Cytofix cytoperm fixation and permeabilization kit

For surface staining of the stimulated cells, anti-CD4-APC (BD Biosciences), anti-CD3-PERCP, anti-CD8-FITC, anti-CD14-FITC (eBioscience, San Diego, CA, USA) were used. Fc receptor binding inhibitor antibody(eBioscience) was used to inhibit the non-specific Fc-gamma receptor (FcgammaR)-mediated binding of mouse monoclonal antibodies. Fix Viability Dye (eBioscience) was used to stain and exclude dead cells from the analyses. For intracellular staining, cells were restimulated on 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 250 ng/mL ionomycin (Sigma-Aldrich) in the presence of 10 µg/mL of brefeldin A (Sigma-Aldrich) for the last 5 h of culture. Cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation and Permeabilization Kit (BD Biosciences), labeled with anti-IL-17-PE (eBiosciences), and analyzed on the Gallios system using the Kaluza software (Beckman Coulter, CA, USA).

Following cell stimulation, 3 × 10⁵ PBMCs were stained for 30 min, 4°C, in the dark with the following anti-human antibodies for surface antigen detection: CD45-BUV395 (HI30), CD3-BUV737 [SK7 (also known as Leu-4)], CD4-APC (SK3 (also known as Leu3a)), CD8-BV605 (SK1), CD56-BB700 (NCAM16.2), and CD19-BV650 (SJ25C1), all purchased from BD Biosciences (BD, San Jose, CA, USA) according to the manufacturer’s information. For intracellular cytokine detection, cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation and Permeabilization Kit (BD) for 10 min at 4°C. Cells were then washed in 1× Perm/Wash Buffer (BD) and stained for 30 min, 4°C, in the dark, with IFNγ-BV480 (B27) and IL4-PE (MP4-25D2). Following doublet exclusion and morphological FSC-A/SSC-A setting, total leukocytes were gated on CD45⁺ cells. Samples were acquired using a BD FACS Fortessa X20 analyzer, equipped with 5 lasers. Cell subsets were identified as CD45⁺ cells: CD3⁺ cells (total T cells), CD3⁺CD4⁺ cells (CD4⁺T cells), CD3⁺CD8⁺ cells (CD8⁺ T cells), CD3^-CD19⁺ cell (B cells), and CD3^-CD56⁺ (total NK cells). Total NK cells were interrogated for CD56 subsets (CD56^bright, CD56^dim). Finally, the production of IFNγ and IL4 was interrogated for CD4⁺ and CD8⁺ T cells, B cells, and total NK cells. Flow data were analyzed using the FlowJo v10 software (Tree Star).

Spleen cells from different groups of BALB/c mice were analyzed for cytokine content as previously described [48 (link)]. Briefly, T cells were seeded at 10⁶/mL in RPMI 1640–10% FBS, and stimulated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) in the presence of monensin (BD) during 4-h incubation at 37 °C, and then treated with Cytofix/Cytoperm fixation and permeabilization kit (BD). For macrophage stimulation, spleen cells were seeded at 10⁶/mL in RPMI 1640–10% FBS, and stimulated with LPS 100 ng/mL (Sigma-Aldrich) in the presence of monensin during 4 h incubation at 37 °C. The permeabilized cells were intracellularly stained with the PE-conjugated cytokine-specific mAbs anti-IFNγ (clone XMG1.2), and anti-TNFα (clone MP6-XT22), all from BD; or isotype-matched irrelevant mAbs from BioLegend.

Flow cytometry was performed on venous peripheral blood collected in heparin-coated vacutainer tubes, on tumor and peritumoral tissues using a FACSCanto II 6-colour flow cytometer, daily calibrated with calibrite beads (Fitc, Pe, PerCP and APC) and compbeads (Pe-Cy7 and APC-Cy7; Becton Dickinson, San Jose, CA, USA). Fluorochrome-labelled monoclonal antibodies (BD Bioscience) for identification of circulating and tissue Treg cells were used: Fitc-anti-FOXP3, Pe-Cy7-anti-CD25, APC-Cy7-anti-CD4, APC-anti-CD45RA, Pe-anti-CD152 (CTLA-4), PercP-anti-CD184 (CXCR4), APC-anti-CD279 (PD-1), Pe-anti-CD278 (ICOS) and APC-anti-CD39 (ENTPD1). Intracellular staining for FoxP3, ICOS and CTLA-4 was performed using a commercially available kit (BD Cytofix/Cytoperm; fixation and permeabilization kit; BD Pharmingen) according to the manufacturer's instructions. A minimum of 100.000 events for each sample were collected and data were analysed using FacsDiva software 6.1.3 (BD Bioscience). The absolute number of CD4 was calculated as follow: [total withe blood cell count (cells/uL) x percent CD4]/100 or [total tumor-infiltrating immune cell count (cells/100 mg tumor) x percent CD4]/100.