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.

Identification of iglb12-like BCRs using anti-iglb12 idiotypes We next assessed whether the anti-iglb12 idiotypes could be used to identify B cells expressing iglb12-like BCRs from within the polyclonal human B cell repertoire of HIV-uninfected individuals using single B cell sorting followed by RT-PCR amplification and sequencing of heavy and light chain genes. In an initial set of experiments, IB1, IB2, and IB3 were biotinylated and tetramerized using streptavidin conjugated to allophycocyanin (APC) and incubated together with 100 million human peripheral blood mononuclear cells (PBMCs) before enrichment using anti-APC microbeads. Using this approach ∼2% of B cells in the APC-enriched fraction bound to IB1/2/3-APC, but not APC-Dylight755 (APC755)-conjugated isotype control tetramers (Fig. 3 A) . Highlighting the efficiency of this enrichment approach, few IB1/2/3-APC + B cells could be detected in the APCdepleted fraction (Fig. 3 A) . Using this approach, single B cells binding IB1/2/3 tetramers were sorted into individual wells of a 96-well plate, and the heavy and light chains expressed by these cells were sequenced using nested RT-PCR (Tiller et al., 2008) . In total, we FACS-purified 91 single IB1/2/3-binding B cells from an individual. This yielded 66 heavy chain and 38 light chain sequences, with 31 heavy and light chain pairs (Table S2) . To confirm the specificity of our flow cytometry approach, antibodies were produced from paired heavy and light chains from 10 cells. 80% of these antibodies bound IB2, but not IB1, IB3, or IB5, when assessed by BLI (Fig. 3 B) . Interestingly, the eight antibodies that bound IB2 all used the same heavy chain variable region as igbl12, V H 1-3, while the two antibodies that failed to bind any of the anti-idiotypes used other V H alleles (Table S2 ). In two of the cloned antibodies, the V H 1-3 + heavy chains were paired with V K 3-20 + light chains, suggesting that the lack of binding to IB1, IB3, and IB5 was not due to the absence of this segment ( Fig. 1 C) . These results highlight the high specificity of our flow cytometry approach and suggest that IB2 is bound by a higher number of cells compared with other anti-idiotypes.