Cytokine and transcription factor analyses show that 1,25(OH)2D3 polarizes human T cells in favor of Th2 rather than the Th1 and Th17 subsets. A) ELISA results for T cells activated with two concentrations of anti-CD3 (10 and 1000 ng/mL) demonstrate that IFNγ and IL-17 concentrations decrease (p < 0.001), while IL-5 concentrations increase (p < 0.05), with the addition of 0.1 nM 1,25(OH)2D3 (denoted as Vit D in figure). Each bar is the mean ± SD of quadruplicate cultures and data displayed is from a single subject. The cytokine ratios were significantly increased between treated and untreated groups (p < 0.001). When analyzed across T cells from 4 subjects, we found that one subject did not alter IFNγ or IL-17 levels with 1,25(OH)2D3 treatment but IL-5 was elevated. Thus, across subjects, the increased ratio of IL-5/IFNγ and IL-5/IL-17 is a consistent outcome for 1,25(OH)2D3 treatment. B) qPCR results for 1000 ng/mL anti-CD3 activated T cells from five human donors (each line represents one donor) demonstrating that mRNA levels for both IFNγ and IL-17 were variably affected by 1,25(OH)2D3, but always increased for IL-5. In all five subjects, the ratio of IL-5 to both IFNγ and IL-17 was elevated by increasing concentrations of 1,25(OH)2D3. These results were reproduced in cells activated by 10 ng/mL anti-CD3 (data not shown). C) qPCR results of transcription factors for T cells from five donors activated with 1000ng/mL anti-CD3 show that while levels of TBX21 (which regulates Th1 polarization) and RORC (Th17) were largely unaltered by 1,25(OH)2D3, GATA-3 (which regulates Th2) transcripts were elevated, resulting in a consistent increase in the ratio of GATA-3 to both TBX21 and RORC levels. These results were reproduced in cells activated by 10 ng/mL anti-CD3 (data not shown).