1678 Vanessa Oliveira et al. cardiac allograft [16, 23–25]. The transplant provides a source of donor alloantigen indispensable for the persistence of the regulatory cells in vivo [26]. In this study, we have addressed whether a similar strategy can be used in vitro to select and expand CD4+ Treg. We show that alloantigen stimulation of total CD4+ or CD25–CD4+ naive cells in the presence of a non-depleting anti- CD4 antibody (YTS177) drives the selection of a population of Treg that express CD25, CD62L, CCR7 and Foxp3 and are capable of suppressing the proliferation and cytokine expression of naive responder T cells, in vitro. Most importantly, these Treg are able to prevent the rejection of an allogeneic skin graft mediated by CD45RBhighCD4+ cells when co-transferred into CBA.Rag–/– recipients. Interestingly, expansion of the in vitro selected CD4+ cells with regulatory activity in the presence of IL-2 resulted in a loss of their capacity to prevent allograft rejection in vivo, although they retained their suppressive properties in vitro. This in vitro approach provides a complementary strategy to in vivo selection of Treg for controlling rejection and illustrates the tolerogenic potential of non-depleting anti-CD4 antibodies. Results Alloantigen stimulation in the presence of anti-CD4 antibody selects CD4+ cells with regulatory properties in vitro and in vivo Here, we asked whether in vitro blockade of CD4 signals to T cells at the time of alloantigen recognition, i.e. in the presence of allogeneic APC, would result in the selection of a population of CD4+ cells with suppressive properties. Purified, unfractionated CD4+ cells from naive CBA mice were cultured with irradiated, allogeneic splenocytes from B10 mice in the presence of 5 lg/mL anti-CD4. After 8 days in culture, their suppressive properties were investigated by coculturing the cells with naive, syngeneic CD4+ cells in the presence of allogeneic B10 splenocytes. As illustrated in Fig. 1A, CD4+ naive T cell responders failed to proliferate when cocultured with CD4+ cells precultured with anti-CD4 (CD4pres). At this ratio, CD4+ cells precultured without anti-CD4 (CD4abs) can also inhibit the proliferation of naive responders, demonstrating that CD4pres or CD4abs could suppress the proliferation of naive CD4+ responders in vitro. Differences between these two populations are detected at lower ratios (1:20) when CD4abs loose their potential to suppress proliferation. In vitro suppression by CD4pres may result from its ability to inhibit IL-2 and IFN-c production by naive CD4+ cells (data not shown), however other mechanisms may be involved as CD4pres express and secrete IL-10 and IFN-c (data not shown). Next, we tested whether CD4pres have lost their potential to act as effector T cells, i.e. the ability to initiate rejection. CD4pres (2 105) or the same number of CD4abs (control culture) were adoptively transferred into CBA.Rag–/– mice and the following day a B10 skin allograft was transplanted. Six out of seven mice reconstituted with CD4pres accepted their skin grafts (n = 7, median survival time (MST) >100 days, Fig. 1B), whereas three Eur. J. Immunol. 2008. 38: 1677–1688 out of five mice reconstituted with CD4abs rejected their skin grafts acutely (n = 5, MST = 24 days, p>0.05, Fig. 1B). Statistically, when compared to CBA.Rag–/– mice reconstituted with CD45RBhighCD4+ naive cells, both CD4pres and CD4abs have lost their potential to act as effector T cells in vivo. To assess the in vivo regulatory potential of the cultured CD4+ cells, CBA.Rag–/– mice were reconstituted with 105 CD45RBhighCD4+ naive T cells and 2 105 CD4pres or CD4abs. CD4pres prevented rejection in seven out of nine mice (n = 9, MST >100 days, p<0.0001 vs. MR), whereas most mice co-reconstituted with CD4abs cells rejected the graft (n = 6, MST = 31 days, p<0.04 vs. 2 105 CD4pres) (Fig. 1C). From these results we can conclude that the presence of anti-CD4 during in vitro culture enhances the regulatory capacity of CD4+ cells. Next, the minimum cell number to achieve in vivo regulation was determined. Therefore, varying cell numbers of CD4pres were co-transferred with 105 CD45RBhighCD4+ naive T cells into CBA.Rag–/– mice the day before transplantation of a B10 skin graft. Co-transfer of 2 105 CD4pres led to permanent acceptance of B10 skin grafts in four out of six mice (n = 6, MST = 94 days, p<0.05 vs. MR, Fig. 1D). Interestingly, transfer of 105 or 5 104 CD4pres could still prevent rejection in a proportion of recipients (n = 5, MST = 49 days; n = 5, MST = 57.6 days, respectively. Fig. 1D). These results indicate that although both CD4abs and CD4pres are unable to induce graft rejection in the absence of an effector population, only CD4pres can regulate skin graft rejection mediated by CD45RBhighCD4+ cells in vivo. CD4pres exhibit antigen specificity It has been shown that regulatory T cells require activation through T cell receptor in order to manifest functional activity but thereafter are nonspecific, i.e. once these cells are activated [27, 28]. Therefore, we wanted to test whether T cells with regulatory activity, CD4pres, could suppress the proliferation of naive T cells in an antigen specific or nonspecific fashion. CD4pres were cultured with CD4+ cells from naive mice in the presence of either B10 or BALB/c allogeneic APC (Fig. 2A). Differential suppression, twofold, of responses to B10 or BALB/c stimulation was only detectable at very low ratios, 1:20 (Fig. 2A). These results showed that the response to third party antigen is not suppressed by CD4pres to the same extent as the response to cognate antigen. Similar results were recently reported by Yamazaki et al. [29]. Next, we investigated the specificity of regulation by CD4pres in vivo. CD4pres precultured in the presence of B10 APC were unable to prevent the rejection of a BALB/c skin graft (n = 6, MST = 26.3 days, BALB/c skin grafts, vs. n = 6, MST = 94 days, B10 skin grafts; p<0.05, Fig. 2B). Rejection of third party BALB/c skin graft in mice co-transferred with CD4pres and CD45RBhighCD4+ cells was not significantly different from rejection in mice reconstituted with CD45RBhighCD4+ cells alone (n = 5, MST = 23.4 days, p = 0.08, Fig. 2B). Hence, CD4pres can suppress the proliferation of naive responders to third party stimulation in vitro but are unable to prevent the rejection of a third party skin graft in vivo. f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu