1. Volker Siffrin, Helena Radbruch, Robert Glumm, Raluca Niesner, Magdalena Paterka, Josephine Herz, Tina Leuenberger, Sabrina M. Lehmann, Sarah Luenstedt, Jan Leo Rinnenthal, Gregor Laube, Hervé Luche, Seija Lehnardt, Hans-Joerg Fehling, Oliver Griesbeck and Frauke Zipp In Vivo Imaging of Partially Reversible Th17 Cell-Induced Neuronal Dysfunction in the Course of Encephalomyelitis

2. Introduction Experimental autoimmune encephalomyelitis (EAE) -> prototype inflammatory demyelinating disease of the central nervous system (CNS) -> model for the human disease multiple sclerosis (MS) Neurodegeneration in inflammatory CNS disease 1.Bystander damage by excitotoxicity, ROS, myelin breakdown products, death ligand TRAIL 2.loss of myelin-derived support and secondary disruption of axonal ion concentrations, => ion channel reorganization processes in demyelinated axons leading to energy failure Th17cells form synapse-like contacts with neurons and their processes and play a dominant role in neuronal injury during EAE (Ivanov et al., 2006)Ivanov et al., 2006

3. Introduction mice (B6.tdRFP→B6.Thy1.EGFP) – (EGFP) expression in neurons and neuronal processes (axons and dendrites) – (tdRFP) expression in bone marrow-derived peripheral immune cells Active EAE –B6.tdRFP→B6.Thy1.EGFP by subcutaneous immunization with myelin oligodendrocyte glycoprotein (MOG 35-55 ) Passive EAE ( adoptive transfer model ) –MOG 35-55 -specific (2d2) and red-fluorescent (tdRFP) in vitro Th17 cell-differentiated 2d2.tdRFP Th17 cells -> B6.Rag1 −/−.Thy1.EGFP mice

4. -Result- CNS-Specific Th17 Cells in EAE Exhibit Characteristic Motility Changes

5. Acitve EAE Passive EAE Immune Infiltrates in Demyelinating Lesions Are Highly Dynamic and Show Different Motility Patterna in Distinct Disease Stages A. B6.tdRFP→B6.Thy1.EGFP B. 2d2.tdRFP Th17 -> B6.Rag1 −/−.Thy1.EGFP C. Movie S1 : B6.tdRFP→B6.Thy1.EGFP E. Movie S2 : 2d2.tdRFP Th17 -> B6.Rag1 −/−.Thy1.EGFP D,F. Automated cell tracking G-I. : Clinical scoore ↑ -> motility of Th17↓  motility changes of CNS-specific CD4 + T cells in particular correlate with the progression and severity of the clinical disease in EAE. Encephaliotogenic T cell confinement

6. Movie S1. TPLSM of CD45 +.tdRFP Cells in a Brainstem EAE Lesion of a B6.tdRFP→B6.Thy1.EGFP Mouse

7. Movie S2. TPLSM of 2d2.tdRFP Th17 in a Brainstem EAE Lesion of a B6.Rag1 −/−.Thy1.EGFP Mouse

8. -Result- Immune-Neuronal Interaction Is a Key Feature beyond Conventional Antigen-Dependent Motility Changes

9. The Interaction of 2d2.tdRFP Th17, OT-2.tdRFP Th17, and 2d2.tdRFP Th1 Cells with Neuronal Pathology A. automated tracking of 2d2.tdRFP Th17 cells B. automated tracking of 2d2.tdRFP Th1 cells -> less invasive C. OT-2.tdRFP Th17 cells enter the CNS lesions similarly as 2d2.tdRFP Th17 cells D,E. 2d2.tdRFP Th17 cells, IL- 17A-enriched 2d2.tdRFP Th17 cells, OT-2.tdRFP Th17 cells ≠ tdRFP Th1 cells G. axonal varicosities and ellipsoid bodies -> signs of a block of axonal transport (Coleman, 2005 ) -> Movie S3Coleman, 2005 H. White : colocalization -> Movie S4 I Contact pattern  Differentiation status of CD4 + T cells influences their capability to interact with the neuronal compartment.

10. Movie S3. 2d2.tdRFP Th17 Closely Interact with Neuronal Pathology in a Brainstem EAE Lesion of a B6.Rag1 −/−.Thy1.EGFP Mouse

11. Movie S4. OT-2.tdRFP Th17 Closely Interact with Neuronal Pathology in a Brainstem EAE Lesion of a B6.Rag1 −/−.Thy1.EGFP Mouse.

12. -Result- Th17 Cells Directly Contact Neurons Irrespective of Their CNS- Antigen Specificity

13. Confocal and Electron Microscopic Analysis of Brainstem EAE Lesions A. 2d2.tdRFP Th17 cells CD4 + T cells (magenta, anti-CD4) ; neurons/neuronal processes (green, Thy1.EGFP); MHC-II + cells (blue) -> Movie S5 B. OT-2.tdRFP Th17 CD4 + T cells OT-2.tdRFP Th17 cells (red), neuronal processes (green, Thy1.EGFP), Hoechst (blue); insert: magenta, anti-CD4. without proximity to MHC-II - expressing cells (insert). vessel cuffs with MHC-II- expressing cells (arrow) C. EM confirmed immediate proximity of CD4 + T cells to neuropil D. Within the cytoplasm of the CD4 + T cells, mitochondria, endoplasmic reticulum, coated vesicles ↓ -> synapse or kinapse-like structural polarization toward compromised neurites. E,F. T cells directly contacting neuronal cell bodies extending a probing process (asterisk) into nonmyelinated presynaptic terminal of an axo- somatic synapse (arrowhead);  CD4 + T cells contact neurons and their processes in EAE, independent of MHC-II expression or antigen- specificity and also in the absence of myelination.

14. Movie S5. 3D-Recombined Image Stack of Confocal Microscopy in a Brainstem EAE Lesion of a B6.Rag1 −/−.Thy1.EGFP Mouse

15. -Result- Th17 Cells Establish Immune-Neuronal Synapses and Induce Neuronal Cell Death In Vitro

16. A.In vitro differentiated CD4 + T cell phenotypes ->induce neuronal damage in cultured hippocampal neurons B. induced substantial neuronal cell death C. reduced the number of NeuN + cells supernatant from Th17 cell cultures -> importance of contact formation for the neuronal damage mechanism T cell microtubular cytoskeleton is rearranged, which leads to a reorientation of the microtubule-organizing center (MTOC) toward the vicinity of the APC-T cell contact area (Dustin and Colman, 2002)Dustin and Colman, 2002 D. Triple-stained Th17 cell- neuronal cocultures for α-tubulin, Hoechst, and CD4 E. MHC-II deficient neurons (H2Ab1-Ea −/− ) -> similar neuronal cell death induction Itgal −/− (LFA-1-deficient) Th17 cells reduced the cell death of neurons  The importance of a functional immune-neuronal synapse formation by these Th17 cells for the induction of cell death

17. -Result- Th17 Cell-Neuronal Interaction Leads to Neuronal Dysfunction as Shown by Sustained Ca 2+ Elevation

18. Th17 Cells Induce Ca 2+ Dynamics in Neurons upon Contact Formation In Vivo A.fluorescence resonance energy transfer (FRET)- TPLSM in B6.Thy1.CertnL15 /Th17 cells (red) B,C. Mean Citrine/Cerulean ratio (YFP/CFP) = intensity- modulated ratio channel  intracellular Ca 2+ increase ( Movie S6 & S7 ) /2d2.tdRFP Th17 cells (green) B6.Thy1.CertnL15 ->Troponin C-based genetically encoded Ca 2+ sensor in neurons (Heim et al., 2007)Heim et al., 2007 -> induced for EAE by adoptive transfer of 2d2.tdRFP Th17 cells blue = low, red = high Axonal Neuronal D. Critical Ca 2+ concentrations were reached in areas of sustained immune- neuronal contact formation normalized to the maximum Ca 2+ increase upon 300μM K + Cl − application E,F. Cotransfer of OT-2.tdRFP Th17 cells into B6.Thy1.CertnL15, -> induced for EAE by subcutaneous immunization with MOG 35-55, -> induced contact-dependent Ca 2+ disturbances -> slightly lower—in neurons and their processes  Ca 2+ disturbances in neurons and their processes occur during contact formation between immune and neuronal cells.

19. Movie S6. Axonal Time-Lapse FRET-TPLSM of a Lesion in 2d2.tdRFP Th17 Cell-Induced EAE in B6.Thy1.CertnL15 Mice

20. Movie S7. Neuronal Time-Lapse FRET-TPLSM of a Lesion in 2d2.tdRFP Th17 Cell-Induced EAE in B6.Thy1.CertnL15 Mice nonmyelinated neuronal cell body

21. -Result- Th17 Cell-Induced Ca 2+ Increase Precedes Irreversible Axonal and Neuronal Injury

22. Th17 Cell-Induced Ca 2+ Dynamics Correlate with Subsequent Morphologic Signs of Neuronal Damage A. sustained immune- mediated Ca 2+ elevation preceded axonal dissection at the site of immune- neuronal contact formation ( Movie S8 ) B. Sustained Ca 2+ increase in neuronal somata -> irreversible damage (integration of PI into the nucleus of these neurons ) C. Largest Ca 2+ increase -> at the border of active demyelinating lesions (disintegrating myelin sheaths and myelin debris ) D,E. substantial decrease of remaining neuronal tissue as defined by GFP loss -> neurodegeneration in the presence of 2d2 Th17 cell F. stained for the amyloid precursor protein (APP) -> accumulate in axons with intra-axonal transport block G. 2d2 Th17 cells tended to promote a nonremitting and more progressive disease in actively immunized B6 mice  immune-mediated Ca 2+ disturbances in neurons and their processes as measured by FRET-TPLSM are an early sign of neuronal dysfunction that can progress to irreversible neuronal damage.

23. Movie S8. Sustained Localized Ca2 + Increase in an EAE Lesion Can Lead to Axonal Dissection Nearby

24. -Result- Immune-Mediated Ca 2+ Oscillations Are Partially Reversible by NMDA Receptor Blockade

25. Immune-Mediated Ca 2+ Oscillations Can be Partially Reverted by Pharmacologic Blockade of Glutamate Receptors and Na + Channels which mechanisms ? glutamate-mediated excitotoxicity -> to test, blocked N-methyl D-aspartate (NMDA) receptors by local application of 300 μM (MK-801) A. Ca 2+ signal of axonal structures with sustained contacts (areas 2 and 3) and one with no visible contact (area 1) before and after the application of MK-801 B-D. 1. The axons without T cell contact maintained low Ca 2+ concentrations 2. the axons with T cell contact exhibited Ca 2+ oscillations E. response to MK-801 with a nearly complete return of Ca 2+ to baseline levels F,G. by MK-801 -Passive EAE-> 59.34% reverted -Active EAE -> 75.64% reversed Na + channel-triggered Ca 2+ elevation? -> using Na + channel blocker phenytoin (PHT, 100μM) F,G. by PHT -Passive EAE-> 14.18% reverted -Active EAE -> 41.98% reversed  Th17 cell-mediated neuronal Ca 2+ disturbances were largely reversible by blocking excitotoxicity whereas a Na + channel blockade was less effective.

26. Graphical Abstract

27. Discussion ► Th17 cells establish immune-neuronal synapses and induce neuronal cell death in vitro ► Th17 cells directly contact neurons irrespective of their CNS-antigen specificity ► Th17 cell-neuronal interaction leads to severe neuronal dysfunction ► Th17 cell-mediated Ca 2+ elevation is partially reversible by blocking excitotoxicity