Vδ2 pe b6

Expansion from the PLWH/ART cohort was assessed for purity of Vδ2 T cells on day 10 or 11. 0.5 × 10⁶ cells were stained with Aqua viability dye (ThermoFisher), Vδ1 APC (TS8.2; ThermoFisher), CD3 BV786 (SK7; Biolegend), CD56 BUV395 (NCAM16.2; BD Biosciences), Vδ2 PE (B6; Biolegend) and TCR αβ PE‐Cy7 (IP36; ThermoFisher). After staining, cells were resuspended in PBS containing 2% FCS before acquisition before acquiring samples on a BD LSR Fortessa using BD FACS Diva. Contaminating cells were magnetically depleted of TCR αβ PE‐Cy7 (IP36; ThermoFisher) and/or Vδ1 PE‐Cy7 (TS8.2; ThermoFisher) binding cells using anti‐PE MicroBeads (Miltenyi Biotec, Sydney, Australia) according to the manufacturer's instructions. Depleted cultures were again checked for Vδ2 T cell purity by staining with an Aqua viability dye (ThermoFisher), CD3 BV786 (SK7; Biolegend), CD56 BUV395 (NCAM16.2; BD Biosciences), Vδ2 PE (B6; Biolegend) and TCR αβ PE‐Cy7 (IP36; ThermoFisher).

On day 14 of expansion, Vδ2 T cells were collected and washed in PBS and stained with Aqua viability dye (ThermoFisher). Next, cells were surface stained for the following antibodies: CD26 FITC (BA5b, Biolegend), CD45RA PerCpCy5.5 (HI100; Biolegend), CD160 Alexa Fluor 647 (BY55; BD Biosciences), TIGIT APC Fire 750 (A15153G; Biolegend), PD‐1 BV421 (EH12.2H7; Biolegend), NKG2D BV650 (1D11; BD Biosciences), CD27 BV786 (L128; BD Biosciences), CD94 BUV395 (HP‐3D9; BD Biosciences), Tim‐3 BUV737 (7D3; BD Biosciences), CD3 BUV805 (SK7; BD Biosciences), Vδ2 PE (B6; Biolegend) and 2B4 PE‐Dazzle (C1.7; Biolegend). After surface staining, cells were washed and resuspended in PBS containing 2% FCS before acquisition on a BD LSR Fortessa using BD FACS Diva.

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.