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 antigenrecognition 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 defined by their V-gene usage. One of those subpopulations are human Vγ9Vδ2 T cells whose eponymous TCRs sense so-called phosphoantigens (PAgs), which are phosphorylated host or microbial metabolites of isoprenoid synthesis. The strongest natural 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 primates 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 BTN3A1 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 involved 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 biochemical 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 tested 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.