TCR sequencing of Treg and Tconv subsets from murine spleen

While unique subsets of Treg cells have been described in some non-lymphoid tissues, their relationship to Treg cells in secondary lymphoid organs and circulation remains unclear. We have identified a recirculating and highly suppressive effector Treg cell subset that expresses the α2 integrin, CD49b, and exhibits a unique tissue distribution. We used TCR sequencing to ask whether CD49b+ activated Treg cells and CD49b− activated Treg cells represent stable states of Treg cell differentiation instructed by distinct TCR repertoires. We did not find a pattern of clonotypes that was specific to either subset, suggesting that essentially all CD49b− Treg cells could give rise to CD49b+ cells. These results shed light on the identity and development of a functionally potent subset of mature effector Treg cells that recirculate through and survey peripheral tissues. Tcra cDNA libraries were prepared from FACS-purified splenic Tconv and Treg cells from Foxp3GFP Tcra+/− DO11.10tg mice. In these mice, the presence of the DO11.10 TCRβ transgene inhibits the recombination of the endogenous Tcrb loci, thus restricting TCR repertoire variability to TCRα chains. Further limitation of the TCR repertoire was achieved by the presence of only one functional Tcra gene. 2 CD4+ T-cell lineages x 3 activation states x 4 replicates = 24 samples.