Name: induction of paralysis and visual system injury in mice by t cells specific for neuromyelitis optica autoantigen aquaporin-4
Uploaded: 2017-08-21T15:00:00.000+00:00
Duration: 9 min 29 s
Description: Watch this Scientific Journal Video about Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4 at JoVE.com

Support was provided to S.S.Z. by the National Institute of Health (RO1 AI073737 and RO1 NS092835-01), National Multiple Sclerosis Society (RG 4768, RG 5179 and RG 5180), Maisin Foundation and Guthy Jackson Charitable Foundation.

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+aquaporin-4+specific+T+cells:+towards+a+model+for+neuromyelitis+optica.">Functional characterization of aquaporin-4 specific T cells: towards a model for neuromyelitis optica.</a> PLoS One. 6 (1), 16083 (2011).</li><li>Pohl, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+T+cell+responses+against+aquaporin+4.">Pathogenic T cell responses against aquaporin 4.</a> Acta Neuropathol. 122 (1), 21-34 (2011).</li><li>Zeka, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+encephalitogenic+aquaporin+4-specific+T+cells+and+NMO-IgG+jointly+orchestrate+lesion+location+and+tissue+damage+in+the+CNS.">Highly encephalitogenic aquaporin 4-specific T cells and NMO-IgG jointly orchestrate lesion location and tissue damage in the CNS.</a> Acta Neuropathol. 130 (6), 783-798 (2015).</li><li>Sagan, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+checkpoint+bypass+permits+emergence+of+pathogenic+T+cells+to+neuromyelitis+optica+autoantigen+aquaporin-4.">Tolerance checkpoint bypass permits emergence of pathogenic T cells to neuromyelitis optica autoantigen aquaporin-4.</a> Proc Natl Acad Sci U S A. 113 (51), 14781-14786 (2016).</li><li>Vita, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+epitope+database+(IEDB)+3.0.">The immune epitope database (IEDB) 3.0.</a> Nucleic Acids Res. 43, 405-412 (2015).</li><li>Harrell, M. I., Iritani, B. 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(82), e51018 (2013).</li><li>Cruz-Herranz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+APOSTEL+recommendations+for+reporting+quantitative+optical+coherence+tomography+studies.">The APOSTEL recommendations for reporting quantitative optical coherence tomography studies.</a> Neurology. 86 (24), 2303-2309 (2016).</li><li>Mendel, I., Kerlero de Rosbo, N., Ben-Nun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myelin+oligodendrocyte+glycoprotein+peptide+induces+typical+chronic+experimental+autoimmune+encephalomyelitis+in+H-2b+mice:+fine+specificity+and+T+cell+receptor+V+beta+expression+of+encephalitogenic+T+cells.">A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells.</a> Eur J Immunol. 25 (7), 1951-1959 (1995).</li><li>Shetty, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodominant+T-cell+epitopes+of+MOG+reside+in+its+transmembrane+and+cytoplasmic+domains+in+EAE.">Immunodominant T-cell epitopes of MOG reside in its transmembrane and cytoplasmic domains in EAE.</a> Neurol Neuroimmunol Neuroinflamm. 1 (2), 22 (2014).</li><li>Molnarfi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+II-dependent+B+cell+APC+function+is+required+for+induction+of+CNS+autoimmunity+independent+of+myelin-specific+antibodies.">MHC class II-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies.</a> J Exp Med. 210 (13), 2921-2937 (2013).</li><li>Jones, M. 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S.A. Sagan, A. Cruz-Herranz, C.M. Spencer, P.P. Ho, L. Steinman, A.J. Green, and R.A. Sobel report no disclosures. S.S. Zamvil is Deputy Editor of Neurology, Neuroimmunology and Neuroinflammation and is a member of the advisory board for the International Society of Neuroimmunology. He has served on the Editorial Board of the Journal of Clinical Investigation, The Journal of Immunology and The Journal of Neurological Sciences, and has been a charter member of the grant review committee for the National Institutes of Health (NIH) Clinical Neuroimmunology and Brain Tumors (CNBT) study section and the National Multiple Sclerosis Society (NMSS). He has served as a consultant and received honoraria from Biogen-Idec, EMD-Serono, Genzyme, Novartis, Roche/Genentech, and Teva Pharmaceuticals, Inc., and has served or serves on Data Safety Monitoring Boards for Lilly, BioMS, Teva and Opexa Therapeutics. Currently, Dr. Zamvil receives research grant support from the NIH, the NMSS, The Maisin Foundation, Biogen and Celgene.

AQP4 was identified as the primary target in NMO IgG in 20053. Then, it was recognized that it would be important to establish an AQP4-targeted animal model of CNS autoimmunity. Such a model could be useful to investigate how AQP4-specific T and B cells participate in development of CNS autoimmunity and to test candidate therapeutics for NMO. Although identification of AQP4-specific T cell epitopes in wild-type mice was first reported in 201013, T cells responding to those epitopes did not cause clinical or histologic disease14,17. The inability to generate a model of CNS autoimmunity based upon immune reactivity to AQP4 remained an enigma until 2015 when Jones, et al.25 discovered that donor AQP4 p135-153-primed T cells from AQP4-/- mice were capable of causing clinical and histologic signs of CNS autoimmunity in WT mice. Of interest, AQP4 p135-153 is predicted to bind MHC II (I-Ab) with high affinity17. Only one other AQP4 amino acid sequence, 201-220, is predicted to bind I-Ab with similar high affinity. Indeed, we observed that AQP4 p135-153 and p201-220 both elicit robust proliferation in AQP4-/-, but not WT, mice. Here, we have shown how one can isolate and expand encephalitogenic Th17 AQP4 p135-153- and p201-220-reactive T cells from AQP4-/- mice. When transferred into WT recipient mice, donor AQP4-reactive Th17 cells induced paralysis, which was accompanied by mononuclear cell infiltrates in spinal cord and optic nerve. Afferent visual system injury is well known in patients with AQP4-seropositive NMOSD26. Here, we observed that optic nerve inflammation induced by AQP4-reactive and MOG-reactive Th17 cells was distinct. Whereas AQP4-specific Th17 cells induced optic perineuritis, MOG-specific Th17 cells induced severe optic neuritis. We have also described the techniques used to monitor optic nerve inflammation induced by AQP4-specific and MOG-specific T cells by serial OCT. Other investigators should now be able to apply the protocols described here to advance their own studies focused on pathogenic mechanisms of ATCA.
One can easily avoid three potential pitfalls in our protocol. First, adoptive transfer of ATCA requires use of AQP4-specific T cells from AQP4-/- donor mice. Specifically, donor WT AQP4 p135-153-specific T cells did not cause ATCA in recipient WT mice. Secondly, it is important to perform a &#34;water-test&#34; with a drop of the peptide/CFA emulsion before immunization of AQP4-/- mice (Protocol Step 1.5). The emulsion should not disperse in water when it is suitable for s.c. injection. If the emulsion disperses, one should mix the emulsion once more, chill again and repeat the water-test. Lastly, activated donor CNS antigen-specific T cells induce CNS autoimmunity more efficiently than resting T cells. One should visually inspect those cultures under the light microscope prior to harvesting the donor T cells for adoptive transfer. Rapidly dividing cells may form clusters, which are easily identified. Also, when cultures contain many activated T cells, the media may transition from pink to orange or even to yellow, due to reduction in pH. One can also assess activation of donor AQP4-primed lymph node T cells for proliferation by 3H-thymidine incorporation, as described in Protocol Step 3.
Our discovery that the two pathogenic AQP4 T cell epitopes are (1) predicted to bind MHC II with high affinity and (2) elicit potent proliferative responses in AQP4-/-, but not WT, mice suggest that T cells targeting those determinants are normally controlled by thymic negative selection17. The TCR repertoires utilized for recognition of AQP4 p135-153 and p201-220 in AQP4-/- mice are unique (Figure 2), which is also consistent with clonal deletion mediated by thymic medullary epithelial cells. Other tolerogenic mechanisms may normally restrain immune responses to AQP4. Subsequent to our initial report17, another group also demonstrated that AQP4 p201-220 contains an encephalitogenic T cell determinant27. When &#945;/&#946; (TCR&#945;-/-) T cell-deficient mice were reconstituted with AQP4-/- CD4+ T cells, it was possible to elicit an encephalitogenic AQP4-specific T cell response, but not an AQP4-specific humoral response, implying that in WT mice AQP4-specific B cell responses, similar to AQP4-specific T cell responses, are subject to negative selection. Indeed, loss of spinal cord axons and RGCs, which was not observed in WT mice with ATCA induced by AQP4-specific T cells alone, may require participation of pathogenic AQP4-specific antibodies. It is clear that mouse models of AQP4-targeted CNS autoimmunity will continue to evolve as we learn more regarding the tolerogenic mechanisms normally controlling AQP4-specific T cell and B cell immunity.
Other models of AQP4-targeted CNS autoimmunity are also being developed6,16,28,29,30. Each one may offer advantages for studying particular aspects that are relevant to NMO pathogenesis. AQP4-specific T cells have been identified in WT rats6,16,29. Those AQP4-specific T cells caused histologic changes of CNS autoimmunity but, similar to observations in mice, WT rat AQP4-specific T cells do not cause significant signs of clinical disease. Therefore, the mechanisms of tolerance restricting T cell and B cell AQP4-specific immune responses in WT mice are also operational in rats. Regardless, one should not underestimate the power in using mouse models for studying mechanisms involved in pathogenesis of disease. The wealth of knock-out, transgenic and reporter mice can be advantageous. It should also be recognized that several fundamental discoveries in autoimmunity were made using mouse EAE models. For example, demonstration that T cell clones specific for a self-antigen can mediate autoimmune disease31,32, identification of the role of T cell costimulation in autoimmunity33 and the discovery of the developmental pathway for Th17 differentiation34 were first described using mouse EAE models. Using the mouse model of ATCA that we have developed, one now has the means to study development and regulation of pathogenic AQP4-specific immune responses in vivo, which should provide important insights related to NMO pathogenesis.

Neuromyelitis optica (NMO) is a central nervous system (CNS) autoimmune inflammatory demyelinating disease that causes recurrent episodes of paralysis and visual loss leading to permanent neurologic disability1. NMO is currently considered primarily to be a humoral autoimmune disease2 as it is associated with antibodies (Igs) targeting aquaporin-4 (AQP4), a water channel expressed abundantly on astrocytes3,4. However, CNS inflammation is a prerequisite for CNS entry of AQP4 Ig5,6. Thus, it has not been possible to establish a model of NMO by transfer of anti-AQP4 Igs alone. The findings that (1) pathogenic AQP4-specific Igs in NMO patients are IgG11,2, a T cell-dependent Ig subclass7 (2) T cells are identified in NMO lesions8,9 (3) NMO is associated with certain MHC II genes (e.g. HLA-DR17 (DRB1*0301))10, and (4) proinflammatory AQP4-reactive DR-restricted Th17 cells are expanded in NMO patients11,12 all indicate that AQP4-specific T cells have a key role in NMO pathogenesis. Thus, it is important to develop animal models to determine how AQP4-specific T cells may contribute to NMO pathogenesis.
Several years ago, multiple AQP4 T cell epitopes were identified in wild-type (WT) mice13,14 and rats15. While it was observed that AQP4-reactive T cells could induce opticospinal inflammation in na&#239;ve recipient rats15,16, significant clinical signs of CNS disease were not observed. Similarly, direct immunization of WT mice with peptides containing AQP4 T cell epitopes14,17, or transfer of proinflammatory T cells targeting those determinants17, did not cause clinical signs or histologic evidence of CNS autoimmunity.
Recently, it was observed that immunization of C57BL/6 AQP4-deficient (AQP4-/-) mice with AQP4 peptide (p) 135-153 or p201-220, two determinants predicted to bind MHC II (I-Ab) with high affinity18, elicited strong CD4+ T cell responses17. In contrast, these two peptides elicited only modest proliferative responses in WT mice. Further, the T cell receptor (TCR) repertoire used for recognition of these determinants by T cells from AQP4-/- mice was unique. Collectively, these findings indicate that T cell recognition of AQP4 is regulated by thymic negative selection. AQP4 p135-153- or p201-220-specific Th17 cells from AQP4-/- donor mice induced paralysis in nearly 100% of na&#239;ve recipient WT mice; this was associated with opticospinal infiltrates of T cells, B cells and monocytes. Serial opticospinal coherence tomography (OCT) demonstrated dynamic visual system involvement. Mice with T cell-mediated AQP4-targeted CNS autoimmunity (ATCA) recovered from paralysis and visual system injury. In contrast to EAE induced by myelin oligodendrocyte glycoprotein (MOG) p35-55-specific T cells, which led to persistent clinical disease, ATCA induced by T cells alone was not associated with axonal loss or reduction in retinal ganglion cells (RGCs). Our results have clearly demonstrated that there are multiple pathogenic AQP4 T cell determinants. This new model of ATCA is useful for studying mechanisms controlling development of pathogenic AQP4-specific T cells, learning how those cells induce CNS inflammation and how they may cooperate with AQP4-specific B cells and antibodies to promote NMO pathogenesis.
In the present report, we describe the protocols used to induce and evaluate T cell-induced ATCA. We begin with the techniques used for immunization, T cell culture and Th17 polarization to generate pathogenic AQP4-specific T cells, flow cytometry analysis to confirm polarization and adoptive transfer of those T cells. We then describe methods used to evaluate clinical and histologic disease and the use of serial OCT to monitor visual system injury in recipient mice.

<table><tbody><tr><td>&lt;em&gt;&lt;span class=&quot;font8&quot;&gt;M. tuberculosis &lt;/span&gt;&lt;/em&gt;&lt;span class=&quot;font6&quot;&gt;H37Ra&lt;/span&gt;</td><td>BD Difco</td><td>231141</td><td>Dessicated, killed &lt;em&gt;&lt;span class=&quot;font8&quot;&gt;M. tuberculosis&lt;/span&gt;&lt;/em&gt;</td></tr><tr><td>Incomplete Freund&#039;s Adjuvant</td><td>BD Difco</td><td>263910</td><td></td></tr><tr><td>AQP4 peptide p135-153</td><td>Genemed</td><td>Custom Synthesis</td><td>Peptide sequence: LVTPPSVVGGLGVTMVHGN</td></tr><tr><td>AQP4 peptide p201-220</td><td>Genemed</td><td>Custom Synthesis</td><td>Peptide sequence: HLFAINYTGASMNPARSFGP</td></tr><tr><td>MOG peptide p35-55</td><td>Genemed / Auspep</td><td>Custom Synthesis</td><td>Peptide sequence: MEVGWYRSPFSRVVHLYRNGK</td></tr><tr><td>3-way Stopcock</td><td>Kimble</td><td>420163-4503</td><td></td></tr><tr><td>HyClone Fetal Bovine Serum (Characterized)</td><td>GE Healthcare Life Sciences</td><td>SH30071</td><td></td></tr><tr><td>Recombinant Mouse IL-23</td><td>R&amp;amp;D Systems (BioTechne)</td><td>1887-ML</td><td></td></tr><tr><td>Recombinant Mouse IL-6</td><td>R&amp;amp;D Systems (BioTechne)</td><td>406-ML</td><td></td></tr><tr><td>Recombinant Mouse IL-12</td><td>R&amp;amp;D Systems (BioTechne)</td><td>419-ML</td><td></td></tr><tr><td>Thymidine [Methyl-&lt;span class=&quot;font12&quot;&gt;&lt;sup&gt;3&lt;/sup&gt;&lt;/span&gt;&lt;span class=&quot;font6&quot;&gt;H]&lt;/span&gt;</td><td>PerkinElmer</td><td>NET027</td><td></td></tr><tr><td>Glass Fiber Filtermats</td><td>PerkinElmer</td><td>1450-421</td><td></td></tr><tr><td>Anti-mouse antibodies</td><td>eBioscience (Affymetrix)</td><td>[various]</td><td></td></tr><tr><td>Anti-mouse TCR V&amp;beta; Screening Panel</td><td>BD Biosciences</td><td>557004</td><td></td></tr><tr><td>LIVE/DEAD Fixable Dead Cell Stain</td><td>ThermoFisher Scientific</td><td>[various]</td><td></td></tr><tr><td>Paraformaldehyde (16%)</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Fixation/Permeabilization Solution Kit with GolgiPlug</td><td>BD Biosciences</td><td>555028</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>Iomomycin (calcium salt)</td><td>Sigma-Aldrich</td><td>I0634</td><td></td></tr><tr><td>Pertussis Toxin from &lt;em&gt;&lt;span class=&quot;font8&quot;&gt;B. pertussis&lt;/span&gt;&lt;/em&gt;</td><td>List Biological Laboratories</td><td>181</td><td></td></tr><tr><td>10% Formalin</td><td>VWR</td><td>89370-094</td><td></td></tr><tr><td>Variable-Flow Peristaltic Pump</td><td>Fisher Scientific</td><td>13-876-2</td><td></td></tr><tr><td>Foam Biopsy Pads, Rectangular</td><td>Fisher Scientific</td><td>22-038-221</td><td></td></tr><tr><td>Isothesia (isoflurane, USP)</td><td>Henry Schein Animal Health</td><td>050033</td><td>NDC : 11695-0500-2</td></tr><tr><td>Tropicamide Ophthalmic Solution, USP (1%)</td><td>Akorn</td><td></td><td>NDC: 17478-102-12</td></tr><tr><td>Spectralis Diagnostic Imaging Platform</td><td>Heidelberg Engineering</td><td></td><td></td></tr></tbody></table>

induction of paralysis and visual system injury in mice by t cells specific for neuromyelitis optica autoantigen aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

In this protocol, we used donor T cells from C57BL/6 AQP4-/- mice. Subcutaneous immunization of these mice with AQP4 p135-153 or p201-220, which contain pathogenic T cell epitopes, elicited strong proliferative T cell responses in draining lymph nodes (Figure 1), whereas these two peptides induced much weaker T cell proliferation in WT mice. In comparison, immunization with AQP4 p91-110, containing a non-pathogenic AQP4 T cell determinant13, or MOG p35-55, a myelin peptide that activates T cells that cause experimental autoimmune encephalomyelitis (EAE)22,23,24, induced similar magnitude of T cell proliferation in AQP4-/- and WT mice. Analysis of TCR utilization by flow cytometry staining for individual V&#946;&#39;s or V&#946; families, demonstrated that p135-153- and p201-220-specific T cells from AQP4-/- mice utilized unique TCR repertoires. Selective hyper-proliferation of AQP4 p135-153 and p201-220 in AQP4-/- mice, as well as the unique TCR utilization (Figure 2), indicated that pathogenic T cell responses to these determinants is normally regulated by thymic negative selection, underscoring the importance for using AQP4-/- donor T cells in this protocol.
Prior to adoptive transfer for induction of ATCA, lymph node cells from AQP4 peptide-primed mice were cultured in vitro in Th17- or Th1-polarizing conditions for three days. The extent of polarization of donor CD4+ T cells was confirmed by intracellular cytokine staining (ICS) and measured by flow cytometry (Figure 3). Na&#239;ve recipient mice were injected intravenously with 2 x 107 donor AQP4 peptide-specific T cells. After approximately six days, nearly 100% of recipient mice developed clinical signs of CNS autoimmune disease, including limp tail and hind limb paralysis (Figure 4). Th17-polarized AQP4-specific T cells induced more severe clinical disease than Th1-polarized AQP4-specific T cells. A representative mouse that received Th17 AQP4-peptide-specific and developed complete hind limb paralysis (paraplegia) is shown in Video 1. In contrast with mice that developed EAE after administration of MOG-specific Th17 cells, recipient mice recovered from clinical disease induced by AQP4-specific Th17 cells. As for EAE induced by MOG p35-55-specific Th17 cells, clinical disease induced by AQP4 p135-153-specific or p201-220-specific Th17 cells was associated with infiltration of mononuclear cells in the CNS parenchyma and meninges (Figure 5). Lesions were more abundant in the meninges than in the parenchyma for CNS autoimmunity induced by AQP4-specific Th17 cells.
Optic nerve involvement was demonstrated by histological evaluation and by serial OCT. AQP4-specific and MOG-specific Th17 cells both induced optic nerve inflammation, which was characterized by presence of mononuclear cells. Whereas AQP4-specific Th17 cells caused optic perineuritis, MOG-specific Th17 cells induced severe optic neuritis (Figure 5). Using serial OCT, optic nerve inflammation was evident by swelling and increased inner retinal layer (IRL) thickness for clinical disease induced by AQP4-specific or MOG-specific Th17 cells (Figure 6). For AQP4 Th17-induced CNS autoimmunity, IRL thickness returned to baseline as mice recovered from clinical disease. In contrast, persistence of EAE induced by MOG-specific Th17 corresponded with IRL thinning and, as we demonstrated previously17, was associated with loss of retinal ganglion cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56185/56185fig1.jpg" /> 
Figure 1: AQP4 p135-153 and p201-220 elicit robust T cell proliferation in AQP4-/- mice, but not WT mice. Mice were immunized s.c. with the indicated peptides in CFA. Eleven days later, lymph nodes were removed, and then cultured with either no antigen, or with the peptide used for immunization. Proliferation was measured by 3H-thymidine incorporation (mean &#177; SEM, representative of 5 experiments). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56185/56185fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56185/56185fig2.jpg" /> 
Figure 2: AQP4-specific T cells from AQP4-/- mice utilize unique TCR repertoires. AQP4-/- and WT mice were immunized with the indicated peptides. Eleven days later, lymph nodes were removed and cultured with peptide used for immunization. Cells were harvested. TCR V&#946; utilization was analyzed by flow cytometry (mean &#177; SEM, n = 5). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56185/56185fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56185/56185fig3.jpg" /> 
Figure 3: Proinflammatory polarization of donor AQP4-specific T cells. Eleven days after immunization with AQP4 p135-153 or p201-220, lymph node cells were harvested and cultured with the peptide used for immunization in non-polarizing conditions, Th1- or Th17-polarizing conditions. Th17 or Th1 polarization was examined by ICS and flow cytometry for IL-17 or IFN-&#947;, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56185/56185fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56185/56185fig4.jpg" /> 
Figure 4: Th17-polarized AQP4-specific T cells induce paralysis in WT recipient mice. WT recipient mice received 2 x 107 donor Th17-polarized AQP4 p135-153 or p201-220-specific T cells from AQP4-/- mice. Th17-polarized MOG-specific T cells served as a positive control. Results are representative of 8 experiments (n = 5/group). * p &#60;0.05, ** p &#60;0.01, *** p &#60;0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56185/56185fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56185/56185fig5.jpg" /> 
Figure 5: AQP4-specific Th17 cells induce opticospinal inflammation in WT mice. Recipient mice received 2 x 107 donor Th17 AQP4 p135-153- or p201-220-primed Th17 cells from AQP4-/- mice or Th17 MOG p35-55-specific cells and were sacrificed 10 days later. Spinal cord and optic nerve tissues were prepared and stained with H&#38;E/LFB to evaluate for evidence of inflammation and demyelination, respectively. Results are representative of 5 mice/group. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56185/56185fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56185/56185fig6.jpg" /> 
Figure 6: Optic nerve inflammation induced by AQP4-specific Th17 cells can be monitored by longitudinal retinal OCT. WT recipient mice received 2 x 107 donor Th17-polarized AQP4 p201-220-specific or MOG p35-55-specific T cells on day 0. They were examined by OCT on day 0 (before administration of T cells) and then on days 4, 7, 8, 9 10, 11 14 and 21. IRL thickness was measured (mean &#177; SEM). Statistics indicate a comparison with na&#239;ve control. Results are representative of 3 experiments (5 mice/group). * p &#60;0.05, ** p &#60;0.01, *** p &#60;0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56185/56185fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4 at JoVE.com

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

induction-paralysis-visual-system-injury-mice-t-cells-specific-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.3K Views.  University of California. Hier presenteren we een protocol om te veroorzaken verlamming en opticospinal ontsteking door overdracht van aquaporin-4 (AQP4)-specifieke T-cellen van de AQP4- / - muizen in WT muizen. Daarnaast tonen we hoe met seriële optische coherentie tomografie controleren visuele systeem dysfunctie.

Video: Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

Medicine

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Neuroscience

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

Biology

Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Tietz, S. M. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59249">Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo.</a> J. Vis. Exp.&#160; (2019)
This video demonstrates the method of inducing experimental autoimmune encephalomyelitis (EAE) by injecting myelin-derived peptides with an adjuvant to activate autoreactive T cells, which migrate to the brain. Pertussis toxin is administered to increase blood-brain barrier permeability, allowing these T cells and other immune cells to enter the brain. These immune cells cause demyelination, leading to impaired nerve transmission and development of EAE.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. EAE ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

Induction of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Schmitz, K., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55321">Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice.</a> J. Vis. Exp (2017).
In this video, a mouse model of multiple sclerosis is established using experimental autoimmune encephalomyelitis (EAE). The mouse is injected with an emulsion containing a neuronal peptide and an adjuvant, triggering an immune response that activates autoreactive T cells. This is followed by an injection of pertussis toxin (PTX) to increase blood-brain barrier permeability, allowing the autoreactive T-cells to infiltrate the brain. This infiltration initiates a signaling cascade that ultimately results in myelin damage, mimicking the demyelinating condition of multiple sclerosis.

1. EAE Induction in SJL/J Mice

	Mice
	
		Use 11-week-old female SJL/J mice and allow them to habituate to the experimental room for about 7 days. Use n = ...

Neuromuscular Disorders

Inducing Paralysis in a Mouse Model via Transfer of Aquaporin-4-Specific Th17 Cells

Source: Sagan, S. A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56185">Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4.</a> J. Vis. Exp. (2017)
This video demonstrates the procedure of inducing paralysis by transferring activated Th17 cells targeting aquaporin-4 into a mouse model. In the central nervous system, the activated Th17 cells interact with aquaporin-4-expressing astrocytes, triggering an immune response that leads to tail and limb paralysis in the mouse.

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

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