1682 Vanessa Oliveira et al. Figure 4. CD4+ cells cultured for 8 days in the presence of anti-CD4 exhibit a higher percentage of CD62L and CCR7 cells. (A) CD4+ cells from naive CBA mice and CD4pres before and after IL-2-mediated expansion were stained with anti-CD4-PerCP, anti-CD62L-PE and anti-CD25-biotin and streptavidin-APC. Plots of CD62L vs. CD25 expression gated on CD4+ cells are shown. The data shown are representative of seven independent experiments. (B) The expression of CCR7 is shown gated in the CD4+CD25+ population. Numbers inside the histogram indicate the percentage of cells that stained positive for CD4, CD25 and CCR7. The data shown in (A) and (B) are representative of two independent experiments. accordance with the regulatory potential found in vivo for each of these cell populations (Fig. 5B). When studying FoxP3 expression in CCR7+CD25+CD4+, CCR7–CD25intCD4+ and CCR7+CD25– CD4+ cells from CD4pres we found similar results (68.4, 33.3 and 0.4%, respectively, Fig. 6A). The same pattern was found for subpopulations sorted from CD4abs (data not shown). Taken together, these results indicate that FoxP3+ cells reside mainly in the CD62L+CD25+CD4+ or CCR7+CD25+CD4+ subpopulation. Despite this, CD62L–CD25intCD4+ cells are also able to regulate rejection of a skin allograft, which may give us the indication that CD25 expression and other factors are more important for in vivo regulation than CD62L. This assumption is further supported by our observation that the CD62L+CD25+CD4+ subpopulation can be found in the draining lymph nodes and in the skin allograft and does not prevent in vivo migration or proliferation of CD45RBhighCD4+ cells (data not shown). Eur. J. Immunol. 2008. 38: 1677–1688 Induced regulatory T cells can be differentiated from CD25–CD4+ naive T cells. We have also performed experiments using CD25–CD4+ cells from naive CBA mice as responders in our anti-CD4 mAb-treated mixed lymphocyte culture. Using our established protocol we were able to generate 7.4% CD62L+CD25+Foxp3+ cells after 8 days of culture (Fig. 6B). In contrast, in control cultures 0.9% of the CD25–CD4+ cells became CD62L+CD25+Foxp3+. We also tested their in vivo regulatory capacity. CBA.Rag–/– mice were recon- stituted with 105 CD45RBhighCD4+ cells either alone or together with 2 105 cells from unfractionated CD4abs or CD4pres cultures set up using CD25–CD4+ T cells. The following day, mice received a B10 skin graft. Co-transfer of CD4pres cells resulted in a prolongation of skin graft survival (MST SD: 24.8 3.9 days, p=0.009) in comparison to recipients reconstituted with CD45RBhighCD4+ cells only (MST SD: 16.5 3.5 days, Fig. 6C). Whereas co-transfer of CD4abs cells resulted in accelerated skin graft rejection (MST SD: 13 0 days). These data clearly indicate that anti-CD4 mAb treatment during mixed lymphocyte cultures facilitates both a selective expansion of initial natural CD4+CD25+Foxp3+ regulatory T cells but also a differentiation of alloantigen-specific induced regulatory T cells, which makes the anti-CD4 mAb treatment-based protocol even more attractive for future clinical applications. Discussion Based on an established in vivo protocol known to select alloantigen-reactive CD25+CD4+ regulatory T cells in vivo [16, 23], we investigated whether a similar strategy would result in the selection of T cells with regulatory potential in vitro. Such an in vitro approach is attractive, as the ex vivo generated regulatory cells could be used throughout the post-transplant course to enhance or sustain the unresponsive state achieved by the in vivo therapy. In this system, mouse CD4+ cells were purified and cultured for 8 days with alloantigen in the form of allogeneic APC and in the presence of a non-depleting anti-CD4 mAb. This in vitro combination led to the selection, of CD4+ cells, designated CD4pres, secreting high amounts of IL-10 and IFN-c (data not shown) that are able to suppress the proliferation of naive syngeneic T cells (Fig. 1A). More importantly, CD4pres are able to prevent rejection mediated by co-transfer of naive T cells (Fig. 1B and C). This (lack of rejection) cannot be explained by an activation induced anergy of CD4pres, as rejection occurs in CBA.Rag–/– reconstituted with CD45RBhighCD4+ naive T cells only. Active regulation is required to prevent rejection mediated by naive T cells. We believe that culture of naive CD4+ cells with alloantigen in the presence of anti-CD4 results in an enrichment and selection of Treg. It is generally thought that protocols generating Treg for clinical application need to have the capacity to produce large numbers of cells that retain their regulatory activity in vivo. When we expanded CD4pres further in vitro, we found that while the expanded population retained potent suppressor function in vitro, f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu