Abstract

The grey matter is a central target of pathological processes in neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. The grey matter is often also affected in multiple sclerosis, an autoimmune disease of the central nervous system. The mechanisms that underlie grey matter inflammation and degeneration in multiple sclerosis are not well understood. Here we show that, in Lewis rats, T cells directed against the neuronal protein β-synuclein specifically invade the grey matter and that this is accompanied by the presentation of multifaceted clinical disease. The expression pattern of β-synuclein induces the local activation of these T cells and, therefore, determined inflammatory priming of the tissue and targeted recruitment of immune cells. The resulting inflammation led to significant changes in the grey matter, which ranged from gliosis and neuronal destruction to brain atrophy. In humans, β-synuclein-specific T cells were enriched in patients with chronic-progressive multiple sclerosis. These findings reveal a previously unrecognized role of β-synuclein in provoking T-cell-mediated pathology of the central nervous system.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request.

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Change history

  • 13 March 2019

    In this Article, owing to an error during the production process, the y-axis label of Fig. 2c should read “Number of Tβ-syn cells” rather than “Number of T1β-syn cells” and the left and right panels of Fig. 4 should be labelled ‘a’ and ‘b’, respectively. These errors have been corrected online.

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Acknowledgements

We thank A. Stas, S. Mole, A. Mönnich, S. Hamann, M. Weig and H. Nguyen for technical assistance, C. Ludwig for text editing, G. Salinas-Riester for her support in performing the transcriptome analyses, K. Raithatha for her help in analysing the transcriptome data, H. Abken, S. Brioschi, L. Flügel, T. Issekutz, P. and W. von der Meide, M. Simons, T. Michaelis and M. Korte for providing reagents and/or technical, scientific or clinical advice and M. Gößwein for help with the artwork. This work was supported by the German Research Foundation (RK-Grant FL 377/3-1, FL 377/2-2; SFB 1328/1 A01, OD 87/1-1), the Federal Ministry for Education and Research (Competence Network Multiple Sclerosis, KKNMS), ERANET consortium MELTRA-BBB and the Ministry of Science and Culture of Lower Saxony (Niedersachsen-Research Network on Neuroinfectiology, N-RENNT).

Author information

Author notes

  1. These authors contributed equally: Dmitri Lodygin, Moritz Hermann

  2. These authors jointly supervised this work: Francesca Odoardi, Alexander Flügel

Affiliations

  1. Institute for Neuroimmunology and Multiple Sclerosis Research, University Medical Center Göttingen, Göttingen, Germany

    • Dmitri Lodygin
    • , Moritz Hermann
    • , Nils Schweingruber
    • , Corinna Schlosser
    • , Arianna Merlini
    • , Henrike Körner
    • , Hsin-Fang Chang
    • , Henrike J. Fischer
    • , Michael Haberl
    • , Francesca Odoardi
    •  & Alexander Flügel
  2. Institute of Functional and Clinical Anatomy, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

    • Cassandra Flügel-Koch
  3. Biomedizinische NMR, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany

    • Takashi Watanabe
    •  & Jens Frahm
  4. Institute for Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany

    • Holger M. Reichardt
  5. Zoological Institute, Division of Cellular Neurobiology, Technical University Braunschweig, Braunschweig, Germany

    • Marta Zagrebelsky
  6. Paracelsus-Elena-Klinik Kassel, Kassel, Germany

    • Brit Mollenhauer
  7. Institute for Neurology, University Medical Center Göttingen, Göttingen, Germany

    • Brit Mollenhauer
    • , Sebastian Kügler
    •  & Dirk Fitzner
  8. Institute for Neuropathology, University Medical Center Göttingen, Göttingen, Germany

    • Christine Stadelmann
  9. Center for Biostructural Imaging of Neurodegeneration, Göttingen, Germany

    • Francesca Odoardi

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Contributions

D.L. designed and cloned the TCR genes for the TCR transgenic rat and analysed the transgenic model. M. Hermann performed most two-photon laser-scanning microscopy experiments and, together with T.W., performed MRI. N.S. collected and analysed the human samples. C.F.-K. with the support of H.K. performed most of the morphological analyses. C. Schlosser analysed the initial grey matter disease model. A.M. performed transcriptome and functional analyses. H.-F.C. established the CAR system. H.J.F. performed oocyte injections. H.M.R. contributed his expertise in rat transgenesis, M.Z. helped to analyse neurodegeneration and B.M. recruited patients with PD. S.K. provided the AAVs. D.F. collected samples from patients with MS. J.F. helped with providing and optimizing MRI technology, C. Stadelmann provided advice on CNS pathology and M. Haberl helped to analyse the NGS data. F.O. performed most of the ex vivo T cell and APC analyses. A.F. together with F.O. designed the study, coordinated the experimental work and wrote the manuscript with inputs from co-authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Francesca Odoardi or Alexander Flügel.

Extended data figures and tables

  1. Extended Data Fig. 1 Characterization of wild-type and TCR-transgenic Tβ-syn cells in vitro and in vivo.

    a, Identification of the immunogenic β-synuclein peptides in Lewis rats. Screening of a β-synuclein peptide library covering the full-length protein for identification of immunogenic peptides. OVA, MBP, concanavalin A (ConA) and a mix of all peptides were used as control. The proliferation of αβTCR+CD4+ T cells isolated from β-synuclein-immunized wild-type rats was measured by flow cytometry. Data are mean + s.e.m. from two independent experiments. b, Antigen response of established effector T cell lines. Wild-type Tβ-syn or TMBP cell lines were cultured in the absence of antigen (No) or stimulated with ConA or the indicated antigens for 72 h. Proliferation was determined by 3H-thymidine incorporation. Data are mean + s.e.m. Representative data of at least four independent experiments. c, Phenotype of Tβ-syn and TMBP cell lines. Expression of αβTCR, CD4, CD8 and of surface activation markers CD134 and CD25 in cultured wild-type Tβ-syn and TMBP cells. Flow cytometry was performed two days (blast T cells) and seven days (resting T cells) after antigen stimulation. IgG, isotype control. d, Histological quantification of Tβ-syn and TMBP cells in cortical grey matter and corpus callosum (white matter) at the peak of T cell infiltration. Data are mean + s.e.m., n = 5 from 2 independent experiments. Unpaired two-tailed t-test. ***P < 0.001. e, Wild-type Tβ-syn cells induce clinical disease. Representative disease course upon intravenous transfer of wild-type Tβ-syn or TMBP cells. Data are mean + s.d., n = 6–8 per group. Representative data of at least six independent experiments. f, Photographs illustrating frontlimb paralysis induced by Tβ-syn cells and classical EAE hindlimb paralysis induced by TMBP cells. g, Expression of Trbv genes in wild-type Tβ-syn cells isolated ex vivo at the onset of Tβ-syn cell brain infiltration. Relative frequency was calculated in Tβ-syn cells from blood and CNS and expressed as ratio of reads mapping to a particular Trbv genomic segment to the number of reads mapping to the constant region of the TCR β chain encoded by Trbc1 and Trbc2 (RNA-seq analysis, n = 3 per group). Data are mean + s.d. h, Production of IFNγ by lymph node cells isolated from naive wild-type and TCR-transgenic (Tg) donors in response to β-syn93–111 measured by ELISA. Data are mean ± s.d. i, Immunization of TCR-transgenic rats with β-syn93–111 results in a monophasic clinical disease. Bars and lines indicate clinical score and relative weight change, respectively (mean ± s.d.). Representative data from two independent experiments (n = 10). j, Expression of surface markers on wild-type Tβ-syn (blue) and transgenic Tβ-syn (green) cell lines. k, TCR repertoire in transgenic Tβ-syn cell lines is dominated by transgenic TCR Vβ8.3 chain expression. j, k, Flow cytometry data. l, Expression of Trbv genes in transgenic Tβ-syn cell lines determined by RNA-seq analysis. Analysis and normalization of data as in g. n = 3 per group. m, n, Proliferative response measured by 3H-thymidine incorporation (m) and IFNγ secretion measured by ELISA (n) in wild-type Tβ-syn (blue) and transgenic Tβ-syn (green) cell lines stimulated with the indicated amount of cognate (β-syn) or non-cognate (MBP, OVA) antigens. Data are mean ± s.d. o, Intracellular staining for IFNγ and IL-17 in TMBP and Tβ-syn cell lines stimulated with anti-CD3 monoclonal antibodies. Representative data from at least three independently established T cell lines. p, Quantification of transgenic Tβ-syn cells infiltrating the cortical grey matter or the corpus callosum (white matter) measured by flow cytometry at the peak of T cell infiltration (n = 5). q, Clinical disease induced by transgenic Tβ-syn effector cells (n = 15). p, q, Data are mean + s.e.m.

  2. Extended Data Fig. 2 Migration properties and differentiation phenotype of Tβ-syn cells.

    a, Tβ-syn and TMBP cells follow similar migratory routes before invading the CNS. Numbers of Tβ-syn and TMBP cells in the indicated organs quantified every 24 h p.t. by flow cytometry. Data are mean + s.e.m. Representative data of three independent experiments including at least three rats per group per time point. b, Tβ-syn and TMBP cells switch from an activated state to a migratory mode. Gene expression changes between migratory T cells (isolated from blood at the onset of CNS infiltration, that is, at day 3 p.t.) and in vitro T cells (24 h after antigen challenge) as measured by RNA-seq analysis. The ratio between the expression in migratory and in vitro activated T cells for the indicated genes is displayed. Data are mean + s.e.m., n = 9–15 per group from 3 independent experiments. c, d, Tβ-syn and TMBP cells display very similar intravascular migration behaviour. Intravital TPLSM recordings were performed upon first arrival of Tβ-syn or TMBP cells in the leptomeningeal brain vessels. c, Representative migratory path of Tβ-syn or TMBP cells over a 30-min recording time. Rolling cells appear as multiple dots. Green, T cell tracks projected over time; red, blood vessels and leptomeningeal phagocytes. Representative data of at least four independent experiments (obtained from individual rats). d, Crawling velocity and track duration of Tβ-syn and TMBP cells. Data are mean ±  s.e.m. The number of analysed T cells is indicated. e, f, Tβ-syn and TMBP cells are similar in integrin and cytokine expression and chemokine responsiveness. e, Relative expression (normalized to Actb) of the indicated integrins, chemokine receptors and cytokines as measured by quantitative PCR in Tβ-syn and TMBP cells isolated from the blood at the onset of T cell CNS infiltration. Data are mean + s.d., n = 3 rats per group. f, Chemotactic response to the indicated chemokines of ex vivo-isolated Tβ-syn and TMBP cells. Data are mean + s.d., n = 2 per group. gi, VLA-4 crucially determines Tβ-syn cell invasion into the brain. g, h, Tβ-syn cell motility was recorded by intravital TPLSM upon first arrival of Tβ-syn cells in the leptomeningeal brain vessels before and immediately after intravenous injection of blocking anti-VLA-4 monoclonal antibody. g, Representative 30-min time-lapse recordings of Tβ-syn cell migratory paths. Green, 30-min Tβ-syn cell track projections; red, Texas red dextran-labelled vessels. Representative data of at least four independent experiments (obtained from individual rats). h, Percentage of crawling or rolling Tβ-syn cells before and after anti-VLA-4 monoclonal antibody treatment (arrow) relative to time point 0. Data are mean ± s.e.m. of n = 3. i, Clinical disease after applications of anti-VLA-4 or isotype monoclonal antibodies (control). Arrows indicate the time point of treatment. Data are mean + s.e.m. of n = 4 per group. Representative data of three independent experiments. jl, LFA-1 integrin does not contribute to Tβ-syn cell invasion in the brain. Intravital TPLSM recordings were performed as in g before and after anti-LFA-1 monoclonal antibody administration. j, Representative 30-min time-lapse recordings of Tβ-syn cell migratory paths. Green, Tβ-syn cell tracks projected over time; red, Texas red dextran-labelled vessels. Representative data of at least four independent experiments (obtained from individual rats). k, Percentage of crawling or rolling Tβ-syn cells before and after monoclonal antibody treatment quantified as in h. Data are mean ± s.e.m. of n = 3. l, Disease course in Tβ-syn cell recipient rats treated either with anti-LFA-1 or isotype monoclonal antibodies (control) at the indicated time points (arrows). Data are mean + s.e.m. of n = 4 per group. Representative data of three independent experiments. m, Saturation of monoclonal antibody binding (in gi and jl experiments) was controlled by staining of blood-derived Tβ-syn cells with anti-VLA-4 or anti-LFA-1 primary monoclonal antibodies followed by secondary detection antibody (red) or with secondary antibody only (black). n, o, IL-17 or IFNγ blockade partially interferes with Tβ-syn cell entry into the brain. n, Disease course in Tβ-syn-cell-transferred rats intrathecally treated at the indicate time points (arrows) with PBS (control), anti-IL-17 or anti-IFNγ monoclonal antibodies. Data are mean + s.e.m. of n = 4 per group. Representative data of three independent experiments. o, Quantification of macrophages (MΦ, CD11b+CD45high), CD4+ and CD8+ T cells infiltrating the brain leptomeninges and the brain parenchyma at the peak of clinical disease. Data are mean + s.e.m. of four rats per group. Representative data of two independent experiments. n, o, Unpaired two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

  3. Extended Data Fig. 3 Tβ-syn cell infiltration into CNS tissue correlates with β-synuclein expression.

    a, Coronal section of naive rat brain. Left, immunostaining for β-synuclein (red) and tyrosine hydroxylase (TH, green). Middle, colour-coded intensity of β-synuclein expression. Right, inflamed brain at the peak of Tβ-syn cell (red) infiltration. Green, TH. Co, cortex; HC, hippocampus; cc, corpus callosum; SN, substantia nigra; CS, colliculus superior; dhc, dorsal hippocampal commissure; cp, cerebral peduncle; dcw, deep cerebral white matter; Ac, aqueduct; MM, mammillary nucleus; pc, posterior commissure. b, Enlarged view of selected regions (i–iii) indicated by frames in a. c, Inflammatory infiltration of the eye. Left, cross-section at the peak of Tβ-syn cell (red) infiltration. Green, CD68+ cells (activated microglia and macrophages). Enlarged view of a selected area (i) is shown in the middle panel. Right, β-synuclein expression within the eye (naive rat). Asterisks indicate the autofluorescent inner and outer segments of the photoreceptor cells. R, retina; CH, choroid; GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; S, sclera.

  4. Extended Data Fig. 4 Tβ-syn cells are reactivated in the brain.

    ae, Tβ-syn cells isolated from their target organ display an activated phenotype. a, Expression of selected sets of genes (grouped in functional classes) determined by RNA-seq analysis of Tβ-syn cells isolated from blood, brain meninges and brain parenchyma at the onset of Tβ-syn cell CNS infiltration. RPKM, reads per kilobase per million mapped reads. Data are mean + s.e.m., n = 9–15 per group from 3 independent experiments. Genes for which the expression in Tβ-syn cells from brain was significantly different from the expression in Tβ-syn cells from blood are highlighted by a red overlay colour. Unpaired two-tailed t-test. b, IFNγ and IL-17 expression (intracellular staining) in Tβ-syn cells isolated from blood and brain at the peak of T cell infiltration. Representative data of three independent experiments. c, Surface membrane activation marker (CD25 and CD134) expression in Tβ-syn cells isolated from the indicated compartments at the onset of Tβ-syn cell CNS infiltration determined by flow cytometry. d, Expression of Ifng and Il17a mRNA in Tβ-syn cells isolated from brain meninges, brain parenchyma, cortical grey matter and corpus callosum (white matter) measured by quantitative PCR (normalized to Actb). Data are mean + s.e.m. Representative data from two independent experiments. Unpaired two-tailed t-test. *P < 0.05, **P < 0.01. e, Top 50 differentially expressed genes in Tβ-syn cells isolated from blood, brain meninges and parenchyma at day 3 p.t. determined by RNA-seq. Heat map shows z-transformed relative expression values. Each column represents a gene, each row represents a biological replicate. Genes known to be regulated by TCR-driven activation are indicated. f, Tβ-syn cells, in contrast to TMBP cells, are strongly activated in the brain. Left, Ifng and Il17a relative expression (normalized to Actb) in Tβ-syn or TMBP cells isolated from blood or from the indicated CNS compartments at the indicated time points p.t. determined by quantitative PCR. Right, expression of the cytokines in the corresponding total CNS tissues. Data are mean + s.d. of three independent experiments. g, Tβ-syn cells do not proliferate in the brain. Histogram plots, Ki-67 expression of in vitro-cultured Tβ-syn cells (two and six days after antigenic stimulation) or of Tβ-syn cells isolated ex vivo from blood and brain at the peak of T cell infiltration. Grey, isotype control. Dot plots, corresponding quantification of BrdU incorporation. Representative data of three independent experiments. h, Kinetics of NFAT translocations in activated Tβ-syn–NFAT cells in vitro. Plot, quantification of T cells with nuclear or cytoplasmic NFAT reporter over the period of 36 h after stimulation. Numbers of counted Tβ-syn–NFAT cells are indicated. Representative fluorescent images showing Tβ-syn–NFAT cells with nuclear (closed arrowheads) or cytoplasmic (open arrowheads) NFAT reporter are shown. ik, Detection of NFAT translocations in situ. i, Histologic analysis of brain tissue at the onset of disease. Top, fluorescent overview images of a coronal brain section with magnified views. bottom, selected region acquired at higher magnification. Green, Tβ-syn–NFAT cells; red, MHC class II+ cells; blue, DAPI (not depicted in the top panel). Filled and open arrowheads indicate T cells with nuclear and cytoplasmic localization of the NFAT–YFP reporter, respectively. Dashed line indicates the position of an area presented in Fig. 3a. j, Confocal images showing representative Tβ-syn–NFAT cells with nuclear NFAT reporter in the parenchyma and meninges of the cortex. Individual z planes. k, In vivo visualization of de novo NFAT reporter translocation. Top, intravital TPLSM recording of NFAT translocation from the cytoplasm (open arrowhead) to the nucleus (closed arrowhead). Bottom, single z planes (shown as single colours and merged channels), maximum intensity projections (MIP) and 3D-rendered images depict the same Tβ-syn–NFAT-CherryH2B cell before (0 min) and after (30 min) NFAT translocation. Histogram profile shows YFP and mCherry fluorescence intensities along the line indicated on the adjacent image.

  5. Extended Data Fig. 5 Activation of Tβ-syn cells within the brain is critical for their infiltration and for secondary immune cell recruitment and clinical disease.

    a, Microglia from CNS grey matter is inefficient to present antigen to TMBP cells. Microglial cells isolated from brain parenchyma (total brain) or from cortical grey matter were cocultured for 48 h with TMBP cells (left) or TOVA cells (right) with or without addition of cognate antigen. Anti-MHC class II blocking monoclonal antibodies or PBS were added for each condition. IFNγ production was measured by ELISA. Representative data from three independent experiments per cell type. Data are mean + s.e.m., n = 3. bd, Blocking antigen presentation in vivo by intrathecal injection of anti-MHC class II monoclonal antibody prevents Tβ-syn cell entry into the brain and local reactivation. b, Number of Tβ-syn cells in blood, brain meninges and brain parenchyma in rats treated with PBS (control) or anti-MHC-II monoclonal antibodies quantified at the onset of disease and the corresponding rate of infiltration in the brain compartments normalized to blood. Flow cytometry data. c, Quantification of the number of macrophages (CD11b+CD45high cells), CD4+ and CD8+ T cells recruited to the indicated brain compartments. b, c, Representative data of two independent experiments including at least three rats per group. d, Relative expression of inflammatory cytokines (normalized to Actb) either in Tβ-syn cells isolated from the indicated brain compartments (left) or in brain tissues (right) assessed by quantitative PCR. Data are mean + s.e.m. of n = 3. Representative data of two independent experiments. eg, Effect of FK506 treatment on the infiltration of Tβ-syn cells into the grey matter of the brain. e, Coronal brain sections (fluorescence overview images and magnified views of selected areas) from control (clinical score 1.5) or FK506-treated (clinical score 0) rats. Red, Tβ-syn cells; green/yellow, tissue autofluorescence. f, Numbers of T β-syn cells and recruited myeloid cells (CD11bhigh) in the indicated compartments determined by flow cytometry. Data are mean + s.e.m., n = 3 per group. g, Expression of CD25 and CD134 on Tβ-syn cells. a, One-way ANOVA with Dunnet’s correction for multiple comparison. bd, f, Unpaired two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

  6. Extended Data Fig. 6 Antigen availability determines T cell infiltration into the brain.

    a, In vitro activation of TOVA–CAR cells. Relative expression of IFNγ in TOVA–CAR cells challenged in vitro with biotin anti-mouse IgG at the indicated doses in the presence or absence of streptavidin. Quantitative PCR performed at the indicated time points after challenge. Stimulation of TOVA–CAR cells at the matching time points with anti-CD3/CD28 is shown as comparison (right plot) (normalized to β-actin). b, In vivo activation prompts TOVA–CAR and myeloid-cell entry into the brain. Plot, number of TOVA–CAR cells in brain meninges and brain parenchyma quantified by flow cytometry 3 days p.t. Rats were injected intrathecally or i.c.v. with biotin anti-mouse IgG (anti-CAR) 6 h before analysis, to trigger T cell activation. Control, PBS-treated animals. Data are mean + s.e.m. Cumulative data of three independent experiments including two animals per group. Intravital TPLSM images of the brain cortex three days p.t. are shown. TOVA–CAR cells show infiltration of CD11b+ cells labelled by intrathecal injection of SeTau647-conjugated anti-CD11b monoclonal antibodies (magenta). Recordings were performed 6 h after i.c.v. injection of PBS (control) or anti-mouse IgG1 (anti-CAR). c, d, Effector T cell entry into the brain is boosted by increased antigen availability. Plots, quantification of TOVA cells (c) or TMBP cells (d) in the indicated brain compartments 3 days p.t. Rats were injected i.c.v. with the cognate antigen 12 h before analysis. n = 2 per T cell specificity. Corresponding intravital TPLSM images are shown. Green, antigen-specific T cells; red, blood vessels and macrophages. eg, Neuronal overexpression of β-synuclein strongly increases Tβ-syn cell invasion into the brain. e, Fluorescence overview images and magnified views of selected areas of coronal brain sections depicting the pattern of Tβ-syn cell infiltration at the peak of the T cell grey matter infiltration. The rats had been neonatally injected with AAVs expressing GFP or β-syn–GFP under the synapsin promoter. Representative data of three independent experiments. Red, Tβ-syn cells; Grey (false colour), GFP- or β-synuclein-expressing neurons. f, Quantitative analyses of Tβ-syn cells by intravital TPLSM in AAV-β-syn-GFP or AAV-GFP rats. Number of grey-matter-infiltrating Tβ-syn cells (left) and their velocity over a 30-min period (right). Note the decreased speed of Tβ-syn cells in rats overexpressing β-synuclein, indicating local T cell activation. n = 2 from 2 independent experiments. g, Neuronal overexpression of β- but not γ-synuclein increases Tβ-syn cell entry into the brain. Fluorescence overview pictures and magnified cortical areas at the peak of Tβ-syn cell infiltration depicting the infiltration pattern of Tβ-syn cells into the brain of rats overexpressing β- or γ-synuclein in neurons. Red, Tβ-syn cells; grey (false colour), neurons overexpressing β- or γ-synuclein. Representative data of two independent experiments. bd, Unpaired two-tailed t-test. f, Mann–Whitney U-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.

  7. Extended Data Fig. 7 Tβ-syn cell activation drives immune cell recruitment.

    a, Recruitment of host T cells and myeloid cells determined by flow cytometry at the peak of disease induced by Tβ-syn cell transfer. Data are mean + s.e.m., n = 3. bd, Tβ-syn cell invasion in the CNS drives recruitment of CNS-ignorant TOVA cells. b, TOVA cell recruitment to the grey matter cortex after transfer of TOVA–Tomato cells alone (top) or after cotransfer with Tβ-syn–Turquoise cells (bottom). Left, fluorescence overview images and magnified views of selected areas of coronal brain sections. Right, TPLSM individual stacks and corresponding migratory paths of Tβ-syn and/or TOVA cells over a 30-min recording time. Data were acquired 3.5 days after T cell transfer. Arrows point to representative TOVA cells. Red, TOVA cells; turquoise, Tβ-syn cells, green, blood vessels (labelled by FITC-conjugated dextran). Representative data of at least three independent experiments (obtained from individual rats). c, Time course of T cell infiltration in the indicated compartments upon single transfer of TOVA–Tomato cells or cotransfer of Tβ-syn–Turquoise and TOVA–Tomato cells, determined by flow cytometry. Data are mean + s.e.m., n = 3 per time point. Representative data of two independent experiments. d, TOVA cells are not reactivated in the brain. Expression level of the indicated cytokines measured by quantitative PCR in Tβ-syn and TOVA cells isolated from the indicated compartments upon cotransfer. Data are mean + s.e.m., n = 3. Representative data of two independent experiments. e, FK506 treatment prevents TOVA-cell recruitment to the grey matter tissue. Fluorescence overview images as in b from control (clinical score 2) or FK506-treated (clinical score 0) rats 3.5 days after cotransfer. Representative images of two independent experiments including three rats per group. f, Blockade of antigen presentation by intrathecal administration of anti-MHCII monoclonal antibodies effectively reduces immune cell recruitment to the brain in the cotransfer experiment. Left, ratio of the numbers of Tβ-syn or TOVA cells in brain tissue to the number of Tβ-syn or TOVA cells in blood. Data are mean + s.e.m., n = 3. Middle and right, absolute number of myeloid cells or T cells in the brain tissue measured by flow cytometry 3.5 days after cotransfer of Tβ-syn and TOVA cells. Data are mean + s.e.m., n = 3. f, Unpaired two-tailed t-test. *P < 0.05.

  8. Extended Data Fig. 8 Inflammatory changes to the brain tissue during acute disease induced by transfer of Tβ-syn cells.

    ad, Tβ-syn cell infiltration into the brain leptomeninges is associated with BBB leakage. a, Representative T1-weighted MRI images highlighting the areas of Gd leakage in meninges and parietal cortex over an EAE course induced by Tβ-syn or TMBP cell transfer. bd, MRI, intravital TPLSM and immunohistochemistry were performed in the same rat at the onset of Tβ-syn-cell-induced disease. b, T1-weighted MRI images before and after Gd administration. c, TPLSM images. Left, infiltration pattern of Tβ-syn cells (green) into the leptomeninges. Red, Texas red dextran-labelled vessels, macrophages. Right, colour-coded (HiLo LUT) images highlighting the areas of dye leakage (arrows). Saturated signal, red; no signal, blue; circles, meningeal phagocytes that took up the dye during the recording time. Shown are tile-scan overview images (top) and magnified views of selected areas (bottom). d, Overview fluorescence image of a sagittal brain section (top) and magnified view of a selected cortical area (bottom) stained for ED1. Note the presence of high numbers of recruited ED1+ myeloid cells (purple) in meninges and adjacent cortex. eh, Migration behaviour of Tβ-syn cells in the cortical grey matter. Intravital TPLSM recording performed at the peak of Tβ-syn cell cortical infiltration reveals a vigorous migratory activity of the Tβ-syn cells within the compact grey matter. e, The 30-min time-lapse trajectories. f, Number of infiltrating Tβ-syn cells. Data are mean + s.e.m., n = 3. g, Tβ-syn cell velocity (n = 340 cells). h, Superimposed trajectories over a 30-min time span recording. Σ, sum of all cell trajectory vectors divided by the number of cells (n = 30 cells, each line represents a cell). ik, Tβ-syn cell infiltration induces neuronal damage. Histological and electron microscope analyses at the peak of Tβ-syn cell infiltration. i, Relative changes in spine density on apical dendrites (cortical layer 2/3) measured by confocal microscopy in brain slices of rats transferred with the indicated antigen-specific T cells. Each dot represents a separate rat. Data are mean + s.e.m. j, Electron micrograph of an apoptotic neuron (highlighted in red, magnified view shown on the right) with a pyknotic and fragmented nucleus (asterisks) next to an intact neuron (green) with euchromatic nucleus. k, Apoptotic pyramidal neuron (N) with numerous apoptotic bodies (i) and pyknotic nucleus (ii) shown at higher magnification. Note the dendritic process (D) that emerges from the strongly altered cell body. l, m, Tβ-syn cell infiltration induces microglial activation. l, Anti-IBA1 staining of cortical tissue performed at the indicated time points after Tβ-syn cell transfer. Overviews pictures with magnified areas and representative 3D-reconstructed images of IBA1+ microglial cells (shown in greyscale). m, Quantification of the morphological parameters of IBA1+ microglial cells. Data are mean + s.d. i, Mann–Whitney U-test. m, One-way ANOVA with Dunnett’s multiple comparison correction for process length and segments and two-way ANOVA with Sidak correction for multiple comparisons for number of intersections. **P < 0.01, ***P < 0.001, ****P < 0.0001.

  9. Extended Data Fig. 9 Consecutive Tβ-syn cell attacks result in cortical grey matter degeneration.

    a, Relapsing-remitting clinical disease bouts induced by repeated Tβ-syn cell transfer. Clinical score (bars) and relative weight changes (lines) in transgenic rats transferred four consecutive times with Tβ-syn or TOVA cells. Data are mean ± s.e.m. Representative data of four independent experiments. b, Consecutive Tβ-syn-cell-mediated grey matter inflammation induces reiterated episodes of BBB leakage. Quantification of Gd enhancement in the indicated brain compartments performed on T1-weighted MRI images acquired at the onset of disease after the first and fourth T cell transfer. Each line represents one individual rat. Representative data of 2 independent experiments (n = 5 per group per experiment). c, Relapsing-remitting clinical disease bouts induced by repeated TMBP cell transfer. Clinical score (bars) and relative weight changes (lines) in transgenic rats transferred four consecutive times with TMBP cells. Data are mean ± s.e.m. d, TMBP-cell-induced disease bouts do not cause BBB leakage. Quantification of Gd enhancement in the indicated brain compartments performed as in b. ei, Repeated Tβ-syn-cell-induced autoimmune inflammation induces long-term damage in the cortical grey matter. Rats were transferred four times with Tβ-syn or TOVA cells. Histological analysis of the cerebral cortex was performed three weeks after the peak of the fourth disease bout. e, Very few residual immune infiltrates are detectable in the recovery stage of relapsing grey matter disease. Top, representative haematoxylin and eosin (HE)-stained coronal paraffin sections of brain cortex showing the presence of very limited infiltrates in the leptomeninges (arrows) of a 4× Tβ-syn cell-transferred rat. Note, however, the thickened leptomeninges, increased vascularization (V) in the cortical layer I (I) and enrichment of cells with small nuclei, representing microglia (examples shown by arrowheads). Bottom, representative CD43 (W3/13) staining on frozen sections of 4× TOVA and 4× Tβ-syn cell-transferred rats. The latter shows few CD43+ lymphocytes within the leptomeninges (arrows) and around cortical vessels (arrowheads). f, Persistent grey matter gliosis induced by 4× Tβ-syn cell transfer. Confocal images of cortical tissue stained with anti-IBA1 (left) or anti-GFAP antibodies (right). g, Rarefaction and disarrangement of neuronal processes. Representative neurofilament stainings of cortical grey matter on 4× Tβ-syn or 4× TOVA-cell-transferred rats. In corresponding regions of the motor cortex, the thickness, length and number of straight filaments (arrowheads) are reduced in rats that have received 4× Tβ-syn cells. h, i, Repeated episodes of grey matter inflammation do not affect white matter thickness. h, Histological analysis of corpus callosum thickness. Mean corpus callosum thickness across all control samples for the analysed anterior–posterior interval was set to 100%. Each data point represents relative corpus callosum thickness for one individual rat measured at the indicated distance to bregma. Lines represent fitting curves (shading, 95% confidence interval) for the respective group of rats. n = 8 for control, n = 6 for 4× Tβ-syn group from 4 independent experiments. Unpaired two-tailed t-test. i, Corpus callosum thickness determined by longitudinal MRI analysis at the indicated time over the disease course. Each line represents one individual rat. Representative data of 2 independent experiments (n = 5 per group per experiment). j, k, Enlargement of the ventricle size induced by relapsing autoimmune grey matter disease. j, Representative z projection of MRI images at the indicated time points after the first and fourth transfer of Tβ-syn or TOVA cells. k, Quantification of changes in ventricular volume over time. Representative data of 2 independent experiments (n = 5 per group per experiment). b, i, k, Two-way ANOVA with Bonferroni’s multiple comparison correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  10. Extended Data Fig. 10 Cytokine and chemokine receptor expression of myelin-reactive and neuronal-antigen-reactive T cells.

    a, Characteristics of the donor cohort. b, Correlation between cytokine production (ELISA) and CD4+ T cell proliferation. PBMCs were stimulated with the indicated antigens. Each dot represents one patient. c, IFNγ and IL-17 production in the proliferating CD4+ T cells (n = 3). d, CD25 and ICOS expression in proliferating (CFSElow) and non-proliferating (CFSEhigh) CD4+ T cells. Flow cytometry seven days after antigenic stimulation. Representative density plots (numbers indicate frequencies as mean + s.d. percentages, n = 3) and quantification of mean fluorescence intensities (MFI, n = 4). e, Gene expression levels of IFNG, IL17A, IL4, CXCR3 and CCR6, determined by quantitative PCR (normalized to RPL13A). PBMCs isolated from patients with MS were stimulated with MBP or β-synuclein. After seven days, cytokine expression was determined in proliferating (CFSElow) and non-proliferating (CFSEhigh) CD3+CD4+ T cells before and after stimulation with CD3 antibodies. Chemokine expression was determined just before CD3 stimulation. Analyses were performed with PBMCs of patients with MS who had a proliferative response to both MBP and β-synuclein. Numbers of analysed patients are indicated. Data are mean + s.e.m. f, Correlation between percentage of CD4+ T cells proliferating in response to the indicated antigens and the grade of disability in patients with MS and Parkinson’s disease. Disability was measured by the expanded disability status scale (EDSS) or the Hoehn and Yahr score for patients with MS and Parkinson’s disease, respectively. g, Correlation between percentage of CD3+CD4+ T cells proliferating in response to the indicated antigens and the disease duration. b, Linear regression analysis. ce, Paired two-tailed t-test. f, g, Pearson’s r2. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Supplementary information

  1. Reporting Summary

  2. Video 1

    Clinical disease induced by TbSYN or TMBP cells. Video showing differences in clinical symptoms after transfer of TbSYN (first part of video) or TMBP cells (second part of video).

  3. Video 2

    Invasion of TbSYN cells from leptomeninges into the adjacent brain GM and of TMBP cells from leptomeninges into the adjacent SC WM parenchyma. Intravital TPLSM recordings of TbSYN (first part of video) or TMBP cells (second part of video) performed on brain cortex and thoracic SC 4 days p.t. 30 min time-lapse recordings and corresponding time-projections. Green: antigen-specific T cells; red: blood vessels and meningeal phagocytes. Scale bar: 100 µm. Time interval: 30 sec. Representative recordings of five independent experiments.

  4. Video 3

    Locomotion behaviour of TbSYN and TMBP cells in the leptomeningeal vessels of brain and SC. Intravital TPLSM recordings of brain cortex (first part of video) and thoracic SC (second part of video) performed 2.5 days p.t. of TbSYN or TMBP cells. 30 min time-lapse videos and corresponding time-projections. During the recording time several T cells of both specificities crawled or rolled (appearing as single dots) on the leptomeningeal vessels of both brain and SC. Green: antigen specific T cells; red: blood vessels and meningeal phagocytes (labelled by i.t. injection of 70 kD Texas red dextran 24 h before imaging). Scale bar: 100 µm. Time interval: 30 sec. Representative recordings of three independent experiments.

  5. Video 4

    Locomotion behaviour of CNS-ignorant TOVA cells in the leptomeningeal vessels of brain and SC. Intravital TPLSM recordings of brain cortex and thoracic SC performed 2.5 days p.t. of TOVA cells. 30 min time-lapse recordings and corresponding time-projections. Scale bar: 100 µm. Time interval: 30 sec. Green: TOVA cells; red: blood vessels. Representative recordings of three independent experiments.

  6. Video 5

    Effect of VLA-4 and LFA-1 blockade on the adhesion of TbSYN cells to brain leptomeningeal vessels. Intravital TPLSM recordings depicting the intravascular motility behaviour of TbSYN cells 2.5 days p.t. before and after injection of the indicated integrin-blocking mAbs. 30 min time-lapse recordings and corresponding time-projections. Scale bar: 100 µm. Time interval: 30 sec. Green: TbSYN cells; red: blood vessels. Representative recordings of three independent experiments.

  7. Video 6

    Real-time detection of TbSYN-cell activation in the cerebral cortex. Intravital TPLSM recording performed on brain cortex 4 days p.t. of β-synuclein specific T cells expressing NFAT biosensor. 30 min time-lapse recordings and corresponding 3D reconstruction. Scale bar: 10 µm. Time intervals: 30 sec. Arrow: NFAT translocation from the cytosol to nucleus, indicated by the red nucleus turning yellow.

  8. Video 7

    Locomotion behaviour of CNS-ignorant TOVA cells in the cerebral cortex in absence or presence of pathogenic TbSYN cells Intravital TPLSM recording performed on brain cortex 4 days p.t. of TOVA cells alone or of TOVA together with TbSYN cells. 30 min time-lapse recordings and corresponding time-projections. Scale bar: 50 µm. Time interval: 60 sec. Red: TOVA cells; turquoise: TbSYN cells; green: blood vessels. Representative recordings of two independent experiments.

  9. Video 8

    Loss of BBB integrity during TbSYN-mediated GM disease. Intravital TPLSM recordings performed 3 min (first part of video) and 30 min (second part of video) after i.v. injection of 70 kD Texas Red dextran dye 3.5 days p.t. of TbSYN cells. In the first part of the video focal leakage of dextran is indicated by yellow arrowheads. In the second part of the video leaked dextran is taken up by meningeal phagocytes (yellow circles). The left frame shows the original composite multicolour TPLSM recording. Green: TbSYN cells; red: blood vessels and meningeal macrophages. The right frame depicts the areas of dye leakage using HiLo LUT colour-code. Red: saturated signal; blue: no signal.

  10. Video 9

    TbSYN cells in contact and engulfed by a neuron of the cortical GM. 3D-reconstruction of TbSYN cells in contact (green), partially engulfed (orange) or completely engulfed (red) by a DiI-labelled neuron (white). 3D-reconstruction from a confocal image of a brain slice labelled by DiI 4 days p.t. of TbSYN cells.

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https://doi.org/10.1038/s41586-019-0964-2

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