Fig 2: 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 3: MBC subsets in SARS-CoV-2 infection(A and B) Confocal images of lung sections from (A and B) K18-hACE2 or (B) wild-type mice infected with 2.103 PFU of SARS-CoV-2. Scale bars, 130 µm.(C and D) Contour plots showing (C) lung and (D) lymph node class-switched resident B cells (left) and MBCs (middle) from mice infected as in (A) and analyzed at day 70. The multicolor bar (right) shows IgH isotype distribution in MBCs.(E and F) Dot plots showing the presence of MBC subsets in (E) lungs and (F) lymph node.(G and H) Contour plots showing MBC binding to Spike protein in (G) lungs and (H) lymph node.(I–K) Confocal images of lung sections at day 70 of the infection. Scale bars: 25 µm in (I), 18 µm in (J), and 20 µm in (K). The quantification in (I) displays the minimal distance of B cell clusters to EpCAM+ cells; dots represent B cell clusters. The quantification in (J) displays the minimal distance (nm) of IgM+ or IgG2b+ cells to the B cell cluster border divided by the cluster’s area (µm2); dots represent individual B cells.(L) Dot plot showing the area and B220 fluorescence for the detected IgG2b+ surfaces in (K) (left). Dot plot showing CCR6 and CXCR3 fluorescence intensity for IgG2b+ MBCs (center). X and Y positions of MBC subsets are laid out in the B220 representation (right).In (E)–(H), bar charts show the quantification of one experiment out of three, mean ± SEM. Each dot represents one mouse. (E and F) Two-way ANOVA test and (G and H) one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.See also Figure S6.

Fig 4: Spatial positioning of MBCs(A–D) Confocal images of (A and D) the lung, (B) mediastinal lymph node, and (C) spleen sections from Aid-EYFP mice treated as in Figure 1A. Scale bars: left 50 µm and right 25 µm in (A), left 300 µm and right 30 µm in (B), left 250 µm and right 55 µm in (C), and 20 µm in (D). The quantification in (A) displays the minimal distance of B cell clusters to the EpCAM+ cells. Bar charts in (B) and (C) show the proportion of MBCs residing in the indicated zones. MZ, marginal zone; PALS, periarteriolar lymphoid sheaths.(E) Dot plot showing CCR6 and CXCR3 fluorescence intensity for MBCs (left). X and Y positions of MBC subsets are laid out in the B220 representation (middle). The quantification displays the distance of each MBC to the cluster’s centroid (right).(F) Lung images of wild-type and CCR6-deficient Aid-EYFP mice treated as in Figure 1A. Scale bars, 30 µm. The quantification displays the density of YFP+ B cells in B cell clusters. Dots represent individual B cell clusters.(G) Strategy to generate mixed bone marrow chimeras.(H and I) Histograms showing (H) CCR6 and (I) CXCR3 expression in the lung MBCs and non-B cells in chimeras.(J and K) Quantification of MBC numbers after 70 days of primary influenza infection in (J) CCR6- or (K) CXCR3-deficient chimeras.(L and M) Enumeration of ASCs after 4 days of re-challenge in (L) CCR6- or (M) CXCR3-deficient chimeras. The quantification of one representative experiment is shown in the bar charts; each dot represents one mouse. In all panels: mean ± SEM, t test: **p < 0.01, ns, not significant.See also Figure S3.

Fig 5: Bona fide and bystander MBCs(A) Contour plots showing Fcer2a expression by the lung MBC subsets after 70 days of influenza infection, paired t test.(B) Contour plots showing the IL-4 GFP expression at day 9 of influenza infection in the mediastinal lymph node (left) and the proportion of GFP+ cells that express PD-1 and CXCR5 (right), one-way ANOVA.(C) Experimental approach for the treatment of wild-type mice with anti-IL4 antibody, PBS, or complexed IL4 during infection.(D) Histograms showing Fcer2a expression in mice treated as in (C), one-way ANOVA.(E and F) Contour plots of (E) lung and (F) lymph node CCR6+ MBCs in mice treated as in (C), one-way ANOVA.(G and H) Contour plots of (G) the lung and (H) lymph node MBCs depicting the proportions of CCR6+CXCR3- and CCR6+CXCR3+ subsets in mice treated as in (C). Quantifications in the left panels show the CXCR3+/CXCR3- MBC ratio, one-way ANOVA. Quantifications in the middle panels show the number of NP+ MBCs, one-way ANOVA. Quantifications in the right panels show the proportion of NP+ MBCs within each subset, two-way ANOVA.(I) Contour plots showing Fcmr expression by the lung MBCs at day 70 of the influenza infection, paired t test.(J) Dot plots showing the binding of lung MBCs to labeled IgM immune complexes (ICs), paired t test.(K and L) Enumeration of ASCs in the lungs of chimeric mice receiving (K) 80% µMT + 20% wild type versus 80% µMT + 20% Fcmr-/- bone marrow or (L) 50% CD45.1 + 50% wild-type CD45.2 versus 50% CD45.1 + 50% Fcmr-/- CD45.2 bone marrow. Mice were infected with influenza PR8, challenged with influenza X31 after 50 days, and sacrificed 4 days later. In (L), CD45.2 cells were sorted before ELISpot, two-way ANOVA test. In all the panels, each dot represents one mouse, mean ± SEM, *p < 0.01, *p < 0.05, ***p < 0.001, and ****p < 0.0001.See also Figure S5.