Vγ9Vδ2 T cells are potent effectors of human immune responses against pathogens and tumors. Their T cell receptors (TCR) recognize small pyrophosphorylated molecules (phosphoantigens) which are products of isoprenoid-synthesis of host or microbes. This recognition requires expression of the cell surface molecule butyrophilin 3A1 (BTN3A1) and its isoforms BTN3A2 and BTN3A3 by the tumor or the antigen-presenting cell. With the exception of primates, most mammalian species including all small animal models lack BTN3 molecules and Vγ9Vδ2 T cells. We have demonstrated that genes for Vγ9Vδ2 TCR and BTN3 co-evolved with the emergence of placental mammals and that the new world camelid alpaca (Vicugna pacos) possesses phosphoantigen-specific Vγ9Vδ2 T cells and only a single BTN3 isoform. In this project we aim at understanding the molecular basis of BTN3 and Vγ9Vδ2 TCR function by analyzing and comparing camelid and human Vγ9Vδ2 T cells. Such knowledge will help to improve Vγ9Vδ2 T cell based therapies, e.g. by engineering cells with tumor specific Vγ9Vδ2 TCR or by modulating overshooting Vγ9Vδ2 T cell responses in infections. We plan to identify general structural features which allow Vγ9Vδ2 TCRs to respond to phosphoantigens and commonalities of Vγ9Vδ2 T cells beyond expression of the Vγ9Vδ2 TCR. To this end we will compare Vγ9Vδ2 TCR in phosphoantigen-responses of alpaca and human blood lymphocytes and gene expression profiles (transcriptomes) between Vγ9Vδ2 T cells of both species. Sites mandatory for BTN3 function and the BTN3/TCR interaction as well as for interaction between BTN3 isoforms will be identified by testing the reactivity of alpaca and human molecule chimeras and BTN3 and TCR mutants with help of newly generated reporter cell lines. Finally, we want to use our expertise in camelid γδ T cells for the analysis of the enigmatic somatic hypermutation (SHM) within γδ TCRs of the dromedary (Camelus dromedarius). Here we aim to learn whether SHM serves as mechanism to increase γδ TCR diversity during γδ T cell development in the thymus or to increase affinity of γδ TCR (antigen receptor) analogous to affinity maturation of antibody producing B cells. As part of the FOR2799 (”Receiving and Translating Signals via the γδ T Cell Receptor”) the project will benefit and contribute from the collaborations with the other projects and finally provide a better insight into the role of γδ T cells in anti-tumor and anti-viral immunity.