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.

To test these predictions, IB2 and IB3 were biotinylated and tetramerized using streptavidin conjugated to APC or R-phycoerythrin (PE). Staining PBMCs with anti-idiotype tetramers revealed that <0.05% and 0.008% of the live B cell population from HIV-uninfected individuals bound IB2-APC or IB3-PE, respectively, but not APC755-or PE-Dylight594 (PE594)-conjugated isotype control tetramers (Fig. 4 A) . To The affinity (K D ) of the anti-idiotype for iglb12 was calculated based upon the on rate (K ON ) and off rate (K OFF ) determined by BLI. (C) The heat map displays the maximum shift (nm) measured by BLI when IB1, IB2, IB3, or IB5 were incubated with the listed antibodies and antibody groups. Detailed information on sequences of the non-V H 1-3 and V H 1-3 + control antibodies can be found in Table S1 . Data are representative of two to three similar experiments. more easily identify rare B cells binding IB2 and/or IB3, tetramer-binding cells were enriched using APC-specific and PE-specific magnetic microbeads from samples of 200 million PBMCs before flow cytometry (Fig. 4 A) . Using this approach, single B cells binding IB2 and/or IB3 tetramers were sorted into individual wells of a 96-well plate, and the heavy and kappa light chains expressed by these cells were sequenced using nested RT-PCR (Tiller et al., 2008) . In total, we FACS-purified 576 single IB2-binding B cells and 150 single IB3-binding B cells from three individuals. From IB2-binding B cells, we obtained man PBMCs that bound a cocktail containing IB1-APC, IB2-APC, and IB3-APC tetramers in fractions enriched or depleted of APC + cells using anti-APC microbeads before flow cytometry. APC755 tetramers containing isotype control antibodies were included in these experiments to exclude B cells specific for the APC, streptavidin, and conserved portions of the anti-idiotypes. The displayed plots were derived from ∼100,000 APC-depleted or ∼400,000 APC-enriched cells derived from 100 million PBMCs and representative of three similar experiments. The mean percentage ± SD of IB1/2/3-APC + APC755 − B cells in the enriched and depleted fractions from three individuals is shown on the plots. (B) The frequency of ten antibodies cloned from IB1/IB2/IB3-APC + APC755 − B cells from an individual were assayed for binding to IB1, IB2, IB3, or IB5 by BLI. Each antibody was assessed for binding in two to three independent experiments. 302 heavy chain and 174 kappa light chain sequences, with 136 heavy and light chain pairs (Table S3 ). IB3-binding B cells yielded 84 heavy chain and 44 kappa light chain sequences, with 27 heavy and light chain pairs (Table S4) . We were unable to recover heavy or light chain sequences from the small number of sorted cells binding both IB2 and IB3 tetramers.