Here

we investigated the mechanism of CD4+CD25+ T-cell-mediated regulation Selleck LBH589 by testing if increased numbers of hapten-presenting DC, including LC, in skin-draining LN accompanies the increased effector CD8+ T-cell development and CHS responses in anti-CD25 mAb treated mice. When anti-CD25 mAb was given before and during sensitization with FITC, the percentages of FITC-bearing DC identified as the CD11c+FITC+ population as well as the percentages of FITC-bearing LC identified as the CD207+FITC+ cells were increased two-fold on day 3 post-sensitization (Fig. 1A, gate R5: 0.54±0.03% of FITC+ DC in control group versus 1.10±0.02% in anti-CD25 mAb-treated group, and, gate R2: 0.22±0.04% versus 0.40±0.05% of FITC+ LC respectively, p<0.02). Similarly, the total numbers of FITC-presenting cells within both total DC and LC populations were increased two-fold in the skin-draining LN of FITC-sensitized mice treated with anti-CD25 mAb (Fig.

Nutlin-3a in vivo 1B, *p<0.05). In contrast, anti-CD25 mAb treatment had no significant impact on the percentages of FITC− DC (Fig. 1A, gates R4 and R3). Therefore, inhibition of regulatory CD4+CD25+ T-cell activity increased the numbers of hapten-presenting DC in the T-cell priming site. Our previous studies indicated that the survival of hapten-presenting DC in skin-draining LN during T-cell priming is restricted through Fas–FasL interactions 1. To begin to study the contribution of CD4+CD25+ regulatory T cells to this mechanism, we tested the expression of Fas on hapten-presenting DC activated during hapten sensitization versus residential DC in the LN. Total DC were purified from the skin-draining LN of FITC-sensitized mice 24 h post-sensitization using positive selection of CD11c+ cells. During co-culture these purified DC activated hapten-specific, but not naïve, CD8+ T cells to produce IFN-γ indicating the presence of hapten-presenting DC in this cell population (data not shown). Purified

DC were stained with PE-labeled anti-Fas mAb and then CD11c+FITC− cells or CD11c+FITC+ cells were gated using CD11c+FITC− cells from naïve mice as a control (Fig. 2A, gates R2 and R3, respectively) and then the levels of Fas expression HAS1 by FITC+ and FITC− DC were quantified as MFI of the PE channel. The majority of DC isolated from the LN of sensitized mice expressed Fas, however, the expression of Fas was increased more than four-fold on FITC-presenting DC when compared with FITC− residential DC (MFI=434.0±11.3 for FITC+ DC versus 92.7±6.9 for FITC− DC, p<0.01). The percentages of DC expressing high levels of Fas were increased three-fold in the FITC+ DC population (67%) in comparison with the FITC− DC (22%) (Fig. 2A). Next, we evaluated the expression of FasL on regulatory CD4+CD25+ T cells versus CD4+CD25− T cells.