Abstract
Although several hundred genetic risk loci have been associated with the development of inflammatory bowel disease (IBD), few have been resolved to a causal biological pathway or to a tractable therapeutic approach. The single nucleotide polymorphism (SNP) rs61839660 was identified through genomewide association studies (GWAS) and subsequently resolved through finemapping as a causal variant in Crohn’s disease (CD). This variant is also associated with other auto-inflammatory and allergic diseases, suggesting a key regulatory node.The rs61839660 SNP lies within the intronic enhancer of IL2RA (CD25), the high affinity receptor for interleukin 2 (IL-2), which is constitutively expressed at high levels on regulatory T cells (Treg), allowing them to respond to low levels of IL-2 in the cellular microenvironment. CD25 is expressed at lower levels on other immune cell subsets, including CD4+ effector T cells (Teff) and natural killer (NK) cells. Binding of IL-2 to the CD25-containing trimeric IL-2R initiates dimerization of intracellular Janus kinase (JAK) proteins, and phosphorylation of signal transducer and activation of transcription (STAT)5.
Crohn’s disease patients of each genotype at the rs61839660 locus were recruited to provide clinical data and peripheral blood samples. Fluorescenceactivated cell-sorting (FACS) and flow cytometry data demonstrate that carriage of the rs61839660 risk ‘T’ allele is associated with increased CD25 expression on resting CD4+ Teff and CD56Dim NK cells, and enhanced pSTAT5 activation in response to IL-2. Functional differences were observed in effector cells from heterozygote subjects in the presence of IL-2, with increased Teff secretion of pro-inflammatory cytokines and enhanced CD56Dim NK cell cytotoxicity compared to wild-type subjects. Preliminary results from flow cytometry performed on colonic biopsy samples suggested an increased proportion of CD4+ Teff in the gut tissue of heterozygote subjects.
The in vitro effects of basiliximab, a monoclonal blocking antibody to CD25, and tofacitinib, a JAK inhibitor, were assessed in sorted immune cell populations from genotyped subjects. Both basiliximab and tofacitinib demonstrated the ability to block the enhanced pSTAT5 activation seen in rs61839660 risk allele carriers. Analysis of a genotyped tofacitinib-treated clinical cohort did not show any difference in clinical response between wild-type and heterozygote subjects.
The data presented in this thesis supports a role for the rs61839660 risk allele in modulating CD25 expression on CD4+ T cell and NK cell subsets, thus altering responsiveness to IL-2 signalling and the balance of pathogenic and protective immunity in Crohn’s disease. Further work is required to establish whether clinically available treatment agents acting on the IL-2 signalling pathway have a role in the personalised treatment of Crohn’s disease in rs61839660 risk allele carriers.
| Date of Award | 1 Aug 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Peter Irving (Supervisor) & Graham Lord (Supervisor) |