Figure 4: NAD⁺ converts naïve CD4⁺ T cells into Th1 and Th2 as well as Th17 cells.

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 Th0 non-polarizing conditions (with α-CD3/α-CD28 and 50 ng ml⁻¹ of recombinant IL-2 cytokine). Cells that were cultured with recombinant IL-2 were stimulated with increasing concentrations of NAD⁺ (0, 5, 50 and 250 μM). After 96 h, percentage of CD4⁺IFNγ⁺, CD4⁺IL-17A⁺, CD4⁺IL-4⁺ and CD4⁺IL-10⁺ cells were assessed by flow cytometry by gating on non-apoptotic 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.