Pd l1 cd274 pe

At 96 h post-irradiation, tumor cells were examined by flow cytometry, as previously described [13 (link)]. Cells were examined on FACSCalibur or FACSVerse cytometers, using the monoclonal antibodies targeting HLA-ABC-FITC, HLA-ABC-PE-Cy7, ICAM-1 (CD54)-PE, CEA (CD66)-FITC, MUC-1 (CD227)-FITC, CD24-PerCP-Cy5.5, CD44-FITC, and the appropriate isotype-matched controls (BD Biosciences, San Jose, CA). The monoclonal antibodies targeting CD70-FITC, CD275 (ICOSL)-PE, CD137L (4-1BBL)-PE, CD252 (OX40L)-PE, PD-L1 (CD274)-PE, CTLA-4 (CD152)-PE, and CD227 (MUC-1)-PE were obtained from BioLegend (San Diego, CA). Antibodies targeting CD133-APC (Miltenyi Biotec, San Diego, CA) and calreticulin-PE (R&D Systems, Minneapolis, MN) were also used. Isotype control staining was < 5% for all samples analyzed. Viability was examined using LIVE/DEAD Fixable Violet Dead Stain Kit (Thermo Fisher Scientific, Rockville, MD). Cell surface expression was evaluated on live cells gated by FSC/SSC and LIVE/DEAD staining.

MC38, YUMM2.1, and YUMM1.1 tumors and spleens were harvested from mice at pre-defined time points. Tumors were digested with collagenase D (Roche), and stained with antibodies to CD3 BV605, Ly6C FITC, PD-L1/CD274 PE, CD8a BV421, CD45RA/B220, CD11b BV785, CD11c PECy7, CD103 PerCP Cyanine 5.5, MHC Class II (I-A/I-E) FITC (Biolegend, San Diego, CA), Ly6G (Gr-1) PerCP Cyanine 5.5, F4/80 Pacific blue/eFluor450, CD25 APC, CD4 FITC (eBioscience, San Diego, CA). Intracellular staining of Foxp3 PE (eBioscience) was done according to manufacturer's recommendations. Cells were analyzed with a LSR-II or FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), followed by Flow-Jo software (Tree-Star, Ashland, OR) analysis (28 (link)).