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Gut inflammation promotes microbiota-specific CD4 T cell-mediated neuroinflammation

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Abstract

The microbiota has been recognized as a critical contributor to various diseases1, with multiple reports of changes in the composition of the gut microbiome in contexts such as inflammatory bowel disease2,3 and neurodegenerative diseases4. These microbial shifts can exert systemic effects by altering the release of specific metabolites into the bloodstream5,6, and the gastrointestinal microbiota has also been reported to exhibit immunomodulatory activity through the activation of innate and adaptive immunity7,8. However, it remains unclear how the microbiota contributes to inflammation in the central nervous system (CNS), where these microorganisms are typically absent. Here we report that T cells that recognize gut-colonizing segmented filamentous bacteria can induce inflammation in the mouse intestine and CNS in the absence of functional regulatory T cells. Gut commensal-specific CD4 T cells (Tcomm cells) that are dysregulated in the inflamed gut can become licensed to infiltrate into the CNS regardless of their antigen specificity and have the potential to be re-stimulated by host protein-derived antigens in the CNS via molecular mimicry, whereupon they produce high levels of GM-CSF, IFNγ and IL-17A, triggering neurological damage. These infiltrated Tcomm cells initiate CNS inflammation by activating microglia through their IL-23R-dependent encephalitogenic programme and their IL-23R-independent GM-CSF production. Together, our findings reveal potential mechanisms whereby perturbation of Tcomm cells can contribute to extraintestinal inflammation.

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Fig. 1: Transferred TCR7B8 CD4 T cells expand and exhibit pathogenic activity in the intestines and CNS in an SFB antigen-dependent manner.
Fig. 2: Gut inflammation promotes TCR specificity-independent T cell CNS infiltration and enables the specific activation of encephalitic TCR7B8 T cells.
Fig. 3: TCR7B8 but not TCR1A2 contributes to CNS inflammation via the recognition of the cross-reactive host proteins ERBB2, TRO, and ANAPC2.
Fig. 4: Gut inflammation accelerates gut commensal-specific CD4 T cell infiltration into the CNS.
Fig. 5: IL-23R-dependent dysregulation and IL-23R-independent GM-CSF production in TCR7B8 CD4 T cells synergistically contribute to CNS inflammation.

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Data availability

All sequencing data generated and assembled for this paper are available at Zenodo (https://doi.org/10.5281/zenodo.15276654 (ref. 60)). The scRNA-seq and TCR repertoire RNA-sequencing data provided in this Article have been deposited at the Gene Expression Omnibus (GEO) under accession GSE295689. Source data are provided with this paper.

Code availability

All code used for computational analysis, and detailed methods on how to generate figure panel are made available at Zenodo (https://doi.org/10.5281/zenodo.15276654 (ref. 60)).

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Acknowledgements

TCR7B8-mediated neurological phenotypes, which consist of hindlimb clasping were initially observed by M. Xu, Y. Yang and T. Sano in the laboratory of D. R. Littman. The authors thank D. Littman, D. Ucker and J. Rehman for valuable discussions throughout the study; the Flow Cytometry Core in the UIC Research Resources Center (RRC) for naive T cell sorting; the Histology core in the RRC for paraffin sections; the Fluorescence Imaging Core in the RRC for their assistance with confocal imaging; and The National Gnotobiotic Rodent Resource Center at the University of North Carolina, University of Michigan Germ-Free Mouse Facility, New York University gnotobiotic core facility, and UIC gnotobiotic Mouse Facility provided spare GF wild-type, Rag1-knockout, Rag2-knockout and Il10-knockout mice when needed. This work was supported by the UIC Department of Microbiology and Immunology Startup fund (T.S.), Schweppe Award in Translational Research (T.S.), UIC COVID Relief funding (T.S.), Michael Reese Pioneer in Research Award (T.S.), University of Illinois startup funds (M.I.), NIH KL2TR002002 (S.E.L.), NIH R01DK126753 (K.-W.K.), NIH DP2-AI145100 (N.C.), U01-AI160418 (N.C.) and the University of Chicago Center for Interdisciplinary Study of Inflammatory Intestinal Disorders (C-IID; P30 DK42086) (N.C.)

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Author notes

  1. Deceased: Akira Yoshii

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Illinois at Chicago College of Medicine, Chicago, IL, USA

    Zachary White, Ivan Cabrera, Andrea Ochoa-Raya, Isabel Kapustka, Qianxun Wang, Takashi Matsuo, Takahiro Kageyama, Benjamin J. Mitchell, Dante Rivera, Pradeep K. Dudeja & Teruyuki Sano

  2. Pritzker School of Molecular Engineering, University of Chicago, Chicago, USA

    Linghan Mei, Margarette Clevenger & Nicolas Chevrier

  3. Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL, USA

    Joseph R. Dominguez, Seung Hyeon Kim & Ki-Wook Kim

  4. Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Jinyan Zhou, Shehata Anwar & Makoto Inoue

  5. Department of Anatomy and Cell Biology, University of Illinois at Chicago College of Medicine, Chicago, IL, USA

    Kevin P. Koster, Shoko Sagoshi, Sarah E. Lutz & Akira Yoshii

  6. Department of Pathology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt

    Shehata Anwar

  7. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Charles Ng

  8. Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL, USA

    Dulari Jayawardena & Pradeep K. Dudeja

  9. Jesse Brown Veterans Affairs Medical Center, Chicago, IL, USA

    Pradeep K. Dudeja

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Contributions

T.S. conceived the project and designed and analysed the data. Z.W. performed most experiment with T.S., I.C., A.O.-R., I.K., K.P.K., Q.W., S.S., T.M., S.H.K., T.K., B.J.M. and D.R., and analysed most of imaging data. L.M performed experiments for TCR repertoire analysis. M.C. performed analysis of scRNA-seq. J.R.D. and K.-W.K. conducted meninges analysis. J.Z., S.A. and M.I. performed immunization studies. C.N., D.J. and P.K.D. performed experiments and analysis for tissue pathology. S.E.L. provided spinal cord samples from EAE-induced mice. A.Y. helped neurological scoring and neurobiological experiments. N.C. supervised all the transcriptomic analysis. M.I. and T.S. performed scRNA-seq experiments. T.S. supervised the research.

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Correspondence to Teruyuki Sano.

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Extended data figures and tables

Extended Data Fig. 1 Transferred TCR7B8 CD4 T cells expand and exhibit pathogenic activity in the intestines and CNS.

(A-C) 1 × 105 naïve TCR7B8 CD4 T cells were retro-orbitally transferred into SFB-colonized Rag2−/− hosts. (A) Body weight. The experiments were conducted twice with multiple littermate controls. Representative ileal and colonic histological images following H&E staining (B) and clasping phenotypes (C) (n = 5/group) at 8 weeks following the transfer. (D) Staining of myelin in the spinal cord (L1) of EAE-induced C57BL/6 WT (Top), TCR7B8 naïve CD4 T cell-transferred C57BL/6 Rag2−/− hosts (Middle), and non-transferred Rag2−/− hosts (Bottom). Stained myelin and cells (DAPI) were represented in yellow and magenta, respectively. (E) Representative images of spinal cord sections from EAE-induced WT mice (bottom), and Rag2−/− hosts 8 weeks post-transfer of TCR7B8 naïve T cells (top), stained for Iba-1 (magenta), CD4 (green), and DAPI (white). This experiment was repeated twice with similar staining results. Similar results were observed in 3 biological replicates (transferred Rag2−/− hosts). EAE scores were as indicated. (F) A representative gating strategy used to analyze the cytokine-producing T cells is shown. (G) Kinetics of TCR7B8 CD4 T cells in the indicated tissues following transfer to Rag2−/− hosts. Representative FACS plots of IFNγ/IL-17A double producing CD4 T cells in the indicated tissues and at the indicated times following transfer. Four biological replicates per time point. Data are given as means ± SEM, with dots corresponding to individual mice. (H) Represented images of H & E staining of spleen, lung, liver, and kidney collected from 1 × 105 SFB TCR7B8 naïve CD4 T cell transferred Rag2−/− mice (n = 6) or non-transferred Rag2−/− (n = 5) 7 weeks following the transfer. The black bar indicates 100 μm. (I) Representative images of TdTomato+ TCR7B8 CD4 T cells in the meninges of 1 × 105 SFB TCR7B8 naïve CD4 T cell transferred Rag2−/− mice 7 weeks following the transfer (n = 4). White: Blood Vessels, Magenta: CD4-TdTomato, and Green: LYVE1 (Lymphatic vessels). (J-K) Cell numbers of total CD4 T cells (J) and IFNγ/IL-17A double producing CD4 T cells in the in the indicated tissues and at the time points labeled following the TCR7B8 naïve CD4 T cell transfer to Rag2−/− hosts (n = 4). (L) Schematic representation of efforts to avoid dual-TCR expression. (M) Neurological scores in SFB-colonized Rag2−/− hosts that received naïve CD4 T cells collected from TCR7B8 Rag2+ /− or TCR7B8 Rag2−/− donors. (A) Two-tailed-Student’s t-test, (M) Two-way ANOVA.

Source Data

Extended Data Fig. 2 SFB TCR7B8 can be re-stimulated in the CNS.

(A-B) Proliferation of TCR7B8 CD4 T cells in TCR7B8 transferred Rag2−/− mice. (A) Flow plots corresponding to Ki67-expressing TCRβ+ CD4+ CD8- T cells in the indicated tissues collected from 30- to 35-week-old TCR7B8 transferred Rag2−/− mice. (B) Frequencies of Ki67-positive cells among TCRβ+ CD4+ CD8- T cells in the indicated tissues. Two experiments using littermates were performed and combined. (C-D) Elimination of SFB following the onset CNS inflammation. (C) Schematic overview of the approach to remove SFB after recipients had developed neurological phenotypes as a treatment intervention. After all transferred SFB-colonized Rag2−/− hosts were confirmed to have developed neurological phenotypes at 7 weeks following the transfer, 9 of the recipients were treated with ampicillin in the drinking water to eliminate SFB from their intestines. As controls, 7 of the recipient mice were instead given regular water. Non-transferred littermate controls were also monitored with or without ampicillin treatment. (D) Neurological score. The numbers of animals used are indicated. Results are presented as the mean ± SEM, with dots representing individual mice. (B) Ordinary one-way ANOVA, (D) Two-way ANOVA.

Source Data

Extended Data Fig. 3 Analysis of regulatory cells in the TCRTg naïve CD4 T cell transferred Rag2−/− hosts.

1 × 105 naïve TCR7B8 CD4 T cells collected from spleen and LNs of TCR7B8 mice, TCR1A2 Rag2−/− mice, or TCROTII TCRα−/− mice, were retro-orbitally transferred into SFB-colonized Rag2−/− hosts. TCROTII transferred Rag2−/− hosts were maintained with 4 mg/ml Ovalbumin containing water. Frequency of natural Tregs (Gated as DAPI-, TCRβ+, CD8α-, CD4+, Foxp3+, and RORγt-) (A) and inducible Treg (Gated as DAPI-, TCRβ+, CD8α-, CD4+, Foxp3+, and RORγt+) (B). Numbers of Foxp3+ IL-10 producing CD4 T cells (C), Foxp3- IL-10 producing CD4 T cells (D), and IFNγ and IL-10 double producing CD4 T cells (Tr1 cells) (E). Two independent experiments were combined. Results are presented as the mean ± SEM, with dots representing individual mice. Two-way ANOVA. (C, D, E) during cell preparation, one brain sample of 1A2 transferred and one spleen samples of OTII transferred mice were lost.

Source Data

Extended Data Fig. 4 In vitro stimulation assay using SFB-specific TCRTg naïve CD4 T cells, BMDCs, and synthesized peptides.

(A) Schematic representation of an in vitro naïve TCR7B8 T cell stimulation assay using BMDCs and synthetic peptides. Synthesized peptides were mixed with 2-4 × 104 BMDCs induced from the BM of CD45.1/CD45.1 C57BL/6 mice together with LPS in 96-well round-bottom plates. After 24 h, equal numbers of magnetically sorted naïve CD4 T cells from TCR7B8 or TCR1A2 animals on the CD45.2/CD45.2 Rag2−/− background were added together with recombinant IL-2. After an additional 24 h, all cells were collected and stained with anti-CD25, anti-CD69, anti-CD45.2, anti-TCRβ, and DAPI. For 72 h culture experiments, naïve CD4 T cells were labeled with CFSE just prior to co-culture with BMDCs. After 72 h, cells were stained with anti-CD25, anti-CD69, anti-CD45.2, anti-TCRβ, and Aqua. After fixation, cells were additionally stained with anti-Nur77 and anti-Ki67 and were then analyzed via FACS. (B) The approach used to gate on CD25/CD69 double-positive proliferating TCRTg CD4 T cells at 24 h following co-culture. (C) Other proliferation and activation makers expressed on CD4 T cells were analyzed at 72 h following co-culture. CFSE-high CD25-negative non-proliferating T cells are highlighted with a black rectangle in non-peptide treated wells and with a green rectangle in the 2.5 nM peptide well. Effectively proliferating cells were defined as CD25+ cells exhibiting CFSE dilution, and these cells were gated with a red square and further assessed for CFSE dilution, CD25, Ki67, and Nur77 expression (bottom).

Extended Data Fig. 5 Screening for potential cross-reactive candidate epitopes capable of stimulating naïve TCR7B8 CD4 T cells in vitro.

(A) A BLAST search was used to identify a list of candidate cross-reactive peptides. Core epitopes for TCR7B8 and matched amino acids in candidate peptides are highlighted in red. Amino acids that did not match core epitopes for TCR7B8 are shown in blue. (B) The percentage of TCR7B8 CD4 T cell activation in response to stimulation with a 100 μM concentration of candidate peptides. T cell activation was determined based upon CD25 and CD69 expression at 24 h following co-culture. (C) The dose-dependency of candidate peptide-induced T cell activation at 24 h following co-culture. In total, 11 candidates selected based on the results in (B) were further tested for their ability to activate TCR7B8 CD4 T cells at concentrations of 10 μM and 0.4 μM. The results shown in (B) were also shown on the same graph in (C). Screening analyses were performed one time. P2X6 (ID3), ANAPC2 (ID4), ErbB2 (ID5), IFT46 (ID6), and TRO (ID11) were further generated as purified peptides and tested in Fig. 3.

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Extended Data Fig. 6 Immune checkpoint blockade promotes TCR7B8-mediated CNS inflammation.

(A-G) The transfer of naïve TCR7B8 CD4 T cells into Rag2−/− hosts with the i.p. injection of 300 μg of Anti-CTLA4 and Anti-PD-1 antibodies (ICB) or control antibodies. (A) Neurological Scores, (B) incidence of neurological disorders, and (C) body weight. (D) Representative FACS plots and (E) numbers of CD4 T cells (left), IFNγ/IL-17A DP CD4 T cells (middle), TNFα-producing CD4 T cells (right) in the brain. (F) Representative FACS plots and (G) numbers of CD4 T cells (left), IFNγ/IL-17A DP CD4 T cells (middle), TNFα-producing CD4 T cells (right) in the Spinal cord. The experiments were conducted twice, and the data were combined except for the body weight data shown in C. (H-I) The transfer of naïve TCR7B8 CD4 T cells into WT hosts with or without the i.p. injection of 300 μg of Anti-CTLA4 and Anti-PD-1 antibodies. (H) Neurological Scores and (I) Faecal LCN2 detection in freshly collected faecal pellets at 7 weeks after transfer. The experiments were conducted two to three times, and the data were combined. (J-K) The transfer of Cd45.2/Cd45.2 TCR7B8 CD4 naïve T cells into CD45.1/Cd45.1 WT and Rag2−/− hosts with or without the i.p. injection of 300 μg of Anti-CTLA4 and Anti-PD-1 antibodies or control antibody. Seven weeks following the transfer, donor and recipient CD4 T cells were analyzed. (J) Representative flow plots of IFNγ and IL-17A producing total CD4 + T cells (including donor- and host-derived CD4 T cells) (left). Frequencies of IFNγ-producing and IFNγ/IL-17A double-producing CD4 T cells in the indicated tissues with or without CPI treatment 7 weeks following the transfer (right). (K) Representative flow plots of TCR7B8 CD4 T cells as CD45.2 + TCRβ + CD4 + T cells in the spinal cord (left). Numbers of CD45.2 + TCR7B8 CD4 T cells were compared (right). Results are presented as the mean ± SEM, with dots representing individual mice. (A, C, H) Two-way ANOVA, (B) Mantel-Cox test (E, G, J, K) Student’s t-test, (I) One-way ANOVA.

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Extended Data Fig. 7 Contributions of Foxp3+ regulatory T cells in TCR7B8-mediated CNS inflammation.

(A) Schematic representation of Treg depletion in SFB TCRTg Foxp3 DTR mice. Adult (8–14 weeks old) TCRTg+ or non TCRTg with or without Foxp3 DTR mice were i.p. injected with 10 μg DT every other day, 4-times. (B) Neurological scores of indicated mouse groups. (C) Efficacy of Treg depletion. Frequencies of Foxp3+ Tregs among CD4 T cells in the colon of indicated mice at Day 8 (left: 7B8 Foxp3 +/+ DT (n = 3), 7B8 Foxp3DTR +DT (n = 4), No TCR Tg Foxp3DTR + DT (n = 3)) and Day 14 (right: 7B8 Foxp3+/+ + DT (n = 4), 7B8 Foxp3DTR +DT (n = 10), No TCR Tg Foxp3DTR + DT (n = 3)), 1A2 Foxp3DTR +DT (n = 5)) following the first DT injection. (D) Frequencies of CD4 T cells (left) and IFNγ/IL-17A double producing CD4 T cells (right) in the brains of indicated mouse groups. (E) Schematic representation of TCR7B8 or TCR1A2 naïve CD4 T cells transfer into the Treg depleted Foxp3 DTR mice carrying polyclonal CD4 T cells. The sorted naïve CD4 T cells (200,000 cells) collected from TCR7B8 or TCR1A2 mice were R.O. transferred Treg depleted Foxp3DTR mice followed by DT injection. (F) Neurological scores of indicated mouse groups. Results are presented as the mean ± SEM, with dots representing individual mice. (B) Two-way ANOVA, (C, D) One-way ANOVA, (F) Two-Tailed-Student’s t-test.

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Extended Data Fig. 8 Gut inflammation accelerates TCR7B8-mediated CNS inflammation.

(A-G) Integrin β7-deficient TCR7B8 CD4 T cells fail to accumulate in the gut, resulting in less inflammation in both the gut and CNS. Comparison between Itgb7 WT and Itgb7-KO TCR7B8 CD4 T cells detected in mix-transferred Rag2−/− hosts. Total CD4 T cell numbers detected in the brain, spinal cord, ileum, colon, and spleen. (A) Schematic representation of Itgb7-sufficient and -deficient naïve TCR7B8 CD4 T cell transfer to Rag2−/− hosts. (B) Represented FACS plots of the detection of two different donor CD4 T cells (Itgb7 +/+ or + /− as CD45.1/CD45.2, and Itgb7−/− as CD45.2/CD45.2) (Top). Frequency of CD4 T cells in the indicated tissues (Bottom) (n = 7). (C) Total CD4 T cell numbers detected in the spinal cord. (D) Representative FACS plots of IFNγ-, IL-17A-, and TNFα-producing Itgb7 WT and KO TCR7B8 CD4 T cells detected in the spinal cord. (E-G) Cell numbers of Itgb7 WT and KO TCR7B8 CD4 T cells producing IFNγ/IL-17A in the spinal cord (E), TNFα in the spinal cord (F), and IL-17A in the Spinal cord (G). Independent experiments were conducted twice using multiple littermate controls (Itgb7 WT TCR7B8 transferred (n = 13) and Itgb7 KO TCR7B8 transferred (n = 12) (C, E, F, G). (H) Transferring dysregulated TCR7B8 CD4 T cells collected from inflamed colon of TCR7B8 Rag2−/− mice into the GF WT hosts. (H) Schematic representation of dysregulated TCR7B8 T cell transfer. CD4 T cells were sorted from the inflamed colons of TCR7B8 Rag2−/− mice. The sorted 1 × 106 CD4 T cells were incubated with 300 μg/ml of Anti-CTLA4 and Anti-PD-1 antibodies or control antibodies for 30 min at room temperature and then R.O. transferred into the GF WT recipients and maintained for 19 days (left). ICB were weekly i.p. injected (300 μg of Anti-CTLA4 and Anti-PD-1 antibodies or control antibodies). Their neurological scores were monitored every day following the transfer (right). (I-J) In vitro polarized TCRTg Th17 cell transfer. Sorted naïve CD4 T cells from TCRTg mice were pathogenically or homeostatically polarized in vitro with IL-6/TGFβ/IL-23/IL1β for pathogenic and IL-6/TGFβ/Anti-IFNγ ab/Anti-IL-4 ab. (K) Neurological score (not EAE score) of immunized SPF WT mice with MOG peptide or SFB 7B8 peptide. Two independent experiments were combined. Results are presented as the mean ± SEM, with dots representing individual mice. (B, C, E, F, G) Two-Tailed-Student’s t-test, (H, I, J, K) Two-way ANOVA.

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Extended Data Fig. 9 IL-23R-dependent dysregulation and IL-23R-independent GM-CSF production in TCR7B8 CD4 T cells synergistically contribute to CNS inflammation.

(A) Faecal LCN-2 detection by ELISA using faecal pellets collected from indicated mice 7 weeks following the transfer. (B-I) Development of spontaneous neurological disorder in Il-23r-sufficient and -deficient TCR7B8 Rag2−/− mice. (B) Neurological scores and (C) Incidence of neurological disorder. (D) Total CD4 T cell numbers detected in the brain and spinal cord. Il-23r-sufficient No TCRTg Rag2+ /− mice (n = 9), Il-23r-sufficient TCR7B8 Rag2−/− mice (n = 16), Il-23r-deficient TCR7B8 Rag2−/− mice (n = 5). (E) Representative FACS plots of IL-17A- and IFNγ-producing cells among CD4 T cells, (F) frequencies of IL-17A/IFNγ DP CD4 T cells (Il-23r-sufficient No TCRTg Rag2+ /− mice (Spleen: n = 6, the other tissues: n = 9), Il-23r-sufficient TCR7B8 Rag2−/− mice (Spleen: n = 11, the other tissues: n = 17), Il-23r-deficient TCR7B8 Rag2−/− mice (n = 6)), (G) representative FACS plots of TNFα and IFNγ among CD4 T cells, (H) frequencies of TNFα/IFNγ DP CD4 T cells (Il-23r-sufficient No TCRTg Rag2+ /− mice (Spleen: n = 6, the other tissues: n = 9), Il-23r-sufficient TCR7B8 Rag2−/− mice (Spleen: n = 11, the other tissues: n = 17), Il-23r-deficient TCR7B8 Rag2−/− mice (n = 6)), and (I) frequencies of GM-CSF producing CD4 T cells in the indicated tissues (Il-23r-sufficient No TCRTg Rag2+ /− mice (n = 6), Il-23r-sufficient TCR7B8 Rag2−/− mice (n = 11), Il-23r-deficient TCR7B8 Rag2−/− mice (n = 6)). Multiple independent experiments were conducted using littermate controls. Results are presented as the mean ± SEM, with dots representing individual mice. (J) UMAP of all cell populations collected from thalamus and brainstem of indicated mice, colored by cluster or mouse treatment. (K) Heat map showing up- and down-regulated genes in five different microglia and macrophage clusters. (A) One-way ANOVA, (B) Two-Tailed-Student’s t-test, (C) Mantel-Cox test, (D, F, H, I) Two-way ANOVA. *P < 0.05, N.S. not significant (p > 0.05). Statistics in B: 9 weeks (p = 0.565947), 10 weeks (p = 0.230589), 11 weeks (p = 0.053358), 12 weeks (p = 0.06618), 13 weeks (p = 0.014725), 14 weeks (p = 0.001189), 15 weeks (p = 0.001511), 16 weeks (p = 0.029699), 17 weeks (p = 0.041539), 18 weeks (p = 0.047768), 19 weeks (p = 0.015624), 20 weeks (p = 0.111945), 21 weeks (p = 0.232965), 22 weeks (p = 0.485166), 23 weeks (p = 0.596934), 24 weeks (p = 0.665319).

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Extended Data Fig. 10 Models for the mechanisms whereby TCR7B8 T cells but not TCR1A2 T cells contribute to CNS inflammation.

Commensal-specific T cells can be generated in the thymus and avoid negative selection. While TCR7B8 T cells can cross-react with host protein-derived peptides, they likely escape negative selection owing to their low affinity for tissue-restricted antigens (TRAs) presented by mTECs. These commensal-specific naïve T cells can then travel to the mLNs wherein they can differentiate into a range of helper T cell lineages upon commensal colonization. These differentiated commensal-specific T cells subsequently migrate to areas where cognate antigens are abundant, most often to the intestines although others can migrate to systemic sites such as the spleen. In the absence of Treg cells, SFB-specific TCR7B8 and TCR1A2 CD4 T cells are further activated in the small and large intestines and produce a range of effector cytokines, whereas Tregs would normally suppress these activities. TCR7B8 CD4 T cells can be found in CNS compartments including the brain and spinal cord in the absence of Tregs. These CNS-infiltrating TCR7B8 CD4 T cells can be re-stimulated by host derived antigens via molecular mimicry and produce higher levels of inflammatory cytokines such as TNFα, GM-CSF, IFNγ, and IL-17A in IL-23R-dependent and -independent fashions relative to cells found in the intestines, thus contributing to CNS inflammation.

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Supplementary Video 1

Neurological phenotype in non transfer Rag2−/− host (score 0). Represented movie of non-transferred Rag2−/− host used in Fig. 1b-c. As negative control, no cells were transferred.

Supplementary Video 2

Neurological phenotype in TCR7B8 naive CD4 T cells transferred Rag2−/− host (score 4). Represented movie of TCR7B8 naive CD4 T cell transferred Rag2−/− host used in Fig. 1b,c. The video was taken at 7 weeks following the transfer.

Supplementary Video 3

Neurological phenotype in TCR7B8 naive CD4 T cells transferred Rag2−/− host (score 6). Represented movie of TCR7B8 naive CD4 T cell transferred Rag2−/− host used in Fig. 1b,c. The video was taken at 7 weeks following the transfer.

Supplementary Video 4

Neurological phenotype inCsf2-sufficient TCR7B8 TCR7B8 naive CD4 T cells transferred Rag2−/− host (score 9). Represented movie of Csf2-sufficient TCR7B8 naive CD4 T cell transferred Rag2−/− host used in Fig. 5q. The video was taken at 10 weeks following the transfer at the same time as the videos in Supplementary Video 5.

Supplementary Video 5

Neurological phenotype in Csf2-deficient TCR7B8 TCR7B8 naive CD4 T cells transferred Rag2−/− host (score 3). Represented movie of Csf2-deficient TCR7B8 naive CD4 T cell transferred Rag2−/− host used in Fig. 5q. The video was taken at 10 weeks following the transfer at the same time as the videos in Supplementary Video S4.

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White, Z., Cabrera, I., Mei, L. et al. Gut inflammation promotes microbiota-specific CD4 T cell-mediated neuroinflammation. Nature (2025). https://doi.org/10.1038/s41586-025-09120-w

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