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 naive mature B cells were prepared from spleens of iglb12 heavy chain transgenic mice using a B cell negative selection kit (Miltenyi Biotec) supplemented with 1.25 µg/ml biotinylated anti-CD93 (AA4.1; eBioscience) to remove transitional B cells. Purified B cells were washed in 1× DPBS, adjusted to a concentration of 5 × 10 7 cells/ml in warm 1× DPBS and incubated with 5 µg CTV (Thermo Fisher Scientific) for 12 min at 37°C before being washed with media containing 10% fetal bovine serum (Thermo Fisher Scientific). Cells were resuspended in warm 1× DPBS and 4 × 10 5 cells per mouse were injected retroorbitally into CD45.1 + recipients. 1 d following the transfer, recipient mice were injected intraperitoneally with 0.2 ml of the following solution: 0.1 ml (25 µg) diluted Sigma Adjuvant System (Sigma-Aldrich) and 20 µg of a humanized version of IB2 containing Fabs fused to the 2W epitope (EAWGALANWAVDSA) heptamerized on the C4B nanoparticle as described previously (Hofmeyer et al., 2013) in 0.1 ml 1× DPBS. A humanized isotype control Fab-C4b in diluted Sigma Adjuvant System was used as a control. 5 d following injection, the spleen, inguinal, brachial, cervical, and mesenteric lymph nodes were harvested into 1× DPBS, minced, and then digested as described previously in dispase (0.8 mg/ml; Thermo Fisher Scientific), collagenase (0.2 mg/ml; Roche), and DNase (0.1 mg/ml; Roche) for 20 min at 37°C. Enzymes were inactivated with 5 mM EDTA, and single-cell suspensions were obtained. Samples were centrifuged and cell pellets resuspended to 0.2 ml in 1× DPBS containing 1% newborn calf serum, 5 µg/ml of anti-CD16/CD3 (2.4G2, BioXCell), and 1.25 µg/ml anti-CD45.2 biotin (104; eBioscience) and enriched using 25 µl anti-biotin microbeads (Miltenyi Biotec) as described for tetramer enrichment.