In mLNs, available MHC class II presented antigen may also comprise considerable proportions of intestinal antigen derived from food and bacterial flora. Therefore, we investigated the TCR sequence overlap of re-isolated donor Treg cells from spleen, pLN, mLN, and LPL (lamina propria lymphocytes) 9 wk after adoptive transfer of WT Treg cells as described for Fig. 2. We were able to analyze several thousands of

recovered Treg cells and revealed strikingly overlapping Tcra rearrangements in mLN and intestinal LPL (Fig. 5A). Comparing the 25 most abundant CDR3 sequences from each tissue, we found that mLN and LPL samples shared 14 out of 25 identical AA sequences, whereas only one was similar between pLN and mLN or pLN and LPL CCR antagonist (Fig. 5B and Table 1). Next, we asked to what extent such organ-specific expansion would be specific for Treg cells as compared with Foxp3− T cells. Therefore, we performed adoptive transfers of either pLN or mLN whole lymphocyte suspensions from CD45.1− WT mice into CD45.1+ TCR-Tg recipients (Fig. 6A). The percentage of input Foxp3+ Treg cells among all CD4+-gated T cells was similar in both cell suspensions. Nine wks after transfer of pLN cells, the frequency of Treg cells among all CD45.1−CD4+ input T cells was assessed. It had increased in spleen, pLN, and mLN (Fig. 6A and B), which is in line

with the Treg-cell expansion after transfer of purified Treg cells shown above. A decreased proportion among LPL may reflect antigen-specific expansion of Foxp3−CD4+ T cells. At the same time, transfer of mLN cells resulted in stable proportions of Treg cells in LPL and elevated frequencies in both mLN and Staurosporine pLN (Fig. 6A and B). Interestingly, expansion of mLN-derived Treg cells was similar in pLN and mLN, although lower than the expansion after transfer of pLN suspensions

(Fig. 6B). In conclusion, these results suggested that, besides homing receptor cues, organ-specific TCR shaping created distinct, highly before diverse but still overlapping TCR repertoires in pLNs and mLNs. After transfer, such locally optimized TCR repertoires supported the maintenance of donor Treg cells in their respective organs of origin. Next, we investigated the impact of Treg-cell repertoire diversity on their genuine function, i.e. their capacity to suppress T-cell activation. In an in vitro system based on T-cell activation with anti-CD3 mAb, Treg cells from TCR-Tg mice were equally efficient as Treg cells from WT mice in suppressing the proliferation of CD8+ and of CD4+ T cells (Fig. 7A and B). In contrast, in an experimental model of acute GvHD 35 less diverse Treg cells were less efficient than WT Treg cells in preventing the lethal disease (Fig. 7C and D). Co-transfer of allogeneic Treg cells derived from OT-II TCR-Tg mice showed only alleviation of the disease but not protection from GvHD (Fig. 7C and D). Taken together, these results suggest that the impact of TCR diversity on Treg-cell function is context dependent.