Fig 2: B7H3-activated MDSC promoted fibroblast activation/myofibroblast differentiation and suppressed T-cell proliferation. BM-derived MDSCs were treated with sB7H3 (4 µg/ml) or SCF (0.1 µg/ml) in presence of GM-CSF (0.01 µg/ml) for 3 days followed by flow cytometric cell sorting. Sorted G-MDSCs or M-MDSCs were co-cultured with primary isolated MLF in 24-well transwell plates (ratio of MDSC : MLF = 2:1). After 48 h of co-culture, RNA from MLF was isolated and analyzed by qPCR for type I collagen (Col1a2) (A), a-SMA (Acta2) (B), and TGFß1 (Tgfb1) expression (C). Cellular RNA from sorted G-MDSCs and M-MDSCs was also extracted and similarly analyzed for expression of TGFß1 (Tgfb1) (D). The 18S RNA was used as internal control for normalization. The data were expressed as fold change relative to the respective control. (E) B7H3 blocking antibody (Ab) was injected intravenously into mice after BLM treatment. The lung single-cell suspensions were obtained 7 days after BLM treatment for flow cytometry analysis of MDSCs. Representative plots (pre-gated by CD45+CD11b+ cells) are shown on the left panel and the quantitative analysis of percentages and absolute cell numbers on the middle and right panels, respectively. (F) Total lung cell numbers were counted using a hemocytometer. (G) Lung tissue RNA was analyzed by qPCR for Tnfa, Col1a1, and Acta2 on day 7 after BLM treatment. (H) BM-derived CD11b+Gr1+ MDSCs with (+) or without (-) sB7H3 activation were co-cultured with CFSE pre-labeled splenocytes in media only (Naïve T cell) or in stimulation medium containing CD3/28 Dynabeads+rmIL2 (Activated T-cell) for 3 days. CD4+ or CD8+ T-cell proliferation was assessed by counting CFSE+ cells with either T-cell marker using flow cytometry. (I) Fresh BM cells were treated with sB7H3 for 72 h and analyzed for CD84 and MDSC markers. The data were shown as the percentage (left) or the absolute numbers per million BM cells (right) of G-MDSC or M-MDSC in the CD84-expressing CD11b+ BM population. Mean ± SD is shown for all. N = 3–8. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. MDSC, myeloid-derived suppressor cell; BM, bone marrow; sB7H3, soluble B7H3; SCF, stem cell factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; MLF, mouse lung fibroblast; BLM, bleomycin; CFSE, carboxyfluorescein succinimidyl ester.

Fig 3: Expression of the costimulatory molecule B7H3 in human OC. (A) Relationship between B7H3 expression level and overall survival of patients with OC. (B) The mRNA expression of CD276 in the tumor and normal ovarian tissue samples analyzed with the TCGA database. (OC vs. normal ovaries, *P <0.05). (C, D) The expression level of B7H3 mRNA expression. B7H3 mRNA expression stratified according to (C) tumor grade (D) tumor stage (P>0.05). (E) Microarrays of human normal tissues were stained for IHC to detect the expression of B7H3. Scale bar, 50 µm. (F) Microarrays of human ovarian tumor-adjacent tissues and OC tissues were stained for IHC to detect the expression of B7H3. High, high grade. Low, low grade. Scale bar, 50 µm. (G, H) Flow cytometry and immunofluorescence staining patterns showed high expression of the B7H3 in OC cell line (SKOV3). Scale bar, 50 µm.

Fig 4: Expression of the immune checkpoint molecule B7-H3 in RMS and normal tissue. (A) Normalized expression (RPKM) of B7 molecules from RNA sequencing of normal muscle (N = 5), fusion negative (N = 66), and fusion positive (N = 35) RMS samples. Each data point represents an individual sample. The median is noted by a horizontal bar and significance denoted by asterisks (*** p < 0.001; ** p < 0.01; * p < 0.05; n.s., not significant, Student’s t-test). (B) Histograms of flow cytometric analysis of B7 molecule expression in RMS and normal muscle cell lines. Bar graph shows mean fluorescence intensity (MFI) fold change in RMS cell lines compared to normal muscle. ND indicates “not detectable” expression (C) Western blot of B7H3 expression in 5 different cell lines. Band intensity for B7-H3 normalized on actin is indicated for each cell line. (D) Tissue Specific (TS) score of protein and RNA expression of B7H3 in each normal tissue (obtained from Jiang et al. [15].

Fig 5: Loss of B7-H3 expression in RMS tumor cells is associated with higher T-cell cytotoxicity. (A) Histogram showing relative proliferation of B7-H3 knockout RH30 cells compared to wild-type cells. (B) Representative pictures of calcein-labeled wild-type and B7-H3 knockout RH30 cells after co-incubation with activated PBMCs for 4 hours. (C) Survival index of wild-type and B7-H3 knockout RH30 cells co-cultured with activated PBMC (N = 10 normal donors) at a ratio target: PBMC of 1:10 or 1:20. (** p < 0.01; * p < 0.05, Paired t-test).