Anti cd3 percp cy5.5 clone ucht1

SARS-CoV-2 specific CD4⁺ T and CD8⁺ T cell immunity against the inactivated COVID-19 vaccine were evaluated by intracellular cytokine staining (ICS) combined flow cytometry among PLWH and HC on day 14 after the second dose, before the booster dose, and on days 14, 30, and 180 after the booster dose vaccination. 1×10⁶ PBMCs were incubated overnight with SARS-CoV-2 Spike peptide pool (1mg/ml) containing 23 peptides from the wild-type SARS-CoV-2 virus in a 200μL final volume medium in round-bottom 96-well plates at 37°C and 5% CO2. Brefeldin A (10μg/ml) (GolgiPlug, BD Biosciences) was added in the last 5 hours of incubation. Surface marker staining was performed with anti-CD3-PerCP-cy5.5 (clone UCHT1; Biolegend), anti-CD8-APC-Cy7 (clone SK1; Biolegend), and anti-CD4-PE-Cy7 (clone RPA-T4; Biolegend) for 30 min at 4°C in the dark. Fixation and permeabilization solution (Cytofix/Cytoperm; BD Biosciences) was added for 45 min at 4°C in the dark. Intracellular cytokines staining with anti-IFN-γ-PE (clone 4S.B3; Biolegend) and anti-TNF-α-APC (clone MAb11; Biolegend) was then performed according to the manufacturer’s instructions. The stained cells were fixed with 2% paraformaldehyde and then analyzed by flow cytometry. Data were analyzed with FlowJo software version 10, and the gating strategy is shown in Supplementary Figure 1.

PBMCs from healthy and untreated HIV-1-infected individuals were gently thawed by adding dropwise complete medium supplemented with 25 U/ml Benzonase Nuclease (Merck Milipore Novagen) followed by a 30 min incubation at 37°C and 5% CO₂. After counting and washing with PBS, cells were incubated with LIVE/DEAD fixable Near-IR Dead Cell staining kit (Invitrogen) and the following antibodies at 4°C: anti-CD3-PerCP-Cy5.5 (clone UCHT1, BioLegend), anti-CD4-BV650 (clone RPA-T4, BioLegend), anti-CD8-AF700 (clone EB6B, BioLegend), anti-CD14-APC-Cy7 (clone HCD14, BioLegend), anti-CD19-APC-Cy7 (clone HIB19, BioLegend), anti-CD56-BUV395 (clone NCAM16.2, BD Optibuild), anti-CD16-BV785 (clone 3G8, BioLegend), anti-CD57-BV510 (clone QA17A04, BioLegend), anti-NKG2A-PE-Vio615 (clone REA110, Miltenyi), anti-NKG2C-BUV563 (clone 134591, BD Optibuild), anti-KIR2DL1/S1-APC (clone EB6B, Beckman Coulter), anti-KIR2DL1/S5-PE (clone 134211, R&D Systems), anti-KIR2DL2/L3/S2-BV711 (clone DX27, BD Optibuild), anti-KIR2DL3-AF488 (clone 180701, R&D Systems), anti-KIR3DL1-AF700 (clone DX9, BioLegend), anti-KIR3DL1/L2-PE-Vio770 (clone 5.133, Miltenyi) and anti-KIR2DS4-Biotin (clone JJC11.6, Miltenyi) with secondary Strepdavidin-BV421 (BioLegend). Cells were washed and then fixed with FluoroFix Buffer (BioLegend). Cells were analyzed by flow cytometry.

After resuscitation and counting, 1 × 10⁶ PBMCs were suspended with a 200μL final volume. For surface marker staining, cells were stained with specific antibodies for 30 min at 4 °C in the dark. The following antibodies were used: anti-CD3-PerCP-cy5.5 (clone UCHT1; Biolegend), anti-CD8-APC-Cy7 (clone SK1; Biolegend), anti-CD4-PE-Cy7 (clone RPA-T4; Biolegend), anti-TCRγδ-FITC (clone B1; Biolegend), anti-Fas-APC (clone DX2; BD Biosciences), Annexin V-PE (Annexin V PE Apoptosis kit, Cat#559763, BD Biosciences), anti-CCR5-PE (clone J418F1; Biolegend), anti-CXCR3-APC (clone G025H7; Biolegend), anti-CXCR4-PE (clone 12g5; Biolegend), anti-CCR7-APC (clone G043h7; Biolegend). The stained cells were fixed with 2% paraformaldehyde and then analyzed by flow cytometry.