Fig 2: Regulation of CD73 and CD39 cell membrane expressions and extracellular adenosine levels by ERs in osteoprogenitor cells.(A) Flow cytometric analyses and (B) quantification of CD73 and CD39 in osteoprogenitors in the absence or presence of E2 (100 nM) for 3 days. (C to E) Single (ESR1 or ESR2) or dual (ESR1 and ESR2) ER knockdown (KD) by siRNA in primary mouse osteoprogenitors and analyzed after 3 days. (C) Flow cytometric analyses of CD73 and CD39 after single knockdown (ESR1 or ESR2) and dual knockdown (ESR1 and ESR2). (D) Percentage of double-positive (CD73/CD39) cells in single knockdown and dual knockdown cells. (E) In vitro adenosine levels normalized by cell number in single knockdown and dual knockdown cells. Control (scrambled) siRNA concentration for single knockdown and dual knockdown are 5 and 10 nM, respectively. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Difference of microenvironment between RSCRC and LSCRC.(A) The surgical specimens of RSCRC (n = 3) with high expression of CD39+?d Tregs and the LSCRC (n = 3) with low expression of CD39+?d Tregs detected by flow cytometry were then analyzed by tandem mass tag–based (TMT-based) quantitative proteomics. The profiling experiments resulted in quantification of 972 differential proteins — among them, 295 high expression proteins were found in RSCRC and 677 in LSCRC. A volcano plot indicating results from protein-level differential analysis comparing RSCRC with LSCRC samples. Dotted lines indicated that the cut-offs used to define regulated proteins (|log2 FC| > 1.2, adjusted P < 0.05). (B) A bubble chart displayed KEGG enrichment pathway of abnormal expression proteins in RSCRC/LSCRC. (C) Bar plot of secondary classification of KEGG pathway annotation highlighted features of RSCRC/LSCRC microenvironment. (D and E) The expression of calcium signaling pathway–related proteins (D) and arachidonic acid metabolism–related proteins (E) in RSCRC and LSCRC analyzed by quantitative proteomics. Data represent mean ± SEM, using 2-way ANOVA followed by Sidak’s multiple-comparisons test. (F) PLA2G4A mRNA expression in colorectal cancer with MSI-high/MSI-low or MSS based on data obtained from the GEPIA database. One-way ANOVA test was used. MSI, microsatellite instability; MSS, microsatellite stable. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 4: Deficient CD73 and CD39 expressions and extracellular adenosine concentration in BM of OVX animals.(A to K) Characterization of healthy (sham) and OVX animals 4 weeks after ovariectomy. (A) Immunofluorescence staining of CD73 (green) and (B) CD39 (red) in vertebrae of OVX animals. Nuclear staining (blue). Scale bars, 100 µm. Inset shows magnified image of bone surface. Yellow arrowheads indicate cells positive for CD73 or CD39 on bone surface. Scale bars, 50 µm. (C) Flow cytometric analysis of CD73 and CD39 membrane expression of hematopoietic cells from mouse BM cells 4 weeks after ovariectomy (OVX) and healthy controls. (D) Percentage and median fluorescence intensity of hematopoietic cells expressing CD73. (E) Percentage and median fluorescence intensity of hematopoietic cells expressing CD39. (F) Flow cytometric analysis of CD73 and CD39 membrane expression of nonhematopoietic cells from mouse BM cells 4 weeks after ovariectomy and healthy controls. (G) Percentage and median fluorescence intensity of nonhematopoietic cells expressing CD73. (H) Percentage and median fluorescence intensity of nonhematopoietic cells expressing CD39. (I) CD73 gene expression and (J) CD39 gene expression of cells from bone chips. (K) Extracellular adenosine concentration in BM plasma of sham and OVX animals. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Suppression of B7-H3 increases intratumoral cytotoxic CD38+CD39+CD4+ T cells.a Experimental design for B7-H3 knockdown Tsc2-/- 105K tumors treated with isotype control, a-CD4, or a-CD8 (Created with Biorender.com). b Images of B7-H3 knockdown Tsc2-/- 105K tumors treated as depicted in a. X indicates that no tumor was detected. n = 10 mice for isotype control, and a-CD8, and n = 7 mice for a-CD4. c Left panel: Suppressed growth of B7-H3 knockdown Tsc2-/- 105K tumors treated with anti-CD8 or anti-CD4 antibodies. Right panel: Tumor volume at 29 days post-cell injection. n = 10 mice for isotype control, and a-CD8, and n = 7 mice for a-CD4. Means ± SD, nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons test, **p < 0.01, ****p < 0.0001. d Uniform manifold approximation and projection (UMAP) plots of 12,820 sorted single CD3+ T cells from sh-NC tumors (n = 80 tumors pooled into 2 independent samples) or sh-B7-H3 (1) tumors (n = 80 tumors pooled into 2 independent samples). CD4+ and CD8+ T cells are indicated. e Expression of CD4 and CD8 based on CITE-seq antibody binding and RNA data. f Percentage of tumor-infiltrating CD4+ and CD8+ T-cells as a proportion of total CD3 T cells in Tsc2-/- 105K control (Ctrl) or B7-H3 knockdown (KD) tumors. n = 80 tumors pooled into 2 independent samples/group. g Volcano plot depicting differentially expressed genes in tumor-infiltrating CD4+ T cells from Tsc2-/- 105K B7-H3 KD versus Ctrl tumors of the CITE-seq RNA data. h GSEA of the CITE-seq RNA data from tumor-infiltrating CD4+ T-cells from Tsc2-/- 105K Ctrl and B7-H3 KD tumors of the CITE-seq RNA data. NES, normalized enrichment score. i Volcano plot depicting differentially expressed genes in tumor-infiltrating CD8+ T cells from Tsc2-/- 105K B7-H3 KD versus Ctrl tumors of the CITE-seq RNA data. j GSEA of the CITE-seq RNA data from tumor-infiltrating CD8+ T cells from Tsc2-/- 105K Ctrl and B7-H3 KD tumors. NES, normalized enrichment score. k UMAP showing minor phenotypic clusters of d. l Dotplot depicting the top 62 upregulated genes in the minor clusters of CD8+ and CD4+ T cells. m Percentage of tumor-infiltrating CD4+ and CD8+ T-cell minor clusters shown as a proportion of total CD3+ T cells isolated from Tsc2-/- 105K Ctrl or B7-H3 KD tumors. n = 80 tumors pooled into 2 independent samples/group. n Epitope and mRNA signals in cells from CITE-seq experiments for selected top upregulated markers in CD4_Teff-Gzmk and CD8_Teff-Gzmk/Gzmb clusters. o Representative images and quantification of CD38+CD39+CD4+ TILs in sh-NC, sh-B7-H3 (1), or sh-B7-H3 (2) Tsc2-/- 105K tumors. White arrows indicate CD38+CD39+CD4+ TILs. n = 6 tumors for sh-NC, and n = 5 tumors for sh-B7-H3 (1), and sh-B7-H3 (2). Mean ± SD, nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons test, *p < 0.05, **p < 0.01. Scale bar = 10 µm. p Quantification of apoptotic cells over time with Tsc2-/- 105K cells cultured alone or with tumor-infiltrating CD38+CD39+CD25-CD4+ T cells. Mean ± SD from 3 independent experiments. q Kaplan-Meier graph from the pan-TCGA data showing improved overall survival of patients with high cytotoxic CD4+ T-cell gene signature. Source data and exact p values are provided in the Source data file.