Fig 2: Effects of GSK445A on CD4+ T cell dynamics in SIV-infected RM on ART.(A) Plasma SIV RNA profiles for 4 RM following SIVmac239M infection, ART initiation on day 56 post-infection (pi) and GSK445A infusion starting day 298 pi. (B) Absolute counts of CD4+ T cells in blood following 3 infusions of GSK445A at 15 µg/kg. Note that the first 2 doses of GSK445A were administered over 5 minutes while the 3rd dose was administered over 20 minutes. (C) Representative flow cytometric analysis of RM RM25866 showing CD69 expression on CD4+ memory and naïve T cells in peripheral blood up to 24 hours following the third infusion of GSK445A at 15 µg/kg. (D) Frequencies of CD69+ CD4+ memory T cells (left panel) and CD69+ CD4+ naïve T cells (right panel) in blood following GSK445A infusion.

Fig 3: Analysis of cytokine milieau in melanoma infiltrated and tumor free lymph nodes(a) Transmissible factors’ influence on CD56 cell subset frequency and CD69 and CCR7 cell surface expression was evaluated in transwell cocultures. TILN and TFLN samples (n=3) were analysed for CD69 and CCR7 expression within CD56dim and CD56bright NK cell subsets. Each specific bar pattern represents the transwell condition with TFLN, TILN or PBL in the upper chamber of the transwell and TFLN or TILN in the lower one. The bar diagram shows the mean of values ± SD (ANOVA, n=3). (b, c) Cytokine production in supernatant of TILN, TFLN and melanoma tumor samples. The histogram reports CXCL8 production by tumor cells (grey column), TILN lymphocytes (black column) and TFLN lymphocytes (white column) as mean±SD of three independent experiments (b). IL-6, CCL2, VEGF release by tumor cells, TILN and TFLN lymphocytes (the results represent mean±SD of three separate experiments, ANOVA) (c). (d) The combination of IL6, CXCL8 and CCL2 can influence CD56bright phenotype on CD56+CD3- NK cells derived from peripheral blood of healthy donors. Bars shows the percentage ± SD of CD56dim (left panel) and CD56bright (right panel) expressing each indicated marker. Results shown are representative of three independent experiments. Statistical analysis is indicated (ANOVA). (e) Frequency of NK and T cell subsets (CD56bright+CD3-; CD56dim+CD3-; CD56-CD3+) expressing the CXCL8 receptor (CXCR2), CCL2 receptor (CCR2) and IL6 receptors in healthy donors (n=8) and melanoma patients peripheral blood (n=9). Each dot represents a different patient or healthy donor. Bars represent mean values. Statistical analysis is indicated (ANOVA).

Fig 4: Ciapavir Does Not Induce Cytokine Release or T Cell ActivationHuman PBMCs and rCD4+ T cells from three healthy donors (n = 3) were treated with Ciapavir at the indicated concentrations, 500 nM vorinostat, 40 nM panobinostat, or combinations thereof, for 24 h. 50 ng/mL PMA and 1 µM ionomycin, or anti-CD3/CD28 antibody-coated beads, served as positive controls.(A and B) Heatmaps represent mean cytokine levels measured in the culture supernatant of PBMCs (A) or rCD4+ T cells (B) from tested donors (see Figure S2 for detailed results).(C and D) rCD4+ T cells treated as indicated were analyzed for CD69 (C) and CD25 (D) expression by flow cytometry.(E) Viability of rCD4+ T cells following treatment was assessed by measuring cellular ATP levels. Values were normalized to untreated cells from each donor. Significance was assessed with a one-way ANOVA using Dunnett’s multiple comparison correction (n = 3).

Fig 5: Viral Loads and RNA Expression in Humanized Mice Treated with CiapavirMice were infected with HIV-1, treated with ART to suppress viral loads, and then administered with Ciapavir or vehicle control.(A and B) Viral loads of control (A) and Ciapavir-treated (B) BLT mice.(C) 3 responding mice showing increases in viral loads 2 days after administration of Ciapavir.(D) Bone marrow HIV RNA levels, with responding mice 1, 5, and 8 indicated.(E–H) Mice described were subjected to flow cytometry analysis at necropsy to analyze immune activation. CD69 expression is shown as percentage gated of all blood CD45+ (human immune) cells (E) and in CD4+ T cells only (F). Bone marrow samples were analyzed for CD69 expression using the same parameters (G and H).See Figure S3 for mean fluorescence intensities. Error bars represent mean and SD (n = 9 for mock, n = 3 for 10 mg/kg Ciapavir, and n = 6 for 20 mg/kg Ciapavir). Statistics represent two-sided Mann-Whitney test (ns, not significant; p > 0.05).