Fig 2: IL-6 neutralization enhances T-cell infiltration into GBM tumors and improves animal survival but does not sensitize tumor to immune checkpoint blockade.GBM was induced in WT B6 mice, followed by injection with control IgG, anti-IL-6 antibody (Ab), immune checkpoint inhibitors (ICIs), or ICIs plus anti-IL-6 Ab. a Schematic approach. b, c Survival and tumor growth analyses (n = 8–12 mice, specific n numbers are shown in the figure). b Mouse survival was monitored for 60 days and subjected to two-sided log-rank Mantel–Cox analysis. MS, median survival. c Tumor volume was analyzed by bioluminescence imaging during days 13–23 (mean ± SEM). Statistical analysis by two-way ANOVA with Dunnett’s test. d–g Tumors were excised 2 days after treatment. Tumor-derived single-cell suspensions were stained with antibodies against CD45, d CD3, e CD11b, f CD4, CD8, CD3, and g Ki67, IFN-?, and CD69, followed by flow cytometry analyses. d Analysis for CD3+ T cells. Left, representative cell sortings. Right, quantified results (n = 6 mice, mean ± SEM). Statistical analysis by one-way ANOVA with Fisher’s LSD test. e–g Quantified results for immune cells (n = 6 mice, mean ± SEM). Statistical analysis by one-way ANOVA with Fisher’s LSD test. Source data are provided as a Source data file.

Fig 3: MEKi suppress M2-like macrophages and MDSCs.a Flow cytometric analysis of the impact of MEKi GDC-0623 and/or anti-CD40 Ab treatment on the macrophage infiltrate in MC-38 and PDA30364 tumors, as performed on four mice per treatment group of the experiments shown in Fig. 3 b–c. Representative dot plots are shown. Cells were pre-gated on living CD45+, CD11b+, F4/80+ cells. One-way ANOVA with post hoc Dunnetts’s test (treatment groups vs. control). Significance levels are indicated by asterisks (*p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001). b Polarization of mouse bone marrow-derived macrophage cultures towards M1- or M2-like phenotype, as assessed by flow cytometry marker analysis. Mean ± s.e.m, n = 6. c Cell viability of polarized macrophages after 3-day treatment with GDC-0623. Left panel: representative dose–response curves. Relative IC50 values were calculated by four-parameter logistic curve fit with a bottom constraint > 0. Mean ± s.e.m, n = 3. Right panel: Ki67/PI-based cell cycle analyses of tumor cells treated with 1 µm GDC-0623 and respective quantification. Mean ± s.e.m, n = 3. Unpaired student t test (medium vs. GDC-0623 for each cell cycle phase; FDR (Q = 1%), two-stage step-up method of Benjamini, Krieger and Yuketieli). d Representative dot plots of myeloid cell populations in MC-38 and PDA30364 tumors. e Representative dot plots of 'Gr1+', Ly6C+Ly6G+, and Ly6C+ Ly6G- myeloid cell populations in PDA30364 tumors. Cells were pre-gated on living CD45+, CD11b+ cells; quantitation normalized to all living cells. Mean ± s.e.m, n = 4. One-way ANOVA with post hoc Dunnett’s test (treatment groups vs. control). F Cell viability of bone marrow-derived MDSC cultures after 3-day treatment with GDC-0623; assay performed as described above. Mean ± s.e.m, n = 3.

Fig 4: IR induces cellular damage and a loss of nerves and SOX2 and SOX2+ progenitors can replenish mouse acinar cells in response to cholinergic mimetics A, BMurine SLG was analyzed for transcriptional changes by qPCR at days 0, 1, 3, and 7 days following 10 Gy of IR.C–E Sox2 CreERT2 ;Rosa26 mTmG murine SLG explants cultured ex vivo for 48 h and immunostained for smooth muscle actin (SMA, C), SOX2 (D) or Ki67 (E) and nuclei. Recombination was induced 24 h before SLG was harvested for culture. Scale bar = 50 µm.Data information: Data in (A and B) were normalized to Rsp29 and the day 0 control. Data in (A and B) (n = 3 per time point) are means + SEM and were analyzed using a one-way analysis of variance with a post hoc Dunnett's test. Bax (D1) **P = 0.00826, Bax (D3) **P = 0.0871, Pmaip1 (D3) *P = 0.0369, p21 (D3) *P = 0.0481, p53 (D1) *P = 0.0337, MKi67 (D1) *P = 0.0319, Sox2 (D1) **P = 0.0072, Sox2 (D3) *P = 0.0418, Aqp5 (D1) *P = 0.0421, Mist1 (D1) *P = 0.0432, Tubb3 (D1) *P = 0.0467, Tubb3 (D3) *P = 0.0470, Tubb3 (D7) *P = 0.0489, Vip (D1) *P = 0.0402. Images in (C, D, and E) are representative of three experiments, n = 3 SLG fragments per experiment.

Fig 5: Muscarinic signaling regulates the acinar lineage and SOX2+ cells.(A–B) E14 mouse SLG epithelia cultured with FGF10 ±CCh for 24 hr. White dashed line outlines acini. Arrows indicate double positive SOX2+EdU+ cells. Scale bar is 50 µm. The number of SOX2+, EdU+ and SOX2+EdU+ cells was quantified in B. (C–E) E14 mouse SLG cultured for 24 hr with DMSO or 4-DAMP (10 µM). The number of SOX2+ and SOX2+Ki67+ cells were counted via FACS, normalized to control and expressed as percentage of total ECAD+ cells (C). In D and E, cultured glands were immunostained for SOX2, nerves (TUBB3), AQP5, KRT19 and Ecadherin (ECAD). Images are 50 µm projections of 5 µm confocal sections. Scale bars are 100 µm. (F–H) E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and immunostained for AQP5 (F) and the number of AQP5+ and KRT19+ cells counted (G). SMG+SLG were also subjected to qPCR analysis (H). Data in B, C, G and H are means ±s.d of three biological replicates and three experiments. Data in B, C and G were subjected a Student’s t-test. *p<0.05 **p<0.01. Data in H were analyzed using a one-way analysis of variance with a post-hoc Dunnett’s test. *p<0.05, **p<0.01. Additional data for this figure in Figure 5—figure supplement 1.DOI: http://dx.doi.org/10.7554/eLife.26620.02210.7554/eLife.26620.023Figure 5—source data 1.Source data relating to Figure 5B.E14 mouse SLG epithelia cultured with FGF10 ±CCh for 24 hr. The number of SOX2+, EdU+ and SOX2+EdU+ cells were quantified. Data are means of three biological replicates and three experiments. s.d. = standard deviation.DOI: http://dx.doi.org/10.7554/eLife.26620.02310.7554/eLife.26620.024Figure 5—source data 2.Source data relating to Figure 5C.E14 mouse SLG cultured for 24 hr with DMSO or 4-DAMP (10 µM). The number of SOX2+ and SOX2+Ki67+ cells were counted via FACS, normalized to control and expressed as percentage of total ECAD+ cells. s.d. = standard deviation.DOI: http://dx.doi.org/10.7554/eLife.26620.02410.7554/eLife.26620.025Figure 5—source data 3.Source data relating to Figure 5G.E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and the number of AQP5+ and KRT19+ cells counted. Counts were normalized to the control (nerves). Data are means of three biological replicates and three experiments. s.d. = standard deviation.DOI: http://dx.doi.org/10.7554/eLife.26620.02510.7554/eLife.26620.026Figure 5—source data 4.Source data relating to Figure 5H.E13 SMG+SLG were cultured ± ganglia and ± CCh (100 nM) for 48 hr and subjected to qPCR analysis. Data were normalized to Rsp29 and control (nerves). Data are means of three biological replicates and three experiments. s.d. = standard deviation.DOI: http://dx.doi.org/10.7554/eLife.26620.026