mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion [CITE-Seq]

Immune checkpoints are often expressed on tumor cells; it remains a prevailing need to identify the mechanisms underlying their regulation and therapeutic benefit. The immune checkpoint molecule B7-H3 is upregulated in many human tumors, including those with high mechanistic/mammalian target of rapamycin complex (mTORC1) activity. However, its role in tumor immunity and the impact of B7-H3-targeted therapy on the tumor immune microenvironment are largely uncharacterized. Here, we used a syngeneic murine model of tuberous sclerosis complex (TSC), a model of mTORC1 hyperactivity, to assess the mechanisms by which B7-H3 remodels the tumor microenvironment. We performed gene and protein expression profiling analysis using data generated from CITE-seq of infiltrating CD3+ T cells isolated by FACS from subcutaneous B7-H3 knockdown and control TSC2-deficient 105K tumors. Overall design: To sort intratumoral live CD3+ T cells, 80 control and 80 B7-H3 shRNA (1) tumors were used. Minced tumor fragments from every 300-400 mm2 of tumor were added to 3 mL of protease solution [5 mM CaCl2, 10 mg/ml Bacillus Licheniformis protease (Sigma Cat#P5380) and 125 U/mL DNase I recombinant (Roche Cat# 4716728001) in PBS and incubated with gentle rotation at 4°C for 10 min. After incubation, the solution was transferred to a Miltenyi C-tube (on ice) and the Miltenyi gentleMACS brain_03 program was run three times in a cold room (4°C). The incubation was repeated with gentle rotation at 4°C for 10 min. Single-cell dissociation was confirmed by microscopic examination. 3 mL 0.25% Trypsin-EDTA was added to the cell suspension and incubated at RT for 1 min. This was followed by the addition of 3 mL ice-cold PBS with 10% FBS. Cells were passed through a 70 µM strainer and the filter rinsed with 3 mL ice-cold PBS with 10% FBS. Tumor-infiltrating immune cells were separated from other cells by centrifugation with the deceleration brake set at 1 at 1,260 g at 4oC for 25 min in a Ficoll gradient (GE Healthcare, Cat#17-1440-03). Cells were stained with Mouse TrueStain Plus FcX (Biolegend, #156604) at 4°C for 10 min followed by staining with an anti-CD3 antibody (Biolegend, Cat#100204) for 30 min at 4°C. Tumors from each group were pooled together, then 1 x 105 live CD3+ T were sorted by flow cytometry and stained with Mouse TotalSeq-A CITE-seq antibodies (Biolegend, # 99833) at 4 °C for 30 min, before being washed in excess PBS with 2% FBS and 2.5 mM EDTA and resuspended in PBS with 0.04% BSA at 1500 cells/ml. 12000-15000 cells were loaded in the 10X Genomics Chromium in duplicate lanes.