Fig 2: Immune Cells and Vasculature of the Murine and Human HGSOC(A) Proportions of immune cell populations estimated from murine tumors and human omental metastases using CIBERSORT. 30200, HGS2, and HGS3, n = 4; 60577 and HGS4, n = 5; HGS1, n = 3; human omental tumors, n = 9. Median values are depicted.(B) Flow cytometric analysis of the immune infiltrate of peritoneal tumors, close to endpoint, from mice injected with 60577, 30200, or HGS cell lines. B cells: CD45+ CD19+, CD4 cells: CD45+ CD3+ CD4+, CD8 cells: CD45+ CD3+ CD8+, monocytes: CD45+ CD11b+ Ly6C+, macrophages: CD45+, CD11b+ F4/80+ (Ly6C/G-), granulocytes: CD45+ CD11b+ Ly6G+, CD11c+ cells: CD45+ CD11b+ CD11c+ (F4/80- Ly6C/G-). A similar analysis of the immune infiltrate in the diseased omentum from patients who underwent upfront surgery is shown for comparison (HuHGS).(C) Quantification of the number of CD3+ cells/mm2 by IHC on peritoneal tumors from mice injected with 60577, 30200, or HGS cell lines. Quantification of CD3+ cells in biopsies from patients (HuHGS) is shown for comparison. Representative images are shown; scale bars set to 100 µm.(D) Quantification of the percentage of an area positive for F4/80 by IHC on peritoneal tumors from mice injected with 60577, 30200, or HGS cell lines. Quantification of CD68+ area in patient biopsies is included for comparison. Representative images are shown; scale bars set to 100 µm.(E) IHC for endomucin (mouse) and CD31 (human) staining, quantified using the Definiens Tissue Studio platform with the blood vessel detection feature. Each dot represents a tumor from an individual mouse or human. Representative images for HGS2 and HuHGS are depicted at left. Scale bars, 100 µm.(F) Heatmap illustrating Gene Ontology biological process angiogenesis gene expression across mouse models and human omental tumors.

Fig 3: Brain T Cells Alter Gene Expression in Microglia, Related to Figure 6(A) Graph of CD4+ T cells in wild-type and MHC II KO mice in the spleen and perfused brain, as a percentage of CD45+ cells (n = 4). (B) Single cell RNaseq data from CD11b+ cells purified from wild-type mice and MHC II-deficient mice were assessed for gene-expression. tSNE dimensionality reduction is used for cluster display, with lineage marker expression indicated by color for Cx3cr1, P2ry12, Tmem119, ApoE, S100a9, Mrc1, Cd74 and S100a4. (C) Gene expression from isolated microglia from adult male wild-type and MHC class II KO mice (n = 4,4) were measured by qPCR. The expression of the indicated genes, selected from the differentially-expressed single cell sequencing dataset (Ccr6 upregulated in MHC II KO microglia, other genes downregulated in MHC II KO microglia) was calculated relative to Ppia rRNA. P values were obtained by two-tailed Mann-Whitney U test. (D) Single cell RNaseq data was generated on microglia collected from wild-type mice and MHC II-deficient mice. Volcano plots of wild-type versus MHC II-deficient clusters 1, cluster 2, cluster 3, cluster 4, cluster 5, cluster 6, cluster 7 and cluster 8 microglia, perivascular macrophages, macrophages, monocytes and granulocytes. Labeled genes with a differential expression of more than 1-log fold change (p < 0.05) are shown in black. (E) Microglia along the trajectory are divided into five groups based on expression of highly variable genes in wild-type and MHC II KO microglia. Cells are colored according to the facet in which they reside. (F) Pseudotime kinetics of genes identified as differentially expressed between wild-type and MHC II KO microglia, colored according to pseudotime trajectory. (G) Single cell RNaseq data from CD11b+ cells purified from wild-type mice and MHC II-deficient mice were assessed for gene-expression. Signature genes previously identified as differentially expressed between yolk sac-derived microglia and peripheral macrophage-derived microglia (Cronk et al., 2018) were assessed. Expression of Sall1, the transcriptional regulator defining authentic microglia differentiation, distinct from macrophage-derived microglia. (left) distribution of reads in normalized UMI counts, (right) marker expression indicated by color on tSNE dimensionality reduction. (H) Average expression of genes constituting the transcriptional signature of genes upregulated in macrophage-derived microglia compared to yolk sac-derived microglia: Abca1, Arhgef10l, Car9, Cln6, Cxcr2, Dclre1c, Dnajb14, Fgf2, Flt3, Fmd6, Fzd7, Galc, Gk, Hoxb4, Ifnar1, Jag1, Kcnn4, Lpar6, Ltbp3, Nrp1, Pcgf2, Pgap1, Pi4k2b, Plekhg5, Pmepa1, Qpct, Rap2a, Rgs1, R1pr1, Sesn1, Sestd1, Sgsh, Slc26a11, Slc44a1, Stab1, Stbd1, Tlr8, Tmem176b, Tpst1, Txndc16, Xylt2, Zdhhc23, Znrf3. (I) Microglia were sorted from control wild-type mice, or wild-type mice treated with anti-CD4 depleting antibody from day 5 or week 3 of age until 7 weeks of age (n = 15,9,9). Quantification of the CD45hiCD11b+Gr1- myeloid compartment of the brain following anti-CD4 depletion, demonstrating no numerical loss of macrophages. (J) Gene expression from isolated microglia was measured by qPCR. The expression of the indicated genes, selected from the downregulated-genes in the single cell sequencing dataset, was calculated relative to Ppia rRNA. P values were obtained by two-tailed Mann-Whitney U test. (K) Brain slices were cultured from neonatal cerebellum and left untreated or seeded with sort-purified CD4+ or CD8+ T cells (n = 2,3,2). Gene expression from isolated microglia was measured by qPCR. The expression of the indicated genes, selected from the downregulated-genes in the single cell sequencing dataset, was calculated relative to Ppia rRNA. P values were obtained by two-tailed Mann-Whitney U test. (L) Mice were i.v. injected with anti-CD45-PE and perfused, following which brains were dissected and analyzed by flow cytometry for the proportion of intravascular CD8 T cells (n = 6). (M) Proportional representation of CD8 T cells among CD45+ leukocytes in mouse (n = 6). (N) Numbers of CD8 T cells per gram in mouse blood and brain (n = 6). (O) CD45.1 and CD45.2 mice were parabiosed and assessed for brain and blood CD8 infiltrate at 2, 4, 8 and 12 weeks post-surgery (n = 12, 12, 18, 16, 14). Curve of best fit for the origin of CD8+ T cells in the blood and brain. (P) Mouse blood and perfused brain were assessed by flow cytometry (n = 6). tSNE of CD8+CD3+CD4-CD45+ cells built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. Colors indicate FlowSOM clusters (Q) which are quantified. (R) Heatmap of protein expression in mouse CD8 T cells. (S) Unaffected human brain tissues removed during brain surgery were compared to peripheral blood mononuclear cells by high-dimensional flow cytometry (n = 8,4), with CD8 T cells quantified as a fraction of CD45+ leukocytes and (T) in absolute number per gram of tissue. (U) Representative tSNE of human CD8+ CD3+CD4-CD14- cells built on ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, ROR?T, CCR4, CTLA-4, CCR7 and CD45RA. Colors indicate FlowSOM clusters (V) which are quantified. (W) Heatmap of protein expression in human CD8 T cells. (X) Isolated microglia were cultured with control conditioned media, conventional T cell conditioned media, Treg conditioned media, IL-4, IFN-?, TGF-ß and Amphiregulin (n = 2/group). Gene expression from isolated microglia was measured by qPCR. The expression of the indicated genes, selected from the downregulated-genes in the single cell sequencing dataset, was calculated relative to Ppia rRNA. P values were obtained by two-tailed Mann-Whitney U test. (Y) Microglia were sorted from TCRa knockout mice or heterozygous littermate controls (n = 7,8). Expression of signature genes (downregulated in MHC II KO microglia) were assessed by qPCR, with individual data-points reflecting the average of quadruplicate technical repeats in each of 19 genes across 7-8 biological replicates. (Z) Gene expression from isolated microglia was measured by qPCR. The expression of the indicated genes, selected from the downregulated-genes in the single cell sequencing dataset, was calculated relative to Ppia rRNA.