Cxcr5 bv421 clone j252d4

PBMC were thawed in complete 10% FBS RPMI media (Millipore Sigma) with Benzonase® Nuclease (Millipore Sigma). CD4 T cells were isolated by magnetic bead negative selection with the EasySep CD4 isolation kit (STEMCell Technologies). Cells were stained for L/D-AQUA (Thermo Fisher) and then extracellular staining was done using: CD4-BV605 (clone RPA-T4, BD Bioscience), CD45RO-PE-Cy7 (clone UCHL1, BD Biosciences), CD8-APC (clone RPA-T8, BD), CXCR5-BV421(clone J252D4, Biolegend), and CD3-PE (SK7, Biolegend). Stained cells were sorted on a BD FACSAria™. 50,000 sorted CD45RO⁺CXCR5⁺ or CD45RO⁺CXCR5^- cells were then plated in a 96 U bottom plate. B cells from a non-related healthy control donor were isolated by magnetic bead negative selection with the EasySep B cell isolation kit (STEMCell Technologies). 50,000 B cells were added to the corresponding 96 U bottom plate in 10% FBS RPMI media (Millipore Sigma) with antiretroviral drugs were added to the culture (200nM raltegravir, 200nM lamivudine) (NIH AIDS reagent program). After 7 days of co-culturing in a 37°C incubator, B-cell differentiation was determined by flow cytometry and absolute cell numbers were quantified using counting beads (Thermo Fisher). Staining for B-cell differentiation was performed using antibodies listed in Supplementary Table 3, panel 5.

Peripheral blood mononuclear cells (PBMC) were isolated from fresh venous blood using Ficoll density gradient. PBMC samples were stained with the following antibodies: CD3-APC-Cy7 (clone HIT3a), CD3-BV510 (clone OKT3), CD4-BV421 (clone OKT4), CD8-PE-Cy7 (clone SK1), CD45RA-BV510 (clone HI100), CCR7-APC (clone G043H7), CD27-FITC (clone MT271), HLA-DR-FITC (clone L243), CXCR5-BV421 (clone J252D4), PD-1-PE (clone EH12.2H7), CXCR3-BV510 (clone G025H7), CCR4-PerCP-Cy5.5 (clone L291H4), CCR6-PE (clone G034E3), CD25-APC (clone BC96), CD127-FITC (clone A019D5), Perforin-PE-Cy7 (dG9), Granzyme B-AF647 (GB11) were purchased from Biolegend (San Diego, CA); CD4-percp (clone SK3), CD38-APC (clone HIT2), Tim-3-PE (clone 7D3), GNLY-AF488 (clone RB1) were obtained from BD Biosciences (San Diego, CA). Granzyme B, Perforin and GNLY were measured de novo, without prior stimulation with PMA/ionomycin. BD Canto II instrument was used for FACS and the data was analyzed using FlowJo software V10 (Tree star Inc. Ashland, OR).