Ifn γ pe cy7

Anti-mouse IL-17A-PE, CD4-APC and IFN-γ-PE-cy7, anti-mouse IgG isotype, anti-human IFN-γ-PE-cy7, CD4-APC and IL-17A-PE and anti-human IgG isotype were obtained from eBioscience. For intracellular expression of IFN-γ and IL-17, CD4+ T cells were incubated for 1 hours with ionomycin (1 μg/mL), PMA (50 ng/mL) and for another 4 hours with brefeldin A (10 μg/ml, Sigma-Aldrich), harvested, washed and fixed before permeabilization.
BMDCs or MD-DCs were stained for 30 minutes at 4 °C with anti-mouse CD86-FITC (eBioscience), CD11c-APC (eBioscience), CD80-PE (eBioscience), CD40-FITC (BioLegend, San Diego, CA, USA), MHCII-PE (eBioscience), anti-human CD86-PE-cy7 (BioLegend), CD40-FITC (BioLegend), HLA-DR-PE-cy5.5 (BioLegend), and CD80-PE (BioLegend).

Cells isolated from spleen, lungs or BAL were cultured with 10 µg/ml Purified Protein Derivative of M. tb (PPD-T) (Statens Serum Institut, Copenhagen, DK), 1 µg/ml anti-CD28 (BD Biosciences) and 10 µg/ml Brefeldin A (Sigma-Aldrich) for 16 h at 37 °C/5% CO₂. Cells were washed (300 g/5 min) and surface stained for 15 min/4 °C with pre-titrated antibodies: PD-1⁻FITC, CD44-BV785, CD8-AF700, KLRG1-PerCP-Cy5.5, CXCR3-BV421, live/dead-Zombie Aqua (all BioLegend, San Diego, CA, USA), CD90.2-eFluor 450 (eBioscience), and CD4-APC-H7 (BD Biosciences). Cells were then washed, treated with BD Biosciences Cytofix/Cytoperm as per the manufacturer’s instructions and stained intracellularly for 30 min/4 °C with IFN-γ-PE-Cy7, IL-2-APC (both eBioscience) and TNF-α-BV605 (BioLegend). Cells were washed again and acquired using an LSRFortessa™ analyser, utilising a 532 nm laser for PE and PE-conjugate excitation, with FACSDiva™ software (BD Biosciences). Final analysis was performed using FlowJo® software (Tree Star, Ashland, OR, USA) on a minimum of 100,000 live lymphocytes (50,000 for BAL).

Mouse cells were stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly-conjugated monoclonal antibodies to CD45-PE-Dazzle594 (Biolegend, 104), TCRβ-APCef780 or APC (Invitrogen, H57-597), CD8-BV510 (Biolegend, 53-6.7), then fixed, permeabilized and stained for the cytokines IL-17A-PerCpCy5.5 or PE (e-Biosciences, 17B7), IFNγ-PE-Cy7 (e-Biosciences, XMG1.2) and TNFα-BV650 (Biolegend, MP6-XT22).

Primary tumor tissues or spleens were dissociated using a gentleMACS Dissociator (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) after enzyme digestion in serum-free RPMI 1640 medium containing DNase I (1 U/mL; Roche, Basel, Switzerland) and collagenase D (1 mg/mL; Roche) for 20 min at 37 °C with gentle agitation. After single-cell dissociation, the cells were filtered through a 40 μm cell strainer, and the red blood cells were eliminated using BD Pharm Lyse buffer (BD Biosciences, San Jose, CA, USA). The cells were stained by incubating at 4 °C for 1 h using the following antibodies: CD45-FITC, F4/80-PE, CD206-APC, CD86-PE/Cy7, CD8-APC, PD-1-PE, and IFN-γ-PE/Cy7 (e-Bioscience, CA, USA). After staining, the cells were analyzed using a BD FACSLyric flow cytometry system (BD Biosciences) and FlowJo software (Treestar, San Carlos, CA, USA).

Cells isolated from spleen, lungs or peripheral blood were cultured with 2 µg/ml of two immunodominant peptides (Pepscan, Lelystad, The Netherlands), [SSTHEANTMAMMARDT] and [AGYAGTLQSLGAEIAV] of the TB10.4 protein, previously demonstrated to stimulate both CD4⁺ & CD8⁺ T cell responses (Kaveh & Hogarth, unpublished); 1 µg/ml anti-CD28 (BD Biosciences) and 10 µg/ml Brefeldin A (Sigma) for 16 h at 37 °C/5% CO₂. Cells were washed at 300 g for 5 min and surface stained with combinations of CD62L-FITC, CD27-PerCP-Cy5.5, CD8-AF700, CD44-BV421, CD127-PE-Cy7, CD69-FITC, CCR7-BV421, live/dead-Zombie Aqua (all BioLegend) and CD4-APC-H7 (BD Biosciences). Cells were then washed, treated with BD Biosciences Cytofix/Cytoperm as per manufacturer’s instructions and stained intracellularly with combinations of IFN-γ-PE-Cy7, IL-2-APC (both eBioscience), IFN-γ-BV605 and TNF-α-BV605 (both BioLegend). Cells were washed again and analysed using an LSRFortessa™ analyser utilising a 532 nm laser for PE and PE-conjugate excitation with FACSDiva™ software (BD Biosciences). Final analysis was performed using FlowJo® software (Tree Star Inc.) on a minimum of 100,000 live lymphocytes (50,000 for peripheral blood).

Whole blood was incubated with 1 μg/mL αCD28, 1 μg/mL αCD49d (BD Biosciences) and stimulated with 20 μg/ml PPD (SSI, Denmark), 5 μg/ml staphylococcal enterotoxin B (Sigma Aldrich), or no antigen (unstimulated). Samples were incubated at 37°C in 5% CO₂ for 6 h, 3 μg/ml Brefeldin-A (Sigma Aldrich) was added, and samples were incubated for another 6 h in a 37°C water bath. Samples were then treated with 2 mM ethylenediaminetetraacetic acid (Gibco), and red blood cells were lysed using FACS Lysing solution (BD Biosciences) and samples were frozen in PBS with 10% DMSO for batched ICS staining. Frozen samples were thawed, permeabilised and incubated with antibodies against CD3 (AF700), IFN-γ (PE-Cy7 (eBioscience); CD4 (APC), CD14 (Pacific blue), TNF-α (PerCP-Cy5.5), and IL-17 (AF488) (BioLegend); CD8 (APC-H7) (Becton Dickinson) and IL-2 (PE) (Beckman Coulter). Samples were acquired on an LSR II flow cytometer (BD Biosciences) and responses analyzed using FlowJo version 8.8.7 (Tree Star Inc., Ashland, USA). Cytokines were measured as the frequency of singlet CD14– CD3+ T cells, CD4+ T cells, and CD8+ T cells. Data are presented as percentages of cytokine-positive cells minus responses in unstimulated cells; the gating strategy is shown in Supplementary Figure 1.