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 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 naïve 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.
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 naïve 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 naïve 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.
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 "water-test" 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 α/β (TCRα-/-) 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.
The authors have nothing to disclose.
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.
M. tuberculosis H37Ra | BD Difco | 231141 | Dessicated, killed M. tuberculosis |
Incomplete Freund's Adjuvant | BD Difco | 263910 | |
AQP4 peptide p135-153 | Genemed | Custom Synthesis | Peptide sequence: LVTPPSVVGGLGVTMVHGN |
AQP4 peptide p201-220 | Genemed | Custom Synthesis | Peptide sequence: HLFAINYTGASMNPARSFGP |
MOG peptide p35-55 | Genemed / Auspep | Custom Synthesis | Peptide sequence: MEVGWYRSPFSRVVHLYRNGK |
3-way Stopcock | Kimble | 420163-4503 | |
HyClone Fetal Bovine Serum (Characterized) | GE Healthcare Life Sciences | SH30071 | |
Recombinant Mouse IL-23 | R&D Systems (BioTechne) | 1887-ML | |
Recombinant Mouse IL-6 | R&D Systems (BioTechne) | 406-ML | |
Recombinant Mouse IL-12 | R&D Systems (BioTechne) | 419-ML | |
Thymidine [Methyl-3H] | PerkinElmer | NET027 | |
Glass Fiber Filtermats | PerkinElmer | 1450-421 | |
Anti-mouse antibodies | eBioscience (Affymetrix) | [various] | |
Anti-mouse TCR Vβ Screening Panel | BD Biosciences | 557004 | |
LIVE/DEAD Fixable Dead Cell Stain | ThermoFisher Scientific | [various] | |
Paraformaldehyde (16%) | Electron Microscopy Sciences | 15710 | |
Fixation/Permeabilization Solution Kit with GolgiPlug | BD Biosciences | 555028 | |
Phorbol 12-myristate 13-acetate (PMA) | Sigma-Aldrich | P8139 | |
Iomomycin (calcium salt) | Sigma-Aldrich | I0634 | |
Pertussis Toxin from B. pertussis | List Biological Laboratories | 181 | |
10% Formalin | VWR | 89370-094 | |
Variable-Flow Peristaltic Pump | Fisher Scientific | 13-876-2 | |
Foam Biopsy Pads, Rectangular | Fisher Scientific | 22-038-221 | |
Isothesia (isoflurane, USP) | Henry Schein Animal Health | 050033 | NDC : 11695-0500-2 |
Tropicamide Ophthalmic Solution, USP (1%) | Akorn | NDC: 17478-102-12 | |
Spectralis Diagnostic Imaging Platform | Heidelberg Engineering |