FIG 1.

NAD⁺ induces T-cell activation in vivo and T-cell differentiation in vitro after TCR activation. A, C57BL/6 mice were treated daily with intraperitoneal injection of 40 mg of NAD⁺ or a placebo solution (PBS). After 7 days, mice were killed, and CD4⁺ T cells were isolated from spleens. Frequencies and total numbers of CD4⁺IFN-γ⁺, CD4⁺IL-4⁺, and CD4⁺IL-17A⁺ cells were analyzed by using flow cytometry. B, CD4⁺ T cells (3 × 10⁶) from 5C.C7 RAG-2–deficient mice were injected into Rag2^−/− (B10.A background) mice as adoptive transfer recipients. Mice were then treated with NAD⁺ or PBS for 7 days and subsequently killed to analyze frequencies and total numbers of CD4⁺IFN-γ⁺ cells by using flow cytometry. C and D, Sorted naive CD4⁺CD44⁻CD62L⁺ T cells were isolated from spleens of C57BL/6 mice and cultured in complete media alone with α-CD3/α-CD28 with or without IL-2 or in the presence of 50 µmol/L NAD⁺. After 96 hours, frequencies of CD4⁺IFN-γ⁺ cells and IFN-γ, IL-4, and IL-17A cytokine secretion were assessed by using flow cytometry (Fig 1, C) and ELISA (Fig 1, D), respectively. Statistics were as follows: n = 15 (Fig 1, A and B) or n = 10 (Fig 1, C and D). Data were derived from 3 independent sets of experiments. The Student t test and ANOVA were used accordingly to compare groups: **P < .01 and ***P < .001. Data are presented as means ± SDs. FACS, Fluorescein-activated cell sorting; FITC, fluorescein isothiocyanate; PE, phycoerythrin.