Cd27 bv650

The CD19⁺ cell fraction was enriched from PBMCs by positive selection with CD19 magnetic microbeads (Miltenyi Biotech) and subsequently stained on ice for 20 min with the following fluorochrome-labeled mouse monoclonal antibodies: CD3-APC/Cy7 (dilution 1:40, clone HIT3a, catalog no. 300317, BioLegend), CD27-Bv650 (dilution 1:50, clone O323, catalog no. 302827, BioLegend), CD19-PE-Cy7 (dilution 1:50, clone SJ25C1, catalog no. 341113, BD Biosciences), HLA-DR-BD Horizon V500 (dilution 1:100, clone G46-6, catalog no. 561224, BD Biosciences) and CD38-PE (dilution 1:100, clone T16, catalog no. IM1832U, Beckman Coulter). Cells were sorted to over 98% purity on a FACSAria III (BD) using the following gating strategy: circulating memory B cells were sorted as CD3^–CD19⁺CD27⁺CD38^−/+ cells, whereas circulating plasmablasts were sorted as CD3^–CD19⁺CD27^hiCD38^hi cells. FACS-sorted cells were collected in 6 μl FCS in Eppendorf tubes that were pre-coated overnight with 2% BSA.

Donor PBMCs was stained with live-dead marker (1:1000; Invitrogen, LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit) to gate out the dead cells for 20 min in 4 °C. After washing with FACS buffer, the cells were stained with respective B, T cell marker panel antibodies diluted in FACS buffer (1xPBS, 2% FBS, 0.1% sodium azide) at 4 °C for 30 min. B cell panel: CD19-BV510 (1:40; Biolegend, Cat. No. 363020), CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD3-APC-Cy7 (1:125; Biolegend, Cat. No. 317342), CD10-PE-Dazzle594 (1:50; Biolegend, Cat. No. 312228), CD21-APC (1:50; Biolegend, Cat. No. 354906), CD27-BV650 (1:40; Biolegend, Cat. No. 302828), CD38-PE-Cy7 (1:40; Biolegend, Cat. No. 356608), PD-1-BV711 (1:20; Biolegend, Cat. No. 329928), IgD-BUV 737 (1:40; BD, Cat. No. 612798), IgG-PerCP (1:40; BD), IgA-PE (1:40; Miltenyi, Cat. No. 130-114-002). T cell panel: CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD19-APC-Cy7 (1:50; BD, Cat. No. 557791), CD3-BUV395 (1:40; BD, Cat. No. 563546), CD4-BUV496 (1:40; BD, Cat. No. 612936), CD8-PE (1:40; BD, Cat. No. 555367), CXCR5-PE-Cy7 (1:100; BD, Cat. No. 624052), CD25-BV421 (1:40; BD, Cat. No. 562442), CD127-FITC (1:20; BD, Cat. No. 557938). The samples were then washed, resuspended in FACS buffer and analysed on BD LSRFortessa flow cytometer (Data availability—Figshare 10.6084/m9.figshare.23549964).

Fresh PBMC isolated from PLWH/ART were rested overnight in RF10 at 37°C 5% CO₂. The next day, 0.5 × 10⁶ PBMC were added to a 96‐well round bottom plate at a 1:1 ratio with murine P815 cells (ATCC, Manassas, USA) and stimulated with 40 ng mL⁻¹ of monoclonal antibodies against either CD3 (OKT3; Biolegend), CD16 (3G8; Biolegend) or an isotype control (MOPC‐21; Biolegend). 5 μg mL⁻¹ of monoclonal antibodies against either CD160 (BY55; Biolegend), 2B4 (C1.7; Biolegend), Tim‐3 (F38‐2E2; Biolegend), TIGIT (MBSA43; eBioscience, San Diego, USA), PD‐1 (EH12.2H7; Biolegend), NKG2C (134522; R&D Systems) or an isotype control (MOPC‐21; Biolegend) were then added to CD3 or CD16 stimulated conditions. CD107a APCH7 (H4A3; BD Biosciences) was added to each well, and the plate was briefly centrifuged before incubation at 37°C with 5% CO₂ for 5 h. After incubation, wells were washed, and cells stained with UV viability dye (ThermoFisher) then a cocktail containing Vδ1 FITC (TS8.2; Invitrogen, Carlsbad, USA), CD3 BV510 (SK7; Biolegend), CD27 BV650 (0323; Biolegend), CD69 PE Dazzle (FN50; Biolegend) and CD56 BUV737 (NCAM16.2; BD Biosciences). After staining, cells were washed and resuspended in PBS containing 2% FCS before acquisition on a BD LSR Fortessa using BD FACS Diva.

Fresh peripheral blood mononuclear cells were isolated from blood obtained from 34 SLE individuals. Cells were stained with LIVE/DEAD Fixable Blue Dead cell stain (Invitrogen, Paisley, UK) to exclude dead cells, Fc receptor blocked (Human TruStain FcX; BioLegend, Oxford, UK) and surface stained using the following markers: IgD-BrilliantViolet(BV)421 (IA6-2), CD19-BV510 (HIB19), CD27-BV650 (O323), CD138-FITC or CD138-PE-Cy7 (MI15), CD24-PerCP-Cy5.5 (ML5), CD95-PE-Cy7 (DX2), CD38-APC (HB7) and CD20-APC-H7 (2H7) from BioLegend or BD. Cells were fixed with BD stabilising fixative reagent. Freshly stained cells were acquired on a 5 laser BD SORP LSRFortessa instrument. BD CS&T beads were used immediately prior to every sample run to maintain instrument consistency throughout the entire study. Data were analysed using FlowJo version 10 (Ashland, OR, USA).

PBMCs from healthy subjects were resuspended in PBS buffer and were incubated with directly conjugated antibodies for 30 min at 4°C. The cells were washed with 1× PBS before flow cytometry analysis. Antibodies used included anti-human CD3-BV786 or CD3-BUV737, CD8-BV510 or CD8-BUV395, CD160-AF488, CD45RA-AF700, CD28-APC, CD95-PE, CD57-BV421, PD-1-BV711, TIM-3-BV650 (BD Biosciences, San Diego, CA, USA), CD4-APC-Fire750, CD244-PE-D594, CCR7-BV421, HLA-DR-AF700, CD38-BV421, CD28-BV711, CD27-BV650, KLRG-1-APC-Fire750, CD95-PE-CY7 (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7, LAG-3-APC (Ebioscience, San Diego, CA, USA) and the corresponding isotype controls. Data were acquired with the LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.5 (Tree Star, Ashland, OR, USA). More information about antibodies is listed in the Supplementary Material (Table S2).