Fig 1: Activated T cells secrete Ccl4 and prime microglia to produce Ccl5.a Activated T cell conditioned medium (act-Tm CM) treatment stimulated WT microglia (MG) to produce a higher level of Ccl5 (ELISA) compared to non-activated T cell-conditioned medium (non-act-Tm CM). Non-activated and activated T cell CM, and CM collected from microglia alone served as controls for these experiments. b WT T cells isolated from the mouse spleen were seeded at the concentration of 2.5 × 106 cells ml−1 in complete PRIM1640 medium followed by 2 days of CD3/CD28 stimulation (activated; act-Tm) or vehicle (PBS) treatment (non-activated; non-act-Tm). CM was collected for chemokine array triplicates. Increased levels of TNF-α, GM-CSF, Ccl2, Ccl1, Ccl3, Ccl4, Ccl5, Il-1ra, and Il-2 expression were observed in activated T cell CM relative to non-activated T cell CM. The fold increases and P values relative to control groups for all three replicates (Supplementary Fig. 1a) are collated in the table. c ELISA assays reveal increased levels of TNFα, GM-CSF, Ccl2, Ccl1, Ccl3, Ccl4, Ccl5, Il-1ra, and Il-2 in the CM of activated, relative to non-activated, T cells. d WT microglia were stimulated with these differentially expressed cytokines [TNF-α (400 pg ml−1), GM-CSF (1000 pg ml−1), Ccl2 (80 pg ml−1), Ccl1 (500 pg ml−1), Ccl3 (8000 pg ml−1), Ccl4 (6000 pg ml−1), Il-1ra (80 pg ml−1), and Il-2 (6000 pg ml−1)] for 24 h at the concentrations detected in the activated T cell CM. Ccl5 production by microglia was increased following Ccl4 (6000 pg ml−1) treatment. Veh: vehicle. e Ccl5 ELISA revealed that activated T cell CM induction of microglial Ccl5 production was reduced following treatment with increasing concentrations of Ccl4 neutralizing antibody. f Microglial Ccr5 and Ccr8 expression was validated using spleen as a positive control. g Increasing concentrations of maraviroc (MCV, Ccr5 receptor inhibitor) and AZ084 (Ccr8 receptor inhibitor) reduced T cell induction of microglial Ccl5 expression. The combination of MCV and AZ084 exhibited the greatest inhibition of microglial Ccl5 expression. All data are presented as the mean ± SEM. a This representative experiment was conducted with n = 3 independent biological samples, and was replicated two additional times with similar results. b n = 3 independent biological samples were examined over three independent experiments, as illustrated in Fig. S1a. c and d Bar graphs represent the means ± SEM of n = 3 independent biological samples. e This representative experiment was conducted with 0 mg ml−1 anti-Ccl4, n = 6; 1, 2, 2.5 mg ml−1 anti-Ccl4, n = 3, independent biological samples, and was replicated two additional times with similar results. f Bar graphs represent the means ± SEM of n = 4 independent biological samples. g This representative experiment was conducted with (from left to right) n = 7, n = 6, n = 6, n = 4, n = 5, n = 4, and n = 5 independent biological samples, and was replicated two additional times with similar results. a, d, e, g One-way ANOVA with Bonferroni post-test correction; b, c, f Two-tailed Student’s t-test. Exact P values are indicated within each panel; N.S.; not significant. From left to right in each panel: a all P < 0.001, c all P < 0.001, d N.S., N.S., N.S., N.S., P = 0.035, P < 0.001, N.S., N.S.; e P = 0.025, P < 0.001, P < 0.001; f N.S.; g P = 0.012, P < 0.001, P < 0.001, P < 0.001, P < 0.001, P < 0.001.
Fig 2: MDK activates T cells to produce Ccl4 through Lrp1/calcineurin/NFAT1 signaling.a T cells expressed only two (Lrp1 and Lrp6) of the putative MDK receptors [protein-tyrosine phosphatase ζ (Ptprz1), neuroglycan-C (Cspg5), low density-lipoprotein receptor-related protein-1 (Lrp1), low density-lipoprotein receptor-related protein-6 (Lrp6) and anaplastic lymphoma kinase (Alk)] by quantitative RT-PCR. Normal mouse cortex was used as an internal positive control. b Lrp1 blocking antibodies (30 µg ml−1) reduced MDK-induced Ccl4 production in T cells. c The 2041C>T neuron conditioned media (N-CM)-mediated Ccl4 production in T cells was attenuated following exposure to Lrp1 receptor blocking antibodies. d Immunoblotting revealed increased NFAT1 nuclear localization in T cells following MDK treatment. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) served as loading controls for the cytoplasm and nuclear fractions, respectively. e Decreased levels of phosphorylated-NFAT1 (p-NFAT) were observed after MDK stimulation of T cells. Lrp1 blocking antibodies (anti-Lrp1, 30 µg ml−1) increased NFAT1 phosphorylation and impaired NFAT1 nuclear localization in MDK-stimulated T cells. f The calcineurin inhibitor FK506 (10 μM) inhibited MDK-induced NFAT1 nuclear localization. g Calcineurin inhibitors, cyclosporin (100 nM) and FK506 (10 μM), inhibited MDK-induced Ccl4 production in T cells. h FK506 (10 μM) reduced 2041C>T neuron conditioned media (N-CM)-induced Ccl4 production in T cells. All data are presented as the mean ± SEM. a Bar graphs represent the means ± SEM of n = 3 independent biological samples. b, c; g, h These representative experiments were conducted with n = 3 independent biological samples,and were replicated two additional times with similar results. b, c, g One-way ANOVA with Bonferroni post-test correction, h Two-tailed Student’s t-test. Exact P values are indicated within each panel; N.S.; not significant. From left to right in each panel: b P = 0.005, P = 0.007; c N.S., P = 0.007; g P < 0.001, N.S., N.S.; h P < 0.001. d–f These are representative images of n = 3 independent biological samples examined over three independent experiments with similar results. Molecular weight markers are denoted at the left side of each blot.
Fig 3: 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 NFκB-dependent microglial Ccl5 expression, culminating in increased glioma growth through Akt/GSK3β/CREB pathway-mediated suppression of cancer (glioma) stem cell apoptosis and increased tumor growth.
Fig 4: NF1-mutant neurons express MDK, which activates T cells to produce Ccl4.a Isogenic hiPSC-induced neurons with heterozygous NF1 patient NF1 gene mutations (2041C>T and 6576C>T) produced higher levels of midkine in the neuron conditioned medium (N-CM) compared to WT (CTL) hiPSC-induced neurons. b Mdk gene expression was higher in the optic nerves of Nf1+/− relative to WT mice. c Increased Mdk expression was observed in optic glioma (OPG)-containing relative to control (CTL) optic nerves. d No change in T cell migration was observed in response to various MDK concentrations. e MDK (50 ng ml−1) stimulation for 48 h increased T cell Ccl4 production. f CM from isogenic hiPSC-induced neurons with NF1 patient NF1 gene mutations (c.2041C>T-N-CM and c.6576C>T-N-CM) exhibited a stronger T cell Ccl4 induction compared to CM from control hiPSC-induced neurons (CTL-N-CM). Anti-MDK neutralizing antibodies reduced T cell Ccl4 production in response to hiPSC-induced neuron CM stimulation. g MDK-activated (50 ng ml−1) T cell CM (mid-treated Tm) increased microglial Ccl5 production relative to non-activated T cell CM (non-act-Tm). h Immunohistochemistry revealed an increased percentage of MDK-immunoreactive cells in the retinal ganglion cell layer of OPG-bearing and Nf1+/− mice relative to control (CTL) or WT mice, respectively. Scale bars, 40 µm. Arrows denote representative immunopositive cells. i MDK gene expression was examined in NF1 pilocytic astrocytomas (NF1-PAs, n = 9), non-NF1-PAs (n = 9), and non-neoplastic brain tissues (n = 4). Increased MDK expression was detected in NF1-PAs compared to normal brain tissue and non-NF1-PAs. Arrows denote representative immunopositive cells. All data are presented as the mean ± SEM. a–c These representative experiments were conducted with a CTL, n = 3, 2041C>T n = 4, 6576C>T, n = 3; b WT, n = 7, Nf1±, n = 6; c CTL, n = 5, OPG, n = 4, independent biological samples, and were replicated two additional times with similar results. d and e Bar graphs represent the means ± SEM of d n = 5, e n = 3, independent biological samples. f This representative experiment was conducted with (from left to right) n = 3, n = 3, n = 3, n = 4, n = 3, n = 5, and n = 3 independent biological samples, and were replicated two additional times with similar results. g Bar graphs represent the means ± SEM of non-activated Tm, n = 3; MDK-treated Tm, n = 4 independent biological samples. h Bar graphs represent the means ± SEM of WT n = 9, Nf1+/−, n = 7, CTL, n = 5, OPG, n = 7, independent biological samples. i Bar graphs represent the means ± SEM of CTL, n = 4, non-NF1, PA, n = 9, NF1, PA, n = 9, independent biological samples. a, d, f, i One-way ANOVA with Bonferroni post-test correction, b, c, h Two-tailed Student’s t-test. Exact P values are indicated within each panel; N.S.; not significant. From left to right in each panel: a all P < 0.00, 1 b P = 0.008, c P = 0.021, d all N.S.; f top P = 0.012, middle P = 0.023, P = 0.030, bottom P = 0.045, P = 0.039; i CTL:NF1 PA P = 0.024, non-NF1 PA:NF1-PA P = 0.007; h all P < 0.001.
Fig 5: CD8+ T cells control Nf1 optic glioma growth.a Immunohistochemistry revealed that anti-CD8 antibody treatment reduced the number of CD3+ T cells, as well as the percentage of Ki67+ cells, Ccl4+ cells, and Ccl5+ cells, in Nf1 optic glioma specimens. Black arrows denote representative immunopositive cells. Scale bar, 40 µm. No differences in microglia (%Iba1+ cells) content were observed in anti-CD8 treated mice compared to the IgG control group. Bar graphs represent the means ± SEM of %Ki67+ cells, IgG, n = 8; anti-CD8, n = 5; CD3+ cells, IgG, n = 8; anti-CD8, n = 5; %Iba1+ cells, IgG, n = 8; anti-CD8, n = 5; %Ccl4+ cells, IgG, n = 6; anti-CD8, n = 5; %Ccl5+ cells, IgG, n = 4; anti-CD8, n = 4, independent biological samples. Two-tailed Student’s t-test. Exact P values are indicated within each panel; N.S.; not significant. From left to right in each panel: a % Ki67+ cells, P = 0.001; CD3+ cells, P = 0.002; %Iba1+ cells, N.S.; %Ccl4+ cells, P = 0.026; % Ccl5+ cells, P = 0.015. b Kaplan–Meier survival analysis (Brain Lower Grade Glioma TCGA Provisional [left panel; P = 6.72e–13] and TCGA PanCancer Atlas [right panel; P = 7.11e−14] datasets) demonstrates that non-overlapping patients with LGG and high tumor CD8 expression have shorter survival time, while c high CD4 expression was not associated with reduced survival time (Brain Lower Grade Glioma TCGA Provisional [left panel; P = 0.371] and TCGA PanCancer Atlas [right panel; P = 0.598] datasets).
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