Antibody staining, flow cytometry and cell sorting

For intracellular cytokine staining, ~10⁶ lymphocytes were stimulated with peptides or peptide pools (5mg/ml/peptide) and Golgiplug (Fisher Scientific, Waltham, MA 1.5 μg/ml) dissolved in DMEM with 2%FBS for 5-6 hours at 37°C. (FAP peptides: FAP1: YSYTATYYI, FAP2: IQYLCWSPV, FAP3: LAYVYQNNI, FAP4: YVYQNNIYL, FAP5: SSWEYYASI, FAP6: RALTLKDIL, FAP7: YDLQNGEFV, FAP8: FAVNWITYL, FAP9: KALVNAQVD, FAP10: IAYSYYGDG, FAP11: TAVRKFIEM, FAP12: LTFWYKMIL, FAP13: SSDYYFSWL, FAP14: SQNHLYTHM, FAP15: IYSERFMGL, FAP16: HLYTHMTHF. MAA peptides: mTrp-1_455-463: TAPDNLGYA, mTrp-1_481-489: IAVVAALLL, mTrp-2_522-529: YAEDYEEL, hTp-2_180-188: SVYDFFVWL, hTrp-2_343-357: STFSFRNAL, mTrp-2_363-371: SQVMNLHNL, hgp100_25-33: KVPRNQDWL, mBraf_594-602: FGLANEKSI). A rabies virus glycoprotein peptide was used as negative control. After stimulation cells were stained as described previously [27, 55]. Cells were stained with Amcyan fluorescent reactive dye (Life technologies, Carlsbad, CA), anti-CD8-Alexa700 or –Brilliant violet (BV) 605 and CD44-FITC or -PercpCy5.5. For intracellular cytokine staining cells were stained with antibodies to IFN-γ(APC or BV421), TNF-α (PE-Cy7, Biolegend, San Diego, California), granzyme B (APC, Life Technologies) and perforin (PE, eBioscience, San Diego, CA) as described [54]. For Trp-1₄₅₅ tetramer staining, cells were stained with PE-labeled Trp-1-specific MHC class I (H-2D^b) tetramer with TAPDNLGYM peptide (NIAID tetramer facility, Atlanta, GA) together with other surface markers including anti-CD8, CD44, and PD-1-BV605 (all from Biolegend, San Diego, CA). For mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (MROS) staining, cells were stained with DioC₆ (40nM) and Mitosox red (5 μM, Life technologies) for 30mins at 37°C before surface markers staining. For staining of other cell populations from tumors, single cell suspensions were blocked with CD16/CD32 Fc receptor blocking antibody (BD Pharmingen, San Jose, CA) for 30 mins at 4°C. Cells were further stained with either sheep anti-FAP antibody (10μg/ml, R&D, AF3715) or normal sheep IgG control antibody (R&D) at 4°C for 1 hour. After washing cells were stained with donkey anti-sheep APC-conjugated secondary antibody for 30 minutes together with anti-CD3-Pacblue, CD14-PercpCy5.5, CD19-FITC, Gr-1-PE, CD11b-PE-Cy7, CD206-BV605, F4/80-Alexa700, Sca-1-PE-Cy7 and CD90.2-FITC (all from Biolegend). For staining of immunosuppressive functions tumor-derived cells were first stained with CellRox green (5mm) for 30 mins at 37°C. After washing, cells were stained with surface markers for 30 mins at 4°C. Cells were then fixed and permeabilized with fixation/permeabilization buffer (Becton Dickinson, Franklin Lanes, NJ) for 30 mins on ice, followed by staining with rabbit polyclonal antibody to iNOS (10mg/ml, Abcam) in 1x permwash buffer (Becton Dickinson) for 30mins on ice. Normal rabbit IgG (R&D) was used as isotype control. Following washing cells were further stained with Alexa647-goat anti rabbit secondary antibody (1:2000, Life Technologies) and Arginase1 PE (R&D) for 30 min at 4°C. To measure expression of phosphorylated STAT, cells were first fixed with pre-warmed Fix Buffer I (pre-warmed to37°C, Fisher) for 10 mins at 37°C. After washing with cell staining buffer (Biolegend), cells were stained with surface markers and permeabilized with PermBuffer III (pre-chilled to −20°C, Fisher) for 30 min at 4°C. Cells were washed twice with cell staining buffer and stained with STAT1(pY701) PercpCy5.5, STAT3(pS727) Alexa647 and STAT6(pY641) antibodies (all from Fisher) diluted in cell staining buffer for 60mins at room temperature. Cells were analyzed by an LSRII (BD Bioscience). Data were analyzed with FlowJo (TreeStar, Ashland, OR).