Eur. J. Immunol. 2008. 38: 1677–1688 Immunomodulation 1681 Figure 3. CD4pres can be expanded in vitro but loose their potential to prevent graft rejection. (A) CD4+ cells from naive CBA mice (2 105) were cultured for 8 days in the presence of anti-CD4 and 5 105 allogeneic splenocytes from B10 mice. Percentage of cell recovery was calculated by dividing the number of viable CD4+ cells obtained after 8 days in culture by the numbers of CD4+ cells at the start of the culture. Data represent the mean SD of more than 40 independent experiments. (B) Cells were harvested and 5 104 cells were restimulated for a further 7 days in the presence of 25 U/mL of human IL-2 (expanded CD4pres) and 5 105 irradiated allogeneic splenocytes from B10 mice. After restimulation, cells were cultured with CFSE-stained CD4+ responder T cells (2 105) from naive CBA mice and irradiated allogeneic B10 splenocytes (5 105). CFSE staining was used to measure proliferation of the naive responder T cells after 7 days in culture. Numbers inside the histograms indicate the percentage of cells that went through, at least, one round of division. The results shown are representative of four independent experiments. (C) CBA.Rag–/– mice were reconstituted with 105 CD45RBhighCD4+ effector T cells alone or together with 2 105 IL-2 expanded CD4pres. The next day, mice received an allogeneic skin graft from B10 mice. MR mice acutely rejected B10 skin grafts (&, n = 8). Co-transfer of 2 105 IL-2 expanded CD4pres did not prevent rejection of B10 skin grafts (*, n = 8). 19.0 days, p = 0.2 vs. MR for CD4pres) or absence (n = 5, MST = 22 days, p = 0.4 vs. MR for CD4abs) of anti-CD4. In contrast, CD62L–CD25intCD4+ cells exhibited different regulatory capacity depending on previous culture with (n = 5, MST >100 days, p=0.055 vs. co-transfer of CD62L+CD25+ T cells) or without anti- CD4 (n = 6, MST = 22 days, p=0.1 vs. MR) (Fig. 5B). Thus, p>0.05, Fig. 5C), indicating that this population can exert nonspecific regulation in vivo. Unfortunately, after expansion in IL-2, it was not possible to test the regulatory properties of the purified CD62L+CD25+CD4+ cells in vivo due to the low numbers of cells recovered from these cultures. Taken together, these results indicate that regulatory properties reside mainly in CD62L–CD25intCD4+ cells from CD4pres cultures could prevent CD62L+CD25+CD4+ cells purified either from CD4pres or CD4pres skin graft rejection mediated by CD45RBhighCD4+ cells in three but also in CD62L–CD25intCD4+ cells of CD4pres cultures. out of five mice. In contrast, none of the skin grafts survived permanently when CD62L–CD25intCD4+ cells from CD4abs cultures were co-transferred with CD45RBhighCD4+ cells. These data indicate that the anti-CD4 treatment results in phenotypic differences (higher frequency of CD62L and CCR7 positive cells) as well as functional changes (in vivo regulatory capacity of CD62L–CD25intCD4+ cells) of allo-stimulated CD4+ cells. Interestingly, even 5 104 CD62L+CD25+CD4+ cells from CD4pres were capable of regulating a skin transplant (data not shown). In fact, 2 105 CD62L+CD25+CD4+ cells were also able to regulate rejection of a skin transplant from BALB/c mice (third party) (n = 5, MST >100 days vs.MST >100 days, B10 skin grafts; The highest percentage of FoxP3+ cells can be found in CD62L+CD25+CD4+ and CCR7+CD25+CD4+ cells FoxP3 expression has been shown to characterize nTreg [38, 39] and to be up-regulated in transgenic T cells cultured with bone marrow dendritic cells and specific antigen [14]. For these reasons we studied FoxP3 expression in subpopulations sorted from CD4pres. As shown in Fig. 6A, CD62L+CD25+CD4+ cells exhibit higher levels of FoxP3 (45.5%) than CD62L–CD25intCD4+ (7.5%) and CD62L+CD25–CD4+ cells (0.9%) cells. These results are in f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu