1684 Vanessa Oliveira et al. Eur. J. Immunol. 2008. 38: 1677–1688 Figure 6. Foxp3 expression and in vivo regulatory capacity when using CD25–CD4+ naive T cells as responders. (A) CD4pres were stained with anti-CD4- FITC, anti-CD62L-PE or anti-CCR7-PE, anti-CD25-biotin and streptavidin-CyChrome and FoxP3-APC. The expression of FoxP3 is shown after gating in the indicated subpopulations from CD4pres. Numbers inside the histogram indicate the percentage of cells that stained positive for CD4, CD25, CD62L or CCR7 and FoxP3. The data shown are representative of two independent experiments. (B) Foxp3 expression gated on CD4+CD25+ cells of CD4pres and CD4abs cultures of CD25–CD4+ naive T cells. (C) CBA.Rag–/– mice were reconstituted with 105 CD45RBhighCD4+ effector T cells from naive animals (control = MR mice) or together with 2 105 CD4pres or CD4abs. MR mice acutely reject B10 skin grafts (&, n = 6; MST = 16.5 3.5 days). Co-transfer of 2 105 CD4pres prolonged skin graft survival (*, n = 5; MST = 24.8 3.9 days) whereas co-transfer of 2 105 CD4abs resulted in accelerated rejection (~, n = 3; MST = 13.0 0.0 days). observations from other groups showing that CD62L+CD25+CD4+ cells expressing CCR7 are able to delay diabetes transfer in NOD mice [36]. Expression of CD62L was also found to be important for the localization of regulatory cells into secondary lymphoid organs in models of cardiac transplantation [33] and graft-versus-host disease [40]. Furthermore, recently it was shown that CCR7 is required for the in vivo function of CD25+CD4+ regulatory T cells [41]. Thus, we can speculate that CD62L and CCR7 expression is required for regulatory cells to migrate into the lymph nodes and regulate the priming of naive T cells, but other factors induced by the anti-CD4 treatment are also important. To increase the amount of antigen-specific regulatory T cells for in vivo cell therapy, a protocol aimed on generating Treg should also enable differentiation of induced Treg from CD25–CD4+ naive precursors. It has been shown that polyclonal stimulation of naive T cells in the presence of TGF-b results in the differentiation of induced Treg [42, 43]. Our results demonstrate that also stimulation of CD25–CD4+ naive T cells with alloantigen in the presence of the non-depleting anti-CD4 antibody YTS177 enables differentiation of CD25+Foxp3+ induced Treg with high regula- tory potential in vivo. The ultimate challenge for our in vitro generation protocol would be to test the regulatory capacity of CD4pres cells upon transfer into fully immunocompetent recipients. Indeed, we are currently performing experiments in which we transfer CD4pres cells into otherwise untreated nave C57BL/6 mice receiving an allograft. f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu