Anti ifn γ clone xmg1

The cell phenotypes were analyzed by FACS Aria II flow cytometer (BD Bioscience), and data were analyzed with the FlowJo V10.0.7 (FlowJo, OR, USA). Cells were collected and washed in PBS, then the single-cell suspensions were labeled with various fluorochrome-conjugated antibodies for 30 minutes within PBS containing 0.5% BSA on ice. The gating strategies for CD4+ central memory T cells, CD8+ central memory T cells, CD4+ effector memory T cells, Tfh cells were CD4+CD62L+CD44+, CD8+CD62L+CD44+, CD4+CD62L-CD44+, CD4+CXCR5+PD-1+, respectively. The following antibodies were used: anti-mouse CD4-AF700 (clone RM4-5, eBioscience), anti-mouse CD8a-BV510 (clone 53-6.7, BD Horizon), anti-mouse CD62L-PE (clone MEL-14, Biolegend), anti-mouse CD44-Percp/Cy5.5 (clone 1M7, Biolegend), anti-mouse CXCR5-APC (clone SPRCL5, eBioscience) and anti-mouse PD-1-PE/Cy7 (clone J43, eBioscience). For ICCS, mice splenocytes were collected and co-stimulated with anti-CD3 antibody and anti-CD28 antibody (Biolegend) for 1 hour at 37 °C, then Cells were incubated with 5 mg/mL brefeldin A (Topscience), 2 mM monensin (Topscience). Cells were then harvested, stained with indicated antibodies with a Fixation/Permeabilization Solution Kit (BD Bioscience). We used these antibodies to detect cytokine secretion of T cells: anti-IFN-γ (clone XMG1.2, Biolegend) and anti-IL-2 (clone JES6-5H4, Biolegend).

Single-cell suspensions were stained with anti-CD45 (clone I3/2,3), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD49b (clone HMα2), anti-CD73 (clone TY/11.8), anti-PD-1 (clone 29F.1A12), anti-Nrp-1 (clone 3E12), anti-IL-10 (clone JES5-16e3), anti-Foxp3 (clone FJK-165), anti-granzyme B (clone QA16A02), and anti-IFN-γ clone XMG1.2) coupled to FITC, PE, PE-Cy7, PerCP, PerCP-Cy7, and APC (all from Biolegend, NJ, USA). For intracellular cytokine staining, cells were stimulated for 5 h at 37°C adding PMA (50 ng/ml) (Sigma, Milano, Italy), ionomycin (1 mg/ml) (Sigma, Milano, Italy), and Brefeldin A (10 mg/ml) (eBioscience, CA, USA). Then, cells were fixed using a permeabilization kit (Biolegend or BD, USA) according to the manufacturer’s procedure and subsequently stained for intracellular markers. Flow cytometric data were acquired using FACS CantoII (BD Immunocytometry System, San Jose, USA) and analyzed with FlowJo software (Tree Star, OH, USA).

Fluorochrome conjugated antibodies specific for mouse, anti-CD3 (clone: 145-2C11), anti-CD4 (clone: RM4-5), anti-Foxp3 (clone: MF-14), anti-IFNγ (clone: XMG1.2) and anti-IL-17A (clone: TC11-18H10.1) were all purchased from Biolegend, San Diego, CA, USA. Anti-CD8 (clone: 53–6.7) was purchased from eBioscience, Frankfurt, Germany.

Tumors were harvested and dissociated into single-cell suspensions. Then, cells were blocked with anti‐FcR (clone 2.4 G2, BD Pharmingen) and labeled with indicated surface markers for 30 min at 4°C. For IFN-γ staining, single-cells were cultured in the presence of a cell activation cocktail (with Brefeldin A) (Biolegend) for 5 h. Cells were permeabilized and stained with intracellular antibodies for 30 min at 4°C as instructed by the manufacturer. Dead cells were excluded using LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). The antibodies used in the flow cytometry analysis were: anti-CD45 (clone 30-F11, Invitrogen), anti-CD3 (clone 145–2C11, Biolegend), anti CD4 (clone RM4-5, Biolegend), anti-CD8 (clone 53–6.7, Biolegend), anti-IFN-γ (clone XMG1.2, Biolegend), anti-CD11C (clone N418, Biolegend), anti-CD80 (clone 16–10A1, Biolegend), and anti-CD86 (clone GL1, Invitrogen). Because the specific reaction with ovalbumin-derived peptide SIINFEKL bound to H-2 Kb of MHC class I, anti–H-2kb bound to SIINFEKL (clone 25-D1.16, Biolegend) was used to recognize the tumor specific immune cells. Flow cytometry was performed on the FACS Aria III platform (BD Biosciences, San Jose, CA, USA) and results analyzed using FlowJo software version 10.4 (TreeStar).

Antigen-specific CD8 T cells were identified by labeling with D^b/NP₃₆₆ or D^b/PA₂₂₄in house prepared tetramers for 1 h at 4°C (22 (link)). Tetramer staining was followed by surface staining with appropriate antibody cocktails for 20 min at 4°C. Surface markers were stained using following antibodies: anti-CD8 (clone 53-6.7, BioLegend), anti-CD90.2 (clone 30-H12, BioLegend), anti-CD45.2 (clone 104, BioLegend), anti-CD103 (clone 2E7, BioLegend), anti-CD69 (clone H.12F3, BioLegend), anti-KLRG-1 (clone 2F1, eBioscience, San Diego, CA, USA), anti-CD127 (clone A7R34, BioLegend), anti-CX3CR1 (clone SA011F11, BioLegend), anti-CXCR3 (clone CXCR3-173, BioLegend), and anti-CD49a (clone Ha31/8, BD Pharmingen). Intracellular cytokine staining was performed using anti-IFNγ (clone XMG1.2, BioLegend), anti-TNF (clone MP6-XT22, BioLegend), and anti-IL2 (clone JES6-5H4, BioLegend) antibodies. Proliferation of CD8 T cells was assessed by intracellular staining with anti-Ki67 (clone MOPC-21, BD Pharmingen). Flow cytometry data were acquired using LSRFortessa (Becton Dickinson, Rutherford, NY, USA) and analyzed using the FlowJo software (Tree Star Inc., Ashland, OR, USA).