Eur. J. Immunol. 2008. 38: 1677–1688 Immunomodulation 1679 Figure 1. Influence of CD4pres and CD4abs on proliferation of naive T cells and skin graft rejection. (A) CD4+ cells precultured in vitro for 8 days with 5 lg/ml of anti-CD4 (YTS 177) (CD4pres) or without anti-CD4 antibody (CD4abs) were cultured with CD4+ responder T cells (2 105) from naive CBA mice and irradiated allogeneic B10 splenocytes (5 105). Proliferation was measured at day 5 by 3H-thymidine incorporation. Data are expressed as mean cpm + SD of triplicate cultures. The results shown are representative of four independent experiments. (B) CBA.Rag–/– mice were reconstituted with 2 105 CD4pres (*; n = 7) or CD4abs (&, n = 5). The next day mice received an allogeneic skin graft from B10 mice. Results were pooled from two independent experiments. (C) CBA.Rag–/– mice were reconstituted with 105 CD45RBhighCD4+ effector T cells from naive animals (control = MR mice; &, n = 9) or together with 2 105 CD4pres (*; n = 9) or CD4abs (~; n = 9). Results were pooled from five independent experiments. (D) CBA.Rag–/– mice were reconstituted with 105 CD45RBhighCD4+ effector T cells from naive animals (control = MR mice; &, n = 7) or together with 2 105 (*; n = 6), 105 (~; n = 5) or 5 104 CD4pres (*; n = 5). CD4pres can be expanded further in vitro but lose regulatory activity Recently, studies by several groups have suggested that regulatory T cells can be expanded in vitro without loss of function using typical T cell growth factors such as IL-2 and IL-15 [30, 31]. The percentage of cell recovery of CD4+ cells after 8 days in culture in the presence of B10 APC and anti-CD4 is around 30% of the starting population (Fig. 3A). Therefore, we tested whether the population of CD4pres could be expanded. After 8 days in culture with anti-CD4, CD4+ cells were restimulated with allogeneic splenocytes (B10) for a further 7 days in the presence of exogenous IL-2 (25 U/mL). Indeed, the addition of IL-2 resulted in an eightfold increase in the number of cells recovered from the cultures and the potential of the expanded CD4pres to prevent proliferation of naive CD4+ cells in vitro was not diminished (Fig. 3B). Furthermore, suppression was still as potent at very low ratios such as 1:50 (8.3% proliferation at 1:50 vs. 9.1% at 1:1, Fig. 3B). In contrast to the non-expanded anti-CD4 Treg, as few as 4000 expanded CD4pres were now sufficient to prevent prolifera- tion of naive CD4+ cells. Next, we investigated whether the expanded CD4pres were able to prevent skin graft rejection in vivo. All mice reconstituted with CD45RBhighCD4+ cells alone rejected their skin grafts acutely grafts (n = 8, MST = 14 days, Fig. 3C). Co-transfer of expanded CD4pres could not prevent skin graft rejection although rejection f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu