Anti ifn γ bv421

Purified NK cells were cultured alone or co-cultured with autologous HIV-infected CD4⁺ T cells in complete growth medium in the presence of GolgiStop (BD) and anti-CD107a PE (clone H4A3) antibody (BD) for 16 h. Following incubation, cells were stained with anti-CD3 BV510 (clone UCHT1) (BD), anti-CD56 PerCP Cy5.5 (clone B159) (BD), anti-IFN-γ BV421 (clone B27) (BD) and anti-Siglec-9 PE-Cy7 (clone K8) antibodies. Analysis of Siglec-9⁺ and Siglec-9^- NK cell subsets were made on CD3^- CD56^dim gated cells. For experiments that involve antibody-sialidase conjugates, PBMC were cultured alone or co-cultured with autologous HIV-infected CD4⁺ T cells that had been treated with Sialidase only (300 nM), isotype-matched antibody (300 nM) and indicated amount of antibody or antibody-sialidase conjugate for 2 h at 37°C. Cells were mixed in the complete growth medium in the presence of GolgiStop (BD) and anti-CD107a PE antibody (BD) for 16 h. Following incubation, cells were stained with anti-CD3 BV510 (BD), anti-CD56 PerCP Cy5.5 (BD), anti-IFN-γ BV421 (BD) and anti-TNF-α (Clone MAb11) (BioLegend). NK cells were defined as CD3^- and CD56⁺. Background NK degranulation (PBMC only control) was subtracted. Analysis of CD107a⁺, IFN-γ⁺, and TNF-α⁺ NK cells was made on CD3^- CD56^dim gated cells.

Dead cells were excluded using a live/ dead fixable dye staining kit (BioLegend). Cells were stained for surface markers and then fixed and permeabilized with CytoFix/Perm Buffer (BD). Intracellular molecules were detected using anti-IFN-γ-BV421 (BD) and anti-caspase 3-AF647 (BD) and anti-Granzyme B AF647 (BioLegend). For intranuclear staining, the True nuclear transcription buffer set from Biolegend was used according to the manufacturer protocol. Antibody against anti-Ki67 AF488 (BioLegend) was used. All samples were acquired on a BD Canto II. All analyses were performed using Flowjo software (Tree Star).

HLA‐B3501‐NY9‐specific CD8⁺ T‐cell clones were stimulated with 2 or 20 μm of NY9 peptide and were incubated for 7 h in the presence of GolgiPlug (BD Biosciences), GolgiStop (BD Biosciences) and anti‐CD107a‐AF488‐FITC (BD Biosciences). Following stimulation, cells were surface stained for 30 min with anti‐CD8‐PerCP‐Cy5.5 (BD Biosciences), anti‐CD4‐BV650 (BD Biosciences) and Live/Dead Fixable Near‐IR Dead Cell Stain (Life Technologies). Cells were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences) and then intracellularly stained with anti‐IFN‐γ‐BV421 (BD Biosciences) as well as anti‐TNF‐PeCy7, anti‐MIP1β‐APC and interleukin‐2‐PE (all BD Biosciences) for a further 30 min. Cells were acquired on a CytoFLEX Flow Cytometer. Post‐acquisition analysis was performed using FlowJo software (version 10.7.1; BD Biosciences). Cytokine detection levels identified in the no‐peptide control condition were subtracted from the corresponding test conditions in all summary graphs to account for nonspecific, spontaneous cytokine production.

CD8⁺ T cell lines were stimulated with 1 μM of the cognate or the homologous peptide and were incubated for 4–5 h in the presence of GolgiPlug, GolgiStop and anti-CD107a-FITC (dilution 1:100) (all BD Biosciences). After stimulation, cells were surface stained for 30 min with anti-CD3-BV480 (1:100), anti-CD8-PerCP-Cy5.5 (1:50) and anti-CD4-BV650 (1:100) antibodies (all BD Biosciences) and Live/Dead Fixable Near-IR Dead Cell Stain (Life Technologies). Cells were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences) and then intracellularly stained with anti-IFN-γ-BV421 (1:100), anti-TNF-PE-Cy7 (1:100), anti-IL2-PE (1:100) and anti-MIP-1β-APC (1:100) antibodies (all BD Biosciences) for a further 30 min. Cells were acquired on the BD FACSymphony A3 system using the FACSDiva software (v.9.0.). Post-acquisition analysis was performed using FlowJo software (v.10). Cytokine detection levels identified in the no-peptide control condition were subtracted from the corresponding test conditions in all summary graphs to account for non-specific, spontaneous cytokine production.

Mouse lung tissue was digested with collagenase to generate a single-cell suspension. Lung cells or cultured cells were stained with CD11c (anti-CD11c-PE-Cy5.5, e Bioscience, USA), MHCII (anti-MHCII-PE, BD Pharmingen, USA) and GITRL (anti-GITRL-BV421, BD Pharmingen, USA) to identify CD11c⁺MHCII⁺GITRL⁺ DCs. For the analysis of Tregs, lung cells or cultured cells were first stained for CD4 (anti-CD4-APC-Cy7, BD Pharmingen, USA) and CD25 (anti-CD25-PE, BD Pharmingen, USA) and then fixed and permeabilized for intracellular Foxp3 (anti-Foxp3-APC, e Bioscience, USA) staining. For detecting Th1, Th2 and Th17 cells, lung cells or cultured cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (1 μg/ml) for 6 h in the presence of Golgi Stop solution. After stimulation, cells were incubated with surface markers for CD4 (anti-CD4-FITC, BD Pharmingen, USA), permeabilized and stained for the intracellular cytokines IL-4 (anti-IL-4-APC, BD Pharmingen, USA), IFN-γ (anti-IFN-γ-BV421, BD Pharmingen, USA) and IL-17 (anti-IL-17-PE, BD Pharmingen, USA). Data were acquired using FACS Canto II (BD Biosciences, USA) and analyzed with FlowJo software.

For assessing HCV-specific CD8 T cell function in SVR and relapsed patients, baseline and EOT PBMC were tested. Cells were thawed and rested overnight to aid in recovery from freezing conditions. At least 1 million PBMC were tested in each condition: unstimulated (negative control) or HCV peptide stimulated (1 μg/ml each peptide). Cells were incubated with respective stimulations along with addition of 10 IU/ml IL-2 for 3 days. On day 4, cells were re-stimulated with HCV peptides for 18 h along with addition of BV650 anti-CD107a antibody and golgi transport blockers (Golgi Stop 0.7 μl/ml and Golgi Plug 1 μg/ml (BD Biosciences). Cells were stained for Live/dead stain and surface markers including HCV tetramers where applicable. Cytokine production was detected by staining with anti-IFNγ BV421, anti-TNFα PE and anti-IL-2 PerCPCy5.5 antibodies following intracellular staining fixation and permeabilization protocol (BD Biosciences) and flow cytometry. Samples were considered positive if the following criteria were met: the percentage of cytokine-positive cells was >2 times background (non-stimulated cells), the percentage of cytokine-positive cells was >0.05% after background subtraction, and the population of positive cells was larger than 10 cells.