Anti tnf α apc clone mab11

This procedure was performed as described before [16 (link)]. PBMCs were incubated for one hour stimulated with anti-CD28/CD49d (1 mg/ml) in a 200μL final volume medium in round-bottom 96-well plates at 37 °C and 5% CO₂. Incubation with Brefeldin A (GolgiPlug, BD Biosciences) for 5 h was performed for further intracellular cytokines staining. Use PBS to wash cells twice and then surface marker staining was performed with with specific antibodies for 30 min at 4 °C in the dark. Fixation and Permeabilization Solution (Cytofix/Cytoperm; BD Biosciences) were added for 45 min at 4 °C in the dark. For intracellular cytokines staining, the following antibodies were used: anti-Granzyme A-PE (clone CB9; Biolegend), anti-Perforin-APC (clone dG9; Biolegend), anti-IFN-γ-PE (clone 4S.B3; Biolegend) and anti-TNF-α-APC (clone MAb11; Biolegend) according to the manufacturer's instructions. The stained cells were fixed with 2% paraformaldehyde and then analyzed by flow cytometry.

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.

The following monoclonal antibodies (mAbs) that cross-reacted with rhesus macaque were obtained from BD Pharmingen (BD Biosciences, CA, USA): anti-CD3-PE/-APC-Cy7 (clone SP34-2), anti-CD4-FITC/-PerCP-Cy5.5 (clone L200), anti-CD8α-PE-Cy7 (clone RPA-T8), anti-CD20-PerCP-Cy5.5 (clone 2H7), anti-CD14-APC (clone M5E2), anti-HLA-DR-APC (clone L243), anti-CD28-APC (clone CD28.2), anti-CD95-FITC (clone DX2), anti-Ki67-PE (clone B56), and anti-IL-4-PerCP-Cy5.5 (clone 8D4-8). Anti-CD38-FITC (clone AT-1) mAb was obtained from STEMCELL. Anti-PD-1-PE (eBioJ105) was obtained from eBioscience (CA, USA). Anti-IL-2-FITC (clone MQ1-17H12), anti-IFN-γ-PE (clone 4S.B3), and anti-TNF-α-APC (clone Mab11) mAbs were all obtained from BioLegend (CA, USA).