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. 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.

免疫检查点常表达于肿瘤细胞，目前仍亟需阐明其调控机制与治疗获益的内在原理。免疫检查点分子B7-H3在多种人类肿瘤中呈高表达，其中包括哺乳动物雷帕霉素靶蛋白复合物（mechanistic/mammalian target of rapamycin complex, mTORC1）活性异常升高的肿瘤。然而，B7-H3在肿瘤免疫中的功能，以及靶向B7-H3的治疗对肿瘤免疫微环境的影响，目前仍未得到充分阐明。本研究使用结节性硬化症（tuberous sclerosis complex, TSC）的同基因小鼠模型——该模型呈现mTORC1活性过高的表型——来解析B7-H3重塑肿瘤微环境的具体机制。本研究通过从皮下B7-H3敲低组及对照组的TSC2缺陷型105K肿瘤中，经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离得到的浸润性CD3+ T细胞的细胞内转录组与表位联合测序（CITE-seq）数据，开展基因与蛋白表达谱分析。为分选肿瘤内活态CD3+ T细胞，本研究共使用80例对照组肿瘤及80例转染B7-H3短发夹RNA（shRNA）#1的敲低组肿瘤。将每块体积300~400 mm²的切碎肿瘤组织块加入3 mL蛋白酶消化液中，该消化液体系为含5 mM氯化钙、10 mg/mL地衣芽孢杆菌蛋白酶（Sigma货号P5380）及125 U/mL重组DNase I（Roche货号4716728001）的磷酸盐缓冲液，随后于4℃条件下轻柔旋转孵育10分钟。孵育结束后，将消化液转移至冰浴的美天旎C管中，于4℃冷室中运行美天旎gentleMACS组织解离仪的brain_03程序，共重复3次。随后再次将样本于4℃条件下轻柔旋转孵育10分钟。通过显微镜观察确认组织已完全解离为单个细胞。向细胞悬液中加入3 mL 0.25%胰蛋白酶-EDTA，于室温（room temperature, RT）下孵育1分钟。随后加入3 mL预冷的含10%胎牛血清（fetal bovine serum, FBS）的磷酸盐缓冲液终止消化。将细胞悬液经70 µm细胞筛过滤，并用3 mL预冷的含10% FBS的磷酸盐缓冲液冲洗滤器。通过聚蔗糖密度梯度离心（GE Healthcare货号17-1440-03）分离肿瘤浸润免疫细胞与其他细胞组分：离心转速设为1260×g，温度4℃，离心时间25分钟，且减速刹车档位调至1档。细胞首先于4℃下用小鼠TrueStain Plus FcX封闭试剂（BioLegend货号#156604）孵育10分钟，随后于4℃下用抗CD3抗体（BioLegend货号#100204）染色30分钟。将每组的肿瘤组织样本混合后，经流式细胞术分选得到1×10^5个活态CD3+ T细胞；随后于4℃下用小鼠TotalSeq-A CITE-seq抗体组合（BioLegend货号#99833）染色30分钟，再用过量的含2% FBS及2.5 mM乙二胺四乙酸（EDTA）的磷酸盐缓冲液洗涤细胞，最后重悬于含0.04%牛血清白蛋白（bovine serum albumin, BSA）的磷酸盐缓冲液中，调整细胞浓度至1500个/mL。将12000~15000个细胞分两孔上样至10× Genomics Chromium单细胞分析系统。