1686 Vanessa Oliveira et al. Eur. J. Immunol. 2008. 38: 1677–1688 (Dynal A.S., Oslo, Norway). CD8– and MHC class II– cells were isolated by magnetic separation, the CD4+-enriched population resuspended and stained for CD4 and CD45RB. CD45RBhighCD4+ cells with 99% purity were obtained by cell sorting using a FACSVantage (Becton Dickinson, San Jose, CA). Cell culture RPMI 1640 culture medium was supplemented with 10% FBS, 2 mM L-Glutamine, 0.5 mM 2-mercaptoethanol, and 100 U/mL each penicillin, streptomycin and kanamycin. Irradiated (3600 rad) allogeneic total splenocytes from B10 mice were used as APC (B10 APC). Purified CD4+ cells (mouse CD4+ isolation kit Miltenyi Biotec, Bergisch-Gladbach, Germany) from naive CBA mice were cultured at 2 105 cells/well in the presence (CD4pres) or absence (CD4abs) of 5 lg/mL of YTS 177 anti-CD4 mAb together with 5 105 APC. Cells were cultured for 8 days, harvested, debris separated from live cells by gradient centrifugation using lymphoprep medium (Axis-Shield PoC AS, Oslo, Norway) and used in a regulation assay. In some experiments, CD4+ cells precultured with anti-CD4 (5 104) were restimulated with 5 105 APC in the presence of 25 U/mL human IL-2 (Roche) (expanded CD4pres). Proliferation of naive responders was assessed by [3H]thymidine (0.5 lCi) incorporation or CFSE dilution after 5 or 7 days in culture, respectively. Cell reconstitution and skin transplantation CBA.Rag 1–/– mice were reconstituted intravenously with syngeneic fractionated T cells. CD45RBhighCD4+ cells (105) purified from naive CBA mice were used routinely to elicit rejection of an allogeneic skin graft [16]. The day after reconstitution, all mice received a B10 skin graft as previously described [16]. Graft rejection was defined as complete destruc- tion of the skin graft. Flow cytometric analysis Cell cultures were incubated with the appropriate antibodies. All incubations were carried out for 20 min at 4 C. Data were acquired using a FACSort (Becton Dickinson, San Jose, CA) and analysed using CellQuest software package (Becton Dickinson). Intracellular staining for FoxP3 studies was performed according to the manufacturer's instructions. Statistics The Log Rank Method was used to compare allograft survival between groups using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). The data were analysed using a two-tailed unpaired Student's t-test. Results are given as the mean SD. p-values of <0.05 were considered significant. Acknowledgements: The authors thank Dr. M. Karim for cell sorting and the BMS-JR staff for expert care of mice used for this study. V. O. received a grant from FCT, Portugal (Fellowship SFRH/BD/5106/2001); B. S. was a Wellcome Trust Travelling Fellow. 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