Cd8 53 6

The antibodies with following specificities were used; CD4 (GK1.5, Tonbo), CD8 (53.67, Tonbo), CD11c (HL3, BD), CD11b (M1/70, eBioscience), TCRβ (H57-597, BD), NK1.1 (PK136, eBioscience), γδTCR (GL3, Biolegend), CD44 (IM7, eBioscience), CD122 (TMβ1, BD), CD62L (MEL-14, eBioscience), IL-15Rα (DNT15Rα, eBioscience), IL-2Rα (3C7, Biolegend), γc (4G3, BD), IL-17 (eBio17B4, eBioscience), IFNγ (XMG1.2, Biolegend), pSTAT5 (clone 47, BD), Foxp3 (MF23, BD). Fluorochrome-conjugated CD1d tetramers loaded with PBS-57 were obtained from the NIH tetramer facility (Emory University, Atlanta, GA).

To determine ex vivo T cell functionality from tumor-bearing mice, single cell suspensions from spleen and tumor were obtained and activated in vitro^{30 (link)}. Briefly, mononuclear cells were restimulated with 1X Cell Stimulation Cocktail (eBioscience) in the presence of Golgiplug and Golgistop (BD) according to manufacturer’s instructions in T cell media. 4–5 hours later, cells were stained with live/dead ghost dye at 1:500 (Tonbo) and the following antibodies at diluted in FACs buffer at 1:200 against CD45 (30F-11, BD), CD3 (17A2, Biolegend), CD4 (RM4.5, Tonbo), CD8 (53–6.7, Tonbo), Klrg1 (2F1, eBioscience), and CD44 (IM7, Tonbo) for 30 minutes at 4°C in the dark. Cells were washed 2X in FACs buffer, fixed/permeabilized using the BD cytofix/cytoperm kit (BD) and stained with anti-IFNγ (XMG1.2, Biolegend) diluted 1:100 in perm/wash buffer for 1 h at 4°C. Cells were washed 2X in perm/wash buffer, resuspended in FACs buffer and stored overnight at 4°C in the dark. Cells were acquired the following day on a Fortessa 1770 flow cytometer following the addition of counting beads (Sigma) and analyzed using FlowJo software (version 10).