Phylogeny and species comparison as tools for understanding antigen recog-nition by human γδ T-cells

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γδ T-cell antigen-receptors (γδTCR) emerged together with the other RAG-dependent antigen receptors and are found in nearly all jawed vertebrate species. There is an increasing interest in clinical use of γδTCR-expressing cells (γδ T cells) but the antigens and the modes of antigen-recognition by γδ T-cell antigen-receptors (TCRs) are poorly understood. γδ T cells vary massively in phenotype and function and subpopulations of γδ T cells are found only in certain species and are often de-fined by their V-gene usage. One of those subpopulations are human Vγ9Vδ2 T cells whose eponymous TCRs sense so-called phosphoantigens (PAgs), which are phos-phorylated host or microbial metabolites of isoprenoid synthesis. The strongest natu-ral PAg is HMBPP which drives Vγ9Vδ2 T-cell-activation and -expansion in infections like tuberculosis and malaria. A rather weak and host cell-produced PAg is IPP which accumulates in some tumor cells or after treatment with amino-bisphosphonates (e.g. zoledronate). This IPP accumulation sensitizes the cells for the tumoricidal activity of Vγ9Vδ2 T-cells. For more than 20 years Vγ9Vδ2 T-cells were believed to be restricted to higher pri-mates but we found such cells in the alpaca although missing in murine rodents. Vγ9Vδ2 TCR do not bind PAgs directly but sense host cell changes induced by PAg -binding to the intracellular domain of BTN (butyrophilin) 3 molecules. In humans BTN3A1 cooperates with its isoforms BTN3A2 and BTN3A3 while alpaca possesses a single BTN3 which integrates the functions of the three isoforms. Apart from BTN3A1, BTN2A1-expression by the target cells is also essential for PAg-mediated activation of Vγ9Vδ2 T cells. However, molecular events and signaling cascades in-volved in the TCR-mediated activation are poorly understood. We will create BTN3-chimeric molecules and mutants to identify minimal molecular requirements of PAg-sensing and to assign protein domains to certain function by analysis with biochemi-cal and imaging techniques. Similarly, we will use species differences to investigate the interaction between BTN3 and BTN2A1 molecules and that of BTNs and TCR. In parallel TCR-BTN interaction shall be studied also in other species. To reconstruct TCR-BTN coevolution BTN-domains will be inserted into human molecules and test-eds for functionality. Compared will be BTNs and TCRs of human, alpaca, camels and the thirteen-lined squirrel, one of the few rodents possessing genes for BTN2, BTN3 and TCR-Vγ9. Finally, we want to learn whether Vγ9Vδ2 T cells from different species, namely human, alpaca and Old-World camelids, share common features apart from their TCR. To this end transcriptomics of Vγ9Vδ2 T cells from different species will be performed aiming to understand what makes a Vγ9Vδ2 T cell a Vγ9Vδ2 T cell apart from its TCR.