DiMeN Doctoral Training Partnership: Deciphering the metabolic signature of CD4+ T helper cells that express dual master transcription factors

Immunotherapy has shown phenomenal success in recent years in the field of both cancer and autoimmunity. In particular, clinical trials that utilise T cells have revolutionised treatment of tumour (Th1 cells) and autoimmune juvenile diabetes (Treg cells). The major pitfall of these trials remains in the area of in-vivo T cell persistence. Recent reports have attributed the lack of persistence to the bioenergetics of T cells utilised in these trials (Sukumar M, et al, Cell Metabolism). Hence there is an immediate need for understanding the bioenergetics profile of inflammatory versus regulatory T cells for improving clinical care.

Background
CD4+ T helper subsets are important mediators of adaptive immunity and are classified into various subtypes based on their transcription factor profile. CD4+ T helper cells that express Tbet transcription factor are called Th1 cells, Th2 (GATA3+), Th17 (RORγt+) or Tregs (FoxP3+) cells. Each subset has a unique function as determined by the respective transcription factor with Tbet driving the production of IFNγ cytokine and hence promoting inflammatory responses and FoxP3 instills suppressive function in Tregulatory cells. Activation of the T cell receptor (TCR) along with co-stimulatory signalling is also critical for T helper cell differentiation, which results in metabolic remodelling and acquiring aerobic glycolysis. Differentiation of CD4+ T helper subsets with diverse effector functions is accompanied by additional changes in metabolism to meet their bioenergetic demands. Hence, Th1 cells have a unique metabolic signature, mitochondrial energetics and glycolytic profile (strongly glycolytic) as compared to Tregs that prefer oxidation of lipids.

We and others have shown that Tbet+Th1 cells can also express FoxP3 and acquire a regulatory phenotype but maintain Tbet expression. Providing co-inhibitory signals through PD-1/PDL-1 (programmed death 1pathway) in Th1 cells stabilizes FoxP3 expression and Tbet expression. The metabolic signature of these cells that express dual opposing transcription factors is unknown.

In this proposal we aim to elucidate the metabolic signature of Tbet+FoxP3+ cells and identify how metabolism co-relates to their function in mouse models of inflammatory diseases.

Methodology
A unique resource that is available to us is a fate mapping reporter murine strain that drives both Tbet and FoxP3 and hence allow isolating Tbet+FoxP3+ T cells based on their transcription factor expression. We aim to isolate these cells from WT mice to analyze their metabolic profile by using the Seahorse XF Analyzer to evaluate real time cellular respiration of the cells and response to metabolic modulators. This analysis takes simultaneous repeated measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) that are representative of mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis respectively (measured by changes in oxygen and proton (pH) concentration respectively).

In addition, CD4+ T helper cells that are Tbet+, will be expanded in vitro in the presence/absence of PDL-1 to become Tbet+FoxP3+ cells. As before we will compare the metabolic profile and in vitro effector phenotype of these expanded cells to day 0 WT Tbet+FoxP3+ sorted cells. The in-vivo function of Tbet+FoxP3+ cells will be tested to determine if function co-relates to specific metabolic signature in this subset.

Training opportunities
The students involved in this project will be trained in cross-disciplinary fields of biochemistry and immunology which include murine models of disease and fate mapping reporters, in vitro cellular immunological and biochemical assays and use of multi-parameter flow cytometry. The strong inter-disciplinary aspect of this PhD will significantly enhance the student’s future career prospects. As well as taking part in the weekly specialist subject research meetings, journal clubs and institute research programs, the student will engage with the Graduate School’s Skills Development Programme and Code of Practice requirements.