Reduced susceptibility of CD4⁺ T cells to HIV-1 infection in the presence of Gal-9. (A) Percentages of p24 suppression in activated CD4⁺ T cells stimulated with Gal-9 (1 μg/mL) 2 hours before HIV-1 infection with X4-tropic (HIV-1_LAI) and R-5 (HIV-1_JR-CSF) isolates. CD4⁺ T cells from 12 HIV-1 seronegative (filled symbols) and 4 HIV-1 seropositive (open symbols) were studied. (B) Percentages of CD4⁺p24⁺ cells in activated CD4⁺ T cells in the presence of Gal-9 (1 μg/mL) 2 hours before HIV-1 infection with X4-tropic (HIV-1_LAI) and R-5 (HIV-1_JR-CSF) isolates. Significance was tested using paired t test. (C-D) Representative flow cytometry dot plots from CD4⁺ T cells stimulated with 0.5 μg/mL or 1 μg/mL Gal-9 before infection with X4-tropic or R5-tropic HIV-1 isolates, respectively. (E-F) Representative flow cytometry dot plots from CD4⁺ T cells stimulated with Gal-9 (1 μg/mL) for 1, 2, and 12 hours before infection with X4-tropic or R5-tropic HIV-1 isolates, respectively. The number of infected cells was quantified by intracellular viral p24 antigen staining using flow cytometry on day 5 after infection. Error bars indicate mean ± SEM from duplicate cell cultures from 12 HIV-1 seronegative and 4 HIV⁺ individuals infected 4 separate times with both X4-and R5-tropic viral isolates in vitro. Each point represents an individual.