Identification of HIV-gp140 Memory B Cells

The HIV-specific memory B cells were identified using the HIV-gp140 protein probe as per the previously reported protocol (25 (link), 26 (link)) with some modifications. In brief, after revival and resting, the PBMCs from the study participants were stained with CD3 BV786, CD19 FITC, and CD27 PE (all from BD Biosciences) and with biotinylated gp140 (5 µg/ml) for 30 min at room temperature and were then stained with streptavidin PE-Cy7 (BD Biosciences) for 30 min at room temperature and acquired on BD FACSAria™ Fusion (BD Biosciences). The total memory B cells were gated as CD3−CD19+CD27+ cells from lymphocytes (Figure 2A) which were further drilled down to identify HIV-gp140-specific memory B cells as CD3−CD19+CD27+gp140+ cells (Figure 2C). These cells were then sorted using BD FACSAria™ Fusion (BD Biosciences) and collected in the FACS tube. The purity of the sorted cells was >95% in all cases.