Fig 2: 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 3: LT-ILC1 cells accumulate in fibrotic area of the human liverRepresentative images of three cirrhotic (A) and two control livers (C) generated with PhenoCycler (formerly Codex) with a selection of markers such as CD56, CD94, aSMA, CD36, and CD32, as well as DAPI for nucleus staining (A, C). An overview (left) and magnification (right) are shown side by side with scale bar and indicated diameters. Fibrotic regions were recognized and delineated with a white dashed line using aSMA-staining. The red arrows indicate LT-ILC1 cells automatically detected by the HALO image analysis software by CD45+CD3-CD56+CD94+EOMES-KLRG1+ definition (see staining in Figure S5). Images in (B) and (D) were artificially generated as cell contours using HALO image analysis and correspond to the respective section from (A) and (C). Only the identified LT-ILC1 cells were highlighted for enhanced visibility (surface is green, nucleus is blue and indicated with red arrows) with visualization of the non-fibrotic (NF) and fibrotic (F) regions.

Fig 4: Liver sinusoidal endothelial cells supported the differentiation of liver-type ILC1s(A) Sorted peripheral blood ILCPs (Figure S5A; Lin-CD45+CD127+CD94-NKp80-CD45RA+CD117+CD294-KLRG1-NKP44-) from four healthy donors were cultured for 2 weeks on OP9-DL4 feeder cells or LSECs with IL-7 +/- TGF-ß1 as shown to the left. The dot plots to the right show the expression patterns of CD56 and CD94 among Lin- lymphocytes derived in the indicated culture conditions.(B) Frequencies of CD200R1+CD49a+EOMES- cells among total Lin-CD45+CD94+CD56+ cells derived in vitro following culture of ILCPs (n = 8) as in (A).(C) Representative flow cytometry analyses of EOMES, T-BET, perforin, CXCR6, NKp80, CD200R1, and CD49a expression by Lin-CD45+CD94+CD56+ cells derived from ILCPs in the indicated culture conditions. Fluorescence minus one (FMO) control with the respective lacking specific antibody is shown as black dotted lines in each histogram.(D) Intracellular flow cytometry analysis of IFN-?, IL-2, GM-CSF, TNF-a, and IL-22 production by in vitro derived Lin-CD45+CD94+CD56+ cells generated from ILCPs as in (A) and following stimulation for 5 h with PMA and ionomycin. Unstimulated cells were set as controls (dotted black line).(E) Representative flow cytometry analyses showing expression of the indicated markers by total Lin-CD45+ cells obtained after 2 weeks culture of primary sorted liver-type hepatic cNK, CD49a+trNK, or CD49a-trNK cells on OP9-DL4 feeder cells or LSECs with IL-7 +/- TGF-ß1 as shown to the left. Data are representative of at least three independent experiments.(F) Representative flow cytometry analyses showing expression of the indicated markers by total Lin-CD45+ cells obtained after 2-week culture of primary sorted liver-type ILC1s (LT-ILC1s) or hepatic NK cells (sorted as Lin-CD56+CD94+NKp80+) with OP9-DL4 cells and IL-7. Data are representative of at least three independent experiments.(G) Frequencies of CD200R1+CD49a+EOMES-cells among total Lin-CD45+CD94+ lymphocytes obtained from cultures initiated with either total hepatic NK cells or liver-type ILC1s (F).*p < 0.05, **p < 0.01, ***p < 0.001, error bars represent SEM. See Figure S6.

Fig 5: 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.