NKG2D ligand expression in pediatric brain tumors

Abstract

Adult brain tumors establish an immunosuppressive tumor microenvironment as a modality of immune escape, with several immunotherapies designed to overcome this barrier. However, the relationship between tumor cells and immune cells in pediatric brain tumor patients is not as well-defined. In this study, we sought to determine whether the model of immune escape observed in adult brain tumors is reflected in patients with pediatric brain tumors by evaluating NKG2D ligand expression on tissue microarrays created from patients with a variety of childhood brain tumor diagnoses, and infiltration of Natural Killer and myeloid cells. We noted a disparity between mRNA and protein expression for the 8 known NKG2D ligands. Surprisingly, high-grade gliomas did not have increased NKG2D ligand expression compared to normal adjacent brain tissue, nor did they have significant myeloid or NK cell infiltration. These data suggest that pediatric brain tumors have reduced NK cell-mediated immune surveillance, and a less immunosuppressive tumor microenvironment as compared to their adult counterparts. These data indicate that therapies aimed to improve NK cell trafficking and functions in pediatric brain tumors may have a greater impact on anti-tumor immune responses and patient survival, with fewer obstacles to overcome.

Keywords: Immune privilege; NK cells; NKG2D ligands; immune surveillance; immunosuppressive.

Figure 1.

NKG2D ligand transcript expression in pediatric low-grade and high-grade glioma samples. Frozen low-grade and high-grade pediatric tumor samples were analyzed for NKG2D ligand transcript expression (A-H). Tumor tissue samples were homogenized with mortar and pestle. mRNA was extracted from cell pellets using RNeasy Mini Kit (Miltenyi). RT-PCR was performed for cDNA synthesis, and qPCR was performed on cDNA samples 3 times in triplicate. Shown as mean +/− SD. Statistical analysis via unpaired t-test, * = p < 0.05.

Figure 2.

Quantification of pediatric TMA IHC NKG2D ligand analysis. TMAs from 5 types of pediatric brain tumors were constructed and verified for validity by pathologist-reviewed H&E staining. TMAs were then stained for expression of the 8 NKG2D ligands (ULBP 1-6, MIC A, MIC B), and analyzed via immunohistochemistry (IHC). Each tissue core was captured by Nuance camera at 10X and analyzed using inForm software (Perkin Elmer). Control sample is normal adjacent brain tissue. (A) Mean OD of positive area and percent cells positive for ULBP-2/5/6. (B) Mean OD of positive area and percent cells positive for ULBP-4. Shown as mean +/− SD. Data was analyzed for statistical significance using One-way ANOVA in conjunction with Dunnet's post-test. * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Figure 3.

Quantitative analysis of infiltrating immune cells in pediatric TMAs by IHC. TMAs from 5 types of pediatric brain tumors were constructed and verified for validity by pathologist-reviewed H&E staining. TMAs were stained for the presence of immune infiltrating cells using antibodies directed against NCR1 (NK cells) and CD163 (myeloid cells), and then analyzed via IHC. Each tissue core was captured by Nuance camera at 10X and analyzed using inForm software (Perkin Elmer). Control samples are normal adjacent brain tissue. (A) Percent cells positive for NCR1 (NK cells). (B) Percent cells positive for CD163 (myeloid cells). Shown as mean +/− SD. Data was analyzed for statistical significance using One-way ANOVA in conjunction with Dunnet's post-test. * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Figure 4.

Phenotypic comparison of peripheral blood lymphocytes between pediatric and adult patients. The phenotype of circulating lymphocytes from pediatric ganglioglioma (WHO grade I), pediatric anaplastic medulloblastoma (WHO grade IV), and adult glioblastoma (WHO grade IV) patients was assessed by flow cytometry. Representative plots showing (A) Percent CD45+ lymphocytes; (B) Delineation of myeloid cells, NK cells, and T cells using antibodies directed against CD14 and CD3; (C) Subgrouping of NK cells using antibodies directed against CD57 and NKG2D; (D) Subgrouping of NK cells using antibodies directed against CD56 and NKG2D; (E) Delineation of myeloid cells using antibodies directed against CD14 and CD33; and (F) Subgrouping of myeloid cells from CD14+CD33+ population using antibodies directed against CD11b and HLA-DR. Percent of population is listed for each gate.

Figure 5.

Phenotypic comparison of tumor-infiltrating lymphocytes between pediatric and adult patients. The phenotype of tumor-infiltrating lymphocytes from pediatric ganglioglioma (WHO grade I), pediatric anaplastic medulloblastoma (WHO grade IV), and adult glioblastoma (WHO grade IV) patients was assessed by flow cytometry. Representative plots showing (A) Percent CD45+ lymphocytes; (B) Delineation of myeloid cells, NK cells, and T cells using antibodies directed against CD14 and CD3; (C) Subgrouping of NK cells using antibodies directed against CD57 and NKG2D; (D) Subgrouping of NK cells using antibodies directed against CD56 and NKG2D; (E) Delineation of myeloid cells using antibodies directed against CD14 and CD33; and (F) Subgrouping of myeloid cells from CD14+CD33+ population using antibodies directed against CD11b and HLA-DR. Percent of population is listed for each gate.