Figure 3. Combination of ART and autophagy inducer rapamycin (Rapa) treatment effectively decreases inflammation, ISG expression, and viral replication in HIV-infected humanized BLT mice.

(A) Eight weeks after immune reconstitution, BLT humanized mice were infected with HIV_NFNSXL9 for 8 weeks. Afterward, mice were treated with ART and rapamycin or DMSO control for 4 weeks before necropsy. (B) PD-1, TIM-3, CD38, and HLA-DR expression was measured by flow cytometry (quantitatively by gating of percentages positive ± SEM) on peripheral blood CD8⁺ T cells from rapamycin or control mice (n = 6–7 per group). (C) Autophagy flux was detected by Western blotting of LC3-I, LC-II, ATG5, and actin using pooled splenocytes from DMSO or rapamycin-treated groups at necropsy (n = 5 per group). The ratio of LC3-II/actin was calculated by ImageJ. (D) Expression levels of human ISGs MX1, OAS1, and IRF7 in multiple lymphoid tissues from humanized BLT mice after treatment were measured by real-time PCR (n = 6–7 per group). (E) Splenocytes from HIV-1–infected mice treated with ART and rapamycin or DMSO were isolated and stained with intracellular Abs against human IRF7 and MX1. MFIs of the ISGs IRF7 and MX1 on human monocytes were measured by flow cytometry (n = 5–7 per group). (F) Relative HIV cellular RNA/HPRT1 expression from multiple lymphoid tissues after the indicated treatment, as compared with control blood. (n = 5–7 per group). Each dot represents an individual mouse; horizontal bars indicate median values. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U test.