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.

<ol>
	<li>Hardy, T. 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=Atypical+inflammatory+demyelinating+syndromes+of+the+CNS.">Atypical inflammatory demyelinating syndromes of the CNS.</a> Lancet Neurol. 15 (9), 967-981 (2016).</li><li>Lennon, V. 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=A+serum+autoantibody+marker+of+neuromyelitis+optica:+distinction+from+multiple+sclerosis.">A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis.</a> Lancet. 364 (9451), 2106-2112 (2004).</li><li>Lennon, V. A., Kryzer, T. J., Pittock, S. J., Verkman, A. S., Hinson, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgG+marker+of+optic-spinal+multiple+sclerosis+binds+to+the+aquaporin-4+water+channel.">IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel.</a> J Exp Med. 202 (4), 473-477 (2005).</li><li>Zamvil, S. S., Slavin, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+MOG+Ig-positive+AQP4-seronegative+opticospinal+inflammatory+disease+justify+a+diagnosis+of+NMO+spectrum+disorder.">Does MOG Ig-positive AQP4-seronegative opticospinal inflammatory disease justify a diagnosis of NMO spectrum disorder.</a> Neurol Neuroimmunol Neuroinflamm. 2 (1), 62 (2015).</li><li>Bennett, J. L., 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=Intrathecal+pathogenic+anti-aquaporin-4+antibodies+in+early+neuromyelitis+optica.">Intrathecal pathogenic anti-aquaporin-4 antibodies in early neuromyelitis optica.</a> Ann Neurol. 66 (5), 617-629 (2009).</li><li>Bradl, 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=Neuromyelitis+optica:+pathogenicity+of+patient+immunoglobulin+in+vivo.">Neuromyelitis optica: pathogenicity of patient immunoglobulin in vivo.</a> Ann Neurol. 66 (5), 630-643 (2009).</li><li>Nurieva, R. I., Chung, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+development+and+function+of+T+follicular+helper+cells.">Understanding the development and function of T follicular helper cells.</a> Cell Mol Immunol. 7 (3), 190-197 (2010).</li><li>Lucchinetti, C. F., 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+pathology+of+an+autoimmune+astrocytopathy:+lessons+learned+from+neuromyelitis+optica.">The pathology of an autoimmune astrocytopathy: lessons learned from neuromyelitis optica.</a> Brain Pathol. 24 (1), 83-97 (2014).</li><li>Zekeridou, A., Lennon, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquaporin-4+autoimmunity.">Aquaporin-4 autoimmunity.</a> Neurol Neuroimmunol Neuroinflamm. 2 (4), 110 (2015).</li><li>Brum, D. G., 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=HLA-DRB+association+in+neuromyelitis+optica+is+different+from+that+observed+in+multiple+sclerosis.">HLA-DRB association in neuromyelitis optica is different from that observed in multiple sclerosis.</a> Mult Scler. 16 (1), 21-29 (2010).</li><li>Varrin-Doyer, 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=Aquaporin+4-specific+T+cells+in+neuromyelitis+optica+exhibit+a+Th17+bias+and+recognize+Clostridium+ABC+transporter.">Aquaporin 4-specific T cells in neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC transporter.</a> Ann Neurol. 72 (1), 53-64 (2012).</li><li>Vaknin-Dembinsky, 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=T-cell+responses+to+distinct+AQP4+peptides+in+patients+with+neuromyelitis+optica+(NMO).">T-cell responses to distinct AQP4 peptides in patients with neuromyelitis optica (NMO).</a> Mult Scler Relat Disord. 6, 28-36 (2016).</li><li>Nelson, P. 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+determinants+of+aquaporin-4,+the+autoantigen+associated+with+neuromyelitis+optica.">Immunodominant T cell determinants of aquaporin-4, the autoantigen associated with neuromyelitis optica.</a> PLoS One. 5 (11), 15050 (2010).</li><li>Kalluri, S. 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. M., Ruddell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+mapping+in+the+mouse.">Lymph node mapping in the mouse.</a> J Immunol Methods. 332 (1-2), 170-174 (2008).</li><li>Ivanova, E., Toychiev, A. H., Yee, C. W., Sagdullaev, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocol+for+retinal+wholemount+preparation+for+imaging+and+immunohistochemistry.">Optimized protocol for retinal wholemount preparation for imaging and immunohistochemistry.</a> J Vis Exp. (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. V., Huang, H., Calabresi, P. A., Levy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+aquaporin-4+reactive+T+cells+are+sufficient+to+induce+mouse+model+of+neuromyelitis+optica.">Pathogenic aquaporin-4 reactive T cells are sufficient to induce mouse model of neuromyelitis optica.</a> Acta Neuropathol Commun. 3, 28 (2015).</li><li>Oertel, F. C., 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=Microstructural+visual+system+changes+in+AQP4-antiboby-seropositive+NMOSD.">Microstructural visual system changes in AQP4-antiboby-seropositive NMOSD.</a> Neurol. Neuroimmunol. Neuroinflamm. 4, 72 (2017).</li><li>Vogel, A. L., 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=Deletional+tolerance+prevents+AQP4+directed+autoimmunity+in+mice.">Deletional tolerance prevents AQP4 directed autoimmunity in mice.</a> Eur J Immunol. , (2017).</li><li>Zhang, H., Verkman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinally+extensive+NMO+spinal+cord+pathology+produced+by+passive+transfer+of+NMO-IgG+in+mice+lacking+complement+inhibitor+CD59.">Longitudinally extensive NMO spinal cord pathology produced by passive transfer of NMO-IgG in mice lacking complement inhibitor CD59.</a> J Autoimmun. 53, 67-77 (2014).</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=Aquaporin+4-specific+T+cells+and+NMO-IgG+cause+primary+retinal+damage+in+experimental+NMO/SD.">Aquaporin 4-specific T cells and NMO-IgG cause primary retinal damage in experimental NMO/SD.</a> Acta Neuropathol Commun. 4 (1), 82 (2016).</li><li>Felix, C. M., Levin, M. H., Verkman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement-independent+retinal+pathology+produced+by+intravitreal+injection+of+neuromyelitis+optica+immunoglobulin+G.">Complement-independent retinal pathology produced by intravitreal injection of neuromyelitis optica immunoglobulin G.</a> J Neuroinflammation. 13 (1), 275 (2016).</li><li>Zamvil, S., 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=T-cell+clones+specific+for+myelin+basic+protein+induce+chronic+relapsing+paralysis+and+demyelination.">T-cell clones specific for myelin basic protein induce chronic relapsing paralysis and demyelination.</a> Nature. 317 (6035), 355-358 (1985).</li><li>Zamvil, S. S., 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=Encephalitogenic+T+cell+clones+specific+for+myelin+basic+protein.+An+unusual+bias+in+antigen+recognition.">Encephalitogenic T cell clones specific for myelin basic protein. An unusual bias in antigen recognition.</a> J Exp Med. 162 (6), 2107-2124 (1985).</li><li>Kuchroo, V. K., 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=B7-1+and+B7-2+costimulatory+molecules+activate+differentially+the+Th1/Th2+developmental+pathways:+application+to+autoimmune+disease+therapy.">B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy.</a> Cell. 80 (5), 707-718 (1995).</li><li>Bettelli, E., 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=Reciprocal+developmental+pathways+for+the+generation+of+pathogenic+effector+TH17+and+regulatory+T+cells.">Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.</a> Nature. 441 (7090), 235-238 (2006).</li></ol>

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 width="1066">
	<colgroup>
		<col style="width:246pt" width="328" />
		<col style="width:162pt" width="216" />
		<col style="width:104pt" width="139" />
		<col style="width:287pt" width="383" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">M. tuberculosis H37Ra</td>
			<td class="xl19" style="width:162pt" width="216">BD Difco</td>
			<td class="xl24" style="width:104pt" width="139">231141</td>
			<td class="xl22" style="width:287pt" width="383">Dessicated, killed M. tuberculosis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Incomplete Freund&#39;s Adjuvant</td>
			<td class="xl19" style="width:162pt" width="216">BD Difco</td>
			<td class="xl24" style="width:104pt" width="139">263910</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">AQP4 peptide p135-153</td>
			<td class="xl19" style="width:162pt" width="216">Genemed</td>
			<td class="xl24" style="width:104pt" width="139">Custom Synthesis</td>
			<td class="xl22">Peptide sequence: LVTPPSVVGGLGVTMVHGN</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">AQP4 peptide p201-220</td>
			<td class="xl19" style="width:162pt" width="216">Genemed</td>
			<td class="xl24" style="width:104pt" width="139">Custom Synthesis</td>
			<td class="xl22">Peptide sequence: HLFAINYTGASMNPARSFGP</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">MOG peptide p35-55</td>
			<td class="xl19" style="width:162pt" width="216">Genemed / Auspep</td>
			<td class="xl24" style="width:104pt" width="139">Custom Synthesis</td>
			<td class="xl22">Peptide sequence: MEVGWYRSPFSRVVHLYRNGK</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">3-way Stopcock</td>
			<td class="xl19" style="width:162pt" width="216">Kimble</td>
			<td class="xl24" style="width:104pt" width="139">420163-4503</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">HyClone Fetal Bovine Serum (Characterized)</td>
			<td class="xl19" style="width:162pt" width="216">GE Healthcare Life Sciences</td>
			<td class="xl24" style="width:104pt" width="139">SH30071</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Recombinant Mouse IL-23</td>
			<td class="xl19" style="width:162pt" width="216">R&#38;D Systems (BioTechne)</td>
			<td class="xl24" style="width:104pt" width="139">1887-ML</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Recombinant Mouse IL-6</td>
			<td class="xl19" style="width:162pt" width="216">R&#38;D Systems (BioTechne)</td>
			<td class="xl24" style="width:104pt" width="139">406-ML</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Recombinant Mouse IL-12</td>
			<td class="xl23" style="width:162pt" width="216">R&#38;D Systems (BioTechne)</td>
			<td class="xl24" style="width:104pt" width="139">419-ML</td>
			<td class="xl22"></td>
		</tr>
		<tr height="25" style="height:18.75pt">
			<td class="xl19" height="25" style="width:246pt;height:18.75pt" width="328">Thymidine [Methyl-3H]</td>
			<td class="xl19" style="width:162pt" width="216">PerkinElmer</td>
			<td class="xl24" style="width:104pt" width="139">NET027</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Glass Fiber Filtermats</td>
			<td class="xl19" style="width:162pt" width="216">PerkinElmer</td>
			<td class="xl24" style="width:104pt" width="139">1450-421</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Anti-mouse antibodies</td>
			<td class="xl19" style="width:162pt" width="216">eBioscience (Affymetrix)</td>
			<td class="xl24" style="width:104pt" width="139">[various]</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Anti-mouse TCR V&#946; Screening Panel</td>
			<td class="xl19" style="width:162pt" width="216">BD Biosciences</td>
			<td class="xl24" style="width:104pt" width="139">557004</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">LIVE/DEAD Fixable Dead Cell Stain</td>
			<td class="xl19" style="width:162pt" width="216">ThermoFisher Scientific</td>
			<td class="xl24" style="width:104pt" width="139">[various]</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Paraformaldehyde (16%)</td>
			<td class="xl19" style="width:162pt" width="216">Electron Microscopy Sciences</td>
			<td class="xl24" style="width:104pt" width="139">15710</td>
			<td class="xl22"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl19" height="42" style="width:246pt;height:31.5pt" width="328">Fixation/Permeabilization Solution Kit with GolgiPlug</td>
			<td class="xl19" style="width:162pt" width="216">BD Biosciences</td>
			<td class="xl24" style="width:104pt" width="139">555028</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Phorbol 12-myristate 13-acetate (PMA)</td>
			<td class="xl19" style="width:162pt" width="216">Sigma-Aldrich</td>
			<td class="xl24" style="width:104pt" width="139">P8139</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Iomomycin (calcium salt)</td>
			<td class="xl19" style="width:162pt" width="216">Sigma-Aldrich</td>
			<td class="xl24" style="width:104pt" width="139">I0634</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Pertussis Toxin from B. pertussis</td>
			<td class="xl19" style="width:162pt" width="216">List Biological Laboratories</td>
			<td class="xl24" style="width:104pt" width="139">181</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">10% Formalin</td>
			<td class="xl19" style="width:162pt" width="216">VWR</td>
			<td class="xl24" style="width:104pt" width="139">89370-094</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Variable-Flow Peristaltic Pump</td>
			<td class="xl19" style="width:162pt" width="216">Fisher Scientific</td>
			<td class="xl24" style="width:104pt" width="139">13-876-2</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Foam Biopsy Pads, Rectangular</td>
			<td class="xl19" style="width:162pt" width="216">Fisher Scientific</td>
			<td class="xl24" style="width:104pt" width="139">22-038-221</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Isothesia (isoflurane, USP)</td>
			<td class="xl19" style="width:162pt" width="216">Henry Schein Animal Health</td>
			<td class="xl24" style="width:104pt" width="139">050033</td>
			<td class="xl22">NDC : 11695-0500-2</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Tropicamide Ophthalmic Solution, USP (1%)</td>
			<td class="xl19" style="width:162pt" width="216">Akorn</td>
			<td class="xl24" style="width:104pt" width="139"></td>
			<td class="xl22">NDC: 17478-102-12</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Spectralis Diagnostic Imaging Platform</td>
			<td class="xl19" style="width:162pt" width="216">Heidelberg Engineering</td>
			<td class="xl24" style="width:104pt" width="139"></td>
			<td class="xl22"></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/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/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/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/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/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/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

Research

JoVE Journal

Immunology and Infection

11.3K Views.  University of California. 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.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...