Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) that is initiated when self-reactive T cells enter the brain and become locally activated after encountering their specific nervous antigens. When and where the disease-relevant antigen encounters occur is unclear. Here we combined fluorescently labeled nuclear factor of activated T cells (NFAT) with histone protein H2B to create a broadly applicable molecular sensor for intravital imaging of T cell activation. In experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, we report that effector T cells entering the CNS become activated after short contacts with leptomeningeal phagocytes. During established disease, the activation process is extended to the depth of the CNS parenchyma, where the cells form contacts with microglia and recruited phagocytes. We show that it is the activation processes during the preclinical phase rather than during established disease that are essential for the intensity and duration of the disease bout.
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We thank N. Meyer, S. Hamann, A. Stas and M. Weig for excellent technical assistance. We thank R. Tsien (Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla) for providing mCherry complementary DNA (cDNA). We also thank C. Ludwig for text editing and M.B. Graeber and F. Lühder for critical reading of the manuscript. D.L., F.O. and A.F. were supported by the Deutsche Forschungsgemeinschaft (FOR 1336, FL 377/2-7; TRR-SFB43 B10 and B11), the Bundesministerium für Bildung und Forschung ('UNDERSTAND MS') and the Hertie Foundation (grant 1.01.1/11/004). The work of H.M.R.'s lab was supported by the Deutsche Forschungsgemeinschaft (RE 1631/10-1; SFB-TRR43 B13).
The authors declare no competing financial interests.
Supplementary Figures 1–10 and Supplementary Methods (PDF 2908 kb)
NFAT translocation in T cells upon interaction with DCs in vitro. Dendritic cells were derived from bone marrow of AsRed-transgenic rats. After stimulation with recombinant rat IFN-γ, DCs were loaded with antigen and co-cultured with resting TMBP-NFAT/YFP-Cherry/H2B cells in a conditioned glassbottom imaging chamber. Representative time lapse epifluorescence recording is shown. Green, NFAT-YFP; red, AsRed (DC) and mCherry-H2B (T cells). White and yellow arrows, T cells before and after nuclear NFAT translocation, respectively. Time interval, 1 min. Scale bar, 10 μm. (MOV 428 kb)
Nuclear NFAT translocation under antigenic challenge in popliteal LNs. Animals received s.c. injection of resting TMBP-NFAT/YFP-Cherry/H2B cells. 20 h later, they were immunized s.c. with either PBS (control, left) or MBP (immunized, right). 2-PM recordings of the draining popliteal lymph nodes were performed 4 h thereafter. Progressive trajectories of individual motile T cells are shown. White and yellow lines depict tracks of T cells with cytosolic (not translocated) or nuclear (translocated) NFAT, respectively. Scale bar, 50 μm. Time intervals, 41.2 s (left), 43.6 s (right). Representative videos of at least 3 independent experiments are shown. (MOV 2027 kb)
Nuclear NFAT translocations in TMBP-NFAT/YFP-Cherry/H2B cells do not occur within CNS blood vessels. 2-PM recordings of TMBP-NFAT/YFP-Cherry/H2B cells within the leptomeningeal vessel during the preclinical phase are shown. Progressive trajectories of intraluminal (left) and extravasated TMBP-NFAT/YFP-Cherry/H2B cells (right) are depicted. Insets: magnified regions with their origins indicated by white frames. White and yellow lines depict tracks of TMBP-NFAT/YFP-Cherry/H2B cells with cytosolic (not translocated) or nuclear NFAT (translocated), respectively. Blood vessels and meningeal phagocytes were labeled by i.v. or i.t. injection of TR-dextran respectively. Scale bar, 50 μm. Time interval, 33.9 s. Representative video of at least 3 independent experiments is shown. (MOV 635 kb)
De novo nuclear translocation of NFAT biosensor in a TMBP-NFAT/YFP-H2B/Cherry cell in leptomeninges of the spinal cord. 2-PM data of the leptomeninges in the preclinincal phase of EAE are shown. Blood vessels and meningeal phagocytes were labeled as described for Supplementary Video 3; yellow line (dotted): perivascular meningeal phagocyte; arrows indicate T cell position and status before (white) and after nuclear translocation of NFAT biosensor (yellow). Scale bar, 50 μm. Time interval, 35.5 s. Representative video of at least 3 independent experiments is shown. (MOV 635 kb)
Induction of nuclear NFAT translocation in TMBP-NFAT/YFP-Cherry/H2B cells after antigen challenge. 2-PM videos were recorded during the acute phase of EAE. During the imaging i.v. injection of MBP was performed. Representative spots before (left) and 2 h after soluble MBP injection (right) are shown. Post-production videos are depicted. Lines: trajectories of TMBP-NFAT/YFP-Cherry/H2B cells with either cytoplasmic (white) or nuclear NFAT (yellow). Circles: stationary TMBP-NFAT/YFP-Cherry/H2B cells with cytoplasmic (white) or nuclear NFAT biosensor (yellow). Red: meningeal phagocytes. Scale bar, 50 μm; Time interval, 47.6 s. Representative videos of at least 3 independent experiments are shown. (MOV 1532 kb)
Visualization of mitosis in a TMBP-NFAT/YFP-Cherry/H2B cell within the CNS. A TMBP-NFAT/YFP-Cherry/H2B cell is shown undergoing mitosis in the CNS during the clinical phase of EAE. Inset: magnified region with its origin indicated by white frames. White arrows: position of an individual T cells; meningeal phagocytes were labeled TR-dextran (red). Scale bar, 50 μm. Time interval, 47.8 s. (MOV 918 kb)
Motility of TMBP-NFAT/YFP-Cherry/H2B cells within the leptomeninges during the acute phase of EAE. TMBP-NFAT/YFP-Cherry/H2B cells were recorded by 2-PM during the acute phase of the disease. Left: trajectories of motile T cells with nuclear or cytoplasmic NFAT biosensor (yellow and white lines, respectively). Right: stationary T cells with nuclear or cytoplasmic NFAT sensor (yellow and white circles, respectively). Scale bar, 50 μm. Time interval, 35.9 s. A representative video of at least 3 independent experiments is shown. (MOV 900 kb)
TOVA-NFAT/YFP-Cherry/H2B cells that cannot recognize CNS antigens are not activated in the leptomeninges. 2-PM movies of TOVA-NFAT/YFP-Cherry/H2B cells (co-injected with unlabeled TMBP cells) during the acute phase of the disease are shown. Trajectories of motile (white lines, left) or stationary (white circles, right) T cells with cytoplasmic NFAT sensor are depicted. Scale bar, 50 μm. Time interval, 38.9 s. A representative video of at least 3 independent experiments is shown. (MOV 253 kb)
Activated TMBP-NFAT/YFP lymphocyte in contact with a resident Iba1+ cell. Maximum intensity projection (left) and animated 3D-reconstruction (right) of a confocal image stack are shown. Images were acquired at the meningeal/subpial white matter interface of the lumbal spinal cord of an animal with preclinical EAE. Tissue section was stained with anti-Iba1 antibody (red) and DAPI. Scale bar, 10 μm. (MOV 253 kb)
De novo nuclear translocation of NFAT biosensor in CNS parenchyma. 2-PM data of acute CNS slices at the peak of clinical EAE are depicted. Arrows indicate T cell position and activation status (white, before nuclear translocation of NFAT biosensor; yellow, after translocation of NFAT). Scale bar, 20 μm. Time interval, 32 s. A representative video of at least 3 independent experiments is shown. (MOV 307 kb)
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Lodygin, D., Odoardi, F., Schläger, C. et al. A combination of fluorescent NFAT and H2B sensors uncovers dynamics of T cell activation in real time during CNS autoimmunity. Nat Med 19, 784–790 (2013). https://doi.org/10.1038/nm.3182
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