Fig 2: B7-H3 expression is regulated by mTORC1.B7-H3 protein expression in Tsc2-/- 105K cells with stable reconstitution of TSC2 or empty vector (EV) (a), TSC2-/- 621-101 angiomyolipoma-derived cells with reconstitution of TSC2 or empty vector (EV) (b), and Tsc2-WT and Tsc2 KO MEFs (c). For all figure legends, n = 3 indicates representative of 3 biologic samples (n = 3). B7-H3 mRNA expression in Tsc2-/- 105K cells with stable reconstitution of TSC2 or empty vector (EV) (d), TSC2-/- 621-101 angiomyolipoma tumor cells with reconstitution of TSC2 or empty vector (EV) (e), Tsc2-WT and Tsc2 KO MEFs (f). Means ± SD, two-tailed unpaired Student’s t-test (d, f) or one-way ANOVA with Dunnett’s multiple comparisons test (e), *p < 0.05, **p < 0.01. n = 3. B7-H3 protein expression in Tsc2-/- 105K cells (g), TSC2-/- 621-101 angiomyolipoma tumor cells (h), and Tsc2 KO MEFs (i) treated with 20 nM rapamycin (Rapa), 500 nM Torin 1 or vehicle for 24 hr (n = 3). B7-H3 mRNA expression in Tsc2-/- 105K cells (j), TSC2-/- 621-101 angiomyolipoma tumor cells (k), and Tsc2 KO MEFs (l) treated with 20 nM rapamycin (Rapa), 500 nM Torin 1, or vehicle for 24 hr. n = 3, means ± SD, one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001. m Immunoblotting analysis of Tsc2-WT and Tsc2 KO MEFs transfected with non-targeting control siRNA (Ctrl) or SMARTpool siRNAs targeting Raptor, Rictor, mTOR, S6K, or 4E-BP1 for 48 h hr (n = 3). n qRT-PCR analysis of Tsc2-WT and Tsc2 KO MEFs transfected with non-targeting control siRNA (Ctrl) or SMARTpool siRNAs targeting either Raptor, Rictor, mTOR, S6K, or 4E-BP1 for 48 hr. n = 3, means ± SD, two-way ANOVA with Dunnett’s multiple comparisons test, ***p < 0.001, ****p < 0.0001. Source data and exact p values are provided in the Source data file.

Fig 3: Inhibition of B7-H3 increases expression of IFN-? signature genes and MHC-II genes via enhancement of effector CD4+ and CD8+ T cell responses.a IFN-? is one of the top activated pathways in B7-H3 knockdown tumors versus control tumors (n = 3/group). NES = normalized enrichment score. b Heatmap of IFN-?-induced gene expression in bulk tumors and sorted tumor cells of the indicated groups (n = 3/group). c Heatmap of unsupervised clustering of MHC-I/II gene expression in sorted tumor cells from each group (n = 3/group). d Immunoblot analysis of tumor lysates derived from the indicated tumors. (n = 3/group). e Representative images of E-cadherin (E-cad) and STAT1 immunofluorescent staining of tumor sections (n = 3/group). Scale bar = 100 µm. f Representative images of E-cad and MHC-II immunofluorescent staining on tumor sections (n = 3/group). White arrows indicate co-expression in the same cells. Scale bar = 100 µm. g Immunoblot analysis of Tsc2-/- 105K cells stably expressing sh-B7-H3 (1) or sh-B7-H3 (2) and control (Ctrl) or CIITA sgRNAs (n = 3). h CIITA knockout increases the subcutaneous growth of tumor cells in (g) in mice. n = 10 mice/group, means ± SEM, two-way ANOVA with Holm-Sidak’s multiple comparisons test, **p < 0.01, ***p < 0.001, ****p < 0.0001. i Growth of subcutaneous sh-NC, sh-B7-H3 (1), or sh-B7-H3 (2) Tsc2-/- 105K tumors in strain-matched WT or IFN-? KO mice. n = 10 mice/group, means ± SD, Mann-Whitney U Test, ****p < 0.0001. Percentage of IFN-?/TNF-a positive tumor-infiltrating CD8+ (j) and CD4+ (k) T cells in the indicated tumors. n = 6, 7 or 8/group. Exact n is indicated in Source data file. Means ± SD, one-way ANOVA with Dunnett’s multiple comparisons test, **p < 0.01, ***p < 0.001. Percentage of IFN-?/TNF-a in WT splenic CD8+ (l) and CD4+ (m) T cells after co-culture with Tsc2-/- control, B7-H3 (1) or B7-H3 (2) shRNA 105K cells. n = 3, means ± SD, one-way ANOVA with Dunnett’s multiple comparisons test, **p < 0.01, ***p < 0.001. n Immunoblot analysis of Tsc2-/- 105K cells stably expressing B7-H3 (1) or B7-H3 (2) shRNA and control (Ctrl) or IFNGR1 sgRNAs (n = 3). o IFNGR1 knockout increases the subcutaneous growth of tumor cells in (n) in mice. n = 10 mice/group, means ± SEM, two-way ANOVA with Holm-Sidak’s multiple comparisons test, *p < 0.05, **p < 0.01. Source data and exact p values are provided in the Source data file.

Fig 4: B7-H3 is highly expressed in adipose tissue and dysregulated with obesity.(A) Normalized consensus human B7-H3 (hB7-H3) RNA expression across human tissues and blood cells by combining the data from the three transcriptomics datasets (HPA, GTEx, and FANTOM5) on the HPA. (B to D) Microarray analysis of hB7-H3 expression in subcutaneous fat biopsies of 770 men [GSE70353, METSIM study (10)] and correlation with BMI (B), percent of fat mass (C), and Matsuda index of insulin sensitivity (D). Red line represents nonlinear regression analysis with straight line fitting. r, correlation coefficient. (E) Microarray analysis of hB7-H3 gene expression in subcutaneous fat from lean nonobese (n = 26) and obese women (n = 30) [GSE25401 (11)]. (F) Microarray analysis of hB7-H3 in subcutaneous fat from obese women before (bBS, n = 16) and after (aBS, n = 16) bariatric surgery and from never-obese lean individuals (lean, n = 16) [GSE59034 (12)]. (G) Microarray analysis of mouse B7-H3 (mB7-H3) gene expression in gWAT from lean mice (n = 6), obese (n = 6), and formerly male obese mice (n = 6) [GSE97271 (13)]. Mice of the obese group received a high-fat high-sucrose diet (Research Diets, D12331; 58% kcal from fat) for 27 weeks, while mice of the lean group were given the low-fat control diet (Research Diets, D12329, 10.5% kcal from fat). After 20 weeks of feeding, a group of HFD-fed mice was switched to the low fat–containing control diet for another 7 weeks. Data are represented as means ± SEM in (E) to (G). Data were analyzed by Pearson correlation (B to D), Student’s t test (E), and one-way analysis of variance (ANOVA) (F and G). *P < 0.05 and **P < 0.01.

Fig 5: Elevated mTORC1 activity and B7-H3/CD276 expression are associated with poorer clinical outcomes and immunosuppressive phenotypes.Kaplan-Meier plot of overall patient survival stratified by high (n = 2076) and low (n = 2185) CD276 expression (a), high (n = 2001) and low (n = 2268) mTORC1 score (b), and high CD276 expression plus high mTORC1 score (n = 1296) and low CD276 expression plus low mTORC1 score (n = 1160) (c). Log-rank test. Forest plot of hazard ratios by tumor type and overall (with 95% confidence intervals) stratified by high and low CD276 expression (d), high and low mTORC1 score (e), and high and low CD276 expression plus high and low mTORC1 score (f). Hazard ratio greater than 1 (red) indicates an association with worse outcome. COX proportional hazards regression with Efron’s approximation. g, h Representative images of strong and weak immunohistochemical staining of phospho-S6 (Ser240/244) (pS6) and B7-H3 in tumor tissue microarrays, which include 12 cancer types: meningothelial meningioma, breast infiltrating ductal adenocarcinoma, colon adenocarcinoma, stomach adenocarcinoma, prostate adenocarcinoma, uterus adenocarcinoma, ovarian adenocarcinoma, esophageal squamous cell carcinomas, kidney clear cell carcinomas, hepatocellular carcinomas, lung adenocarcinoma/squamous cell carcinomas, and bladder transitional/papillary cell carcinomas. Scale bar = 50 µm (g). Correlation analysis between the phospho-S6 immunoreactivity score and B7-H3 expression score in the tumors in g (Pearson correlation coefficient test) (h). i, j Representative images of strong and weak immunohistochemical staining of phospho-S6K (Thr389) (pS6K) and B7-H3 in tumor tissue microarrays, which include 12 cancer types indicated in (g). Scale bar = 50 µm (i). Correlation analysis between the phospho-S6K immunoreactivity score and B7-H3 expression score in the tumors in i (Pearson correlation coefficient test) (j). mTORC1 score (k) and CD276 expression (l) among the six immune subtypes. Bars indicate median line, boxes denote interquartile range, whiskers denote 1–99 percentile and outliers are shown. Nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons test. n = 5429, ****p < 0.0001. m Heatmap indicates mean scores of immune-cell of all patients in high CD276 and high mTORC1 tumors (left) and low CD276 - low mTORC1 tumors (right). Source data is provided in the Source data file.