1− donor cells nor to a significant conversion into Foxp3+ WT Treg cells (Fig. 2B, right panel). These results

could be confirmed in a different experimental system by employing luciferase-expressing Treg cells 36 with a WT TCR repertoire. Monitoring Foxp3-specific light emission clearly showed similar WT Treg-cell expansion in OT-II TCR-Tg hosts over time (Fig. 2C). In addition, the same effect of efficient competition with the less diverse endogenous host Treg cells was observed after transfer into a different strain of TCR-Tg hosts, namely OT-I (Fig. 2C). Furthermore, WT donor Alvelestat supplier Treg-cell frequency in TCR-Tg hosts correlated to the input dose (Fig. 2D) and their expansion was associated with higher proliferation rates and an activated phenotype (Fig. 2E and F). Re-analysis of recovered donor Treg cells 2 months after transfer revealed a reduced but still highly diverse TCR repertoire when compared with similar numbers of the control group (Supporting Information Fig. 2). Therefore, sustained survival and expansion was not restricted to a small number of clones but included a broad set of donor Treg cells. This suggests that TCR diversity and continuous self-antigen recognition control the total size of the peripheral Treg-cell pool independently of homeostatic factors such as IL-2. Of note, exogenous administration of recombinant IL-2 increased Treg-cell proliferation and absolute numbers in both

WT and TCR-Tg mice (Supporting Information Fig. 3). Taken together, these adoptive transfer experiments revealed a hitherto unappreciated role for TCR diversity in Treg-cell homeostasis and imply that find more Cyclooxygenase (COX) it is probably a combination of TCR specificity and TCR-independent factors that determine on the one hand the competitive/homeostatic fitness of Treg cells and on the other hand the total pool/niche size. In principle, endogenous rearrangements in TCR-Tg mice were able to produce any potential TCR chain combination and thus there were no distinct gaps in their Treg-cell repertoire. However, we still observed a few qualitative differences on the Treg-cell population level. Jα-usage of the analyzed Vα8 family sequences in

Treg cells from TCR-Tg mice showed an increased proportion of the elements TRAJ5*01 and TRAJ34*02 (Supporting Information Fig. 4), while Jα-element usage was consistent in independent experiments for both types of Treg cells (Supporting Information Fig. 4). It is likely that this biased Jα-usage reflects preferential selection of Tcra rearrangements that can efficiently pair with the clonotypic Tcrb chain. Furthermore, productive VJ rearrangements in TCR-Tg mice included on average more non-templated N-nucleotides compared with WT Treg cells (6.679±0.079 versus 5.89±0.050 N-nucleotides; p<0.0001). Also, we found lower isoelectric point (pI) values of pH 9.289±0.029 for the Treg cells from TCR-Tg mice versus pH 9.473±0.021 (p<0.0001) in WT.