Figure 5.

Effect of in vivo administration of NAD⁺ on the immune response against EL4 and EG7 tumors. (A–D) 2 × 10⁵ EL4 (A) or EG7 (B–D) tumor cells were inoculated s.c. on day 0 into the backs of CD38^−/− or WT mice and tumor growth was followed. Groups of mice (n = 7–8) received systemic injections of PBS on day −1 (control), anti-CD25 (PC-61) antibody on days −4 and −1, 10 mg (CD38^−/−) or 60 mg (WT) NAD⁺ on day −1, or s+16a-Fc on day −2 followed by NAD injection on day −1. (C) WT mice that had been treated with 60 mg NAD⁺ on day −1 and had rejected the primary EG7 tumor received a secondary tumor challenge 2 mo later with a fivefold higher dose of EG7 tumor cells (10⁶ cells). Control group of untreated mice were injected in parallel with the same number of EG7 cells. (D) Flow cytometry analyses of lymphocytes from blood samples taken on days 12–14 after inoculation of EG7 tumor cells. Cells were stained for the indicated markers before flow cytometry analyses and gating on CD4⁺ or CD8⁺ cells. Data are representative of at least three independent experiments for each tumor model (with n = 7–8 mice per group). Statistical Mann-Whitney U tests were used and showed significant differences between tumor volumes in all experiments when comparing PBS to NAD-treated animals after day 15. Horizontal lines show the mean. **, P < 0.01; ***, P < 0.001. Error bars represent SEM.