Fig 2: Control of DC maturation by manipulating SMG1 activity. (A) Chemical structure of SMG1 inhibitor. (B) Immunoblot analysis of P-T28-UPF1 and P-S1078/S1096-UPF1 in BMDCs cultured with Pam3CSk4 (1 ng/ml) and/or SMG1 inhibitor (0.3 µM) for 2 h. (C) RNA expression levels in BMDCs. The BMDCs from C57BL/6J mice were treated with SMG1 inhibitor (0.3 µM) and stimulated with Pam3CSK4 (1 ng/ml). (D) BMDCs from C57BL/6J mice were treated with SMG1 inhibitor (0.3 µM) and stimulated with Pam3CSK4 (10 ng/ml), LPS (100 ng/ml), R848 (100 nM) or CpG DNA (ODN1668, 100 nM) for 24 h. Production of IL-6 and TNF in the culture supernatant were measured by ELISA. (E) Expression levels of CD40 and CD80 on BMDCs. The BMDCs from C57BL/6J mice were cultured with Pam3CSK4 (1 ng/ml) and/or inhibitor (0.3 µM SMG1) for 48 h and expression of CD40 and CD80 on BMDCs were analyzed by Flow cytometry. (F) Luciferase activity of HEK293T cells transfected with indicated luciferase reporter plasmids, together with control plasmid (Mock) or Regnase-1 expression plasmids. (G) Allogeneic mixed lymphocyte reaction of splenic CD4+ T cells from C57BL/6J mice with BMDCs from BALB/c mice cultured with SMG1 inhibitor (0.3 µM) and Pam3CSK4 (1 ng/ml) for 48 h. See also Supplementary Figure S7.

Fig 3: High IL-6 expression and low CD40 expression correlate with poor survival in human GBM patients.a, b Correlation of CD40 expression with a IL-6 and b IL-4 expression was subjected to linear regression analyses using GlioVis/TCGA GBM-RNA-seq (n = 160 patients) and low-grade glioma (n = 513 patients) data sets. Statistical analysis by linear regression analysis. c, d Correlation of IL-6 and CD40 expression (high/low cutoff of 40%) with overall survival was analyzed using TCGA-Firehose data set. Statistical analysis by two-sided log-rank test. e A schematic model. IL-6 induces anti-inflammatory and pro-inflammatory functions in Mϕs, through IL-10 and CD40 expression, respectively. Combination therapy by anti-IL-6 neutralization and CD40 activation reverses Mϕ-mediated tumor immunosuppression and promotes T-cell infiltration and activation, sensitizing tumor to checkpoint inhibition treatment.

Fig 4: Addition of GM-CSF blocks the differentiation and fusion of osteoclasts. Knockout of NRP2 in osteoclastic precursors isolated from NRP2 Fl/Fl; CSF1R-Cre transgenic mice by addition of 4-HydroxyTamoxifen in vitro and differentiated into osteoclasts in the presence of RANKL and M-CSF, LNCaP C4-2B CM and PC3 CM. Under these conditions, one set as control and the other set treated with GM-CSF at 600 pg·mL−1. TRAP staining to compare the osteoclast differentiation in NRP2WT and NRP2KO condition in RANKL+M-CSF at a Day 2. b Day 3. in LNCAP C4-2B CM e Day 2 f day 3. Quantification and comparison of the number of TRAP-positive multinucleated osteoclast per well in NRP2WT and NRP2KO osteoclasts with and without GM-CSF in RANKL and MCSF c Day 2 d Day 3 and in LNCaP C4-2B CM g Day 2 h Day 3. All values reported as mean± SEM from two independent experiments. Statistically significant P value denoted as *** (0.001), **** (0.000 1), ***** (0.000 001). i Flow cytometric analysis of surface markers of mononucleated bone marrow cells differentiated by PC3 CM

Fig 5: GY161 could induce BMDC maturation. Maturation of BMDCs was evaluated by measuring the expression of the surface molecules CD40, CD80 and CD86 by flow cytometry after 24 h of coculture with 0.1% DMSO (control) or compounds (10.0 µM). Significant differences among the groups were analyzed using one-way ANOVA with Bonferroni post hoc test. The data are presented as the mean ± SE, where *P < 0.05, **P< 0.01, ***P<0.001. Combo: a physical mixture of ibrutinib with GY100 (1:1 molar ratio).