Fig 8. NIH45-46-STSia induces NK degranulation towards autologous primary HIV-infected CD4⁺ T cells.

(A) A schematic representation of the workflow to evaluate effector NK degranulation against autologous HIV-infected CD4⁺ T cells in the presence of bNAb or bNAb-STSia conjugate. CD4⁺ T cells were isolated from fresh PBMC and exposed to HIV-1 IIIB for 72 h. On the third day, virus-infected CD4⁺ T cells were treated or not with Sialidase, NIH45-46, NIH45-46-STSia, or isotype-matched control antibody. PBMC from the same donor were co-cultured with autologous HIV-infected CD4⁺ T cells in the presence of CD107a antibody for 16 h. Following overnight incubation, the mixtures were stained with a cocktail of antibodies for CD3, CD56, IFN-γ, and TNF-α. Percent NK cell positive for CD107a, IFN-γ, and TNF-α expression was derived after gating on CD3^-CD56^dim NK cells. Control = data from the NK cells + HIV-infected CD4⁺ T cells condition. All other conditions contain NK cells + HIV-infected CD4⁺ T cells, in addition to the indicated reagent. (B) Data from donor 1. (C) Data from donor 2. (D) Data from donor 3. (E) Average data from all donors. Assay from each donor was performed in 4 replicate wells (E:T 10:1; n = 3 donors). Statistical analyses for panels B-D were performed using unpaired ANOVA with post-hoc Dunnett T3 method (to correct for multiple comparisons) comparing the indicated groups. Statistical analysis for panel E was performed using paired ANOVA with post-hoc Holm-Sidak method (comparing the indicated conditions).