Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes

Bancroft, Tara; DeBuysscher, Blair L.; Weidle, Connor; Schwartz, Allison; Wall, Abigail; Gray, Matthew D.; Feng, Junli; Steach, Holly R.; Fitzpatrick, Kristin S.; Gewe, Mesfin M.; Skog, Patrick D.; Doyle-Cooper, Colleen; Ota, Takayuki; Strong, Roland K.; Nemazee, David; Pancera, Marie; Stamatatos, Leonidas; McGuire, Andrew T.; Taylor, Justin J.

Single-cell sorting Single human B cells were isolated using a FACSARIA II cell sorter with Diva configuration (BD Biosciences) following tetramer enrichment and cell surface marker staining using human PBMCs as described above. Single B cells from defined subpopulations were sorted according to surface marker expression patterns. Specifically, IB2-or IB3-specific B cells were sorted using side scatter-based doublet discrimination, followed by gating on FVD − CD19 + CD20 + CD3 − CD14 − CD16 − B cells that bound IB2-PE or IB3-APC tetramers but not PE650 or APC755 tetramers containing isotype control antibodies. We also sorted 576 control FVD − CD19 + CD20 + CD3 − CD14 − CD16 − CD27 − IgM/D + B cells from two individuals that were not stained with an antiidiotype. Single cells were sorted into individual wells of 96-well PCR plates (Eppendorf) containing 10 µl/well ice-cold lysis buffer containing 0.25 µl (12.5 U) RNaseOUT (Thermo Fisher Scientific), 2.5 µl 5× SuperScript IV First Strand Buffer (Thermo Fisher Scientific), 0.625 µl 0.1 M dithiothreitol (Thermo Fisher Scientific), 0.3125 µl 10% Igepal detergent (Sigma-Aldrich), and 6.625 µl diethyl pyrocarbonate-treated water. Plates were sealed with adhesive PCR plate seals (Thermo Fisher Scientific), centrifuged briefly, and immediately frozen on dry ice before storage at −80°C.