Antigen recognition and thymic maturation of human TCR Vgamma9-Vdelta2 cells

T cells are divided into two populations according to the type of TCR used for antigen recognition. One population uses a TCR heterodimer, which is composed by the non-covalently associated alpha and beta chains. This TCR recognizes protein and lipid antigens, which are presented by MHC and CD1 antigen-presenting molecules, respectively. A second population uses a TCR heterodimer composed by the gamma and delta chains and recognizes nonpeptidic ligands in the absence of MHC and CD1 restriction. In humans the major population of TCR gd cells uses the Vg9-Vd2 TCR. This is a unique population because it is present only in primates and constitutes >50% of peripheral TCR gd cells. TCR Vg9-Vd2 cells are activated by microbial phosphorylated metabolites and by so far unknown ligands expressed by a group of tumor cells. The principal microbial antigen is (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), an intermediate metabolite generated in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoids biosynthesis. Despite these cells were described in 1986, many aspects remain unclear, including the nature of the stimulatory ligands present in tumor cells, the mechanisms of their activation during infection, the molecular mechanisms involved in antigen presentation, and the requirements for thymic maturation. In this dissertation we have addressed these important issues using ex vivo cells, biochemical approaches for ligand identification, T cell activation assays and generation of transgenic mice expressing this human TCR. We have identified endogenous metabolites generated in the mevalonate pathway as the tumor ligands which stimulate TCR Vg9-Vd2 lymphocytes. We have found that tumor cells show altered mevalonate pathway which leads to accumulation of intermediate metabolites. This is novel mechanism utilized by the immune system to monitor the metabolic integrity of cells and to react to those which have a dysregulation of this important metabolic pathway. In a second series of studies we have investigated how TCR Vg9-Vd2 cells are activated during bacterial infections. Despite published studies identified HMB-PP as a potent stimulatory ligand in vitro, there was no formal evidence that this compound participates in cell activation during infection. Unexpectedly, we found that HMB-PP is not the major stimulatory ligand during infection and instead endogenous mevalonate metabolites are the stimulatory ligands. We describe how infection modifies the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), which is the key enzyme of the mevalonate pathway, and promotes increased synthesis of stimulatory metabolites. We show that infection induces a transient increase in HMGR protein levels and dephosphorylation, leading to increased enzymatic activity. This alteration occurs already within 1 hour after infection, thus representing a rapid mechanism reacting to infection. Thus, like with recognition of tumor cells, also during infection, the immune system of primates utilizes a mechanism which detects alterations of an important metabolic pathway. We also investigated the mechanisms how mevalonate metabolites traffic within cells. We found that these ligands, which are generated within the cytoplasm, are transported to the cell surface, where they interact with the TCR gd, by the MRP5 transporter. We showed that MRP5-blocking drugs inhibit presentation to TCR gd cells that over expression and knocking down of MRP5 protein increase and inhibit ligand presentation, respectively. These results show that like peptides, which are transported from cytoplasm to the ER through the ABC transporters TAP1 and TAP2, also TCR gd ligands utilize ABC transporters to become immunogenic. We also found that MRP5 is not involved in forming complexes presented to the TCR gd and that other unknown ubiquitous and nonpolymorphic molecules are involved in this process. In the last part of these studies we investigated the requirements for thymic maturation and peripheral expansion of TCR Vg9-Vd2 cells. We generated a transgenic (Tg) mouse model in which T cells express a TCR composed by human Vg9-Vd2 chains. Tg thymocytes express molecules characteristic of partially mature thymocytes together with high levels of Tg TCR. Tg cells do not acquire a mature phenotype and do not exit the thymus in the absence of TCR triggering. However, upon injection of TCR-specific mAbs, Tg thymocytes undergo maturation and colonize peripheral lymphoid organs. Mature Tg T cells remain in the periphery for up to 6 months, with a phenotype of naive T cells and strongly react to physiological ligands when stimulated by human antigenpresenting cells, which express the restriction element. Thus, Tg T cells expressing the human TCR Vg9-Vd2 resemble TCR ab cells since they also require selection events during thymic maturation.