Human peripheral blood CD4+ T cells were stimulated with anti-CD3, IL-2, and IL-4 under conditions previously determined to optimally induce IL-10-Treg cells [12]. The expression of Foxp3 and IL-10 in the presence or absence of 1α25VitD3 was determined by flow cytometry. 1α25VitD3 at 10−6 M led to an increase in Foxp3 expression as compared with control cultures (No VitD3), whereas lower doses of 1α25VitD3 minimally affected Foxp3 expression. In contrast, Selleckchem Seliciclib maximal IL-10 induction was observed, as expected, at 10−7 M and 10−8 M 1α25VitD3 [12]. This response was confirmed

using a panel of donors. A statistically significant increase in the frequency of Foxp3+ T cells was observed at 10−6 M, but not at 10−7 M 1α25VitD3, while significant induction of IL-10+ T cells was seen at 10−7 M, but not at 10−6 M (Fig. 1A). In summary, 1α25VitD3 enhances both the percentage of Foxp3+ cells and the expression of Foxp3 transcripts (data not shown), but at a distinct concentration of 1α25VitD3 than required for optimal IL-10 induction.

In our early studies, cells were analyzed for expression of Foxp3 and IL-10 independently by flow cytometry. To confirm and extend the finding of differential effects of 1α25VitD3 on these molecules, a protocol for costaining was developed. CD4+ T cells were cultured with 10−6 M or 10−7 M 1α25VitD3 and then restimulated with anti-CD3 and IL-2 for 16 h and analyzed for expression of IL-10 and Foxp3 by secretion assay and then intranuclear staining. In two representative see more donors, shown in Figure 1B, virtually no (≤0.2%) cells stained positive for both Foxp3 and IL-10 in the presence of 10−7 M 1α25VitD3. When cells from a panel of healthy donors were screened, we observed that cell cultures preferentially expressed a high frequency of Foxp3+ cells and low levels of IL-10, or conversely Mephenoxalone low Foxp3 and a high frequency of IL-10+ cells in response to culture with 1α25VitD3 (Fig. 1B and C). Since Foxp3 expression may not always reflect inhibitory function, the functional consequences of 1α25VitD3 modulation of

Foxp3 versus IL-10 expression by human CD4+ T cells was next investigated. CD4+ T-cell lines generated from the same donor in the presence of either high (10−6 M; Foxp3-promoting Treg conditions) or lower (10−7 M; IL-10-Treg favoring conditions) concentrations of 1α25VitD3 were tested for their capacity to inhibit the proliferation of autologous, naïve CFSE-labeled responder T cells. Both populations showed comparable inhibitory activity (Fig. 2A). The suppression by cells generated with 10−7 M 1α25VitD3 could be diminished by the addition of anti-IL-10 receptor antibody to the co-culture, while in T-cell cultures generated with 10−6 M 1α25VitD3, the antibody had little effect (Fig. 2B), suggesting both IL-10-dependent and IL-10-independent mechanisms of suppression existed in the two different populations.