The impact of gamma-delta TCR sequences on selection and peripheral repertoire shaping of gamma-delta T cells.

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Three conserved lineages of lymphocytes rearrange clonal antigen receptors during their development, namely B cells, alpha-beta T cells and gamma-delta T cells. These gamma-delta T cells are rather oligoclonal T lymphocyte populations, with variable TCR diversity depending on their localization within tissues. Since the ligands of gamma-delta T cell receptor (TCR) remain largely unknown it is currently not established whether the gamma-delta TCR recognizes canonical self or foreign structures. We have previously described that developing gamma-delta T cells undergo TCR quality control selection and can differentiate into mature effector cells in the thymus. However, it is currently unclear whether engagement of specific TCR ligands is required for the differentiation of gamma-delta T cells into effector T cell subsets. Moreover, it is not even clear whether the gamma-delta TCR has any function in mature gamma-delta T cells. Thus, the central aim of this proposal is to define how clonal selection and peripheral homeostasis of gamma-delta T cells is instructed by their individual gamma-delta TCR sequences. To achieve this goal, we need comprehensive gamma-delta TCR sequence information. To overcome the limitations of classical sequencing approaches, our lab has established methods for deep-sequencing of very diverse antigen receptor chains. For example, we recently analyzed the alpha-beta TCR repertoire of CD4+Foxp3+ regulatory T cells. There, we suggested that continuous competition of peripheral regulatory T cells for limited self-antigen shapes an organ-optimized, yet highly diverse, local TCR repertoire. In this research project proposal, we intend to adopt our high-throughput sequencing protocols to gamma-delta T cells. In addition, we are modifying our sequencing approach to include rapid amplification of cDNA ends (RACE) to generate unbiased templates comprising all gamma-delta TCR. Also, we are establishing further protocols to sequence gamma-delta TCR chain amplicons derived from genomic DNA samples. Once we are able to define the complete antigen receptor range of gamma-delta T cells, we will monitor thymic selection and peripheral shaping of their TCR. To this end, we will chose and compare templates from lymphoid and mucosal tissues to clarify how the gamma-delta TCR repertoire is shaped locally, and how it may adapt during ageing and in response to commensal microbiota. At the same time, we will rigorously test the null hypothesis that the gamma-delta TCR has no specific post-thymic function. This question can only be answered by the use of genetic models to conditionally induce a loss of functional gamma-delta TCR in mature gamma-delta T cells. In summary, we expect to draw stringent conclusions how thymic and peripheral shaping of the gamma-delta TCR repertoire impacts on gamma-delta T cell function and homeostasis.