Abstract

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue1,2. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical3,4,5,6. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

Data deposits

The data discussed in this publication have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus and are accessible through GEO Series accession number GSE75488.

References

  1. 1.

    , , , & Immunophenotyping of cerebrospinal fluid cells in multiple sclerosis: in search of biomarkers. JAMA Neurol . 71, 905–912 (2014)

  2. 2.

    et al. Phenotypic and functional analysis of T cells homing into the CSF of subjects with inflammatory diseases of the CNS. J. Leukoc. Biol. 73, 584–590 (2003)

  3. 3.

    et al. Human cerebrospinal fluid central memory CD4 + T cells: evidence for trafficking through choroid plexus and meninges via P-selectin. Proc. Natl Acad. Sci. USA 100, 8389–8394 (2003)

  4. 4.

    et al. C-C chemokine receptor 6–regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE. Nature Immunol. 10, 514–523 (2009)

  5. 5.

    & The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends Immunol. 26, 485–495 (2005)

  6. 6.

    , & Involvement of the choroid plexus in central nervous system inflammation. Microsc. Res. Tech. 52, 112–129 (2001)

  7. 7.

    et al. Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature 462, 94–98 (2009)

  8. 8.

    et al. Recruitment of beneficial M2 macrophages to injured spinal cord is orchestrated by remote brain choroid plexus. Immunity 38, 555–569 (2013)

  9. 9.

    & CNS-specific T cells shape brain function via the choroid plexus. Brain Behav. Immun. 34, 11–16 (2013)

  10. 10.

    et al. A combination of fluorescent NFAT and H2B sensors uncovers dynamics of T cell activation in real time during CNS autoimmunity. Nature Med. 19, 784–790 (2013)

  11. 11.

    et al. The activation status of neuroantigen-specific T cells in the target organ determines the clinical outcome of autoimmune encephalomyelitis. J. Exp. Med. 199, 185–197 (2004)

  12. 12.

    et al. Chemokines enhance immunity by guiding naive CD8 +  T cells to sites of CD4 +  T cell–dendritic cell interaction. Nature 440, 890–895 (2006)

  13. 13.

    et al. Generalized Lévy walks and the role of chemokines in migration of effector CD8 + T cells. Nature 486, 545–548 (2012)

  14. 14.

    et al. Environmental context explains Lévy and Brownian movement patterns of marine predators. Nature 465, 1066–1069 (2010)

  15. 15.

    et al. Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator. Nature Med. 19, 778–783 (2013)

  16. 16.

    et al. Localizing central nervous system immune surveillance: meningeal antigen-presenting cells activate T cells during experimental autoimmune encephalomyelitis. Ann. Neurol. 65, 457–469 (2009)

  17. 17.

    , , & Rapid leukocyte integrin activation by chemokines. Immunol. Rev. 186, 37–46 (2002)

  18. 18.

    et al. T cells become licensed in the lung to enter the central nervous system. Nature 488, 675–679 (2012)

  19. 19.

    et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453, 51–55 (2008)

  20. 20.

    et al. Structural and functional features of central nervous system lymphatic vessels. Nature 523, 337–341 (2015)

  21. 21.

    et al. A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J. Exp. Med. 212, 991–999 (2015)

  22. 22.

    et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. Sci. Transl. Med. 4, 147ra111 (2012)

  23. 23.

    , , , & Cerebrospinal fluid dendritic cells infiltrate the brain parenchyma and target the cervical lymph nodes under neuroinflammatory conditions. PLoS ONE 3, e3321 (2008)

  24. 24.

    et al. Inspiration is the major regulator of human CSF flow. J. Neurosci. 35, 2485–2491 (2015)

  25. 25.

    , , & Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nature Rev. Immunol . 7, 678–689 (2007)

  26. 26.

    , , , & Blood-borne soluble protein antigen intensifies T cell activation in autoimmune CNS lesions and exacerbates clinical disease. Proc. Natl Acad. Sci. USA 104, 18625–18630 (2007)

  27. 27.

    et al. Autoimmune CD4 + T cell memory: lifelong persistence of encephalitogenic T cell clones in healthy immune repertoires. J. Immunol. 175, 69–81 (2005)

  28. 28.

    et al. Inflammatory cortical demyelination in early multiple sclerosis. N. Engl. J. Med. 365, 2188–2197 (2011)

  29. 29.

    & T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341, 619–624 (1989)

  30. 30.

    , , & Gene transfer into CD4 +  T lymphocytes: green fluorescent protein-engineered, encephalitogenic T cells illuminate brain autoimmune responses. Nature Med. 5, 843–847 (1999)

  31. 31.

    et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature Biotechnol. 24, 461–465 (2006)

  32. 32.

    , & Myelin basic proteins. Methods Enzymol. 32, 323–341 (1974)

  33. 33.

    et al. The N-terminal domain of the myelin oligodendrocyte glycoprotein (MOG) induces acute demyelinating experimental autoimmune encephalomyelitis in the Lewis rat. J. Neuroimmunol. 63, 17–27 (1995)

  34. 34.

    et al. Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis. Immunity 14, 547–560 (2001)

  35. 35.

    et al. Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain. J. Biol. Chem. 271, 11067–11075 (1996)

  36. 36.

    et al. Differential immune cell dynamics in the CNS cause CD4 +  T cell compartmentalization. Brain 132, 1247–1258 (2009)

  37. 37.

    et al. Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion. J. Exp. Med. 201, 1805–1814 (2005)

  38. 38.

    Inhibition of in vivo lymphocyte migration to inflammation and homing to lymphoid tissues by the TA-2 monoclonal antibody. A likely role for VLA-4 in vivo. J. Immunol. 147, 4178–4184 (1991)

  39. 39.

    & CXCR3 blockade inhibits T-cell migration into the CNS during EAE and prevents development of adoptively transferred, but not actively induced, disease. Eur. J. Immunol. 40, 2751–2761 (2010)

  40. 40.

    et al. Treatment of experimental autoimmune encephalomyelitis with the chemokine receptor antagonist Met-RANTES. J. Neuroimmunol. 128, 16–22 (2002)

  41. 41.

    , , , & CXCL12 (SDF-1alpha) suppresses ongoing experimental autoimmune encephalomyelitis by selecting antigen-specific regulatory T cells. J. Exp. Med. 205, 2643–2655 (2008)

  42. 42.

    et al. Vagal withdrawal and susceptibility to cardiac arrhythmias in rats with high trait aggressiveness. PLoS ONE 8, e68316 (2013)

  43. 43.

    , & Protein and synthetic polymer injection for induction of obstructive hydrocephalus in rats. Cerebrospinal Fluid Res. 4, 9 (2007)

  44. 44.

    & A procedure for direct lumbar puncture in rats. Brain Res. Bull. 59, 245–250 (2002)

  45. 45.

    et al. An RNA sequencing transcriptome analysis reveals novel insights into molecular aspects of the nitrate impact on the nodule activity of Medicago truncatula. Plant Physiol. 164, 400–411 (2014)

  46. 46.

    et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013)

  47. 47.

    et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)

  48. 48.

    , & HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015)

  49. 49.

    & Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010)

  50. 50.

    et al. BioMart and Bioconductor: a powerful link between biological databases and microarray data analysis. Bioinformatics 21, 3439–3440 (2005)

  51. 51.

    , & Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols 4, 44–57 (2009)

  52. 52.

    et al. Instant effect of soluble antigen on effector T cells in peripheral immune organs during immunotherapy of autoimmune encephalomyelitis. Proc. Natl Acad. Sci. USA 104, 920–925 (2007)

  53. 53.

    et al. Fiji: an open-source platform for biological-image analysis. Nature Methods 9, 676–682 (2012)

Download references

Acknowledgements

The authors thank S. Hamann, A. Stas, N. Meyer, S. Mole, and M. Weig for excellent technical assistance. We thank G. Salinas-Riester for her support in performing the transcriptome analyses, T. Lingner for his help in analysing the transcriptome data and W. Lühder for contributing to the mathematical T-cell locomotion analyses. We are grateful to C. Ludwig for text editing. This work was supported by the Deutsche Forschungsgemeinschaft (TRR-SFB43 project B10, FORR 1336 project B1 and RK-Grant FL 377/3-1), the Bundesministerium für Bildung und Forschung (‘UNDERSTAND MS’), the Hertie Foundation (grants 1.01.1/11/004 and 1130072), the Ministry of Science and Culture of Lower Saxony (Niedersachsen-Research Network on Neuroinfectiology, N-RENNT) and the European Commission ERA-NET NEURON (MELTRA-BBB).

Author information

Author notes

    • Christian Schläger
    • , Henrike Körner
    • , Francesca Odoardi
    •  & Alexander Flügel

    These authors contributed equally to this work.

Affiliations

  1. Institute of Neuroimmunology, Institute for Multiple Sclerosis Research, University Medical Centre Göttingen, 37073 Göttingen, Germany

    • Christian Schläger
    • , Henrike Körner
    • , Michael Haberl
    • , Dmitri Lodygin
    • , Francesca Odoardi
    •  & Alexander Flügel
  2. Max-Planck-Institute for Experimental Medicine, 37075 Göttingen, Germany

    • Alexander Flügel
  3. Institute of Anatomy, University of Leipzig, 04103 Leipzig, Germany

    • Martin Krueger
    • , Elke Brylla
    •  & Ingo Bechmann
  4. Department of Structural and Geotechnical Engineering, University of Rome La Sapienza, 00185 Rome, Italy

    • Stefano Vidoli
  5. Department Neurosurgery, University Medical Centre Göttingen, 37075 Göttingen, Germany

    • Dorothee Mielke
    •  & Veit Rohde
  6. Division of Immunology, Department of Pediatrics Dalhousie University, Halifax B3H 4R2, Canada

    • Thomas Issekutz
  7. Departamento de Biología Celular e Inmunología, Centro de Biología Molecular Severo Ochoa, 28049 Madrid, Spain

    • Carlos Cabañas
  8. Medical Clinic and Policlinic IV, Ludwig-Maximilians-University of Munich, 80336 Munich, Germany

    • Peter J. Nelson
  9. Department of Neurology, University Hospital, 01307 Dresden, Germany

    • Tjalf Ziemssen

Authors

  1. Search for Christian Schläger in:

  2. Search for Henrike Körner in:

  3. Search for Martin Krueger in:

  4. Search for Stefano Vidoli in:

  5. Search for Michael Haberl in:

  6. Search for Dorothee Mielke in:

  7. Search for Elke Brylla in:

  8. Search for Thomas Issekutz in:

  9. Search for Carlos Cabañas in:

  10. Search for Peter J. Nelson in:

  11. Search for Tjalf Ziemssen in:

  12. Search for Veit Rohde in:

  13. Search for Ingo Bechmann in:

  14. Search for Dmitri Lodygin in:

  15. Search for Francesca Odoardi in:

  16. Search for Alexander Flügel in:

Contributions

C.S. performed most intravital TPLSM imaging studies. H.K. performed most of the CSF analyses, fluorescence microscopy and T-cell-CSF-transfers. M.K., E.B. and I.B. performed and analysed immunofluorescence and electron microscopic analyses. S.V. performed the mathematical cell motility analyses. M.H. performed antibody labelling and contributed to TPLSM imaging. D.M. and V.R. designed the operative strategy, D.M. performed the plexus preparation. C.C. provided HUTS4 antibody and contributed with technical advice. T.I. provided the anti-VLA-4 and anti-CXCR3 antibodies and contributed with technical advice. P.J.N. provided CCR5 blocker and contributed with scientific advice. T.Z. contributed with T-cell characterization in the CSF, D.L. designed and produced genetic retroviral sensors and contributed to the analysis of NSeq data. F.O. performed most ex vivo T-cell analyses, i.e. cytofluorometric characterizations, quantitative PCR and NSeq 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 financial interests.

Corresponding author

Correspondence to Alexander Flügel.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    T-cell activation, but not differentiation, adhesion or cell motility-related genes are differentially regulated in TMBP cells in the CSF - RNA-Seq data depicting groups of genes categorized according to their function. Red: genes differentially regulated (≥2.5-fold) in TMBP cells in the CSF compared to CNS parenchyma. Gene names in blue: master transcription factors that determine the differentiation of CD4+ T-helper cell subsets. Mean ±s.d.

  2. 2.

    Supplementary Table 2

    Genes differentially regulated between the CSF and the CNS parenchyma cluster in pathways related to T-cell activation - Pathway enrichment analysis using the KEGG database on the genes significantly regulated between CSF and CNS parenchyma in TMBP cells.

  3. 3.

    Supplementary Table 3

    Genes differentially regulated between the CSF and the leptomeninges cluster in pathways related to T-cell activation - KEGG pathway analysis on genes significantly regulated between CSF and leptomeninges in TMBP cells.

Videos

  1. 1.

    TMBP cell extravasation from leptomeningeal blood vessels

    Intravital TPLSM recording was performed on the thoracic SC during the pre-invasion phase of leptomeningeal TMBP cell infiltration, where the vast majority of both TMBP cells (blue) and CD11b+ myeloid cells (magenta) were located within the vascular compartment. Depicted is an example of a TMBP cell (white arrowhead) transgressing the vascular wall after having crawled within the lumen of a leptomeningeal blood vessel. Blue: TMBP-LifeAct-Turquoise2 cells. Red: blood vessels. Magenta: CD11b+ myeloid cells. Scale bars: 25 µm. Time interval: 32 sec.

  2. 2.

    Detachment of TMBP cells from the leptomeninges into the CSF

    Intravital TPLSM recording of the thoracic SC was performed during established leptomeningeal TMBP cell infiltration. 1st half: Representative TMBP-GFP cells (white circles) detaching from the pial surface and subsequently being dragged away within the CSF. 2nd half: Examples of TMBP-GFP cells rolling along the pial surface (white arrows). Green: TMBP-GFP cells. Red: blood vessels, meningeal phagocytes. Blue: collagen fibers. Scale bar: 50 µm. Time interval: 32 sec. Representative recording of five independent experiments.

  3. 3.

    T-cell detachment from the leptomeninges verified by fast-acquisition fluorescence video microscopy

    Intravital fluorescence video microscopy of the thoracic SC was performed during established leptomeningeal TMBP cell infiltration. Example of a TMBP-GFP cell detaching from the pial surface (false color: orange) and subsequently being dragged away with the CSF is depicted. Green: TMBP-GFP cells. Scale bar: 50 µm. Time interval: 5.8 sec. Representative recording of 5 independent experiments.

  4. 4.

    Detachment of photoconverted TMBP-Dendra2 cells from the leptomeninges into the CSF

    Intravital TPLSM recording of the thoracic SC was performed 30 min after photoconversion of TMBP-Dendra2 cells during established leptomeningeal TMBP cell infiltration. Depicted are examples of photoconverted TMBP cells (white circles) detaching from the pial surface and subsequently being dragged away with the CSF. White circle (dotted line): example of a T cell re-attaching from the CSF to the pial surface. Red (auto-fluorescence): leptomeningeal blood vessels, meningeal phagocytes. Yellow: photoconverted TMBP-Dendra2 cells. Green: non-photoconverted TMBP-Dendra2 cells. Scale bar: 50 µm. Time interval: 52 sec. Representative recording of three independent TPLSM recordings.

  5. 5.

    TMBP cells are, for the majority of their crawling time, in direct contact with meningeal macrophages

    Surface-rendered 3D reconstruction of an intravital TPLSM recording performed on the thoracic SC during established leptomeningeal TMBP cell infiltration. TMBP-GFP cells in contact with meningeal macrophages are depicted in yellow (magenta-colored circles), when not in contact in green (white-colored circles). Red: meningeal macrophages. Scale bar: 50 µm. Time interval: 32 sec. Representative recordings of three independent experiments.

  6. 6.

    Integrin blockade results in a massive release of TMBP cells from the leptomeninges into the CSF

    Intravital TPLSM recordings during established leptomeningeal TMBP cell infiltration were performed before (top) and 4 h after combined infusion of anti-VLA-4/LFA-1 mAbs (bottom). Note that after infusion of blocking antibodies a substantial fraction of TMBP-GFP cells is floating within the CSF appearing as green streaks (white circles) or rolling along the pial surface looking like pearl-strings (white arrows). Blue: collagen fibers. Scale bars: 100 µm. Time interval: 48 sec. Representative recordings of three independent experiments.

  7. 7.

    Chemokine blockade via PTX induces TMBP cell detachment from the leptomeninges.

    Intravital TPLSM recordings of the thoracic SC were performed during established leptomeningeal TMBP cell infiltration before (left) or 4 h after infusion of PTX (right). Orange (false color): examples of TMBP cells being released from meningeal phagocytes (Mɸ, dotted lines) into the CSF. Green: TMBP-GFP cells. Gray (false color): vessel lumen, meningeal phagocytes. Scale bars: 50 µm. Time interval: 32 sec. Representative recordings of five independent experiments.

  8. 8.

    Hyperventilation induces an increase of rolling/floating TMBP cells in the CSF

    Fluorescence video microscopy recordings were performed during established leptomeningeal TMBP cell infiltration under steady state conditions (control, respiratory rate: 81 bpm) and during hyperventilation (respiratory rate: 100 bpm). Green: TMBP-GFP cells. Arrows point to representative rolling/floating TMBP-GFP cells. Time interval: 8.4 sec. Scale bar: 50 µm. Representative recordings of three independent experiments.

  9. 9.

    Re-attachment of TMBP cells from the CSF to the leptomeninges

    Intravital TPLSM recordings on thoracic SC were performed during established leptomeningeal TMBP cell infiltration. Depicted are examples of TMBP-GFP cells re-attaching from the CSF to the pial surface (yellow circles). Note that after re-attachment T cells show either short, intermittent adhesive interactions with the pial surface (rolling; pattern 1, 1st half), or they stably adhere to the leptomeningeal structures (capture) followed by subsequent crawling (pattern 2, 2nd half). Green: TMBP-GFP cells. Red: blood vessels, meningeal phagocytes. Blue: collagen fibers. Scale bars: 50 µm. Time interval: 32 sec. Representative recordings of 5 independent experiments.

  10. 10.

    Re-attachment of TMBP cells from the CSF to the leptomeninges visualized by fast-acquisition fluorescence video microscopy

    Intravital fluorescence video microscopy was performed on the thoracic SC during established leptomeningeal TMBP cell infiltration. Depicted is an example of a TMBP-GFP cell (false color: orange) that after a brief period of rolling arrests (capture). Subsequently, the cell continues its way by further rolling along the pial surface. Green: TMBP-GFP cells. Scale bar: 50 µm. Time interval: 9.8 sec. Representative recording of three independent experiments.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature16939

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.