Characterisation of human antigen-specific CD4+CD39+ regulatory T cells

Human antigen-specific CD4+ T cells can be detected through the dual expression of CD134 (OX40) and CD25 after 44 hours stimulation with cognate antigen. Work in this thesis demonstrates that differential surface expression of CD39 on these cells enables isolation of a CD39+ Treg-enriched cell population. These Treg-enriched CD4+CD25+CD134+CD39+ T cells originate from peripheral memory CD4+CD45RO+CD127lowCD25highCD39+ Tregs and across recall responses to a wide range of antigens they consistently form a substantial proportion of the CD4+ T cell response. Characterisation of these antigen-specific CD39+ T cells in the context of chronic viral infection revealed they were hypoproliferative in vitro and their depletion did not substantially affect the in vitro proliferation of effector CD4+ T cells. TCR repertoire analysis of CMV peptide-specific CD39+ T cells revealed a bias towards common TRBV usage within a highly polyclonal repertoire. The CMV-specific Treg-enriched CD25+CD134+CD39+ T cells were also expanded in vitro as T cell clones that stably retained their antigen specificity and a Treg phenotype, with high FOXP3, CD25, CTLA-4 and CD39 expression. Importantly, these clones could suppress autologous effector T cell proliferation and the magnitude of suppression positively correlated with CD39, FOXP3 and CTLA-4 expression. Coeliac disease was used as a model of autoimmune disease and the OX40 assay methodology was used to detect gliadin-specific CD4+ T cell responses in the peripheral blood of coeliac individuals following oral gluten challenge. The frequency of gliadin-specific CD4+CD25+CD134+ T cells peaked at day 6 post-gluten challenge and these cells were overwhelmingly comprised of CD39+FOXP3+ cells. Upon expansion as T cell lines and clones, the gliadin peptide-specific CD39+ T cells expressed a Th3 cell-like phenotype and exerted in vitro suppression of autologous effector T cell proliferation. Molecular analysis of the TCR repertoire revealed oligoclonal bias within gliadin peptide-specific CD4+ T cells in coeliac disease and detected several public clonotypes. This work describes and validates methodology for the identification and isolation of antigen-specific CD39+ Tregs, enabling the role of Tregs in memory responses to be further defined, allowing a greater understanding of disease pathogenesis and paves the way for developing this methodology as a novel immunotherapy