Fig. 3.

Ex vivo NMN treatment suppresses resting CD4⁺T cell reactivation in cART-treated people living with HIV by reducing CD25⁺cells. Frozen PBMCs from four independent cART-treated people living with HIV (PLWH) and four independent HIV-uninfected donors (HUD) were used for NAD level detection via NAD/NADH-Glo™ Assay. (a) The intracellular NAD level in PBMCs between PLWH and HUDs was displayed. Data represent Mean with Mix to Max in floating bars. Fresh or frozen PBMCs from six independent cART-treated PLWH were used for resting CD4⁺ T cell isolation. Purified resting CD4⁺ T cells were treated with PMA (50–500 ng/mL) plus Ionomycin (1 μg/mL) (in short as PMA/Iono), PMA/Iono plus 10 mM NMN or mock, under treatment of 10 nM EFV and 10 U/ml IL-2. On day 4 or 7 after treatment, cells were collected for FACS analysis, whereas viral RNA in the supernatant was extracted for HIV-1 real-time PCR assay. (b) The experimental flowchart was displayed. Normalized HIV-1 mRNA levels to PMA/Iono activation control were compared on day 4 (c; n = 4, frozen PBMCs) and day 7 (d; n = 3, frozen PBMCs) after reactivation. For FACS analysis on activation markers, representative FACS plots (e) and histogram plots (g) were displayed. The percentage (f) and MFI (h) of CD69⁺, CD25⁺, and HLA-DR⁺ cells on CD4⁺ T cells was compared (n = 6). For (c and d), (f) and (h), data represent Mean ± 95% CI. For (c–d), data passed normality test, and statistics were calculated using a paired Student's t-test; for (f) and (h), data did not pass normality test, and statistics were calculated using a Friedman test with post-hoc multiple comparison tests.