Figure 1. Susceptibility of CD4⁺ T cell subsets to the Natural Killer (NK) immune response.

The susceptibility of different cell subpopulations that compose the HIV-reservoir to Natural Cytotoxicity (NC) and Antibody-Dependent Cell Cytotoxicity (ADCC) mediated by NK cells was measured by performing functional assays. (A) Percentage of gp120-coated cells killed by ADCC after being exposed to HIV-specific immunoglobulins (Igs) in the presence of NK cells. The intrinsic susceptibility to ADCC was measured in Naïve (T_NA), Stem Cell Memory (T_SCM), Central Memory (T_CM), Effector Memory (T_EM), T_CD32^dim, and T_CD20^dim subsets. Statistical comparisons were performed using the ANOVA Friedman with Dunn’s multiple comparison test. Median with interquartile range is shown. (B) Percentage of total gp120-coated CD4⁺ T cells from different cohorts of patients killed by ADCC. Healthy donors (HD), Elite Controllers (EC), and antiretroviral-treated (ART) PLWH. Statistical comparisons were performed using one-way ANOVA with Tukey’s multiple comparison test. Median with interquartile range are shown. (C) Percentage of cell subsets killed by ADCC in cells from HD, EC, and ART. Statistical comparisons were performed using the one-way ANOVA with Tukey’s multiple comparison test. Median with interquartile range is shown. (D–E) Spearman correlations between the size of the HIV-reservoir measured as total HIV-DNA in samples from ART-suppressed PLWH, and the potency of autologous NK cells to kill (D) total CD4⁺ T cells or (E) T_CD32^dim cells by ADCC. (F) Representative flow cytometry gating strategy used to quantify HIV infection after ex vivo infection with BaL or NL4.3. Fluorescence minus one (FMO) control was used to determine CD32 expression. Cells were infected for 5 days and the frequency of expression of CD32 on HIV-infected cells was measured for each condition. (G) Percentage of killing by NC of ex vivo HIV-infected T_CD32^dim and T_CD32⁻ cells mediated by autologous NK cells from ART-treated PLWH (n=14). Killing was calculated by normalizing the proportion of each subset within the p24⁺ fraction in the co-culture condition to the basal condition. (H) Percentage of ADCC killing of ex vivo HIV-infected T_CD32^dim and T_CD32⁻ cells mediated by autologous NK cells from ART-treated PLWH (n=14). Killing was calculated by normalizing the proportion of each subset within the p24⁺ fraction in the co-culture condition with plasma to the co-culture without plasma. Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test. *p<0.05; **p<0.01.

Figure 1—figure supplement 1. Gp120 cell coating efficiency, gating strategy for the NK-killing assays, and percentages of antibody-dependent cell cytotoxicity (ADCC) killing in elite controllers (EC), and healthy donors (HD).

(A) Cell coating with recombinant gp120 HIV protein. Detection was performed by flow cytometry using the anti-gp120 antibody A32, and a FITC-labeled anti-human secondary antibody. Mean Fluorescence Intensity (MFI) values are shown. (B) Gating strategy used to identify cell killing after the ADCC assay in the different cell subsets. Cell doublets were excluded by forward and side scatter signals and B and myeloid cells discarded based on their high expression of CD20 and CD32. Beads for absolute cell counting were included to calculate ADCC killing by measuring the disappearance of cells in each cell subset. (C) Frequency of expression measured by flow cytometry of CD32 on CD4⁺ T cells from healthy donors (HD, n=8), Elite controllers (EC, n=7) and ART-treated and virologically-suppressed PLWH (ART, n=15). Median with interquartile range are shown. Statistical comparisons were performed using the Mann-Whitney test. ***p<0.001; ****p<0.0001. (D–E) Correlations of the HIV-reservoir size and CD32 expression. Spearman correlations are shown in samples from HIV-infected individuals. ART-treated and EC are shown in blue and green dots, respectively. (D) Spearman correlation between the total HIV DNA reservoir size and the frequency of expression of CD32 in CD4⁺ T cells. ART-treated and EC are shown in blue and green dots, respectively. (E) Spearman correlation between HIV RNA levels in CD4⁺ T cells and the frequency of expression of CD32 in CD4⁺ T cells. (F–G) Intrinsic susceptibility to NK-mediated ADCC of Naïve (T_NA), Stem Cell Memory (T_SCM), Central Memory (T_CM), Effector Memory (T_EM), T_CD32^dim, and T_CD20^dim subsets in (F) EC, and (G) HD. Median with interquartile range is shown. Statistical comparisons were performed using the ANOVA Friedman test. *p<0.05; **p<0.01.

Figure 1—figure supplement 2. Percentage of CD32 expression on uninfected cells (p24⁻) and HIV-infected cells (p24⁺) with BaL (n=26) in (A) or with NL4.3 (n=11) in (B) after 5 days of infection.

Median with interquartile range are shown. Statistical analyses consisted of the Wilcoxon matched-pairs signed-rank test. (C) Spearman correlation between the percentage of ADCC-killing of the total HIV-infected CD4⁺ T cells and the percentage of ADCC-killing of the T_CD32^dim subset is shown.