Fig 2: A Conserved Residency Profile for CD4 T Cells and Regulatory T Cells in the Healthy Mouse and Human Brain, Related to Figure 2(A) Healthy perfused mouse brains were compared to blood by high-dimensional flow cytometry. Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4+CD3+CD45+CD8-Foxp3- T cells or (B) CD4+CD3+CD45+CD8-Foxp3+ Tregs (n = 5). (C) Transcription profile of CD4+ T cells purified from the murine brain, with analysis through the 10X single cell pipeline and filtering for known cytokines. Naive, activated and regulatory cells were defined based on tSNE clusters and the relative expression of CD44, CD62L and Foxp3 within each cluster. (D) CD4 T cells were assessed in the perfused mouse brain by high-dimensional flow cytometry. Wild-type mice were sampled across the late embryonic (day 19), post-natal development (day 5, 10, 21, 30) and during healthy aging (weeks 8, 12, 30 and 52). n = 9,3,3,5,2,8,5,6,5. Quantification of CD4+ cells per gram of brain tissue, (E) CD4+ T cells, as percentage of CD45+ cells, (F) CD4+Foxp3- conventional T cells and (G) CD4+Foxp3+ regulatory T cells. (H) tSNE of CD4+Foxp3- T cells gated on CD4+Foxp3-CD3+CD8-CD45+ cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. Dendrogram represents cross-entropy distance between samples. (I) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4+CD3+CD45+CD8-Foxp3- T cells at different ages. (G) tSNE of CD4+Foxp3+ T cells gated on CD4+Foxp3+CD3+CD8-CD45+ cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4. FlowSOM clusters identified in color. P values refer to cross-entropy difference between age-matched blood and brain samples. (J) Comparison of expression levels of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 on blood versus brain CD4+CD3+CD45+CD8-Foxp3+ T cells at different ages. (K) Brain regions were surgically dissected and resident CD4 T cells were characterized by high-dimensional flow cytometry at 10 days, 30 weeks, 60 weeks and 90 weeks of age (n = 6,4,4,5). (L) Numbers and frequencies of CD4 T cells across brain regions in pups and adult mice. (M) tSNE of CD4+ Foxp3- T cells gated on CD4+Foxp3-CD3+CD8-CD45+ cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions in adult mice. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (N) Dendrogram showing the relationship in Tconv across the brain regions based on cross-entropy differences in tSNE. (O) CD69 expression in Tconv from the brain regions in pups and adult mice. (P) Heatmap showing expression of markers in brain region Tconv. (Q) tSNE of CD4+ Foxp3+ T cells gated on CD4+Foxp3+CD3+CD8-CD45+ cells and built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4, shown for blood and different brain regions. The adjusted P value reflects the cross-entropy difference between tSNE plots in brain region versus blood. (R) CD69 expression in Treg from the brain regions in pups and adult mice. (S) Heatmap showing expression of markers in brain region Treg. (T) Unaffected human brain tissues removed during brain surgery were compared to peripheral blood mononuclear cells by high-dimensional flow cytometry (n = 4). Representative histograms for CD4+Foxp3- T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (U) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, ROR?T, CCR4, CTLA-4, CCR7 and CD45RA on CD4+Foxp3- T cells. (V) tSNE of CD4+Foxp3+ T cells gated on CD4+Foxp3+CD3+CD8-CD14- cells and 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. Dendrogram showing the relationship across the brain regions based on cross-entropy differences in tSNE. (W) Representative histograms for CD4+Foxp3+ T cells from human peripheral blood mononuclear cells, white matter, gray matter and meninges for CCR2, CXCR3, PD-1 and CD69. (X) Expression of ICOS, CD28, CD69, Ki-67, CD95, CD31, HLA-DR, CCR2, CXCR5, CD25, PD-1, CXCR3, ROR?T, CCR4, CTLA-4, CCR7 and CD45RA on CD4+Foxp3+ T cells. (Y) Single cell RNaseq data from sorted CD4+CD3+CD8- cells from the human brain and PBMCs. Quality control filtering and gating based on expression markers identified 86 CD4+ T cells from the brain and 567 CD4+ T cells from the blood. tSNE dimensionality reduction is used for cluster display, with lineage marker expression indicated by color for CD3D, CD4, IL7R, IL2RA, FOXP3, CD44, SELL, AREG, CD69, KLRG1 and NR4A1. (Z) Differentially expressed genes were assessed for pathway by GSEA.

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.

Fig 4: A Conserved Residency Program for CD4 T Cells and Tregs in the Healthy Mouse and Human Brain(A) Healthy perfused mouse brains were compared to blood by high-dimensional flow cytometry. n = 5. t-Distributed Stochastic Neighbor Embedding (t-SNE) of conventional T cells built on key markers (CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, and CTLA4). Colors indicate FlowSOM clusters, quantified in side panel.(B) Representative histograms for conventional T cells from wild-type mouse blood and brain.(C) t-SNE of Tregs built on key markers. Colors indicate FlowSOM clusters, quantified in side panel.(D) Representative histograms for Tregs from mouse blood and brain.(E) Unaffected human brain tissues were compared to peripheral blood mononuclear cells (PBMCs) by high-dimensional flow cytometry, n = 4. t-SNE of conventional T cells built on key markers (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.(F) Dendrogram showing the relationship across the brain regions based on cross-entropy differences in t-SNE.(G) 10× single-cell sequencing was performed on sorted CD4 T cells from the human brain and PBMCs. Quality control filtering and gating based on expression markers identified 86 CD4+ T cells from the brain and 567 CD4+ T cells from the blood. t-SNE visualizing cell clusters built on the combined population of 653 CD4+ cells. Clusters are identified with different colors and labeled based on signature expression of transcriptional markers (left) or tissue origin (right).(H) Volcano plot of differential expression between brain and PBMC CD4 T cells. Indicated cut-offs are used for pathway analysis.See also Figure S2.

Fig 5: Differential PBMC activation in co-cultures from two patients with GBM with similar tumor Delta24-RGD sensitivity(A) Median fluorescence intensity (MFI) of CD69 on CD8+ T cells, CD4+ T cells, and NK cells in mock or Delta24-RGD-infected co-cultures derived from GS.1091 or GS.1191.(B) MFI of CD192, CD64, CD163, and CD206 on monocyte-derived macrophages in mock or Delta24-RGD-infected co-cultures derived from GS.1091 or GS.1191.(C) Average cytokine production of IFNγ, granzyme B, IL-6, CCL4, CXCL9, and CXCL10 in mock or Delta24-RGD-infected co-cultures derived from GS.1091 or GS.1191.Statistical significance between groups was evaluated by two-way ANOVA with correction for multiple comparisons using Sidak method (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001; mean with standard deviation).