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.

Purified anti-idiotype antibodies were biotinylated using an EZlink Sulfo-NHS-LC-Biotinylation kit (Thermo Fisher Scientific) using a 1:1 molar ratio of biotin to anti-idiotype. Unconjugated biotin was removed by centrifugation using a 50-kD Amicon Ultra size exclusion column (EMD Millipore). To determine the average number of biotin molecules bound to each anti-idiotype molecule, streptavidin-PE (ProZyme) was titrated into a fixed amount of biotinylated anti-idiotype in increasing concentrations and incubated at room temperature for 30 min. Samples were run on an SDS-PAGE gel (Bio-Rad Laboratories) and transferred to nitrocellulose and incubated with streptavidin-Alexa Fluor 680 (1:10,000; Thermo Fisher Scientific) to determine the point at which there was excess biotin available for the streptavidin-Alexa Fluor 680 reagent to bind. Biotinylated antiidiotypes were mixed with streptavidin-PE or streptavidin-APC at the ratio determined above to fully saturate streptavidin and incubated for 30 min at room temperature. Unconjugated antiidiotypes were removed by several rounds of dilution and concentration using a 300K Nanosep centrifugal device (Pall Corporation). Control PE594 and APC755 tetramers were created by mixing isotype control antibodies with streptavidin-PE preconjugated with Dylight 594 (PE594) or streptavidin-APC preconjugated with Dylight 755 (APC755; both from Thermo Fisher Scientific) following the manufacturer's instructions. On average, PE594 and APC755 contained 4-8 Dylight molecules per PE/ APC. The concentration of each anti-idiotype tetramer was calculated by measuring the absorbance of PE (565 nm, extinction coefficient = 1.96 µM −1 cm −1 ) or APC (650 nm, extinction coefficient = 0.7 µM −1 cm −1 ) and the lot-specific ratio of streptavidin-PE or streptavidin-APC. Figure 10 . Transgenic B cells expressing the iglb12 heavy chain enter germinal centers despite competition from WT cells. Data from two experiments in which 4 × 10 5 purified B cells from CD45.2 + iglb12 heavy chain transgenic mice were labeled with CTV and adoptively transferred retro-orbitally into WT CD45.1 + recipients 1 d before intraperitoneal immunization with 20 µg huIB2-C4b or control-C4b and 25 µg Sigma Adjuvant System. 14 d later, spleen and lymph nodes from individual mice were pooled and enriched for IB2-PE + and CD45.2-biotin + iglb12 heavy chain transgenic donor cells using anti-PE and anti-biotin microbeads before analysis by flow cytometry. (A) Representative flow cytometric analysis of live CD19 + CD3 − Gr-1 − F4/80 − B cells binding IB2-PE tetramers, but not isotype control PE594 tetramers. (B) Representative gating of GL7 + CD38 − germinal center B cells in the IB2 + population described in A.