Pediatric glioma immune profiling identifies TIM3 as a therapeutic target in BRAF fusion pilocytic astrocytoma
Shashwat Tripathi, … , Amy B. Heimberger, Michael DeCuypere
Shashwat Tripathi, … , Amy B. Heimberger, Michael DeCuypere
Published August 13, 2024
Citation Information: J Clin Invest. 2024;134(19):e177413. https://doi.org/10.1172/JCI177413.
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Research Article Immunology Oncology

Pediatric glioma immune profiling identifies TIM3 as a therapeutic target in BRAF fusion pilocytic astrocytoma

Abstract

Despite being the leading cause of cancer-related childhood mortality, pediatric gliomas have been relatively understudied, and the repurposing of immunotherapies has not been successful. Whole-transcriptome sequencing, single-cell sequencing, and sequential multiplex immunofluorescence were used to identify an immunotherapeutic strategy that could be applied to multiple preclinical glioma models. MAPK-driven pediatric gliomas have a higher IFN signature relative to other molecular subgroups. Single-cell sequencing identified an activated and cytotoxic microglia (MG) population designated MG-Act in BRAF-fused, MAPK-activated pilocytic astrocytoma (PA), but not in high-grade gliomas or normal brain. T cell immunoglobulin and mucin domain 3 (TIM3) was expressed on MG-Act and on the myeloid cells lining the tumor vasculature but not normal brain vasculature. TIM3 expression became upregulated on immune cells in the PA microenvironment, and anti-TIM3 reprogrammed ex vivo immune cells from human PAs to a proinflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven, low-grade gliomas, anti-TIM3 treatment increased median survival over IgG- and anti–PD-1–treated mice. Single-cell RNA-Seq data during the therapeutic window of anti-TIM3 revealed enrichment of the MG-Act population. The therapeutic activity of anti-TIM3 was abrogated in mice on the CX3CR1 MG–KO background. These data support the use of anti-TIM3 in clinical trials of pediatric low-grade, MAPK-driven gliomas.

Authors

Shashwat Tripathi, Hinda Najem, Corey Dussold, Sebastian Pacheco, Ruochen Du, Moloud Sooreshjani, Lisa Hurley, James P. Chandler, Roger Stupp, Adam M. Sonabend, Craig M. Horbinski, Rimas V. Lukas, Joanne Xiu, Giselle Lopez, Theodore P. Nicolaides, Valerie Brown, Nitin R. Wadhwani, Sandi K. Lam, Charles David James, Ganesh Rao, Maria G. Castro, Amy B. Heimberger, Michael DeCuypere

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Figure 5

Anti-TIM3 effectively reprograms the immune environment.

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Anti-TIM3 effectively reprograms the immune environment. (A) Immunocompe...
(A) Immunocompetent GEMM were treated with anti-TIM3 (300 μg/mouse) or IgG (100 μg/mouse) once per week or anti–PD-1 (200 μg/mouse) 3 times per week starting at day 28. (B) Histological evaluation of the GEMM displaying features of a low-grade glioma such as a loose microcystic pattern. Scale bar: 100 μm. (C) H&E-stained images demonstrating heterogeneity in a region of greater cellular density. Scale bar: 100 μm. (D) H&E-stained image demonstrating that glioma cells were monotonous and lacked mitosis (higher magnification of the image in C). (E) H&E-stained image demonstrating perineuronal satellitosis at infiltrating edges of the tumors. Scale bar: 100 μm (B and C) and 50 μm (D and E). (F) Immunofluorescence imaging of TIM3 expression on MG in the GEMM (n = 4). GFAP: purple; P2RY12: green; TIM3: yellow. (G) Immunofluorescence imaging p-ERK1/2 expression in the GEMM (n = 4). GFAP: purple; p-ERK: white. Scale bar: 50 μm (F and G). (H) Survival of low-grade glioma GEMM mice using Kaplan-Meier analysis. IgG: n = 20 mice (median survival [MS]: 110.5 days), anti–PD-1: n = 20 mice (MS: 134.5 days), anti-TIM3: n = 21 mice (MS: 253 days). Statistics (log-rank test): control versus anti–PD-1, P = 0.64; control versus anti-TIM3, P < 0.01; anti-TIM3 versus anti–PD-1, P = 0.02. (I) Survival of low-grade glioma CX3CR1-KO GEMM mice using Kaplan-Meier analysis. IgG: n = 20 mice (MS: 121 days), anti-TIM3: n = 28 mice (MS: 129.5 days). P = 0.51, by log-rank test for control versus anti-TIM3. (J) Representative immunofluorescence imaging of brains from the murine LGG model. Tumors were demarcated using H&E by a neuropathologist. Scale bar: 100 μm. (K) scRNA-Seq UMAP plot of the myeloid cells. n = 3 per group. (L) scRNA-Seq UMAP plot of the lymphoid cells. (M) Strip plot showing the differential abundance of cell types in the WT GEMM with treatment, log2 FC. Cell types with a P value of 0.1 or less are shown in gray. Cell types were ranked by the mean log2 FC of anti-TIM3 versus IgG. MG-Homo, MG-homeostatic; MG-Inflam, inflammatory MG; MG-Phago, phagocytic MG; Mono, monocytes.