Multi-color flow cytometric analysis was performed on mononuclear cells isolated from iliac LN samples. The following antibodies were used: LIVE/DEAD dead cell stain kit (Invitrogen); anti-CD8a (clone RPA-T8), anti-CD4 (clone OKT4), anti-PD-1 (clone EH12.2H7), anti-ICOS (clone C398.4A), anti-CD25 (clone BC96), anti-CXCR3 (clone G025H7) (BioLegend); anti-CXCR5 (clone MU5UBEE), anti-FOXP3 (eBioscience); anti-Bcl-6 (clone K112-91), anti-CD95 (clone DX2), anti-CD3 (clone SP34-2), and anti-Ki-67 (clone B56) (BD Biosciences); and anti-CD20 (clone B9E9), IgG (clone G18-145), IgM (G20-127) (Beckman Coulter).
For each BG505 SOSIPv5.2 Env trimer probe analysis, the biotinylated probes were individually premixed with fluorochrome-streptavidin conjugates (SA-Alexa647 and SA-BV421, Thermo Fisher Scientific and BioLegend) at room temperature (RT) for 20 min. After surface staining followed by washes, cells were fixed and permeabilized using FoxP3/Transcription Factor Staining Buffer kit (Thermo Fisher Scientific) according to manufacturer's protocols. Upon permeabilization, cells were stained with intranuclear Abs, washed twice and acquired on an LSR Fortessa Cell Analyzer (BD Biosciences). Flow cytometry data were analyzed with FlowJo (Tree Star).
For each BG505 SOSIPv5.2 Env trimer probe analysis, the biotinylated probes were individually premixed with fluorochrome-streptavidin conjugates (SA-Alexa647 and SA-BV421, Thermo Fisher Scientific and BioLegend) at room temperature (RT) for 20 min. After surface staining followed by washes, cells were fixed and permeabilized using FoxP3/Transcription Factor Staining Buffer kit (Thermo Fisher Scientific) according to manufacturer's protocols. Upon permeabilization, cells were stained with intranuclear Abs, washed twice and acquired on an LSR Fortessa Cell Analyzer (BD Biosciences). Flow cytometry data were analyzed with FlowJo (Tree Star).