Clone im7

Lung single-cell suspensions from vaccinated or Mtb-infected mice were generated as before4 (link). For LNs and spleens, organs were passed through a 70 μm cell strainer and then processed as for lungs4 (link). For flow cytometric analysis, cells were either stained immediately, or stimulated for 18 h in the presence of Ag85B_240–254 peptide, with GolgiStop (5 μl ml⁻¹; BD Biosciences) and Brefeldin A (1 μg ml⁻¹; BioLegend, San Diego, CA, USA) added for the last 5 h. Intracellular cytokine staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences). Cells were collected using a BD FACS Jazz or a BD LSR Fortessa, and analysis was performed using FlowJo (Treestar, Ashland, OR, USA). Antibodies used include anti-mouse CD11c (clone HL3; BD Biosciences; dilution: 1/200), anti-mouse MHC-II (clone M5/114.15.2; Tonbo Biosciences, San Diego, CA, USA; dilution: 1/150), anti-mouse CD3 (clone 500A2; BD Biosciences; dilution: 1/200), anti-mouse CD4 (clone RM4-5; BD Biosciences, dilution: 1/200), anti-mouse CD44 (clone IM7; eBioscience, dilution: 1/400), anti-mouse IL-17 (clone TC11-18H10; BD Biosciences, dilution: 1/100), anti-mouse IFN-γ (XMG1.2; BD Biosciences; dilution: 1/100), anti-mouse Thy1.1 (clone OX-7; BD Biosciences; dilution: 1/100) and anti-mouse CD103 (clone 2E7, eBioscience; dilution: 1/200).

Tumours were digested in sterile Epicult media (Stem Cell Technology), minced with sterile razor blades and incubated for 3 h in the presence of Collagenase/Hyaluronidase (1,000 units per sample). Cells were washed with sterile filtered PBS supplemented with 1% bovine serum albumin (PBS-BSA 1%) and filtered through a 40-μM nylon mesh (BD Biosciences). For the detection of CD44 and CD133 antigens, cells were stained in a volume of 100 μl (PBS-BSA 1%) with each antibody CD44-APC (100 ng per 10⁶–10⁸ cells, Clone IM7, eBiosciences) and CD133/1-PE (100 ng per 10⁶–10⁸ cells, clone AC133, Miltenyl Biotech). Cells were labelled on ice for 30 min and analysed (BD FACS Aria III, Flow Core). Samples were analysed for cell population distribution and sorted for GFP/viability (GFP+/DAPI−) and CD133/CD44 expression. For flow plot analyses, samples were run using the FlowJo 7.5 software (Tree Star).