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 naïve 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.