Dynamic fibroblast-immune interactions shape recovery after brain injury

Abstract

Fibroblasts and immune cells coordinate tissue regeneration and necessary scarring after injury. In the brain, fibroblasts are border-enriched cells whose dynamic molecular states and immune interactions after injury remain unclear¹. Here we define the shared fibroblast-immune response to brain injury. Early profibrotic myofibroblasts develop from pre-existing brain fibroblasts and infiltrate brain lesions, orchestrated by fibroblast TGFβ signalling, profibrotic macrophages and microglia, and perilesional glia. Myofibroblasts transition into several late fibroblast states, including lymphocyte-interactive fibroblasts. Interruption of the early myofibroblast state exacerbated sub-acute brain injury, tissue loss and secondary neuroinflammation, with increased mortality in the transient middle cerebral artery occlusion stroke model. Disruption of late lymphocyte-fibroblast niches via selective loss of fibroblast chemokine CXCL12 led to late brain-specific innate inflammation and lymphocyte dispersal with increased IFNγ production. These data indicate the response to brain injury is coordinated by evolving temporal and spatial fibroblast states that limit functional tissue loss and chronic neuroinflammation.

Fig. 1. Identification of a dynamic and persistent fibroblast response to brain injury.

a, Homeostatic brain fibroblasts are labelled in Col1a2^creER; Rosa26^tdT+ mice. Pial/perivascular fibroblasts (top right), periventricular/choroid plexus fibroblasts (bottom right). Mice were treated with tamoxifen from day −9 to day −7 before mice were euthanized for imaging. Scale bars: 2,000 μm (left), 200 μm (right). b, Lesional fibroblasts are labelled in Col1a1^GFP+; Col1a2^creER; Rosa26^tdT+ mice after PT cortical brain injury (image of GFAP⁺ gliosis (far right) is from a separate mouse). Mice were treated with tamoxifen from day −16 to day −14 before injury. Scale bars, 200 μm. c, A fibrotic lesion labelled for collagen 1 (COL1) and ER-TR7 (a fibroblast marker monoclonal antibody) within a glial scar (GFAP⁺) in the controlled cortical impact (CCI) model of TBI. Scale bars: 500 μm (left), 100 μm (right). d, Lesional fibroblasts after tMCAO stroke. Mice were treated with tamoxifen on days −7 to −5 before injury to track ontogeny. Scale bar, 250 μm. e, Time course showing collagen 6 (COL6) expression and persistence after endothelin-1-induced (ET-1) ischaemic stroke in adult marmosets. Scale bars, 250 μm. f,g, Time course in the PT injury model, showing fibroblasts (f; Col1a1-GFP⁺) and/or associated ECM (g; ER-TR7). Scale bars: 500 μm (f), 200 μm (g). h, Immunofluorescence showing astrocyte-dense (GFAP⁺) and fibroblast-dense regions (ER-TR7⁺) (top) and spatial plots (bottom, from same tissue) with 55-μm spot-based clusters. Early necrosis-associated autofluorescence is prominent; white dotted lines denote lesion borders (10X Genomics, Visium). i,j, Uniform manifold approximation and projection (UMAP) plots showing spot-based clusters (i) or fibroblast-enriched spots (j; Col1a1⁺). Four main fibroblast-enriched clusters are highlighted (black dashed line). k, Time course showing fibroblast-enriched spots in UMAP (top) or spatial (bottom) plots. l,m, UMAP and spatial plots (l) and volcano plot (m) comparing 7 dpi (early) fibroblast-enriched signature (cluster 10_0, red) and 21 dpi (late) fibroblast-enriched signature (cluster 8, blue). n,o, TGFβ score (n; genes upregulated in lung adventitial fibroblasts cultured with TGFβ) and proliferation score (o; 23-gene signature) across early and late fibroblast signatures. n = 118 (early) and n = 160 (late) spots. MAST (model-based analysis of single-cell transcriptomics) test (hurdle model with likelihood ratio test, false discovery rate (FDR)-adjusted; m); two-way Mann–Whitney test (n,o). Slice thickness: 200 μm (a), 14 μm (b,f,g), 30 μm (c), 50 μm (d), 40 μm (e) and 10 μm (h). Images represent two or more mice.

Fig. 2. Myofibroblasts spatiotemporally correlate with lesional profibrotic macrophages and disease-associated microglia.

a, Schematic of the snRNA-seq experiment showing sample time points and microanatomy resulting in a library of 28,187 nuclei. b–d, UMAP with 8,096 fibroblasts and 189 mural cells, showing fibroblast subclusters (b), microanatomy (c) or time point (d). Dotted lines (b) highlight early myofibroblast/proliferative clusters (red) and multiple late states (blue). e, Heat map of select fibrosis-related genes expressed in proliferative and myofibroblast clusters (red dashed line). f,g, Gene set enrichment analysis (f) and ligand–transcriptional-network analysis (g; NicheNet) of myofibroblasts. Tgfb1 is highlighted as the top predicted driving ligand. P_adj, adjusted P value. h,i, Schematics showing Cthrc1^creER; Rosa26^tdT lineage tracing²⁸ (h) and hypothesized Cthrc1 expression trajectory (i; with tamoxifen (Tam) induction regimens for j–n). j–n, Confocal microscopy showing Cthrc1-lineage⁺ lesional fibroblasts after PT injury (tamoxifen induction and collection days indicated), with robust injury-induced Cthrc1–tdT expression (j) but lack of lineage-traced fibroblasts (k). Active Cthrc1 expression is reduced but present at 7 dpi (l; shown at 14 dpi) and absent by 14 dpi (m; shown at 21 dpi). Labelled fibroblasts persist to 365 dpi (n). Scale bars, 200 μm. o, Confocal microscopy showing time course of lesional accumulation of myeloid cells (IBA1^hi surface). Scale bars, 500 μm. p,q, Annotated UMAP of reclustered myeloid cells (p) and violin or spatial plots of profibrotic SAM score (q; Trem2, Cd9, Spp1, Gpnmb, Fabp5 and Cd63). q, Left, score by cluster; DAM: n = 1,267, SAM: n = 816, PVM/BAM: n = 305, ECM macrophages (ECM_Macs): n = 173, monocyte/dendritic cell (Mono/DC): n = 116, dendritic cells (DCs): n = 20, microglia: n = 110, osteoclasts: n = 19 nuclei. Right, score by time point within DAM and SAM lesional clusters; 7 DPI DAM: n = 168, 21 dpi DAM: n = 1,080, 7 DPI SAM: n = 247, 21 dpi SAM: n = 506. Bottom, score mapped onto spatial transcriptomic Visium data. r, Schematic showing potential ligand–receptor interactions between SAM and DAM and myofibroblasts, derived from Extended Data Fig. 5x. Over-representation test (one-sided Fisher’s exact test, FDR-adjusted) (f); Kruskall–Wallis test, Dunn’s multiple comparisons correction (q, left; relevant comparisons shown); two-way Mann–Whitney test, Bonferroni correction (q, right). Slices thickness, 14 μm. Images represent two or more mice.

Fig. 3. Distinct late fibroblast states include lymphocyte-interactive fibroblasts associated with T cell persistence.

a, Fibroblast cluster abundance over time, with corresponding early and late spatial signatures. b, Co-expression of pial, arachnoid and dural genes among corresponding clusters. c, snRNA-seq signatures of meningeal fibroblast subsets mapped onto spatial transcriptomic data. d,e, Fluorescence microscopy (d) and cartoon (e) of late fibroblast subset topography and protein expression. Scale bars: 500 μm (main images), 100 μm (enlarged views). f, Gene set enrichment analysis among lymphocyte-interactive fibroblasts. g, Heat map of chemokines expressed in ≥0.5% of any cluster; lymphocyte-interactive fibroblasts are highlighted (blue). h–j, Total T cells (h; CD3ε⁺), CD4⁺ and CD8⁺ T cells (i) and or T_H1 (TBET⁺), T helper type 2 (T_H2; GATA3⁺) and T helper type 17 (T_H17; RORγt⁺) CD4⁺ T cells (j; 14 dpi) after PT injury (cortical flow cytometry). Rest: n = 5, contralateral: n = 46, 7 dpi: n = 6, 14 dpi: n = 38, 21 dpi: n = 18, 60 dpi: n = 11 mice (h); n = 18 mice per time point (i); n = 15 mice (j). k,l, Native microscopy (k; 21 dpi) and time course of T cell surfaces (l; Cd4^cre; Rosa26^tdT+; Cd3e⁺) near fibroblast-rich lesions (ER-TR7⁺). Scale bars: 50 μm (k), 500 μm (l). m, Schematic of proximity analysis between T cells (CD4^cre; Rosa26^tdT+; Cd3e⁺) or myeloid cells (IBA1^hi, macrophages and reactive microglia) and fibroblast ECM (ER-TR7) or astrocytes (GFAP). n,o, Median T cell distance from nearest fibroblast ECM or astrocyte surface (n) and T cell or myeloid cell distance from nearest fibroblast surface (o). 21 and 60 dpi. n = 5 mice per time point (2 slices per mouse; lighter green or grey dots and P values represent tissue slices; darker dots and P values are per mouse). p,q, Image (p; 21 dpi) and quantification (q) of T cell proximity to CD80⁺ lymphocyte-interactive fibroblasts at 14, 21 and 60 dpi. n = 5 mice per time point (2 slices per mouse). Scale bar, 50 μm. r,s, Schematic for ex vivo lesional coculture (r) and quantification of T cell survival (s; 21 dpi lesions). Alone: n = 36, contralateral: n = 34, 21 dpi lesion: n = 44, beads: n = 33 wells. t, T cell survival after coculture with dural or lung fibroblasts. T cells alone (T alone): n = 3, T cells plus meningeal fibroblasts (men. fib.): n = 3, lung: n = 4 wells. Over-representation test (one-sided Fisher’s exact test, FDR-adjusted) (f); one-way ANOVA, Tukey post-test (h,q,s,t); multiple two-way t-tests, Holm–Sidak correction (i; paired per point, unpaired per slice (n,o)); one-way repeated-measures ANOVA, Tukey post-test (j). Slice thickness, 14 μm. Images represent two or more mice. Source data

Fig. 4. Brain lesional myofibroblasts are coordinated by TGFβ signalling, profibrotic myeloid cells and perilesional glia to drive wound healing and limit chronic inflammation.

a–c, Treatments with control or clodronate (Clod) liposomes and images of fibroblasts and astrocytes in lesions (a), ECM (ER-TR7) coverage (b) and lesion size (c) at 14 dpi. b, Control: n = 7, clodronate: n = 6 mice. c, Control: n = 15, clodronate: n = 10 mice. Two slices per mouse. Scale bars, 500 μm. d, Schematic of ablated fibroblast TGFβ signalling (Col1a2^creER; Tgfbr2^flox; tamoxifen treatment on days −14 to −12, −7, 0 to 2, 5, 8 and every 3 days subsequently until sample collection). e–h, Images of control and Tgfbr2-cKO (cKO) lesions with thresholded ER-TR7 (e) or Col1a2^creER; Rosa26^tdT+ fibroblasts (f), ER-TR7 coverage (g) and lesion size (h) at 14 dpi. Control (ctrl): n = 20, Tgfbr2-cKO: n = 12 mice; 2 slices per mouse. Scale bars, 500 μm. i, Cortical neutrophils in controls and Tgfbr2-cKO lesions. 0 dpi control (non-littermate-sham): n = 8, 2, 4 and 7 dpi control and 2, 7 and 21 dpi Tgfbr2-cKO: n = 6, 14 dpi control: n = 12, 21 dpi control: n = 9, 0 dpi Tgfbr2-cKO: n = 3, 4 dpi Tgfbr2-cKO: n = 5, 14 dpi Tgfbr2-cKO: n = 7 mice. j–l, Images of lesions in control (cre-negative or vehicle) and myofibroblast deleter (Cthrc1^creER; Rosa26^DTA) mice (j), and ER-TR7 coverage (k) and lesion size (l) at 21 dpi. Control: n = 19, deleter: n = 22 mice; 2 slices per mouse. Scale bars, 200 μm. m, Lesion size at 28 dpi in controls and Tgfbr2-cKO mice after late tamoxifen treatment (days 14–16, 19, 22 and 25). Control: n = 6, Tgfbr2-cKO: n = 7 mice per group; 2 slices per mouse. n, Cortical neutrophils at 21 dpi after late or continuous tamoxifen treatment. Control late: n = 7, Tgfbr2-cKO late: n = 6, Tgfbr2-cKO continuous: n = 6, control continuous: n = 9 mice. o–q, Images of cortical lesions in control (IgG) mice or mice with α_vβ₈ blockade (ADWA11) at 14 dpi (o), ER-TR7 coverage (p) and cortical neutrophils (q). IgG: n = 11, ADWA11: n = 9 mice (p; 2 slices per mouse); IgG: n = 7, ADWA11: n = 8 mice (q). Scale bars, 500 μm. r,s, Cortical lesions in control and GFAP^cre; Itgb8^flox mice at 14 dpi (r) and ER-TR7 coverage (s). Control: n = 12(control), Tgfbr2-cKO: n = 7 mice; 2 slices per mouse. Scale bars, 200 μm. t, Fibroblast snRNA-seq cluster abundance in control, Tgfbr2-cKO (Col1a2^creER; Tgfbr2^flox), and ADWA11-treated mice at 7 dpi (early) and 21 dpi (late). u, Expression of myofibroblast genes in myofibroblasts across time points. v–x, Myeloid cluster abundance (v), SAM frequency (flow cytometry) (w; 14 dpi) and perilesional T cell numbers (x; 14 dpi). n = 4 mice per group (w); control: n = 9, Tgfbr2-cKO: n = 6 mice (x; 2 slices per mouse). Quantification normalized to controls in each experiment (c,h,k,l). Two-way Student’s t-test (b,c,g–i (0 dpi), k–m,p,s,w,x); two-way repeated-measures ANOVA, Sidak’s post-test (i) (7–21 dpi, per time point; bold: lesioned control versus lesioned Tgfbr2-cKO, grey: lesioned control versus contralateral control, italic: lesioned Tgfbr2-cKO versus contralateral Tgfbr2-cKO); two-way repeated-measures ANOVA, Sidak’s post-test (q); two-way ANOVA, Sidak’s post-test (n). Slice thickness: 14 μm. Dotted lines indicate lesion boundary (a,e,f,j,o,r); images represent two or more mice. Source data

Fig. 5. Discrete beneficial functions of early myofibroblasts and late lymphocyte-interactive fibroblasts.

a,b, tMCAO-induced fibrotic lesion at 14 dpi (a) and mouse survival (b). Control stroke: n = 18, Tgfbr2-cKO stroke: n = 21, control sham/rest: n = 10, Tgfbr2-cKO sham/rest: n = 15 mice. Mice were treated with tamoxifen at −3 to −1, 2, 5, 8 and 11 dpi. Scale bars: 500 μm (main images), 200 μm (enlarged views). c–f, Ipsilateral hemisphere area (c), brain midline shift (d), oligodendrocyte density (e) and FluoroJade-C⁺ (degenerating) neuron density (f) at 3 days after tMCAO. Control, n = 4, Tgfbr2-cKO, n = 6 mice. Two slices per mouse (c,d); light grey or red dots and text represent values and P values per tissue slice, dark colors per mouse (c); normalized to contralateral (c,e,f, per group). g,h, Heart rate (g) and mean arterial pressure (h) 1 to 3 days after tMCAO. Control groups: n = 7, Tgfbr2-cKO groups: n = 4 mice (g); control uninjured: n = 8, Tgfbr2-cKO uninjured: n = 5, control stroke: n = 7, Tgfbr2-cKO stroke: n = 4 mice (h). n = 1 Tgfbr2-cKO stroke recorded at 1 dpi, n = 1 Tgfbr2-cKO stroke recorded at 2 dpi; remaining mice recorded at 3 dpi. i, Schematic showing loss of CXCL12 production in Col1a2^creER; Cxcl12^flox fibroblasts with tamoxifen treatment at 0 to 2,7 to 9 and 14–16 dpi. j–l, Surfaced T cells (CD3ε⁺CD45⁺, processed via Imaris software ‘surface’ function) near lymphocyte-interactive fibroblasts (CD80⁺) in control (j) or Cxcl12-cKO mice at 21 dpi (k) with quantification (l). n = 7 mice per group; 2 slices per mouse. Scale bars: 500 μm (left), 100 μm (right). m–p, IFNγ expression in CD8⁺ T cells (m), IFNγ (n) and IL-17A (o) expression in CD4⁺ conventional T (T_conv) cells, and IL-17A expression in γδ T cells (p) at 21 dpi. Control: n = 11, Cxcl12-cKO: n = 14 mice. q, Cortical neutrophils in control and Cxcl12-cKO mice at 21 dpi. Control, n = 21, Cxcl12-cKO: n = 16 mice. r, Schematic of snRNA-seq experiment. Control (wild-type (WT)) and Cxcl12-cKO mice were collected at 21 dpi (2 mice per genotype), lesions and parenchyma were micro-dissected and multiplexed, and nuclei were sorted and sequenced (19,668 nuclei). s, Global UMAP analysis of samples depicted in r. t, Type 1 T cell abundance (180 nuclei). u, IFNγ response score among lesional myeloid cells. Control: n = 1,659, Cxcl12-cKO: n = 2,056 nuclei. v, UMAP of neuronal cells (5,862 nuclei). w, IFNγ response score of excitatory and inhibitory neurons. Wild-type excitatory: n = 2,392, wild-type inhibitory: n = 576, Cxcl12-cKO excitatory: n = 2,229, Cxcl12-cKO inhibitory: n = 665 nuclei. x, Schematic showing a model of T cell regulation via late lesional fibroblast-derived CXCL12. log-rank (Mantel–Cox) test, Bonferroni correction (k = 4) (b); two-way Student’s t-test (c–e,l–p); two-way repeated-measures ANOVA, Sidak’s post-test (f,q (repeated-measures),g,h); two-way Mann–Whitney test (u,w (Bonferroni correction)). Slice thickness: 14 μm. Images represent two or more mice. Source data

Extended Data Fig. 1. Additional characterization of the CNS fibroblast response to PT injury, related to Fig. 1.

a, Homeostatic dural meningeal fibroblasts (Col1a2^creER; Rosa26^TdT+; whole-mounted dura, tamoxifen days ⁻11–⁻7 before harvest). b-c, fibroblast infiltration (Col1a1^GFP+, b, or Col1a2^creER; Rosa26^TdT+, c) after CCI model of TBI (tamoxifen days ⁻18–⁻16). d, Fibroblast expansion near lesion borders (white dotted line) by 4dpi (tamoxifen days ⁻14–⁻12,⁻7,0–2dpi). e-f, Flow plot (e) and quantification (f) of fibroblast nuclei in Pdgfrα^GFP mice (marking fibroblasts and oligodendrocyte precursor cells [OPC]). n = 7(resting cortex), n = 11(ipsilateral cortex/lesion, 14dpi), or n = 8(dura, resting or 14dpi) mice. g-h, Expanded perilesional vasculature and associated ECM after PT injury, with Pdgfrα^GFP+ nuclei (g) or fibroblasts (h) visualized in lesional perivascular spaces. i-l, Resting fibroblasts (Col1a1^GFP+) partially recombined by Col1a2^creER (i.e., TdT⁺; i), quantification of GFP expression within recombined cells (j), 21dpi lesional fibroblasts (Col1a1^GFP+) with a fibroblast origin (TdT⁺) (k), and quantification of recombination efficiency before and after injury (l). Tamoxifen days ⁻16–⁻14 (before injury, “uninjured”/“lineage-traced”) or 7–9dpi (“post-injury induction”). n = 6(uninjured), n = 5(lineage traced), or n = 3(post-injury) mice (1 40μm slice [uninjured] or 2 14μm slices/mouse). m, Lineage-traced fibroblasts in Gli1^creER; Rosa26^TdT mice (preferentially recombining fibroblast subsets in multiple tissues). Tamoxifen days ⁻16–⁻14 before injury. n, Time course showing stromal/fibroblast (Twist2^cre; Rosa26^TdT+) expression of fibroblast markers (Dcn, Col6a1, Postn) and pericyte marker (Desmin). Serial sections; images representative of n = 2–3 (2dpi), n = 2–6 (7dpi), or n = 2–7 (21dpi) mice. o-q, Lack of lineage-traced lesional cells (with extra-lesional accumulation) in Ng2^creER; Rosa26^TdT mice (o), images of Ng2-lineage⁺ pericytes (Desmin⁺) or oligodendrocyte-lineage cells (SOX10⁺; p), and quantification of recombination efficiency within each lineage (q). n = 4 mice. Tamoxifen days ⁻16–⁻14 before injury. r, Lack of lineage-traced lesional cells (with sparse smooth muscle visible) in Acta2^creER; Rosa26^TdT mice. Tamoxifen days ⁻16–⁻14 before injury. s, Quantification of lesional recombined cells in Acta2^creER, Ng2^creER, Gli1^creER, and Col1a2^creER mice. n = 3(Acta2^creER, Gli1^creER, Col1a2^creER) or n = 4(Ng2^creER) mice. t-u, Lineage traced fibroblasts (t, Col1a2^creER; Rosa26^TdT) but not pericytes (u, Atp13a5^creER; Rosa26^TdT) accumulating within fibrotic lesions (Col1⁺) after distal Middle Cerebral Artery Occlusion (dMCAO), 14dpi. Tamoxifen given for 3 days, 6–8 weeks prior to injury. v-w, Images of Atp13a5-lineage⁺ pericytes (v, PDGFRβ⁺, NG2⁺) and quantification of recombination efficiency (w). n = 3 mice (3 slices/mouse). x, Quantification of lesional or contralateral recombined pericytes or fibroblasts (TdT⁺ area in Atp13a5^creER or Col1a2^creER; Rosa26^TdT mice) after dMCAO, 14dpi. n = 6(Atp13a5^creER) or n = 5(Col1a2^creER) mice (2–3 slices/mouse). y-ac, Schematic (y) showing transcranial 4-hydroxy-tamoxifen (4-OHT) induction (top) and intraperitoneal tamoxifen induction (bottom). Transcranial 4-OHT sparsely recombines dural (z) but not perivascular or leptomeningeal (pial or arachnoid) fibroblasts (aa), highlighted with arachnoid markers ALDH1A2 and E-Cadherin (ab, top and bottom) and quantified in ac. Shown 2–10 days after induction; n = 3(intraperitoneal), n = 5(transcranial-perivascular/leptomeningeal), or n = 6(transcranial-dural) mice. ad-ae, Image (ad) and quantification (ae) of lesional fibroblasts recombined via transcranial 4-OHT. n = 6(lesion [2 slices/mouse] and dura-transcranial) or n = 3(dura-intraperitoneal) mice. One-way ANOVA, Tukey post-test (f,l,s); two-way ANOVA, Sidak’s post-test (x [repeated measures], ae); multiple two-way T-tests, Holm-Sidak correction (ac). 14μm (b-d,h,k,m-p,r,ad), 200μm (g), 40μm (i,n [desmin],aa,ab), or 10μm (t,u,v) slices; images represent two or more mice. Source data

Extended Data Fig. 2. Spatial transcriptomics characterization of PT injury, related to Fig. 1.

a, Spatial transcriptomics plot showing all spot-based clusters. Slices shown in original Visium slide configuration with two slices/capture area: rest (top left), 2dpi (bottom left), 7dpi (middle, 2 biological replicates), and 21dpi (right, 2 biological replicates). b-c, Dot plots showing marker genes (b) or fibroblast-associated genes (c) for all spot-based clusters. c3 (“resting brain border”), c8 (“late fibroblast”), c10_0 (“early fibroblast”), and c13 (“late lesion border”) are highlighted (colored boxes). c10_0 was selected from c10 for further analysis based on high Pdgfra expression. d, UMAP showing spot-based clusters colored by timepoint, highlighting divergence among fibroblast-containing clusters. e-g, UMAP and/or spatial plots showing the “resting brain border” signature near the cortical boundary (e, cluster 3, brown) and the “late lesion border” signature (f, cluster 13, cyan) overlying astrocyte-containing spots (g, Gfap⁺). h-i, Spatial plots showing distribution of “fibroblast TGFβ score” (h) or “proliferation score” (i).

Extended Data Fig. 3. Additional transcriptomic characterization of fibroblasts across species and injury models, related to Fig. 2.

a, Gating strategy for sorting DAPI⁺ nuclei for snRNAseq. b-c, Global UMAP (b) and Col1a2 feature plot (c) showing fibroblast clusters (black dotted line) along with immune, oligodendrocyte lineage, endothelial, astrocyte, and neuron clusters. d-e, Global UMAPs showing barcode-based microanatomical metadata (d) or timepoint metadata (e). Neurons show parenchymal origin and broad temporal distribution, dural fibroblasts (dissected only at rest) show meningeal origin and resting distribution, and lesional fibroblasts, immune cells, and glial cell subsets show lesional origin and temporal heterogeneity. f-g, Dot plots showing marker genes for all cellular clusters (f) or all fibroblast subclusters (g). h, Violin plots mapping each lesional fibroblast cluster identity (via marker-DEGs) onto spatial transcriptomic clusters (Fig. 1), with spatial “fibroblast-enriched signatures” highlighted. i, Adult marmoset annotated snRNAseq data (Boghdadi et al.) from endothelin-1-induced stroke (cortex, 7dpi). Fibroblasts highlighted (red dotted line) based on COL1A2 expression. j-k, UMAPs showing integrated fibroblasts from 7dpi (described in i) and resting adult marmoset dataset, showing annotated fibroblast subclusters (j) and timepoint (k). Black dotted lines highlight 7dpi-enriched clusters 1 and 4. l-m, Bar plot showing relative cluster abundance (l, as proportion of fibroblast nuclei), and heatmap of select fibroblast/myofibroblast-related genes across fibroblast clusters (m). Black dotted lines highlight 7dpi-enriched clusters 1 and 4. n, Violin plot showing myofibroblast-associated TGFβ score. n = 529(rest) or n = 663(7dpi) fibroblasts. o, Human TBI annotated snRNAseq data (Garza et al.), including control tissues (varying timepoints; controls from subjects with non-neurological causes of death). Fibroblasts highlighted (red dotted line) based on COL1A2 expression. p-q, Heatmap and violin plot showing elevated expression of selected fibroblast/myofibroblast genes (p) or myofibroblast-associated TGFβ score (q) in post-TBI fibroblasts. n = 54(control) or n = 23(post-TBI) fibroblasts. r-t, Human glioblastoma multiforme (GBM) tissue snRNAseq data (Jain et al.), shown as UMAP (r) and feature plots of COL1A1 (s) or CTHRC1 (t). Cancer-associated fibroblasts are highlighted (r, red dotted line). u-w, UMAP showing reclustered fibroblasts (u), and heatmap or violin plot showing fibroblast expression of select fibroblast/myofibroblast genes (v) or myofibroblast-associated TGFβ score (w), enriched in cluster 0. n = 235(c0), n = 67(c1), n = 66(c2), n = 63(c3), n = 38(c4), or n = 25(c5) fibroblasts. Two-way Mann-Whitney test (n,q). Source data

Extended Data Fig. 4. Additional in vivo validation of early TGFβ-associated myofibroblasts, related to Fig. 2.

a-b, Nuclear flow cytometry showing elevated pSMAD3 expression within lesional fibroblasts (Pdgfrα^GFP-int) relative to dural/parenchymal fibroblasts (a) or other lesional cells (b), 7dpi. n = 4 mice. c-e, Image of αSMA⁺ expression among fibroblasts (Col1a2^creER; Rosa26^TdT+) at 7/21dpi (c), and quantification of the number (d) and percentage (e) of αSMA⁺ fibroblasts. n = 3 mice/group. f, Confocal microscopy showing Cthrc1^creER; Rosa26^TdT+ fibroblasts in whole-mounted dural meninges at indicated timepoints. Tamoxifen days ⁻16–⁻14 prior to injury (rest); days ⁻1–1 (2dpi); days 0–2,5 (7dpi); or days 0–2,5,12 (14dpi). g, Schematic showing tamoxifen induction for (h-l) and hypothesized trajectory for Acta2 (gene for αSMA) expression. h-l, Confocal microscopy showing Acta2-lineage⁺ lesional fibroblasts after PT injury (tamoxifen induction and harvest days indicated), with robust injury-induced Acta2 (TdT) expression (h) but lack of lineage-traced fibroblasts (i). Active Acta2 expression is reduced but present at 7dpi (j, shown at 14dpi) and absent by 14dpi (k, shown at 21dpi). l (inset from k) shows expected Acta2^creER; Rosa26^TdT+ vascular smooth muscle cells (white arrows) near CD31⁺ vessels. m-n, Image (m) and quantification (n) of fibroblast proliferation (EdU incorporation) 2 h after EdU pulse (“Pulse”; 7dpi, 14dpi, or 21dpi) or after EdU injection every other day (“Continuous”, and shown in m; 14dpi). In insets (m), arrows mark proliferating fibroblasts. Fibroblasts defined as Col1a2^creER; Rosa26^{TdT+/Sun1GFP+}. Tamoxifen days 0,1,2,5,12. n = 3 mice/timepoint (2 slices/mouse). o-p, Cthrc1-lineage⁺ fibroblasts within fibrotic scar after tMCAO (o) or TBI (p). Tamoxifen days 0,1,2,5,12 (o) or 0,1,2,5 (p). One-way repeated-measures ANOVA, Tukey post-test (a); paired two-way Student’s T-test (b); two-way Student’s T-test (d,e); one-way ANOVA, Tukey post-test (n). 14μm slices. Source data

Extended Data Fig. 5. Additional characterization of injury-responsive profibrotic CNS myeloid cells, related to Fig. 2.

a-b, Quantification of Fig. 2o showing number of myeloid cells (IBA1^hi, macrophages and reactive microglia) per slice (a) and median myeloid cell distance from the nearest fibroblast-associated ECM (ER-TR7⁺) surface (b). n = 3(2dpi), n = 4(7dpi), or n = 5(14/21/60dpi) mice (2–4 slices/mouse). c, Schematic showing myeloid cell distribution by ontogeny. d-f, Immunofluorescent imaging from microglia lineage tracer mice (d, P2ry12^creER; Rosa26^TdT; tamoxifen days ⁻14–⁻12 before injury), with quantification of lineage traced cell density (e) and proportion (f) in inner core, fibrotic border, or outer glial regions at late timepoints (14/21dpi). n = 6 mice. g-i, Imaging from monocyte lineage tracer mice (g, Ccr2^creER; Rosa26^TdT; tamoxifen 0–2dpi), with quantification of density (h) and proportion (i) by region, as above. n = 5 mice. j-k, Time course showing Ccr2-lineage traced cells differentiating into both lesional macrophages and dendritic cells, shown as normalized counts (j) or as a proportion of the indicated population (k). Tamoxifen 0–2dpi. n = 4(0/2dpi), n = 3(7/21dpi), or n = 5(14dpi) mice. l-n, Imaging from BAM/PVM lineage tracer mice (l, PF4^cre; Rosa26^TdT), with quantification of density (m) and proportion (n) by region, as above. n = 4 mice. o-q, UMAP of reclustered myeloid cells (o, colored by timepoint; major resting populations labelled), dot plot showing myeloid subcluster marker genes (p), and violin plot of Disease Associated Microglia (“DAM”) score (q, an aggregate of 30 published DAM-specific markers). n = 1267(DAM), n = 816(SAM), n = 305(PVM/BAM), n = 173(ECM_Macs), n = 116(Mono/DC), n = 20(DCs), n = 110(Microglia), or n = 19(Osteoclasts) nuclei. r, DAM/SAM snRNAseq identities mapped onto spatial transcriptomic data. s-u, Imaging of FABP5⁺ cells near Cthrc1^creER; R26^TdT+ myofibroblasts at 7dpi (s), and surfaced images of FABP5⁺ DAM (t, microglia-derived) or SAM (u, monocyte-derived) in perilesional/border regions at 7dpi (with inset) or 21dpi. v-w, Density plots showing expression of SAM score among myeloid cells after stroke in marmosets (v) or TBI in humans (w). x, Ligand-receptor interactions between SAM or DAM and myofibroblasts (CellPhoneDB). Left half: ligand (first/purple molecule in labelled pair) expressed by SAM/DAM, receptor (grey) expressed by myofibroblast. Right half: ligand expressed by myofibroblast, receptor expressed by SAM/DAM. Dotted lines (left) and schematic (right) highlight modules with macrophage-expressed ligands (blue) or myofibroblast-expressed ligands (orange). y, Expression of DAM markers (CD9/CD63) on sorted homeostatic microglia cultured for 72 h alone or with 7/21dpi lesions. n = 7 wells/condition. One-way ANOVA, Tukey post-test (a,b,y); one-way repeated-measures ANOVA, Tukey post-test (e,f,h,i,m,n); Kruskall-Wallis test, Dunn’s multiple comparisons correction (q, relevant comparisons shown). 14μm slices; images represent 2 or more mice. Source data

Extended Data Fig. 6. Additional characterization of late lesional fibroblasts, related to Fig. 3.

a, Single nuclear signatures of late lesional fibroblast states mapped onto spatial transcriptomic data, showing expected localization. b, Heatmap of selected late fibroblast state markers used for imaging. c, Late fibroblast state markers shown at 7dpi. d, Quantification of c and Fig. 3d, showing the number of subset marker-positive fibroblasts at 7/21dpi. n = 3 mice/timepoint. e-i, Violin plots (e) showing ECM (Col1a1) and smooth muscle-related^, (Cdh18, Sema3c) genes (enriched among late inner fibroblasts), with imaging and quantification of CDH18⁺ fibroblasts (f,g) and SEMA3C within the inner core (h,i, 21dpi). n = 3 mice/timepoint. j, Phylogenetic tree showing lesional stromal cell cluster relationships. k, Imaging (left) and quantification (right) of subset marker expression among Cthrc1-lineage⁺ fibroblasts (tamoxifen continuously, i.e. days 0–2,5,8,11, etc.; fibroblast- vs. non-fibroblast-adjacent FGF13/LAMA1 quantified). n = 3 mice. l, Fibroblast subset marker staining after tMCAO (14 dpi) and cartoon of fibroblast subset topography. m, Perilesional T cell time course (CD4^cre; Rosa26^TdT+; CD3ε⁺). n = 3(2dpi), n = 4(7dpi), or n = 5(14/21/60dpi) mice (2−5 slices/mouse). n, Lesional type 1 lymphocytes (Tbet^zsGreen+, 21dpi). o, Tissue resident memory CD8⁺ T cell distribution (CD44⁺ CD69⁺; 60dpi). n = 17 mice. p-r, T cell proportion <10μm from nearest fibroblast-associated ECM (ER-TR7⁺) or astrocyte (GFAP⁺) surface (p), T cells within or outside fibroblast-dense lesion (q), and T or myeloid cell (IBA1^hi) proportion within lesion (r). n = 5 mice/timepoint (2–4 slices/mouse; slice values/statistics shown with lighter colors). s, T cell proportion <10μm from nearest CD80⁺ fibroblast. n = 5 mice/timepoint (2 slices/mouse; slice values/statistics shown with lighter colors). t-v, T cell counts (t), proliferation (u, CTV dilution), and activation (v, CD44⁺) after coculture (21dpi lesions). n = 36(t,u, alone), n = 33(v, alone), n = 31(contralateral), n = 43(t, 21dpi lesion), n = 44(u,v, 21dpi lesion), n = 33(t,u, beads), or n = 30(v, beads) wells. w, T cell survival after coculture with 7dpi or 21dpi lesion. n = 18(7dpi) or n = 24(21dpi) wells. x, T cell survival counts after coculture with dural meningeal or lung fibroblasts. n = 3(alone, meningeal) or n = 4(lung) wells. y, Conventional dendritic cell (cDC) infiltration and persistence after PT injury (cortical flow cytometry). n = 6(sham/60dpi), n = 7(1/2dpi), n = 10(7dpi), n = 5(14 dpi), or n = 3(21dpi) mice. Two-way repeated-measures ANOVA, Sidak’s post-test (d,q,v); two-way Student’s T-test (g,w); paired two-way Student’s T-test (i,o); one-way ANOVA, Tukey post-test (m,s-u,x,y); multiple two-way T-tests, Holm-Sidak correction (p [per slice]); multiple paired T-tests, Holm-Sidak correction (p [per point], r). 14μm slices; images represent two or more mice. Source data

Extended Data Fig. 7. Profibrotic macrophages promote CNS fibroblast expansion, related to Fig. 4.

a-b, 14 dpi splenic immune cells (a) or 4 dpi cortical phagocytic myeloid cells (b) after treatment with control or clodronate liposomes, as frequency (top) or counts (bottom). n = 11/8 (control, a/b) or n = 9/7 (clodronate, a/b) mice. Within each experiment, clodronate liposome-treated mice were normalized to control liposome-treated mice. c-e, Perilesional myeloid cell (IBA1) staining at 14dpi, with images (c) and quantification via thresholding (d) and normalization to reactive astrocyte (GFAP)-traced lesion area (e). n = 7(control) or n = 6(clodronate) mice (2 slices/mouse). f-h, Quantification of lesional Col1a1^GFP+ fibroblast total density (f) and density within in inner core (g) or fibrotic border (h) in control or clodronate-treated mice. 14dpi; n = 8(control) or n = 4(clodronate) mice (2 slices/mouse). i, Spatial transcriptomic feature plots from Visium dataset (Fig. 1) showing Ccr2 and Tgfb1 transcripts in perilesional distribution at 2dpi. j-l, Feature plot of Tgfb1, plotted in order of expression, from mouse PT injury snRNAseq (j), marmoset stroke snRNAseq (k), or human TBI snRNAseq (l); dotted lines highlight myeloid cells. m, Representative image showing recombined macrophages and microglia (TdT⁺) in a Cx3cr1^creER; Rosa26^TdT mouse (tamoxifen as in Fig. 4d; 14dpi). n-o, Lesional images (n; dotted line shows lesion boundary) and ER-TR7 coverage (o, normalized per experiment) following genetic targeting of macrophage and microglial Tgfb1 in Cx3cr1^creER; Tgfb1^flox/GFP-KO mice, 14dpi. Controls were littermate Cx3cr1^creER; Tgfb1^flox/+ mice; tamoxifen as in Fig. 4d. n = 17(control) or n = 15(Tgfb1 cKO) slices (2 slices/mouse; slice values/statistics shown with lighter colors). Multiple T-tests, Holm-Sidak correction (a,b); two-way Student’s T-test (d-h, o). 14μm slices; images represent two or more mice. Source data

Extended Data Fig. 8. TGFβ drives CNS wound healing and resolution of inflammation, related to Fig. 4.

a, Intact fibroblast topography in uninjured cKO (Col1a2^creER; Tgfbr2^flox) parenchyma (insets: leptomeningeal/perivascular/periventricular fibroblasts). b, Lesional images showing ER-TR7 in controls or cKOs, 14dpi (tamoxifen as in Fig. 4d; dotted line shows lesion boundary). c-g, Quantification of 14dpi lesional Col1a2^creER; Rosa26^TdT+ fibroblast total density (c) and density within in inner core (d) or fibrotic border (e), with cKOs showing overall and inner fibroblast reduction with an intact but thinner lesional border (f), summarized via schematic (g), 14dpi. n = 6(control) or n = 5(cKO) slices/group (2 slices/mouse). h-i, ER-TR7 coverage (h) and lesion size (i, normalized per experiment) in control or cKO mice after tamoxifen pre-treatment only (tamoxifen days ⁻16–⁻14) without follow-up induction, 14dpi; n = 6(control) or n = 7(cKO) mice. j, S100A8⁺ neutrophils (PMN; left, surfaced; right, native) in controls (top) or cKOs (bottom), 14dpi (tamoxifen as in Fig. 4d). k, Time course showing cortical monocytes in controls/cKOs. n = 8(0dpi-control [non-littermate sham]), n = 6(2/4/7dpi-control, 2/7/21dpi-cKO), n = 12(14dpi-control), n = 9(21dpi-control), n = 3(0dpi-cKO), n = 5(4dpi-cKO), or n = 7(14dpi cKO) mice. l-q, Neutrophil or monocyte counts in meninges (l-m), blood (n-o), or 14dpi spinal cord (p-q) of controls/cKOs. n = 5(0dpi control [non-littermate sham], 7dpi cKO), n = 3(0dpi cKO), n = 6(2/4/7dpi control, 2/21dpi cKO), n = 12(14dpi control), n = 9(21dpi control), n = 5(l-m, 4dpi cKO), n = 6(n-o, 4dpi cKO), or n = 7(14dpi cKO) mice (l-o); n = 4 mice/group (p-q). r, Control/cKO spinal cords showing intact neurons without fibrosis/gliosis, 14dpi. s-t, Image (s) and quantification (t) of cCasp3⁺ puncta within corpus callosum in control or cKO mice, 14dpi. n = 4(sham-control), n = 6(sham-cKO), n = 8(stroke-control), or n = 7(stroke-cKO) mice (2–4 slices/mouse). u, Evans Blue extravasation in uninjured (resting or sham) or contralateral/lesioned hemispheres of controls/cKOs, 4dpi. n = 6(uninjured-control), n = 7(uninjured-cKO), or n = 9(contralateral/lesioned-control and cKO) mice. v, Quantification of overt bleeding in controls/cKOs, 4dpi. n = 9(control) or n = 7(cKO) mice. w, ER-TR7 coverage in control or cKO mice induced with late tamoxifen (beginning 14dpi), 28dpi. n = 6(control) or n = 7(cKO) mice. x, Co-expression of TGFβ-activating integrin pairs Itgav and Itgb1, Itgb6, or Itgb8 (snRNAseq). y-aa, Quantification of lesional Col1a1^GFP+ fibroblast total density (y) and density within in inner core (z) or fibrotic border (aa) in IgG- or ADWA11-treated mice, 14dpi. n = 3(IgG) or n = 4(ADWA11) mice (2 slices/mouse). ab-ae, Lesion size (ab, normalized per experiment), cortical monocytes (ac), and meningeal (ad) or blood neutrophils (ae) in IgG- and ADWA11-treated mice, 14dpi. n = 14(IgG) or n = 13(ADWA11) mice (ab, 2 slices/mouse); n = 7(IgG) or n = 8(ADWA11) mice (ac,ae); n = 6(IgG) or n = 7(ADWA11) mice (ad). af, Itgb8^TdT mouse, with perilesional/astrocytic TdT, 14dpi (insets: GFAP/TdT colocalization, yellow). ag, Lesion size in hGfap^cre; Itgb8^flox mice and controls (normalized per experiment), 14dpi. n = 12(control) or n = 7(cKO) mice (2 slices/mouse). ah-aj, Control or Emx1^cre; Itgb8^flox lesional images (ah), ER-TR7 coverage (ai), and lesion size (aj, normalized per experiment), 14dpi. n = 7 mice/group (2 slices/mouse). Two-way Student’s T-test (c-f,h,i,k [0dpi],p,q,v,w,y-ae,ag,ai,aj); two-way ANOVA, Sidak’s post-test (k [repeated measures, 7–21dpi, per timepoint; bold=lesioned-control/lesioned-cKO, grey=lesioned-control/contralateral-control, italics=lesioned-cKO/contralateral-cKO], t,u); multiple two-way T-tests, Holm-Sidak correction (l-o). 14μm slices; images represent two or more mice. Source data

Extended Data Fig. 9. Additional molecular characterization of injury response after fibroblast impairment, related to Fig. 4.

a, snRNAseq schematic. Control (WT), cKO (Col1a2^creER; Tgfbr2^flox), and α_vβ₈-blocking antibody (ADWA11)-treated mice were harvested at 7 and 21dpi (2 mice per timepoint/condition), lesions were micro-dissected, and nuclei were sorted (DAPI⁺) and sequenced (final library 60,070 nuclei). b-c, Global UMAPs from snRNAseq data showing indicated cell types (b) or Col1a2 expression (c). d-e, UMAPs (18,455 fibroblasts and 496 mural cells) with fibroblast subclusters (d) or timepoint (e). f, Violin plots mapping “CNS fibroblast signatures” (derived from Fig. 2, x-axis) onto clusters identified in d (y-axis). g-i, Characterization of “Altered dural/lymphocyte-interactive” fibroblasts, shown as highlighted cluster in Cxcl12 feature plot (g), UMAP with subclusters (h), and violin plots showing subcluster expression of “Lymphocyte-interactive” and “Altered-dural” signatures (i, derived from Fig. 2), highlighting 3 lymphocyte-interactive-like and 1 altered-dural-like subcluster(s). j, Fibroblast UMAP separated by condition, with pan-fibroblast reduction in cKO mice and late-inner fibroblast reduction in α_vβ₈-blocked (ADWA11) mice. k-p, Images (top) and quantification (bottom) of fibroblast subset marker expression among Col1a2^creER R26^TdT fibroblasts in control or cKO mice. 7dpi cKOs show a decrease in αSMA⁺ myofibroblasts (k, quantified as myofibroblast density [left] or proportion [right]). 21dpi cKOs show a trending decrease in FGF13⁺ late inner fibroblasts (l) and decreases in CD80⁺ lymphocyte interactive fibroblasts (m), ALPL⁺ altered dural fibroblasts (n), and LAMA1⁺ pial fibroblasts (p), though ALDH1A2⁺ leptomeningeal/arachnoid fibroblasts are preserved (o). Blue dotted lines show lesion border. n = 3(control) or n = 4(cKO) mice. q, Violin plots showing expression of myofibroblast-related genes among the myofibroblast cluster. r, Gene set enrichment among the myofibroblast cluster, with controls showing ECM-process enrichment. s-t, UMAPs showing myeloid cell subclusters (s) or timepoint (t). u, Violin plots mapping expression of “CNS myeloid cell signatures” (derived from Fig. 2, x-axis) onto clusters identified in s (y-axis). v-w, Pseudotime (v, Monocle3) and UMAP plots separated by condition (w) showing potential myeloid differentiation trajectories, including monocyte-to-SAM (top) and microglia-to-DAM (bottom), with monocytes and microglia as root states. x, Schematic showing effect of TGFβ-activated myofibroblasts on SAM/DAM formation. y-z, Violin plots (y) or feature plots (z) showing “dysmature” transcriptional signature across conditions. n = 1841/1281(Ctrl/ADWA11 DAM), n = 573/5551(Ctrl/ADWA11 SAM), or n = 1724/3181(Ctrl/ADWA11 PVM/BAM) nuclei (y). aa, Surfaced T cells (CD3ε⁺; CD45⁺) in control and cKO mice, with fibroblast-rich (ER-TR7⁺) lesion. ab-ac, UMAP showing lymphocyte subclusters (ab) or genotype (ac, Ctrl and cKO). Dotted black lines highlight CD8 and γδT cells. ad, Violin plot of lymphocyte Itgae expression. n = 495(WT) or n = 336(cKO) nuclei. Two-way Student’s T-test (k-p); over-representation test (one-sided Fisher’s exact test, FDR-adjusted, r); two-way Mann-Whitney test (y [Bonferroni correction], ad). 14μm slices; images represent two or more mice. Source data

Extended Data Fig. 10. Additional characterization of Tgfbr2 and Cxcl12 cKO mice, related to Fig. 5.

a, Hematoxylin and eosin staining of liver, kidney, and lung tissue in control and cKO (Col1a2^creER; Tgfbr2^flox) mice without brain injury. Images representative of n = 2 mice/group. b-d, Area positive for mouse IgM (b) or IgG (c), or MFI of Ter119 (d) in control or cKO mice (3dpi, tMCAO). n = 4(control) or n = 6(cKO) mice. e-f, Systolic (e) or diastolic (f) blood pressure in control or cKO mice (1−3dpi, tMCAO). n = 8(control-uninjured), n = 5(cKO-uninjured), n = 7(control-stroke), or n = 4(cKO-stroke) mice; 1 cKO-stroke recorded 1dpi, 1 cKO-stroke recorded 2dpi, remaining mice recorded 3dpi. g, Serum levels of ALT in control or cKO mice (3dpi, tMCAO). Dotted lines indicate bounds of normal range. n = 8(control-uninjured), n = 3(cKO-uninjured), or n = 6(control-stroke, cKO-stroke) mice. h-i, Representative images (h) and quantification (i) of cCasp3⁺ liver cells/puncta in control or cKO mice (3dpi, tMCAO). n = 5(control-uninjured), n = 3(cKO-uninjured), n = 8(control-stroke), or n = 7(cKO-stroke) mice (5 FOVs averaged per mouse; FOV values/statistics shown with lighter colors). j, Serum levels of creatinine in control or cKO mice (3dpi, tMCAO). Dotted lines indicate bounds of normal range. n = 9(control-uninjured), n = 3(cKO-uninjured), n = 7(control-stroke), or n = 3(cKO-stroke) mice. k, O₂ saturation (pulse oximetry) in control or cKO mice (1–3dpi, tMCAO). n = 7(control-uninjured), n = 4(cKO-uninjured, cKO-stroke), or n = 8(control-stroke) mice. 1 cKO-stroke recorded 1dpi, 1 cKO-stroke recorded 2dpi, remaining mice recorded 3dpi. l-m, Feature plot (l) or violin plot (m) showing Cxcl12 expression among late fibroblast subsets including lymphocyte interactive, pial, and altered dural fibroblasts. n-p, Surfaced images showing T cells (CD3ε⁺; CD45⁺) near ER-TR7⁺ lesion in control or Cxcl12 cKO mice (n), with quantification of lesion size (o, normalized per experiment) and ER-TR7 coverage (p), 21dpi. n = 7 mice/group (2 slices/mouse, o). q, Volume of surfaced CD80⁺ cells in control or Cxcl12 cKO mice, 21dpi. n = 7 mice/group. r, MHCII⁺ cells within 500 µm of lesion, 21dpi. n = 7 mice/group. s-z, Quantification of total cortical CD8⁺ T cells (s), CD4⁺ T cells (t), or γδ T cells (u); IFNγ⁺ CD8⁺ T cells (v); IFNγ⁺ CD4⁺ conventional T (T_conv) cells (w) or IL17⁺ CD4⁺ T_conv cells (x); IL17⁺ γδ T cells (y); and IL10 + CD4⁺ T_conv cells (z), 21dpi. n = 26(control) or n = 18(Cxcl12 cKO) mice (s,t); n = 11(control) or n = 14(Cxcl12 cKO) mice (u-z). aa-af, Expression of IFNγ among CD8⁺ or CD4⁺ T cells and neutrophil counts in meninges (aa-ac) or blood (ad-af). n = 11(control) or n = 14(Cxcl12 cKO) mice (aa,ab,ad,ae); n = 20(control) or n = 16(Cxcl12 cKO) mice (ac,af). ag, Fibroblast UMAP (7,954 nuclei) showing subclusters annotated according to fibroblast identity mapping (e.g., Extended Data Fig. 9f). ah, Fibroblast cluster abundance in control and Cxcl12 cKO mice. ai-ak, T cell UMAP plots showing type 1 and 17 clusters (ai) or microanatomy (aj), and dotplot showing expression of relevant distinguishing marker genes (ak). al-am, Myeloid UMAP plots (3,792 nuclei), annotated according to myeloid identity mapping (e.g., Extended Data Fig. 9u) with subclustered SAM (al), or annotated by microanatomy (am). an, Dotplot showing expression of inflammatory/M1-like genes across SAM subsets, with higher expression in SAM_2. ao, Myeloid cluster abundance, including SAM subclusters, in control and Cxcl12 cKO mice. ap, Gene set enrichment among control (vs. Cxcl12 cKO) neurons. Two-way Student’s T-test (b,c,o-af); two-way ANOVA, Sidak’s post-test (d [repeated measures]; e-g,i-k); over-representation test (one-sided Fisher’s exact test, FDR-adjusted, ap). 14μm slices; images represent two or more mice. Source data