Tumors obtained at necropsy were digested in media containing 1 mg/mL collagenase and 20 μg/mL DNAse I (Sigma) for 2 hours at 37°C, and passed through a 100 μm screen to obtain a single-cell suspension. CD8+ T cells were isolated (STEMCELL Technologies, Vancouver, BC Canada) and then stained with anti-CD3 (17A2, eBioscience), anti-CD8 (53-6.7, eBioscience) and anti-CD69 (H1.2F3, eBioscience) antibodies and DAPI. For PD-L1 quantification, tumor suspensions were stained with anti-CD45 (30-F11, BD Bioscience), anti-CD11b (M1/70, eBioscience), anti-GR1 (1A8, BD Bioscience), anti-F4/80 (BM8.1, Tonbo Biosciences), anti-PD-L1 (MIH5, eBioscience) and DAPI.
For PD-1 quantification on antigen-specific T cells, splenocytes obtained from immunized animals were enriched for CD8+ T cells and stained as above, along with tetramers specific for the SSX2 p41 or p103 epitopes (NIH Tetramer Core Facility, Atlanta GA), anti-PD-1 (J43, BD Bioscience, San Jose CA) and Ghost Dye–780 (Tonbo Bioscience, San Diego, CA).
For in vitro culture studies, cells were treated for 18 hours with 1 μg/mL recombinant mIFNγ (Shenandoah Biotechnology, Warwick PA), or cultured for 48 hours with CD8+ splenocytes isolated from immunized animals, and then collected using non-enzymatic cell dissociation solution (Sigma), and stained as above.
For PD-1 quantification on antigen-specific T cells, splenocytes obtained from immunized animals were enriched for CD8+ T cells and stained as above, along with tetramers specific for the SSX2 p41 or p103 epitopes (NIH Tetramer Core Facility, Atlanta GA), anti-PD-1 (J43, BD Bioscience, San Jose CA) and Ghost Dye–780 (Tonbo Bioscience, San Diego, CA).
For in vitro culture studies, cells were treated for 18 hours with 1 μg/mL recombinant mIFNγ (Shenandoah Biotechnology, Warwick PA), or cultured for 48 hours with CD8+ splenocytes isolated from immunized animals, and then collected using non-enzymatic cell dissociation solution (Sigma), and stained as above.