Gorbachev, unpublished observations). Anti-CD25 mAb treatment of mice receiving CYC202 nmr WT DC increased hapten-specific CD8+ T-cell activation, while blockade of CD4+CD25+ T-cell activity did not affect hapten-specific CD8+ T-cell activation in recipients of lpr DC. Finally, CD4+CD25+ T cells suppressed the activation of hapten-specific CD8+ T cells cultured with WT but not lpr DC, indicating that negative regulation of effector CD8+ T-cell activation was mediated through effects on Fas-expressing DC but not on Fas-expressing CD8+ T cells. Together these results indicate

that CD4+CD25+ T cells regulate the priming functions of hapten-presenting DC in CHS through Fas–FasL interactions. The ability of regulatory CD4+CD25+ Akt inhibitor T cells to express FasL and kill Fas-expressing target cells has been previously reported 19, 26, 27. This report is the first, to our knowledge, demonstrating the ability of these regulatory cells to restrict DC priming

functions in CD8+ T-cell-mediated immune responses through a Fas–FasL-dependent mechanism. Furthermore, CD4+CD25+ T cells suppress CD8+ T-cell-mediated CHS responses in a non-specific manner. CD4+CD25+ regulatory T cells activated by hapten sensitization restricted the ability of LC activated by subsequent sensitization with a non-related hapten to activate CD8+ T cells specific to the latter hapten. These results are consistent with studies demonstrating non-antigen-specific suppression of T-cell-mediated autoimmune gastritis and viral responses by CD4+CD25+ regulatory T cells 28, 29. The current report further supports the hypothesis that previously activated CD4+CD25+ regulatory T cells can exert non-specific suppressor functions 28. Collectively, these studies reveal the restriction of cutaneous DC priming functions in the skin-draining LN through Fas–FasL interactions as a mechanism employed by CD4+CD25+ T cells to regulate

effector CD8+ T-cell development and expansion during cutaneous immune responses. The findings may be also applicable to the understanding of immunoregulation of other T-cell-mediated immune responses. WT and lpr female mice on the C57BL/6 background were purchased from The Jackson Laboratory G protein-coupled receptor kinase (Bar Harbor, ME). All animal experiments were performed according to the National Institutes of Health Guides for the Care and Use of Laboratory Animals and all protocols were approved by the Institutional Animal Care Use Committee (IACUC) of The Cleveland Clinic. DNFB and FITC were purchased from Sigma (Sigma Chemical, St. Louis, MO). mAb for the capture and detection of IFN-γ in ELISPOT assays, PE-labeled and biotin-labeled hamster isotype control Ab, anti-CD11c, anti-Fas and anti-FasL mAb MFL3, and streptavidin-APC, streptavidin-PE and streptavidin-FITC were purchased from BD Bioscience (San Diego, CA). AlexaFluor 647-labeled mAb RMUL.