Pe cy7 conjugated anti ifn γ clone b27

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.

PBMCs collected prior to AC were stimulated for 4 hours with 1 μM of HLA-optimal peptides of clade B HIV consensus and autologous sequences observed in plasma before and after loss of viral control. BV711-conjugated anti-CD107A (clone H4A3, Biolegend) was included during stimulation to measure degranulation. GolgiStop and GolgiPlug (BD Biosciences) were added 2 hours post-stimulation to enable intracellular cytokine staining. Cells were stained with Live/Dead Violet, BV605-conjugated anti-CD3 (clone SK7, Biolegend) and BUV395-conjugated anti-CD8 (clone RPA-T8, BD Biosciences), fixed and permeabilized using Cytofix/Cytoperm (BD Biosciences), stained for intracellular PE/Cy7-conjugated anti-IFN-γ (clone B27, Biolegend) and analyzed via flow cytometry. Recognition was considered maintained if the frequency of CD107A⁺ IFN-γ ⁺ CD8⁺T cells upon variant peptide stimulation was greater than 50% that of baseline autologous peptide stimulation.