Supplementary Figure 1. Patient characteristics. (A) Venn diagram showing matching samples from patients for PBMCs, tumor biopsies for RNA-seq analysis and flow cytometry. (B) Heatmap showing the Z score of subtype gene expression in the patient cohort and the molecular and clinical characteristics of the patients. IDH mut= IDH 1/2 mutant, MGMT-M= methylated MGMT promoter and in red AIII= grade III astrocytoma (all other patients are GBM, MGMT promoter unmethylated and IDH wildtype).
Supplementary Figure 2. Immunophenoscore in poor and good prognosis patients.
Immunophenograms for individual patients (A) and bar chart showing the median and SE of their weighted Z score grouped according to prognosis (B). * indicates a p-value < 0.05. MHC= antigen processing, EC= effector cells, SC=suppressor cells, IPS=immunophenoscore.
Supplementary Figure 3. Expression of the gene signature according to genotype and grade.
Heatmap showing Z score of expression of the signature in TCGA dataset for different combinations of genotype and grade.
Supplementary Figure 4. Flow cytometry analysis of tumor-infiltrating myeloid cells. Gating strategy to define tumor-infiltrating myeloid cells. After doublet exclusion on CD45+CD11b+ cells, myeloid cells are defined as Draq7-CD3-CD19- cells.
Supplementary Figure 5. Flow cytometry analysis of tumor-infiltrating lymphoid cells. Gating strategy to define tumor-infiltrating lymphoid cells. After doublet exclusion on CD45+CD11b- cells and gating on Draq7- live cells, cells are defined as T cells: CD3+, B cells: CD19+, NK cells: CD3- CD16/56+, CD4 T cells: CD3+CD4+, CD8 T cells: CD3+CD8+, and regulatory (Reg) T cells:
CD3+CD4+CD25+CD127-. CD69 (or isotype) expression by T, B and NK cells is shown with bars denoting CD69+ cells.
Supplementary Figure 6. Immune cell markers expression correlates with measured cell counts in matching samples from the same patient. Comparison between log10 average normalized counts per million of CD3E, CD3G, CD3D (RNAseq) and log10 counts per million of total cells (flow
cytometry) for T cells (R2 = 0.9189, p-value=0.04) (A). Comparison between log10 average normalized counts per million of BLK, MS4A1 (RNAseq) and log10 counts per million of total cells (flow cytometry) for B cells (R2 = 0.9209, p-value=0.04) (B). Comparison between log10 average normalized counts per million of ITGAM, IDO1, CD209, CLEC4A (RNAseq) and log10 counts per million of total cells (flow cytometry) for myeloid cells (R2 = 0.9029, p-value=0.0498) (C).
Supplementary Figure 7. Flow cytometry analysis of glioma infiltrating leukocyte density according to tumor grade. Quantitative analysis of myeloid cells (A), CD8 T cells (B), CD4 T cells (C), regulatory T cells (D), B cells (E), NK cells (F), CD69-positive T cells (G), CD69-positive B cells (H) and CD69-positive NK cells (I) in patients with grade I, III or IV glioma. Data are represented as counts per million of total cells and depicted as box (25th to 75th percentiles) and whisker (10th to 90th percentiles) with the middle line representing the median. Numbers of glioma samples (n), as well as Kruskal-Wallis test (α = 0.05) derived p values are indicated. (J) Quantitative analysis of plasmatic IL- 6 (left) and CXCL3 (right) in patients with poor (mOS < 24 months) and good (mOS ≥ 24 months) prognosis and among patients with grade II, III or IV glioma (right). Data are expressed as pg per g of total plasmatic proteins and depicted as box (25th to 75th percentiles) and whisker (10th to 90th percentiles) with the middle line representing the median. Numbers of glioma samples (n), as well as Kruskal-Wallis test-derived p values are indicated in the graph.
