Fig 2: The Nef protein from T/F HIV-1 strains modulate Tim-3 expression.A and B, purified CD4+ T cells from healthy donors were isolated using negative selection, activated using PHA/IL-2 stimulation, and infected with T/F viruses CH58 or CH77 and their ΔNef mutants. Forty-eight hours after infection, CD4+ T cells were stained using the Tim-3 antibody. The infected population was gated by intracellular p24 staining. C and D, the Tim-3 mean fluorescence intensity (MFI) in the uninfected (p24−) or the p24+ population is plotted graphically. These data were obtained in at least five independent experiments. (∗∗p < 0.01; ∗∗∗p < 0.001). T/F, transmitted/founder; Tim-3, T-cell immunoglobulin mucin domain-3.

Fig 3: Nef and Tim-3 are in close proximity within cells.A, HeLa CD4+ cells were transfected with Tim-3-FLAG-Vn (Tim-3-Vn) and NefWT-Vc or NefLLAA-Vc. Twenty-four hours later, cells were fixed, immunostained for FLAG (white) and HA (red), and mounted on DAPI-containing media for nuclear staining (blue), and BiFC (green) was observed using a Zeiss 880 Airyscan microscope. Images shown are representative cells from three independent experiments and 30 cells total. The scale bar is 10 μm. B, graph summarizing the BiFC signal intensity in HeLa CD4+ cells transfected with Tim-3-Vn and NefWT-Vc or with NefLLAA-Vc mutant. The values are normalized to Nef expression (HA staining). The dots represent three independent repeats each for a total of 30 cells. The bar graph shows the mean ± SD (∗p < 0.05). C, correlation of the BiFC signal intensity and Nef expression in HeLa CD4+ cells transfected with Tim-3-Vn and NefWT-Vc or with NefLLAA-Vc. The p-value indicates the significance for the difference in slope between NefWT and NefLLAA. n = 3, 30 cells. D, correlation between BiFC and Tim-3 expression in HeLa CD4+ cells transfected with Tim-3-Vn and NefWT-Vc or with NefLLAA-Vc mutant. The p-value indicates the significance for the difference in the slope between NefWT and NefLLAA, n = 3, 30 cells. BiFC, bimolecular fluorescence complementation; DAPI, 4′,6-diamidino-2-phenylindole; NefWT-VC, Venus fluorophore was fused to Nef; Tim-3, T-cell immunoglobulin mucin domain-3; Tim-3-VN, FLAG-tagged Tim-3.

Fig 4: Tim-3 receptor fate after Nef-mediated upregulation.A, schematic depicting the fate of Tim-3 (soluble Tim-3 [sTim-3]). B, purified CD4+ T cells from healthy donors were isolated using negative selection and infected with an eGFP-expressing NL4-3 or various Nef mutants (ΔNef or NefLLAA). Forty-eight hours after infection, CD4+ T cells were stained at 4 °C using an anti-Tim-3 antibody and either fixed immediately or incubated at 37 °C for 15 or 90 min before fixation. The expression of labeled Tim-3 was analyzed using flow cytometry. Infected cells were gated based on eGFP expression. The mean fluorescence intensity of cell surface Tim-3 is indicated within each gate. C, graphical summary for the kinetics of Tim-3 receptor expression. The percentage of Tim-3 remaining on the cell surface was calculated by dividing the mean fluorescence intensity of the Tim-3 receptor at the 15- or the 90-min time point by the 0-min time point and multiplying the resulting value by 100. The 0-min time point was set to 100. ±SD of the mean is indicated (n ≥ 3 experiments; ∗p < 0.05, ∗∗p < 0.01). Tim-3, T-cell immunoglobulin mucin domain-3.

Fig 5: Nef increases Tim-3 shedding from the cell surface.A, purified CD4+ T cells from healthy donors were isolated using negative selection and infected with an eGFP-expressing NL4-3 or the Nef mutants (ΔNef or NefLLAA). Forty-eight hours after infection, cell culture supernatants were analyzed for soluble Tim-3 using a MAGPIX assay. Values are sTim-3 concentrations (pg/ml) ± SD of the mean (∗p < 0.05). B, purified CD4+ T cells from healthy donors were isolated using negative selection and infected with an eGFP-expressing NL4-3. Cells were left untreated or treated with 25 μM BB-94 soon after infection and cell surface Tim-3 analyzed 48 h after infection. The number indicates the mean fluorescence intensity of surface Tim-3. C, graph summarizing fold increase in Tim-3 cell surface levels after BB-94 treatment. The values were derived from the mean fluorescence intensity ratio between treated and untreated samples for the Tim-3 receptor. ±SD of the mean is indicated (n ≥ 3 experiments; ∗p < 0.05). D, purified CD4+ T cells from healthy donors were isolated using negative selection and infected with an eGFP-expressing NL4-3, and cells were left untreated or treated with 25 μM BB-94. Cell culture supernatants were analyzed for soluble Tim-3 using a MAGPIX assay. Values are sTim-3 concentrations (pg/ml) ± SD of the mean (n ≥ 3 experiments; ∗∗p < 0.01). BB-94, Batimastat; sTim-3, soluble Tim-3; Tim-3, T-cell immunoglobulin mucin domain-3.