Anti mouse thy1.1 clone ox 7

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).

Cell surface staining was performed according to standard procedures using antibodies against anti-mouse CD4 (clone GK1.5; BD Pharmingen), anti-mouse Thy1.1 (clone OX-7; BD Pharmingen), anti-mouse Thy1.2 (clone 53-2.1; BD Pharmingen), anti-mouse LAG-3 (clone C9B7W; BD Pharmingen), anti-mouse TNFR2 (clone TR75-89; BD Pharmingen), anti-mouse CD25 (clone PC61; BD Pharmingen), anti-mouse CTLA-4 (clone UC10-F10-11; BD Pharmingen), anti-mouse PD-1 (clone J43; eBioscience), anti-mouse GARP (clone YGIC86; eBioscience), anti-mouse CD38 (clone 90; eBioscience), anti-mouse GITR (clone DTA-1; eBioscience), anti-mouse neuropilin-1 (clone 3DS304M; eBioscience), and anti-mouse CD73 (cloneTY/11.8; Biolegend). Foxp3 was detected using the GFP reporter or by intracellular cytokine staining using a anti-mouse Foxp3 (clone FJK-16 s; eBioscience) antibody. All flow cytometry was performed on a BD LSRII or BD FACSCalibur and analyzed using FlowJo (TreeStar). Surface stain was performed by incubating cells with antibody cocktail mix for 20 minutes at 4°C. For intracellular cytokine Foxp3 staining, cells were first stained for surface receptors, fixed in 4% formyl saline, permeabilized (0.5% BSA, 0.1% Triton, and 2 mM EDTA in PBS) for 45 minutes at room temperature. Cells were incubated overnight with the anti-cytokine antibodies and analyzed by flow cytometry.

Pmel-1 transgenic (Thy1.1+Vβ13+) mice were bred in house. BMDC were generated from C57BL/6 mice as described above. On day 3, DC were transduced at an MOI of 50 with Ad5-gp100. On day 4, DC were with transduced at an MOI of 50 with Ad5-GFP, Ad5-GFP + Mimic maturation cocktail, or Ad5-LMP1 in the presence of 1ug/ml doxycycline. On day 5, 1x10⁶ DC were injected intradermally into the flank of C57BL/6 mice that had been adoptively transferred with 1x10⁶ purified CD8+ T cell isolated from a Pmel-1 mouse spleens 24 hours previously by positive bead selection (Miltenyi). For a positive control, LPS (Sigma Aldrich) + gp100 peptide (KVPRNQDWL) (American Peptide) were injected subcutaneously into C57BL/6 mice. After 5 days, spleens were harvested and processed for single cell suspensions. Spleen preps were stained with anti-mouse CD3e clone 500A2, anti-mouse CD8a PerCP clone 53–6.7, anti-mouse Thy-1.1 clone OX-7 (BD Bioscience). Data represented as percent of CD3+ CD8+ T cells that were Thy1.1+.