Pe cf594 anti granzyme b clone gb11

CD8⁺ T cells were isolated from peripheral blood via EasySep Human CD8⁺ T Cell Isolation Kit (StemCell Technologies) and stimulated with 50 nM pH LA tetramer at 37°C for 4 hours in the presence of BV711-conjugated anti-CD107A (clone H4A3, Biolegend) to stain for transient surface expression during cellular degranulation. GolgiStop and GolgiPlug (BD Biosciences) were added after 2 hours to block cytokine secretion. Unstimulated cells were instead stained with pHLA tetramer at 4°C for 15 minutes before surface staining. Cells were stained with BUV395-conjugated anti-CD8 (clone RPA-T8, BD Biosciences) and Live/Dead Violet before fixation and permeabilization with Cytofix/Cytoperm (BD Biosciences) followed by staining with intracellular PE/Cy7-conjugated anti-IFN-γ (clone B27, Biolegend), PerCP/Cy5.5-conjugated anti-TNF-α (clone Mab11, Biolegend), PE-conjugated anti-Perforin (clone B-D48, Biolegend), PE/CF594 anti-granzyme B (clone GB11, BD Biosciences), and BV605-conjugated anti-IL-2 (clone MQ1-17H12, Biolegend) for flow cytometry. Lytic degranulation was calculated as the frequency of CD107A⁺ Perforin⁺ Granzyme B⁺ cells within the viable pHLA tetramer⁺ CD8⁺ T cell population. Gating was established using fluorescence-minus-one controls for pHLA tetramer, CD107A, perforin and granzyme B or using unstimulated controls for IFN-γ, TNF-α and IL-2.

Ex vivo (d0) and peptide-expanded (d6) CD8⁺ T cells were isolated from PBMCs and LNMCs using EasySep Human CD8⁺ T Cell Isolation Kit (StemCell Technologies) and stimulated with 4 nM APC-pHLA tetramer at 37°C for 4 hours in R10 with BV711-anti-CD107A (clone H4A3, Biolegend) to measure cytolytic degranulation. Cells were stained with BUV395-anti-CD8 (clone RPA-T8, BD Biosciences), Live/Dead Blue (Thermo Fisher), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained for intracellular PE-anti-perforin (clone B-D48, Biolegend) and PE-CF594-anti-granzyme B (clone GB11, BD Biosciences). FMO controls were used to establish gating. Experimental negative controls were pre-treated with 50 nM dasatinib 30 minutes before tetramer stimulation to prevent activation, and experimental positive controls were treated with PMA and ionomycin (eBioscience Cell Stimulation Cocktail, Thermo Fisher). Samples were analyzed by flow cytometry. Representative gating and controls are shown in fig. S5A and C. Samples with fewer than 40 live HIV-specific pHLA-tetramer⁺ CD8⁺ T cells at d0 in both PB and LN were excluded from downstream analyses.