Figure 5: NAD⁺ promotes IL-10 under Th1 conditions.

Sorted naïve CD4⁺ T cells were isolated from spleens of 5C.C7 Rag2^−/− mice (C57BL/6 background) and were activated with α-CD3/α-CD28 antibodies only or in Th1 polarizing conditions (with α-CD3/α-CD28, 20 ng ml⁻¹ of recombinant IL-12, 50 ng ml⁻¹ of recombinant IL-2 cytokine and 10 μg ml⁻¹ of anti-IL-4). Cells in Th1 polarizing conditions were cultured in the presence of increasing concentrations of NAD⁺. After 96 h, percentage of CD4⁺IFNγ⁺IL-10⁺ and CD4⁺ IFNγ⁺IL-17A⁺ cells was measured by flow cytometry by gating on non-apoptotic CD4⁺ cells. To set the gates, flow cytometry dot plots were based on comparison with isotype controls, fluorescence minus one (FMO), permeabilized and unpermeabilized unstained cells (n=15; the data derived from three independent experiments). Data represent mean±s.d. NS, not significant *P<0.05; **P<0.01; ***P<0.001 as determined by Student’s t-test, comparing the indicated groups.