We and others have previously reported that the frequency of T follicular helper cells (Tfh) is increased in blood from patients with type 1 diabetes (T1D) [1,2]. Tfh cells play a critical role in the selection and maturation of high-affinity B cells and are one of the main sources of the pro-inflammatory cytokine IL-21 in humans. However, one major limitation to the functional characterisation of Tfh cells in humans has been the limited access to tissue resident cells. Current studies of Tfh cells in humans have been limited to the characterisation of cells in blood, which are known to phenotypically differ from bona fidetissue-resident Tfh cells.
In collaboration with the translational gastroenterology unit (TGU) and Oxford University Hospitals, we have now access to fresh biopsy tissue from patients undergoing large bowel resection surgery. This provides a unique opportunity to isolate and characterise the tissue-resident T cell subsets, including Tfh cells, which are particularly abundant in the gut. Using flow cytometric analysis, we have shown that a Tfh cell population displaying a prototypical CXCR5+PD-1+ICOS+phenotype can be clearly identified in the small intestine. This is in marked contrast with blood Tfh cells, which are very limited in blood and lack a defined expression of the markers PD-1 and ICOS (Fig. 1).
The aim of this project will be to utilise state-of-the art single cell genomic approaches to characterise the transcriptional profile of tissue-resident T cells. This will include utilising single-cell RNA-sequencing (scRNA-seq) and scATAC-seq to define and compare the transcriptional landscape of Tfh cells from derived from tissue and blood. An additional emphasis of this project will be in the investigation of the regulation of the IL-2 signalling pathway in tissue resident T cell subsets, by taking advantage of the expertise of the lab in designing and implementing IL-2 trials in humans. In addition, the development of the analytical and computational methods to analyse complex genomic datasets will be essential for the success of the project.
To undertake the experimental characterisation of T cell subsets in blood and tissue, and to develop the statistical and analytical tools to analyse these data. Integrate the results with the currently ongoing clinical and experimental medicine trials of low-dose IL-2 in T1D children to develop new model to investigate the regulation of the disease-associated IL-2 / IL-21 locus in isolated tissue resident T cell subsets and the mechanisms driving the association with human disease.
There will be opportunity to learn and develop skills in polychromatic flow cytometry, single-cell genomics, bioinformatics, statistics and a wide range of molecular, metabolic and immunological techniques.
We have close links to the biopharmaceutical industry and there may be opportunities within this project for collaboration.
Project reference number: 879
|Professor John A Todd FRS FMedSci||Wellcome Trust Centre for Human Genetics||Oxford University, Henry Wellcome Building of Genomic Medicine||GBRfirstname.lastname@example.org|
|Professor Linda Wicker||Wellcome Trust Centre for Human Genetics||Oxford University,||email@example.com|
|Dr Ricardo Ferreira||NDM, WHG||University of Oxford||GBRfirstname.lastname@example.org|
|Dr Melanie Dunstan||NDM, WHG||University of Oxford||GBRemail@example.com|
AIMS/HYPOTHESIS: Type 1 diabetes results from the autoimmune destruction of insulin-secreting pancreatic beta cells by T cells. Despite the established role of T cells in the pathogenesis of the disease, to date, with the exception of the identification of islet-specific T effector (Teff) cells, studies have mostly failed to identify reproducible alterations in the frequency or function of T cell subsets in peripheral blood from patients with type 1 diabetes. METHODS: We assessed the production of the proinflammatory cytokines IL-21, IFN-γ and IL-17 in peripheral blood mononuclear cells from 69 patients with type 1 diabetes and 61 healthy donors. In an additional cohort of 30 patients with type 1 diabetes and 32 healthy donors, we assessed the frequency of circulating T follicular helper (Tfh) cells in whole blood. IL-21 and IL-17 production was also measured in peripheral blood mononuclear cells (PBMCs) from a subset of 46 of the 62 donors immunophenotyped for Tfh. RESULTS: We found a 21.9% (95% CI 5.8, 40.2; p = 3.9 × 10(-3)) higher frequency of IL-21(+) CD45RA(-) memory CD4(+) Teffs in patients with type 1 diabetes (geometric mean 5.92% [95% CI 5.44, 6.44]) compared with healthy donors (geometric mean 4.88% [95% CI 4.33, 5.50]). Consistent with this finding, we found a 14.9% increase in circulating Tfh cells in the patients (95% CI 2.9, 26.9; p = 0.016). CONCLUSIONS/INTERPRETATION: These results indicate that increased IL-21 production is likely to be an aetiological factor in the pathogenesis of type 1 diabetes that could be considered as a potential therapeutic target. Hide abstract
The strong genetic association between particular HLA alleles and type 1 diabetes (T1D) indicates a key role for CD4+ T cells in disease; however, the differentiation state of the responsible T cells is unclear. T cell differentiation originally was considered a dichotomy between Th1 and Th2 cells, with Th1 cells deemed culpable for autoimmune islet destruction. Now, multiple additional T cell differentiation fates are recognized with distinct roles. Here, we used a transgenic mouse model of diabetes to probe the gene expression profile of islet-specific T cells by microarray and identified a clear follicular helper T (Tfh) cell differentiation signature. Introduction of T cells with a Tfh cell phenotype from diabetic animals efficiently transferred diabetes to recipient animals. Furthermore, memory T cells from patients with T1D expressed elevated levels of Tfh cell markers, including CXCR5, ICOS, PDCD1, BCL6, and IL21. Defects in the IL-2 pathway are associated with T1D, and IL-2 inhibits Tfh cell differentiation in mice. Consistent with these previous observations, we found that IL-2 inhibited human Tfh cell differentiation and identified a relationship between IL-2 sensitivity in T cells from patients with T1D and acquisition of a Tfh cell phenotype. Together, these findings identify a Tfh cell signature in autoimmune diabetes and suggest that this population could be used as a biomarker and potentially targeted for T1D interventions. Hide abstract