Dominant tolerance to self-antigen requires the presence of sufficient numbers of CD4(+) Foxp3(+) Treg cells with matching antigen specificity. However, the size and role of TCR repertoire diversity for antigen-specific immuno-regulation through Treg cells is not clear. Here, we developed and applied a novel high-throughput (HT) TCR sequencing approach to analyze the TCR repertoire of Treg cells and revealed the importance of high diversity for Treg-cell homeostasis and function. We found that highly polyclonal Treg cells from WT mice vigorously expanded after adoptive transfer into non-lymphopenic TCR-transgenic recipients with low Treg-cell diversity. In that system, we identified specific Treg-cell TCR preferences in distinct anatomic locations such as the mesenteric LN indicating that Treg cells continuously compete for MHC class-II-presented self-, food-, or flora-antigen. Functionally, we showed that high TCR diversity was required for optimal suppressive function of Treg cells in experimental acute graft versus host disease (GvHD). In conclusion, we suggest that efficient immuno-regulation by Treg cells requires high TCR diversity. Thereby, continuous competition of peripheral Treg cells for limited self-antigen shapes an organ-optimized, yet highly diverse, local TCR repertoire.
