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.

Tetramer enrichment For human PBMC analysis, 200 × 10 6 frozen cells were thawed into RPMI with 10% fetal bovine serum (Thermo Fisher Scientific), 100 U/ml penicillin (Thermo Fisher Scientific), 100 µg/ml streptomycin (Thermo Fisher Scientific), and 0.0275 mM 2-mercaptoethanol (Sigma-Aldrich). Cells were centrifuged and resuspended to 0.2 ml in ice-cold FACS buffer composed of 1× Dulbecco's PBS (DPBS) with 1% newborn calf serum (Thermo Fisher Scientific) containing 2% rat and mouse serums. PE594-and APC755-conjugated control antiidiotype tetramers were added at a final concentration of 5-15 nM and incubated on ice for 5 min to allow binding to B cells of unwanted specificities. IB2-PE and IB3-APC or IB1-APC, IB2-APC, and IB3-APC tetramers were added at a final concentration of 5 nM and incubated on ice for 25 min, followed by a 10-ml wash with ice-cold FACS buffer. 25 μl of both anti-PEand/or anti-APC-conjugated microbeads (Miltenyi Biotec) were added and incubated on ice for 30 min. 3 ml of FACS buffer was then added to the cell mixture and passed over a magnetized LS column (Miltenyi Biotec). The column was washed once with 5 ml ice-cold FACS buffer and then removed from the magnetic field. 5 ml ice-cold FACS buffer was pushed through the unmagnetized column twice using a plunger to elute the bound cell fraction.