Cd107a apch7 h4a3

Cryopreserved PBMC were thawed in RF10, then rested overnight at 37°C 5% CO₂. The next day, 1.0 × 10⁶ PBMC were added to a 96‐well round bottom plate. For ICM blocking experiments, 4 μg mL⁻¹ of blocking antibodies against 2B4 (eBioPP35; eBioscience), PD‐1 (EH12.2H7; Biolegend), CD160 (688327; Biolegend), Tim‐3 (F38‐2E2; Biolegend) or an isotype control (MOPC‐21; Biolegend) were added to wells and incubated for 30 min at 37°C 5% CO₂. HMB‐PP (Sigma‐Aldrich) was added to wells at a final concentration of either 20 ng mL⁻¹, 2 ng mL⁻¹, 0.2 ng mL⁻¹ or 0.02 ng mL⁻¹. Some wells were left unstimulated to assess background activation. CD107a APCH7 (H4A3; BD Biosciences) was added to each well before incubation at 37°C 5% CO₂ for 5 h. After incubation, cells were washed then stained with Aqua viability dye (ThermoFisher) and a cocktail containing CD69 FITC (FN50; Biolegend), CD3 BUV805 (SK7; BD Biosciences), plus either Vδ2 PE (B6; Biolegend) for blocking experiments or Vγ9 PE (B3; Biolegend) and Vδ2 BV786 (B6; BD Biosciences) for HMB‐PP titrations. After staining, cells were resuspended in PBS containing 2% FCS before acquisition on a BD LSR Fortessa using BD FACS Diva.

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.