Fig 2: In vitro validation of selected modulators in primary mouse astrocytes.(A) Schematic mechanism of action of afatinib, UNC2025, and pemigatinib. (B) Ccl2, Ccl5, Tnf, Nos2, Lif, and Ngf expression, quantified by reverse transcriptase qPCR (RT-qPCR) in primary mouse astrocytes after stimulation (stim) with TNF-a and IL-1ß, IL-6, IFN-?, or GM-CSF with or without afatinib, UNC2025, and pemigatinib; n = 4 per group. Two-way ANOVA with Dunnett’s multiple comparisons test. (C) iNOS, TNF-a, GM-CSF, and Ki-67 were quantified by intracellular flow cytometry of primary mouse astrocytes after stim with TNF-a and IL-1ß with or without afatinib, UNC2025, and pemigatinib. Data are representative for n = 2 independent experiments with n = 4 per group. One-way ANOVA with Dunnett’s multiple comparisons test; data are reported as mean ± SD. (D) ELISA measurement of NGF and BDNF produced by primary mouse astrocytes after stim with TNF-a and IL-1ß with or without afatinib, UNC2025, and pemigatinib; n = 2. One-way ANOVA with Dunnett’s multiple comparisons test; data are reported as mean ± SD. (E) CCL2, NOS2, and LIF expression quantified by RT-qPCR in human astrocytes with or without stim with TNF-a and IL-1ß with or without afatinib, UNC2025, and pemigatinib; n = 4. One-way ANOVA with Dunnett’s multiple comparisons test; data are reported as mean ± SD. (F) Relative expression of Il6, and Ccl2 in primary mouse microglia quantified by RT-qPCR after stim with ACM derived from unstimulated and stim (TNF-a and IL-1ß) primary mouse astrocytes with or without afatinib, UNC2025, and pemigatinib; n = 3. One-way ANOVA with Dunnett’s multiple comparisons test; data are reported as mean ± SD. (G) Annexin V propidium iodide apoptosis assay of neuronal N2A cells stimulated with ACM; n = 3. Cells were categorized as in early or late apoptosis. One-way ANOVA with Dunnett’s multiple comparisons test; data are reported as mean ± SD. (H) Migration assay of CD11b+ myeloid cells stimulated with ACM; n = 3. One-way ANOVA with Dunnett’s multiple comparisons test; data are reported as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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: A booster immunization enhances the B cell response to ChAdOx1 nCoV-19 immunization in aged mice(A) Scheme of the prime-boost immunization protocol.(B) Percentage of Ki67+ B cells in the draining lymph node.(C and D) Percentage (C) and number (D) plasma cells in the iliac lymph node.(E) Pie charts showing the proportion of IgM+IgD– (orange) and switched IgM–IgD– (blue) plasma cells from (B) and (C).(F and G) Percentage (F) and number (G) of germinal center B cells in the iliac lymph node.(H) Pie charts showing the proportion of IgM+IgD– (orange) and switched IgM–IgD– (blue) germinal center cells from (F) and (G).(I and J) Number of T follicular helper (I) and T follicular regulatory (J) cells in the draining lymph node.(K) Percentage (K) of splenic germinal center B cells.(L–O) Serum anti-spike IgM (L), IgG (M), and IgG subclasses (N and O) prior to boost (day 29) and 9 days after boost immunization.(P and Q) SARS-CoV-2 neutralizing antibody titers in sera were determined by micro neutralization test, expressed as reciprocal serum dilution to inhibit pseudotyped virus entry by 50% (IC50). Samples below the lower limit of detection (LLoD) are shown as half of the LLoD.(R) Linear regression of serum dilution to inhibit pseudotyped virus entry by 50% (IC50 Log10) and serum anti-spike ELISA titer (Log10) in 22-month-old mice, 9 days post-boost. For all bar graphs, the bar height corresponds to the median and each circle represents one biological replicate. The Shapiro-Wilk normality test was used to determine whether the data are consistent with a normal distribution, followed by either an ordinary one-way ANOVA test for data with a normal distribution or a Kruskal-Wallis test for non-normally distributed data alongside a multiple comparisons test. For ELISA, data analyses were done on log transformed values. In (B)–(O), data are shown from one of two independent experiments (n = 4–8 per group/experiment); in (P)–(R), the data are pooled from two experiments.

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.