisor) & David D'Cruz (Supervisor) |