Fig. 6.

NMN treatment plus cART significantly increases CD4⁺T cell percentage in HIV-infected huPBL mouse model in vivo. (a) Schematic diagram of humanized mice experiment using human PBL reconstituted NSG mice (HuPBL). (b) Plasma NAD level was detected via luciferase assay on Day 28 post-infection. Plasma viral load (c), CD4/CD8 ratio (d), the percentage of CD4⁺ T cells in CD3⁺ cells (e), and the percentage of CCR5⁺ on CD4⁺ T cells (f), were monitored weekly before or post-infection. Among splenocytes collected on Day 28 post-infection, the percentage of CD4⁺ T cells in CD3⁺ cells (g), the percentage of Annexin V⁺ cells in CD4⁺ T cells (h), the percentage of CD25⁺ cells in CD4⁺ T cells (i), the percentage of HLA-DR⁺CD38⁺ cells in CD4⁺ T cells (j), and the percentage of p24⁺ cells in CD4⁺ T cells (l) from splenocytes were analysed by flow cytometry. The MFI values of CD25 (m) and HLA-DR (n) in p24⁺CD4⁺ T cells were also analysed by flow cytometry. (k) Splenocyte p24 DNA level was detected via real-time PCR. By immunohistochemistry (IHC) analysis on spleen tissue sections, IHC slides were scanned via PerkinElmer Vectra Polaris™ Automated Quantitative Pathology Imaging System and analysed by Inform Software. (o) The cytoplasm p24 max intensity (normalized counts) in CD25⁺CD4⁺ cells was calculated using Inform Software and compared among groups. Data represent Mean ± SD. For (c-f), the area under curve (AUC) was calculated and used for statistical analysis by an unpaired Student's t-test, and the p values reported in black text are the AUC analysis comparing ART alone to ART-plus-NMN; for (b) and (i), data did not pass normality test, and statistics were calculated using Mann–Whitney test; for (g–h) and (j–o), data passed normality test, and statistics were calculated using unpaired Student's t-tests. # represents the significant difference between experimental groups and the control group, and the corresponding p value was reported in the corresponding group colour (cART group in red, while cART-plus-NMN group in purple); ∗ represents the significant difference between cART group and cART-plus-NMN group, and the corresponding p value was reported in black colour. Two batches of mice were conducted in this experiment. Each dot represents one individual mouse. Circles show mice from Batch 1, whereas rectangles show mice from Batch 2.