Eur. J. Immunol. 2008. 38: 1677–1688 Immunomodulation 1683 Figure 5. In vivo regulation is enriched within the CD62L+CD25+CD4+ subpopulation. (A) CD4pres or CD4abs were fractionated into three subpopulations based on surface expression of CD25 and CD62L. (B) CBA.Rag–/– mice were reconstituted with 105 CD45RBhighCD4+ effector T cells alone (control = MR mice; &, n = 9) or together with 2 105 of the following purified cell populations: CD62L+CD25+CD4+ cells from CD4pres (*; n = 11), CD62L–CD25intCD4+ cells from CD4pres (*; n = 5), CD62L+CD25–CD4+ cells from CD4pres (&; n = 5), CD62L+CD25+CD4+ cells from CD4abs (*; n = 5), CD62L–CD25intCD4+ from CD4abs (*; n = 6) and CD62L+CD25–CD4+ cells from CD4pres (&; n = 5). The next day the mice received an allogeneic skin graft from B10 mice. These results were pooled from four independent experiments. (C) CBA.Rag–/– mice were reconstituted with 105 CD45RBhighCD4+ syngeneic T cells from naive animals (MR mice) or together with CD62L+CD25+CD4+ cells from CD4pres cultures. 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 CD62L+CD25+CD4+ cells prevented rejection in five out of five mice receiving B10 skin grafts (*; n = 5) and three out of five of the mice receiving BALB/c skin grafts (*; n = 5). Results were pooled from three independent experiments. they completely lost regulatory capacity in vivo (Fig. 3B and C). This loss of functional activity in vivo may result from the dramatic decrease in the percentage of cells co-expressing CD25 and CD62L and functional changes of the CD62L–CD25intCD4+ population after expansion with IL-2 (Fig. 4A). In vivo regulatory potential resided mainly in the CD62L+CD25+CD4+ subpopulation purified from either the CD4pres or CD4abs cultures, but there was also evidence for regulatory activity in CD62L–CD25intCD4+ of CD4pres cultures (Fig. 5B). The percentage of CD62L+ or CCR7+ cells in the CD25+CD4+ population is higher in cultures in the presence of anti-CD4 (Fig. 4B) and the anti-CD4 treatment results in functional changes of CD62L–CD25intCD4+cells, which again demonstrates the important role of this antibody for the in vitro enrichment and selection of Treg. CD62L+CD25+CD4+ cells from CD4pres cultures exhibited higher percentages of FoxP3 expression than either CD62L+CD25–CD4+ or CD62L+CD25intCD4+ cells (Fig. 6A). The percentage of FoxP3+ cells found in CD62L–CD25intCD4+ varied between experiments, but it was always lower than that found in CD62L+CD25+CD4+ cells. In vivo, CD62L+CD25+CD4+ cells from CD4pres cultures were detected both in the draining lymph node and in the skin graft 7 days after transplantation, but were not detected in the spleen of reconstituted CBA.Rag–/– mice (data not shown). These results are in accordance with f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu