Cd4 bv605 clone rpa t4

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) from healthy subjects were isolated from heparinized whole blood by density gradient centrifugation and 1x10⁶ PBMCs were stained with viable and dead cells LIVE/DEAD™ Fixable Aqua dead cell stain kit (Life Technologies) for 15 min followed by an extracellular staining. First CCR7 PerCp Cy 5.5 was stained (clone G043H7, Biolegend) for 15 min at 37 °C followed by CD3 AF700 (clone OKT3, Biolegend), CD4 BV605 (clone RPA-T4, BD Biosciences), CD8 PB (clone SK1, Biolegend), CD45 RA FITC (clone HI100, Biolegend), CD56 APC-Cy7 (clone HCD56, Biolegend), CD19 PE-Cy7 (clone HIB19, Biolegend), CD26 APC (clone BA5b, Biolegend) for 15 min at 4°C. Non-specific binding of Fc-receptors was blocked by 2% polyclonal IgG (Flebogamma). CD26 surface expression was measured with CytoFLEX LX (Beckman Coulter) and data was analyzed with FlowJo software 10.0.08. Gating strategies are shown in the Supplementary Information.

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood via Ficoll–Hypaque density gradient centrifugation (Pharmacia, Uppsala, Sweden). Multicolor flow cytometry with fluorescent conjugated antibodies obtained from BioLegend (San Diego, USA) were as follows: CD3-APC-Cy7 (clone SK7), CD8-Percp/Cyanine5.5 (clone SK1), CD56-BV421 (clone HCD56), CCR5-FITC (clone J418F1), and CXCR4-APC (clone RG5). CD4-APC-H7 (clone RPA-T4) and CD4-BV605 (clone RPA-T4) were obtained from BD Biosciences (New Jersey, USA). PBMCs were first stained at 4°C for 30 min with fluorescent antibodies under dark conditions. Cells were then permeabilized using a Cytofix/Cytoperm Kit (BD Bioscience) and stained with p24-PE (clone KC57) or p24-FITC (clone KC57) from Beckman Coulter (Eurocenter S.A, California, USA). The cells were fixed in 0.5% formaldehyde and analyzed using a FACSCanto^TM flow cytometer (BD Biosciences). The data were analyzed using the FlowJo software (TreeStar).