Figure 7: NAD⁺ does not induce cell death.

Sorted naïve CD4⁺ T cells were isolated from spleens of wild-type C57BL/6 mice and cultured (0.5 × 10⁶ cells per well) in Th1 (with α-CD3/α-CD28, 50 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; a) and Th2 (with α-CD3/α-CD28, 50 ng ml⁻¹ of recombinant IL-4, 50 ng ml⁻¹ of recombinant IL-2, 10 μg ml⁻¹ of anti-IL-12 and anti-IFN-γ antibodies; b) polarizing conditions for 1 week, washed and followed by and incubation period of 72 h with NAD⁺ (100 μM). Cells were then rested for 72 h and a 2^d Th1 or Th2 stimulation was performed. After 10 days, total cell numbers and frequencies of CD4⁺IFNγ⁺ and CD4⁺IL-4⁺IL-10⁺ cells were measured by flow cytometry by gaiting on CD4⁺ T cells; cell proliferation was assessed by cell counting after each incubation period (n=10; the data derived from two 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.