Fig 2: GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 µm. Zoom-in factor: 4

Fig 3: IL-6 induces Mϕ-mediated immunosuppression but stimulates CD40 expression.a–e Bone marrow (BM)-derived Mϕs were isolated from mice and treated with 50 ng/ml IL-4 and IL-6 for 2 days, followed by RNA-seq analysis (n = 3 mice). Genes were mapped and subjected to a principal component and b volcano plot analyses. c Heatmap of secretome genes. d Expression of immunosuppressive cytokines (top) and M2 Mϕ activation-associated genes. Left, heatmap. Right, means of fold expression of control. e, f BM-derived mouse Mϕs were treated with IL-4 and IL-6 for 2 days, and analyzed by flow cytometry. e IL-10 expression. Left, representative sortings. Right, quantitative results (n = 3 mice, mean ± SEM). Statistical analysis by one-way ANOVA with Dunnett’s test. f CD206 expression (n = 3 mice, mean ± SEM). Statistical analysis by one-way ANOVA with Dunnett’s test. g Expression of Mϕ activation-associated receptor genes. Left, heatmap. Right, quantitative results (n = 3 mice, mean ± SEM). Statistical analysis by two-way ANOVA with Dunnett’s test. h BM-derived mouse Mϕs were treated with IL-4 and IL-6, and analyzed by flow cytometry. Left, representative sortings. Right, quantitative results (n = 3 mice, mean ± SEM). Statistical analysis by one-way ANOVA with Dunnett’s test. i GBM was induced in control WT or IL-6-ΔEC mice. Two weeks after tumor implantation, tumor-derived single-cell suspensions were analyzed by flow cytometry (mean ± SEM, n = 3 mice for control group and n = 4 mice for IL-6-ΔEC group). Statistical analysis by two-tailed Student’s t-test. j GBM was induced in mice. Two days after treatment with IL-6 Ab and ICIs or with control Ab, tumors were analyzed by flow cytometry (n = 5 mice, mean ± SEM). Statistical analysis by two-tailed Student’s t-test. Source data are provided as a Source data file.

Fig 4: Genetic ablation of IL-6 reverses GBM immunosuppression.GBM was induced by RCAS-mediated genetic engineering in Ntv-a;Ink4a-Arf−/−;Ptenfl/fl;LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5-CreERT2;Il6fl/fl recipient mice that were pretreated with (IL-6-ΔEC) or without (Control) tamoxifen. Two weeks after tumor implantation, tumors were excised. a Schematic approach. b, c Tumor-derived single-cell suspensions were analyzed by CyTOF. b Representative CyTOF sorting. c Quantitative results (mean ± SEM, n = 4 mice). Statistical analysis by two-tailed Student’s t-test. d–f Tumor-derived single-cell suspensions were analyzed by flow cytometry. d Analysis for CD3+ T cells. Left, representative cell sortings. Right, quantified results (n = 6 mice, mean ± SEM). Statistical analysis by two-tailed Student’s t-test. e, f Analysis for e CD4+/CD8+ T cells or f myeloid cells (n = 6 mice, mean ± SEM). Statistical analysis by two-tailed Student’s t-test. g, h Tissue lysates from normal brains and tumors were subjected to ELISA analysis for g IL-10 and h TGF-β expression (mean ± SEM, n = 4 mice for IL-6-ΔEC GBM group and n = 3 mice for other groups). Statistical analysis by two-way ANOVA with Sidak’s test. Source data are provided as a Source data file.

Fig 5: IL-6 neutralization and CD40 stimulation plus immune checkpoint blockade synergistically reverses Mϕ-mediated immune suppression and activates GBM-associated T cells.GBM was induced in mice, followed by different treatment and endpoint analyses. a Experimental procedure. b Tumor volume was analyzed pre- and post treatment by bioluminescence imaging. Left, representative images. Right, quantified results (n = 6 mice, mean ± SEM). Statistical analysis by two-way ANOVA with Dunnett’s test. c–f Tumor-derived single-cell suspensions were analyzed by flow cytometry. c, d Cells were probed with c, anti-F4/80 and anti-IL-10, or d anti-CD45 and anti-CD8 antibodies. Left, representative sortings. Right, quantified results (n = 5 mice, mean ± SEM). Statistical analysis by one-way ANOVA with Dunnett’s test. e, f Cells were probed with e anti-CD8 and anti-Ki67, or f anti-CD8 and anti-IFN-γ antibodies. Quantified results are shown (n = 5 mice, mean ± SEM). Statistical analysis by one-way ANOVA with Dunnett’s test. g, h Tumor lysates were subjected to g IL-10 and h TGF-β ELISA analysis (mean ± SEM, n = 4 mice for ICI plus IL-6 Ab treatment group, n = 6 mice for ICIs, CD40 Ab, plus IL-6 Ab treatment group, and n = 5 mice for other groups). Statistical analysis by one-way ANOVA with Dunnett’s test. Source data are provided as a Source data file.