1680 Vanessa Oliveira et al. Eur. J. Immunol. 2008. 38: 1677–1688 Figure 2. In vitro and in vivo third party responses of CD4pres. (A) CD4pres were cultured at the indicated ratios with CD4+ responder T cells (2 105) from naive CBA mice and irradiated allogeneic splenocytes either from B10 mice or from third party (BALB/c) mice (5 105). Cells were cultured for 5 days and proliferation was measured by [3H]-thymidine incorporation. Data are expressed as mean cpm SD of triplicate cultures and are representative of three independent experiments. (B) CBA.Rag–/– mice were reconstituted with 105 CD45RBhighCD4+ syngeneic T cells from naive animals (MR mice) or together with 2 105 CD4pres. The next day, mice received an allogeneic skin graft from B10, or third party BALB/c, mice. MR mice acutely reject B10 (&, n = 7) and BALB/c skin grafts (&, n = 5). Co-transfer of 2 105 CD4pres prevented rejection in four out of six mice receiving B10 skin grafts (*, n = 6) whereas none of the BALB/c skin grafts were accepted (*, n = 7). Results were pooled from two independent experiments. occurred with a delayed kinetic (n = 8, MST = 11 days, p>0.05, Fig. 3C). These results indicate that although the expanded CD4pres show a high regulatory potential in vitro, they were unable to prevent skin graft rejection in vivo. Cells with highest regulatory potential are present within the CD62L+CD25+ CD4+ subpopulation Lymphocyte extravasation from the blood circulation into high endothelial venules and subsequent entry into the secondary lymphoid organs involves a series of step-wise events, which involve CD62L and CCR7 [32]. The mAb, which block the interaction of CD62L with its ligands, prevent indefinite allograft survival and highlight the importance of this molecule for alloantigen tolerance [33]. To understand the differences in regulatory potential in vivo between expanded and non-expanded CD4pres, the profile of cell-surface expression of both T cell populations was analysed. Cells were harvested after 8 days in culture (non-expanded CD4pres) or after a further 7 days in culture in the presence of IL-2 (expanded CD4pres) and stained with antibodies recognising CD62L and CD25. Of all non-expanded CD4pres, 36% expressed both CD25 and CD62L, whereas only 11% of the expanded population could be stained with both antibodies (Fig. 4A). These findings together with published studies from other groups [34–37] prompted us to investigate if this difference in CD62L expression could explain the results obtained in vivo (Fig. 1C and 3C). We analysed CD62L expression in CD25+CD4+ cells from CD4pres and CD4abs and found that 83 and 68% of cells, respectively, expressed CD62L. This surface marker is known to be necessary for cells to migrate in and out of lymph nodes. Following this observation, we decided to study CCR7 expression in CD25+CD4+ cells from CD4pres and CD4abs and found that, whereas 58% of CD25+CD4+ cells from CD4pres express CCR7, this percentage decreased to 17% in CD25+CD4+ cells from CD4abs (Fig. 4B). Based on these observations, we investigated whether CD62L+CD25+CD4+ cells purified from CD4pres or CD4abs showed regulatory activity in vivo. Cells were separated by cell sorting into three cell populations according to their surface expression of CD25 and CD62L: CD62L+CD25+, CD62L–CD25int and CD62L+CD25– (Fig. 5A). CBA.Rag–/– mice were reconstituted with 105 CD45RBhighCD4+ cells either alone or together with 2 105 of each purified population from CD4pres or CD4abs (Fig. 5B). The following day, mice received a B10 skin allograft. In vivo regulatory properties resided in the CD62L+CD25+CD4+ popula- tion, regardless of their previous culture with (n = 11, MST >100 days, p<0.0001 vs. MR; CD4pres) or without anti-CD4 (n = 3, MST >100 days, p<0.001 vs. MR, CD4abs), as seen by long-term graft survival in all mice reconstituted with this population and CD45RBhighCD4+ cells. In contrast, all mice receiving CD62L+CD25–CD4+ cells and CD45RBhighCD4+ cells rejected their skin allografts with the same tempo as MR mice (Fig. 5B), irrespective of previous culture in the presence (n = 5, MST = f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu