Fig 2: Phenotypic Profile of Brain-Resident TCR Transgenic CD4 T Cells, Related to Figure 4CD4 T cells from perfused brains of TCR transgenic mice and wild-type controls (n = 5) were assessed by high parameter flow cytometry for CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. (A) tSNE of CD4+Foxp3- conventional 2D2 T cells, gated on CD4+Foxp3-CD3+CD8-CD45+ cells and subdivided into transgene-expressing (Va3.2+Vß11+) and transgene-non-expressing (Va3.2-Vß11-) cells. The tSNE was built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. FlowSOM clusters are illustrated in color and (B) quantified. (C) Heatmap of expression changes between transgene-expressing (Va3.2+Vß11+) and transgene-non-expressing (Va3.2-Vß11-) conventional 2D2 CD4+ T cells. (D) tSNE of CD4+Foxp3+ 2D2 regulatory T cells, gated on CD4+Foxp3+CD3+CD8-CD45+ cells and subdivided into transgene-expressing (Va3.2+Vß11+) and transgene-non-expressing (Va3.2-Vß11-) cells. The tSNE was built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. FlowSOM clusters are illustrated in color and (E) quantified. (F) Heatmap of expression changes between transgene-expressing (Va2+Vß5+) and transgene-non-expressing (Va2-Vß5-) regulatory T cells. (G) tSNE of CD4+Foxp3- conventional OT-II T cells, gated on CD4+Foxp3-CD3+CD8-CD45+ cells and subdivided into transgene-expressing (Va2+Vß5+) and transgene-non-expressing (Va2-Vß5-) cells. The tSNE was built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. FlowSOM clusters are illustrated in color and (H) quantified. (I) Heatmap of expression changes between transgene-expressing (Va2+Vß5+) and transgene-non-expressing (Va2-Vß5-) conventional OT-II CD4+ T cells.

Fig 3: Altered Activated Conventional Brain CD4 T Cells following Exposure to a Wild Microbiome or Broad-Spectrum Antibiotic Treatment, Related to Figure 5(A) The microbial diversity of caecal contents in specific pathogen-free (SPF) and wild-cohoused (CoH) mice was compared through 16S rRNA analysis. When comparing the microbial communities from SPE and CoH mice groups, we detect differences in richness but not in the Shannon diversity index (p < 0.03 and p > 0.05, respectively, Wilcoxon test) in the CoH compared to SPF mice. 32% of the variation on the microbial composition can be explained by this grouping (adonis test, R2 = 0.31681, p = 0.022). (B) Principal coordinate analysis (PCoA) of the mice microbiota community variation based on Bacteria and Archaea genus-level Bray-Curtis dissimilarity distances revealed two different clusters. (C) Phylum and genus distribution barplots. Only the top 14 genera are displayed. The body of the boxplot represents the first and third quartiles of the distribution and the median line. The whiskers extend from the quartiles to the last data point within 1.5 × IQR, with outliers beyond. ‘uc_f’: unclassified genus of the family and ‘uc_o’: unclassified genus of the order. Differential abundance analysis at the phylum level identified Deferribacteres and Proteobacteria enriched and Tenericutes depleted in CoH mice (q-values < 0.1, Wilcoxon test). (D) Lymph nodes, spleen, blood, perfused brain and perfused lung were dissected from gnotobiotic, SPF and dirty cohoused mice (n = 6, 10, 12) and assessed by flow cytometry. Total leukocyte counts, and (E) absolute numbers of CD4 T cells or (F) Tregs. (G) Proportion of CD4 T cells within the CD45+ compartment. (H) Proportion of Tregs within the CD4+ compartment. (I) Proportion of naive or (J) activated cells within the conventional T cell compartment. (K) Dendrogram showing the relationship of conventional T cells and (L) Tregs across the samples, based on cross-entropy differences in tSNE for expression of CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, CTLA4. (M) Representative histograms shown for ICOS, PD-1, CTLA-4 and CD44 on conventional CD4 T cells. (N) Expression of assessed markers on conventional CD4 T cells. (O) Representative histograms shown for ICOS, PD-1, CTLA-4 and CD44 on Tregs. (P) Expression of assessed markers on Tregs. (Q) Wild-type mice were placed on broad-spectrum antibiotics for 2, 4 or 6 weeks (n = 5, 8, 6) and were compared to untreated control mice (n = 8) by high parameter flow cytometry of the perfused brain. CD4 T cells in perfused brain, as a percentage of CD45+ cells, and (R) absolute numbers of conventional and regulatory CD4 T cells in the brain. P value refers to comparison of conventional T cells. (S) 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. P values represent cross-entropy comparison to control mice. FlowSOM clusters are illustrated in color and (T) quantified. (U) Expression heatmap and (V) marker expression rate for CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 levels in CD4+Foxp3- T cells. (W) 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. P values represent cross-entropy comparison to control mice. FlowSOM clusters are illustrated in color and (X) quantified. (Y) Expression heatmap and (Z) marker expression rate for CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet and CTLA4 levels in CD4+Foxp3+ T cells.

Fig 4: Brain-Resident CD4 T Cells Acquire a Residency Phenotype In Situ during a Prolonged Brain Transit(A) Schematic of parabiosis experiments (n = 12,12,18,16,14).(B and C) Curve of best fit for the origin of conventional (B) T cells or (C) Tregs showing CD69- and CD69+ in the blood and brain.(D) Derived median dwell times.(E and F) t-SNE of conventional (E) T cells and (F) Tregs built on CD62L, CD44, CD103, CD69, CD25, PD-1, Nrp1, ICOS, KLRG1, ST2, Ki67, Helios, T-bet, and CTLA4. FlowSOM clusters represented in color. Host and incoming cells were defined on CD45.1 versus CD45.2 expression, and are shown at the 2-, 4-, and 8-week time points.(G and H) CD69 histograms for CD4 conventional (G) T cells and (H) Tregs.(I and J) Population flow diagrams for conventional (I) T cells and (J) Tregs, in homeostatic state. Circle areas represent population frequencies, calculated independently for blood and brain. Small black circles represent cell death. The size of arrow ends is proportional to the rate of population flow. Numbers display the corresponding entry or exit rate, in events/1,000 cells/day. Numbers with asterisk denote rates with high estimation uncertainty. Population transitions with rates lower than 0.1/1,000 cells/day are not shown.See also Figure S3.

Fig 5: 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.