Fig 2: T cell-induced Ccl5/CD44-mediated cell survival underlies Nf1 optic glioma growth.a, b Immunohistochemistry revealed that anti-CD8 antibody treatment reduced the percentage of p-AKTSer473-expressing and p-CREBSer133-expressing cells in mouse Nf1 optic glioma specimens. Black arrows denote representative immunopositive cells. Scale bars, 20 µm. Bar graphs represent the means ± SEM of n = 4 independent biological samples. Two-tailed Students-t test. Exact P values are indicated within each panel; a P < 0.001; b P = 0.002. c, d Kaplan–Meier survival curves (Brain Lower Grade Glioma TCGA Provisional [2 left panels; c P = 1.29e-6, d P = 2.06e-3] and TCGA PanCancer Atlas [2 right panels; c P = 4.69e-13, d P = 9.86e-3] datasets) demonstrate that non-overlapping patients with LGG who harbor high CCL5 expression or CD44 expression have reduced survival time. e Schematic representation of the neuron–immune–cancer axis in NF1-LGG. Meningeal T cells infiltrate into the optic glioma in an integrin (VLA-4)-dependent manner, and are activated by MDK produced by Nf1-mutant retinal ganglion cells (neurons) through a RAS-dependent mechanism. This neuron-mediated T cell activation increases CD8+ T cell Ccl4 production through increased Lrp1/calcineurin signaling, and results in increased NF?B-dependent microglial Ccl5 expression, culminating in increased glioma growth through Akt/GSK3ß/CREB pathway-mediated suppression of cancer (glioma) stem cell apoptosis and increased tumor growth.

Fig 3: MEKi/anti-CD40 Ab combination therapy increases CD8/Treg ratio and effector function of tumor-infiltrating T cells.a Flow cytometric analysis of the impact of MEKi GDC-0623 and/or anti-CD40 Ab treatment on the T cell infiltrate in B16-OVA, MC-38, and PDA30364 tumors, as performed on four mice per treatment group of the experiments shown in Fig. 3a–c. Representative dot plots are shown. Cells were pre-gated on living, CD45+, CD3+, Thy-1.2+ cells. b–d Quantitation of CD8+ T cell to CD4+ CD25+ Foxp3+ regulatory T cell ratio, regulatory T cell frequencies among CD4+ T cells, and CD8+ T cell frequencies among living cells. #Two animals without Tregs. One-way ANOVA with post hoc Dunnett’s test (treatment groups vs. control). Mean ± s.e.m, n = 4. e TILs isolated from PDA30364 tumors were restimulated ex vivo with PMA/Ionomycin in presence of GolgiPlug and stained for intracellular IFN? and TNFa. Mean ± s.e.m., n = 4. One-way ANOVA with post hoc Tukey’s test. Significance levels are indicated by asterisks (*p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001).

Fig 4: Combination of MEK inhibition with agonist anti-CD40 Ab mediates control of established tumors.a–c Outgrowth of B16-OVA, MC-38, and PDA30364 tumors upon treatment with MEKi GDC-0623 and/or anti-CD40 Ab. Mice were treated daily with 30 mg kg-1 GDC-0623 or vehicle for ~2 weeks (gray rectangle). Anti-CD40 or control Ab were administered on treatment days 3, 5, 7 as well as 1 day prior to biomarker analyses (green arrows). Each group consisted of at least six mice (nB16-OVA = 10, nMC38 = 10, nPDA30364 = 10). Four mice per group were killed at end of treatment for biomarker analyses. Two-way ANOVA with post hoc Dunnett’s test; treatment groups vs. control. Significance levels are indicated by asterisks (*p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001). d Cumulative data of several experiments with the PDA30364 tumor model. e PDA30364 tumor growth in mice treated with monotherapies (anti-CD40 Ab, anti-PD-1 Ab, or GDC-0623) and combination therapies (anti-CD40 Ab + GDC-0623 and anti-PD-1 Ab+GDC-0623). Mice were treated daily with 30 mg kg-1 GDC-0623 or vehicle for ~5 weeks. Anti-CD40, anti-PD-1, and control Ab were administered twice weekly (arrows). Each group consisted of at least nine mice (nControl = 9, nCD40 = 9, nGDC-0623 = 12, nCD40+GDC-0623 = 10, nPD-1 = 10, nPD1-GDC-0623 = 11). Four mice per group were killed 2 weeks after treatment start for biomarker analyses. f Impact of T cell depletion on efficacy of GDC-0623 and/or anti-CD40 Ab treatment against PDA30364 tumors. Where indicated, groups received depletory anti-CD4, anti-CD8 or anti-CD4+ anti-CD8 Abs twice per week. Each group consisted of at least five mice.

Fig 5: Intermittent BAY1082439 treatment induces prolonged T cell-inflamed phenotype even after drug withdrawal.A-B BAY-I treatment induced prolonged T cell-inflamed phenotype after drug withdrawal. Castrated Pten-null mice were treated with vehicle (n = 24), BAY-I (n = 32), or BAY-I then drug withdrawal for 4 (n = 15) weeks, BAY-I then drug withdrawal then S1PR modulator Fingolimod for 4 weeks (n = 6) or BAY-I then drug withdrawal for 10 weeks (n = 5) before the analyses. Prostate tumor sizes and weight were presented in (A), and prostate tumor-associated CD8+ T cell number and the percentages of CD8+ in CD45+ cells were determined by FACS analysis (B). C IHC analysis showed that CD8+ T cells remained in the cancer acini 4 weeks after drug withdrawal. Red dash line marks the boundary between cancer acini and stroma areas. The same staining was performed with 6 mice in vehicle and BAY-I cohort and 8 mice in BAY-I withdraw cohort and similar results were observed. D RNA-seq analysis shows T cell inflamed phenotype after drug withdrawal. RNAs were extracted from tumor tissues in (A, B), and RNA-seq analyses were performed. The relative expression levels of indicated genes in each sample were determined and the statistical analysis was performed based on the average of expression levels of each cohort. n = 6, 7 and 7 for vehicle, BAY-I and BAY-I withdraw 4 weeks group. p was calculated by two-sided T-test. E The relative expression levels of the Ifn-? gene were determined by RT-PCR analysis. n = 6 at each treatment cohort. F The enrichment of T cell receptor pathway was determined by GSEA based on RNA-seq data in E. Statistical test was performed by GSEA. A and B, data were presented as dot plots with mean as the central lines; E Data were presented as mean ± SEM; N.S p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 by two-sided T-test. Source data and exact p values are provided in the Source Data file.