Fig 2: HCMV positivity in GBM determined comparatively by serology, qPCR and IHC. GBM patients were investigated for presence of HCMV pp65 and IE-1 expression by (A) qPCR, IHC and serology (dark gray bars = positive samples and light gray bar = negative samples) and (B) IE-1 expression on macrophage/myeloid derived cells (left panel) and on tumor cells (right panel). Magnification 400X, Scale bar 100 μm. (C) Venn diagram showing comparative HCMV serology (IgG), qPCR (pp65), qPCR (IE-1) and IHC (IE-1) detection in GBM tissue and blood. (D) Venn diagram showing comparative HCMV pp65 and IE-1 qPCR in GBM patients' blood and tumor. (E) Representative dotplots showing (left to right) macrophages (CD45brightCD11bbright) and microglia (CD45dimCD11bbright) within tumor biopsies cells; pp65 vs. CD45 within macrophages of tumor biopsies; pp65 vs. CD31 within CD45− tumor biopsy cells; and pp65 vs. CD45 within CD45− tumor biopsy cells. (F) % mean ± SEM of pp65+ cells within CD45brightCD11bbright macrophages, CD45dimCD11bbright microglia, CD45-CD31+ endothelial cells and CD45- tumor cells. (2-way ANOVA, Bonferroni's multiple comparison, ****P < 0.0001). IHC = immunonohistochemistry; IE-1 = immediate early -1.

Fig 3: Tumor-associated MΦ express enhanced FPN protein. (A) FACS panel how to gate CD45+ immune cells and CD326+ epithelial/tumor cells. Immune cells were further gated for CD33+/HLA-DR+ cells of which CD64+ and MerTK+ MΦ were sub-selected. Cells were subsequently analyzed for their FPN protein expression, displayed as MFI (mean fluorescence intensity). (B) FPN protein expression as MFI of CD45-/CD326- stroma cells (n = 26). (C) FPN protein expression as MFI of MΦ in tumor tissue compared to adjacent healthy tissue (n = 26). (D) Representative pictures for the MΦ marker CD163 and FPN protein expression in tumor tissue compared to healthy adjacent tissue applying confocal laser scanning miscroscopy. DAPI was used as nuclear stain. Scale bar: 200 µm. FPN mRNA expression normalized to 18S expression correlated to (E) low (G1–G2) and high (G3–G4) tumor grade and (F) low (pT1–pT2) and high (pT3–pT4) tumor pT-stage in our patient cohort (upper panels; n = 48) compared to the TCGA data base, applying the ccRCC-KIRC data set (lower panels; n = 70). (G) Kaplan-Meier curve of high or low FPN expression from the R2 bioinformatics platform using the ccRCC-KIRC data set. Graphs are displayed as means ± SEM with * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: Increased CD4+ and CD8+ TEM cells in HCMV+ GBM tumor with higher CTLA4/CD28 ratio compared with donor peripheral blood. (A) Representative dotplots showing lymphocytes in dissociated GBM tumor gated on FSC vs. SSC; singlets, live dead and within CD45+ cells, left to right. Representative dotplots showing (B) CD4+ (Th) vs. CD8+ (CTLs) T cell populations and (C) CD45RO vs. CD62L populations within the Th cell and CTL gates. % mean ± SEM of (D) Th (CD3+CD4+) and (E) CTLs (CD3+CD8+) naïve (N: CD45RO−CD62L+CCR7bright), central memory (CM: CD45RO+CD62L+CCR7bright), effector (E: CD45RO−CD62L−CCR7low) and effector memory (EM: CD45RO+CD62L−CCR7low) in peripheral blood (n = 22) and tumor (n = 19) of HCMV+ and HCMV− patients compared with seropositive vs. seroponegative donors (n = 16) (Two way ANOVA, Bonferroni's multiple comparison *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001). Mean MFI ± SEM of CCR7 within (F) the Th (CD3+CD4+)and (G) CTLs (CD3+CD8+) T cell subsets, showing low CCR7 expression in TEM and TE subsets (Two way ANOVA, Bonferroni's multiple comparison, *P < 0.05, and ****P < 0.0001). Mean MFI ± SEM of (H) CD28 and (I) CTLA-4 within the Th (CD3+CD4+)TEM; (J) CD28 and (K) CTLA-4 within the CTLs (CD3+CD8+)TEM (Two way ANOVA, Bonferroni's multiple comparison, **P<0.01, ***P < 0.001 and ****P < 0.0001). (L) % mean ± SEM of CTLA-4+ cells within the CTLs (CD3+CD8+)TEM (Two way ANOVA, Bonferroni's multiple comparison, ***P < 0.001 and ****P < 0.0001).

Fig 5: Gating strategy for FACS analyses of Liver immune cellsA representative of 60 individual human samples.(A) Forward and side scatter gating on lymphocytes to exclude cellular debris.(B) Singlets gating to exclude doublets.(C) Live cell positive gate.(D) CD45+ cells positive gating.(E) CD3+ and CD4+ gating for CD4+ T cells.(F) CD3+ and CD8+ positive gating for CD8+ T cells.(G) from the second staining panel we gated forward and side scatter gating on lymphocytes to exclude cellular debris then in singlets to exclude doublets followed by live cell and CD45+ cells positive gating. (G) CD11b and CD15 positive gating for macrophages/monocytes CD11b+CD15low and neutrophils CD11bhighCD15high.(H) HLA-DR high and CD3/CD16/CD19 negative gating for dendritic cells.(I and J) (I) CD11c vs CD123 for plasmacytoid DC (CD11c-CD123+) and myeloid DC (CD11c+CD123-) and (J) CD11c vs CD14 for monocyte exclusion.