Rotation 1: CD8+ Tregs: the key to poor vaccine responses in the aged?

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BBSRC strategic theme: Bioscience for an integrated understanding of healthT cells are highly migratory cells that continually circulate between lymphoid organs and the blood in a largely quiescent state. Upon activation of T cells through their T cell receptor (TCR), T cells undergo a profound phenotype switch, entering an activated state of high functionality. These activated T cells have a further expansion of their migratory capacity, with elevated ability to enter non-lymphoid tissues, including both internal (e.g. liver) and barrier (e.g. gut) tissues. Across these highly diverse microenvironments, T cells need to maintain highly efficient function, at a high energetic state. Indeed, immune activation is among the most energetically expensive activity the body is capable of. The activation of T cells is known to change their metabolic profiles and reliance; it is likely that further changes in metabolic profiles/reliance will occur in non-lymphoid tissues, however this is poorly studied due to the technical limitations of studying T cells within the tissue environment.This project brings together expertise in tissue immunology (Liston lab) and metabolism (Trefely lab) to study the metabolic requirements of tissue T cells. We will use the FlowCode system, recently developed by the Liston lab for massively parallel flow cytometry-based CrispR screens in vivo. This system allows testing of small CrispR libraries (several hundred genes) in vivo, using flow cytometry phenotypic assays, providing a high degree of sensitivity, unique functional read-outs, and enabling the work to be performed on the small tissue-resident population. A metabolic-focused CrispR library will be cloned into the FlowCode retrovirus library and transduced into mature T cells. Transfer in vivo provides for a protein epitope-barcoded set of T cells, each with a unique metabolic gene knockout. I will test the relative impact of the metabolic deficiencies on different T cell subtypes (CD4 Tconv, CD8, Treg; and naïve/activated/memory subsets) within the circulating lymphoid compartment, to identify cell type-specific requirements. I will also use tissue-based extraction and barcoding to determine which metabolic genes alter tissue residency, across a diverse range of tissues. Finally, I will challenge mice with dietary (high fat diet) and infectious (flu) challenges, to determine whether these changes to the metabolic context of the tissues alters the molecular reliance of metabolic pathways. Identified genetic control networks across the cell type / tissue type / metabolic context matrix will then be dissected at the metabolite level using cutting-edge liquid chromatography-mass spectrometry (LC-MS) based methods for metabolomic analysis.These findings will unravel the gene-metabolism rules of tissue T cells, and inform as to the molecular requirements of effective T cell-based therapeutics in metabolically-challenged tissues.