Buv395 conjugated anti cd8 clone rpa t8

Peptide-HLA monomers were obtained from ImmunAware (Copenhagen, Denmark) with the exceptions of A*11:01-ACQGVGGPSHK (Dr. Masafumi Takiguchi, Kumamoto University, Japan) and A*26:01-EVIPMFSAL (MBL International). Tetramers were produced by multimerization with APC-conjugated streptavidin (Biolegend) as per manufacturer’s protocol and stored at 4°C for a maximum of 4 weeks prior to use. Staining was performed using individual pH LA tetramers at 4°C prior to surface marker staining to prevent cell activation and steric interference. Where indicated, cells were also stained with BUV395-conjugated anti-CD8 (clone RPA-T8, BD Biosciences), APC/Cy7-conjugated anti-CD45RA (clone HI100, Biolegend) and FITC-conjugated anti-CD62L (clone DREG-56, Biolegend) and Live/Dead Violet viability dye (Thermo Fisher); or with BUV395-conjugated anti-CD8 (clone RPA-T8, BD Biosciences), BV605-conjugated anti-PD1 (clone EH12.2H7, Biolegend), AlexaFluor 488-conjugated anti-TIM3 (clone 344823, R&D Systems), PE/Dazzle 594-conjugated anti-TIGIT (clone A15153G, Biolegend), PE/Cy7-conjugated anti-CD160 (clone BY55, Biolegend), PerCP/Cy5.5-conjugated anti-2B4 (clone C1.7, Biolegend), BV650-conjugated anti-CD39 (clone TU66, BD Biosciences), BV510-conjugated anti-CD57 (clone HNK-1, Biolegend) and APC/Cy7-conjugated anti-CD28 (clone CD28.2, Biolegend) and analyzed by flow cytometry.

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.