Monocyte-mediated phagocytosis of HIV-1 antigens

Monocyte-mediated phagocytosis was measured with a bead-based assay and the THP-1 cell line, as previously described^{64 (link)}. Briefly, biotinylated (EZ-Link Sulfo-NHS-LC-LC-Biotin, Thermo Fisher) BG505 SOSIP trimer was coupled to 1 μm yellow fluorescent neutravidin beads (Thermo Fisher) for 2 h at 37 °C. Excess antigen was removed in two washes with 0.1% BSA in PBS for blocking. Then, saturated beads (1.82 × 10⁸ beads/well) were incubated with 10 μL of mAb (RM19R, VRC01, or PGT145 at various concentrations, starting at 5 μg/mL, 2-fold dilutions) in PBS for 2 h at 37 °C. ICs were washed and 2.5 × 10⁴ THP-1 cells (American Type Culture Collection) were added per well and incubated for 16 h at 37 °C. Cells were fixed in 4% paraformaldehyde (PFA) and sample acquisition was performed via flow cytometry (IntelliCyt, iQue Screener plus). Events were gated on single cells and bead-positive cells. A phagocytosis score was calculated as the percent of bead-positive cells × GMFI/1000. All samples were run in duplicate.