Subtype and grade-dependent spatial heterogeneity of T-cell infiltration in pediatric glioma

Abstract

Brain tumors are the leading cause of cancer-related mortality in children and have distinct genomic and molecular features compared with adult glioma. However, the properties of immune cells in these tumors has been vastly understudied compared with their adult counterparts. We combined multiplex immunofluorescence immunohistochemistry coupled with machine learning and single-cell mass cytometry to evaluate T-cells infiltrating pediatric glial tumors. We show that low-grade tumors are characterized by greater T-cell density compared with high-grade glioma (HGG). However, even among low-grade tumors, T-cell infiltration can be highly variable and subtype-dependent, with greater T-cell density in pleomorphic xanthoastrocytoma and ganglioglioma. CD3+ T-cell infiltration correlates inversely with the expression of SOX2, an embryonal stem cell marker commonly expressed by glial tumors. T-cells within both HGG and low-grade glioma (LGG) exhibit phenotypic heterogeneity and tissue-resident memory T-cells consist of distinct subsets of CD103+ and TCF1+ cells that exhibit distinct spatial localization patterns. TCF1+ T-cells are located closer to the vessels while CD103+ resident T-cells reside within the tumor further away from the vasculature. Recurrent tumors are characterized by a decline in CD103+ tumor-infiltrating T-cells. BRAF^V600E mutation is immunogenic in children with LGG and may serve as a target for immune therapy. These data provide several novel insights into the subtype-dependent and grade-dependent changes in immune architecture in pediatric gliomas and suggest that harnessing tumor-resident T-cells may be essential to improve immune control in glioma.

Keywords: brain neoplasms; pediatrics; t-lymphocytes; tumor microenvironment.

Figure 1

Subtype-dependent and grade-dependent changes in T-cell infiltration in pediatric gliomas. Tumors from patients with LGG (including PXA (n=5), GG (n=7) and PA (n=6)) and HGG (including anaplastic astrocytoma (n=2) and GBM (n=5)) were examined using multiplex immunohistochemistry. (A) CD3 T cells as a percentage of total cells in LGG (PXA, GG and PA) versus those in HGG (AA and GBM). (B) T-cell density in LGG and HGG tumor tissue. (C) Percentage of CD3+ T cells in different LGG subtypes (PXA, GG and PA). (D) CD3+ T-cell density in different LGG subtypes (PXA, GG and PA). (E) Percentage of T cells in clusters in PXA and GG, and PA and HGG. (F) Representative sections from patients with PXA, GG, PA and GBM showing distribution of T cells in tumor tissue. All graphs show mean, and SEM dots represent individual patient values. GBM, glioblastoma multiforme; AA, anaplastic astrocytoma; GG, ganglioglioma; HGG, high-grade glioma; LGG, low-grade glioma; PA, pilocytic astrocytoma; PXA, pleomorphic xanthoastrocytoma.

Figure 2

Phenotypic heterogeneity of glioma-infiltrating T cells. (A to D) Single-cell mass cytometry was performed on freshly isolated tumor tissue from pediatric glioma samples to characterize the tumor infiltrating T cells. (A) CD3-gated density plot showing expression of CD45RO and CD69 on tumor-infiltrating T cells. Bar graph shows CD69+, CD45RO+ and TRM cells as percent of total CD3, CD4 and CD8 T cells in all patients (n=9). (B) Heatmap showing expression of CD103, TCF1, CD27, CD127, PD-1, GZMB, TIGIT and LAG3 in CD103+, TCF1+ and TCF1/CD103^neg T cells (n=4). (C) Bar graph showing distribution of CD4 and CD8 T cells within CD103+, TCF1+ and TCF1/CD103^neg T cells (n=4). (D) Bar graph shows expression of CD27 and CD127, inhibitory immune checkpoints PD-1, TIGIT and LAG3, and GZMB in CD103+, TCF1+ and TCF1/CD103^neg T cells (n=4). (E and F) Panels show multiplex IHC was used to characterize the expression of TCF1 and CD103 on tumor-infiltrating T cells. (E) Major subtypes of T cells identified by multiplex IHC of paraffin-embedded tumor tissue included CD3+ and TCF1+ T cells, CD3+ and CD103+ T cells, and CD3+, CD103^neg and TCF1^neg T cells (n=26). (F) CD103+ and CD3+ T cells as percent of total CD3+ T cells in PXA/GG as well as in patients with PA and HGG as determined by multiplex IHC. All graphs show mean and SEM dots represent individual patient values. GG, ganglioglioma; GZMB, granzyme B; HGG, high-grade glioma; IHC, immunohistochemistry; PA, pilocytic astrocytoma; PXA, pleomorphic xanthoastrocytoma.

Figure 3

Spatial heterogeneity of T-cell subsets in glioma tissue. (A) Bar graph showing the distance of TCF1+ and CD103+ T-cells from CD31+ endothelial cells (in microns). Bar graph shows mean and SEM (n=25). (B to D). Representative images showing differential spatial distribution of TCF1+ and CD103+ T-cells in glioma tissue. The endothelium is labeled by CD31+ cells.

Figure 4

Recurrent pilocytic astrocytoma is characterized by increased vascularity and a decline in CD103+ T_RM cells. Comparison of CD103+ T-cells and CD31+ endothelial cells in paired tissues at initial diagnosis and at relapse in three patients with pilocytic astrocytoma. (A) Changes in CD103+ T-cells at recurrence. (B) Changes in CD31+ endothelial cells at recurrence.