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

γδT细胞抗原受体(γδTCR)与其他依赖RAG的抗原受体一起出现，几乎存在于所有有颌骨脊椎动物中。γδT细胞受体(γδT细胞)的临床应用越来越受到人们的关注，但对γδT细胞抗原受体(TCR)的抗原及其识别方式却知之甚少。γδT细胞在表型和功能上有很大的差异，γδT细胞亚群仅存在于某些物种中，通常由其V基因的使用来定义。其中一个亚群是人类Vγ9Vδ2 T细胞，其同名TCR感应所谓的磷酸抗原(PAG)，即合成异戊二烯的磷酸化宿主或微生物代谢物。最强的天然PAg是hMBPP，它在结核病和疟疾等感染中驱动Vγ9Vδ2 T细胞的激活和扩增。一种相当弱的宿主细胞产生的PAG是IPP，它积聚在一些肿瘤细胞中或在氨基双膦酸盐(例如唑来膦酸盐)治疗后积聚。这种IPP的积聚使细胞对Vγ9Vδ2 T细胞的杀瘤活性敏感。20多年来，V-γ-9V-δ-2 T细胞被认为仅限于高等灵长类动物，但我们在羊驼中发现了这种细胞，尽管这种细胞在小鼠啮齿动物中是缺失的。Vγ9Vδ2TCR不直接与PAG结合，但能感应PAG与BTN3分子胞内区结合所引起的宿主细胞变化。在人类中，BTN3A1与其亚型BTN3A2和BTN3A3协同作用，而羊驼只有一个BTN3，整合了这三种亚型的功能。除BTN3A1外，靶细胞表达BTN2A1对于PAG介导的Vγ9Vδ2T细胞活化也是必不可少的。然而，TCR介导的激活过程中涉及的分子事件和信号级联却知之甚少。我们将创建BTN3嵌合分子和突变体，以确定PAG传感的最低分子要求，并通过生物化学和成像技术的分析将蛋白质结构域分配给特定的功能。同样，我们将利用物种差异来研究BTN3和BTN2A1分子之间的相互作用，以及BTN和TCR之间的相互作用。同时，还应研究TCR-BTN在其他物种中的相互作用。为了重建TCR-BTN共同进化，BTN-结构域将被插入到人类分子中并测试其功能。我们将比较人类、羊驼、骆驼和十三纹松鼠的BTN和TCRs，十三纹松鼠是少数几种拥有BTn2、BTN3和TcR-Vγ9基因的啮齿动物之一。最后，我们想要了解来自不同物种的Vγ9Vδ2 T细胞，即人、羊驼和东半球骆驼的TCR，除了TCR外，是否有共同的特征。为此，我们将对不同物种的Vγ9Vδ2 T细胞进行转录转录，以了解是什么使Vγ9Vδ2 T细胞成为Vγ9Vδ2 T细胞。

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.