Figure 4.

Gating strategy used to assess killing of reactivated HIV-1 latent reservoirs by the generated ADCC antibodies. To assess the ability of ADCC antibodies to lyse reactivated HIV latent reservoirs, the FACS-sorted resting memory CD4+ T cells (target) were reactivated with Env protein and co-cultured with CD4−CD8− cell (effector) in the presence of generated antibodies. The reduction in the frequencies of p24-expressing resting memory CD4+ T cells was used as the final readout of the assay. The dot plots depict the representative graphs for (A) the frequencies of p24-expressing resting memory CD4+ T cells. The live cells were gated from lymphocytes by using the live-dead dye. From the live cells, CD3+CD4+ cells were gated which were further drilled down to gate CD4+CD45RO+ resting memory T cells. These cells were then assessed for the expression of p24 in different culture conditions, viz., uninfected, unstimulated, Env-stimulated, and after the addition of antibodies from R-ADCC and NR-ADCC LTNPs. The uninfected cells were kept as a control to set the gate. (B) The dot plots show the gating strategy used to assess the simultaneous activation of CD3−CD56^dim NK cells (measured in terms of secretion of cytokine IFNγ and degranulation marker CD107a) in different culture conditions, viz., uninfected, unstimulated, Env-stimulated, and after the addition of IgGs from R-ADCC and NR-ADCC LTNPs.