Immunodominant T cell determinants of aquaporin-4, the autoantigen associated with neuromyelitis optica

Autoantibodies that target the water channel aquaporin-4 (AQP4) in neuromyelitis optica (NMO) are IgG1, a T cell-dependent Ig subclass. However, a role for AQP4-specific T cells in this CNS inflammatory disease is not known. To evaluate their potential role in CNS autoimmunity, we have identified and characterized T cells that respond to AQP4 in C57BL/6 and SJL/J mice, two strains that are commonly studied in models of CNS inflammatory diseases. Mice were immunized with either overlapping peptides or intact hAQP4 protein encompassing the entire 323 amino acid sequence. T cell determinants identified from examination of the AQP4 peptide (p) library were located within AQP4 p21-40, p91-110, p101-120, p166-180, p231-250 and p261-280 in C57BL/6 mice, and within p11-30, p21-40, p101-120, p126-140 and p261-280 in SJL/J mice. AQP4-specific T cells were CD4+ and MHC II-restricted. In recall responses to immunization with intact AQP4, T cells responded primarily to p21-40, indicating this region contains the immunodominant T cell epitope(s) for both strains. AQP4 p21-40-primed T cells secreted both IFN-γ and IL-17. The core immunodominant AQP4 21-40 T cell determinant was mapped to residues 24-35 in C57BL/6 mice and 23-35 in SJL/J mice. Our identification of the AQP4 T cell determinants and characterization of its immunodominant determinant should permit investigators to evaluate the role of AQP4-specific T cells in vivo and to develop AQP4-targeted murine NMO models.     

Identification of two major conformational aquaporin-4 epitopes for neuromyelitis optica autoantibody binding

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease characterized by the presence of anti-aquaporin-4 (AQP4) antibodies in the patient sera. We recently reported that these autoantibodies are able to bind AQP4 when organized in the supramolecular structure called the orthogonal array of particles (OAP). To map the antigenic determinants, we produced a series of AQP4 mutants based on multiple alignment sequence analysis between AQP4 and other OAP-forming AQPs. Mutations were introduced in the three extracellular loops (A, C, and E), and the binding capacity of NMO sera was tested on AQP4 mutants. Results indicate that one group of sera was able to recognize a limited portion of loop C containing the amino acid sequence (146)GVT(T/M)V(150). A second group of sera was characterized by a predominant role of loop A. Deletion of four AQP4-specific amino acids ((61)G(S/T)E(N/K)(64)) in loop A substantially affected the binding of this group of sera. However, the binding capacity was further reduced when amino acids in loop A were mutated together with those in loop E or when those in loop C were mutated in combination with loop E. Finally, a series of AQP0 mutants were produced in which the extracellular loops were progressively changed to make them identical to AQP4. Results showed that none of the mutants was able to reproduce in AQP0 the NMO-IgG epitopes, indicating that the extracellular loop sequence by itself was not sufficient to determine the rearrangement required to create the epitopes. Although our data highlight the complexity of the disease, this study identifies key immunodominant epitopes and provides direct evidence that the transition from AQP4 tetramers to AQP4-OAPs involves conformational changes of the extracellular loops.     

Complement-dependent cytotoxicity in neuromyelitis optica requires aquaporin-4 protein assembly in orthogonal arrays

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which binding of pathogenic autoantibodies (NMO-IgG) to astrocyte aquaporin-4 (AQP4) causes complement-dependent cytotoxicity (CDC) and inflammation. We previously reported a wide range of binding affinities of NMO-IgGs to AQP4 in separate tetramers versus intramembrane aggregates (orthogonal arrays of particles, OAPs). We report here a second, independent mechanism by which CDC is affected by AQP4 assembly. Utilizing lactate dehydrogenase release and live/dead cell cytotoxicity assays, we found in different cell lines, and with different monoclonal and patient-derived NMO-IgGs, that CDC was greatly (>100-fold) reduced in cells expressing M1- versus M23-AQP4. Studies using a M23-AQP4 mutant containing an OAP-disrupting mutation, and in cells expressing AQP4 in different M1/M23 ratios, indicated that NMO-IgG-dependent CDC requires AQP4 OAP assembly. In contrast, antibody-dependent cell-mediated cytotoxicity produced by natural killer cells did not depend on AQP4 OAP assembly. Measurements of C1q binding and complement attack complex (C9neo) supported the conclusion that the greatly enhanced CDC by OAPs is due to efficient, multivalent binding of C1q to clustered NMO-IgG on OAPs. We conclude that AQP4 assembly in OAPs is required for CDC in NMO, establishing a new mechanism of OAP-dependent NMO pathogenesis. Disruption of AQP4 OAPs may greatly reduce NMO-IgG dependent CDC and NMO pathology.     

Intravenous neuromyelitis optica autoantibody in mice targets aquaporin-4 in peripheral organs and area postrema

The pathogenesis of neuromyelitis optica (NMO) involves binding of IgG autoantibodies (NMO-IgG) to aquaporin-4 (AQP4) on astrocytes in the central nervous system (CNS). We studied the in vivo processing in mice of a recombinant monoclonal human NMO-IgG that binds strongly to mouse AQP4. Following intravenous administration, serum [NMO-IgG] decreased with t_1/2 ∼18 hours in wildtype mice and ∼41 hours in AQP4 knockout mice. NMO-IgG was localized to AQP4-expressing cell membranes in kidney (collecting duct), skeletal muscle, trachea (epithelial cells) and stomach (parietal cells). NMO-IgG was seen on astrocytes in the area postrema in brain, but not elsewhere in brain, spinal cord, optic nerve or retina. Intravenously administered NMO-IgG was also seen in brain following mechanical disruption of the blood-brain barrier. Selective cellular localization was not found for control (non-NMO) IgG, or for NMO-IgG in AQP4 knockout mice. NMO-IgG injected directly into brain parenchyma diffused over an area of ∼5 mm² over 24 hours and targeted astrocyte foot-processes. Our data establish NMO-IgG pharmacokinetics and tissue distribution in mice. The rapid access of serum NMO-IgG to AQP4 in peripheral organs but not the CNS indicates that restricted antibody access cannot account for the absence of NMO pathology in peripheral organs.     

Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1/M23 isoforms and orthogonal arrays

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ± 7 nm. Different NMO-rAbs and NMO patient sera showed a wide variation in NMO-IgG binding to M1 versus M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 versus M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 versus M23 was due to OAP assembly rather than to differences in the M1 versus M23 N termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, result in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4 and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity.     

Neuromyelitis optica: aquaporin-4 based pathogenesis mechanisms and new therapies

Neuromyelitis optica (NMO) is an autoimmune 'aquaporinopathy' of the central nervous system that causes inflammatory demyelinating lesions primarily in spinal cord and optic nerve, leading to paralysis and blindness. NMO lesions show loss of aquaporin-4 (AQP4), GFAP and myelin, infiltration of granulocytes and macrophages, and perivascular deposition of activated complement. Most patients with NMO are seropositive for immunoglobulin autoantibodies (AQP4-IgG) against AQP4, the principal water channel of astrocytes. There is strong evidence that AQP4-IgG is pathogenic in NMO, probably by a mechanism involving complement-dependent astrocyte cytotoxicity, causing leukocyte infiltration, cytokine release and blood-brain barrier disruption, which leads to oligodendrocyte death, myelin loss and neuron death. Here, we review the evidence for this and alternative proposed NMO pathogenesis mechanisms, such as AQP4-IgG-induced internalization of AQP4 and glutamate transporters, complement-independent cell-mediated cytotoxicity, and AQP4-IgG inhibition of AQP4 water transport function. Based on the initiating pathogenic role of AQP4-IgG binding to astrocyte AQP4 in NMO, selective blocker therapies are under development in which AQP4-targeted monoclonal antibodies or small molecules block binding of AQP4-IgG to astrocytes and consequent downstream pathology.     

Evidence against cellular internalization in vivo of NMO-IgG, aquaporin-4, and excitatory amino acid transporter 2 in neuromyelitis optica

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are thought to be pathogenic in neuromyelitis optica (NMO). Prior work has suggested that a key component of NMO autoantibody (NMO-IgG) pathogenesis is internalization of AQP4 and the associated glutamate transporter EAAT2, leading to glutamate excitotoxicity. Here, we show selective endocytosis of NMO-IgG and AQP4 in transfected cell cultures, but little internalization in brain in vivo. AQP4-dependent endocytosis of NMO-IgG occurred rapidly in various AQP4-transfected cell lines, with efficient transport from early endosomes to lysosomes. Cell surface AQP4 was also reduced following NMO-IgG exposure. However, little or no internalization of NMO-IgG, AQP4, or EAAT2 was found in primary astrocyte cultures, nor was glutamate uptake affected by NMO-IgG exposure. Following injection of NMO-IgG into mouse brain, NMO-IgG binding and AQP4 expression showed a perivascular astrocyte distribution, without detectable cellular internalization over 24 h. We conclude that astrocyte endocytosis of NMO-IgG, AQP4, and EAAT2 is not a significant consequence of AQP4 autoantibody in vivo, challenging generally accepted views about NMO pathogenesis.     

Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-1(26-35) multimers at frequencies that are in the range of 10(-4) CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.     

Detection and characterization of T cells specific for BDC2.5 T cell-stimulating peptides

Nonobese diabetic (NOD) mice expressing the BDC2.5 TCR transgene are useful for studying type 1 diabetes. Several peptides have been identified that are highly active in stimulating BDC2.5 T cells. Herein, we describe the use of I-Ag7 tetramers containing two such peptides, p79 and p17, to detect and characterize peptide-specific T cells. The tetramers could stain CD4(+) T cells in the islets and spleens of BDC2.5 transgenic mice. The percentage of CD4(+), tetramer(+) T cells increased in older mice, and it was generally higher in the islets than in the spleens. Our results also showed that tetAg7/p79 could stain a small population of CD4(+) T cells in both islets and spleens of NOD mice. The percentage of CD4(+), tetramer(+) T cells increased in cells that underwent further cell division after being activated by peptides. The avidity of TCRs on purified tetAg7/p79(+) T cells for tetAg7/p79 was slightly lower than that of BDC2.5 T cells. Although tetAg7/p79(+) T cells, like BDC2.5 T cells, secreted a large quantity of IFN-gamma, they were biased toward being IL-10-producing cells. Additionally, <3% of these cells expressed TCR Vbeta4. In vivo adoptive transfer experiments showed that NOD/scid recipient mice cotransferred with tetAg7/p79(+) T cells and NOD spleen cells, like mice transferred with NOD spleen cells only, developed diabetes. Therefore, we have generated Ag-specific tetramers that could detect a heterogeneous population of T cells, and a very small number of NOD mouse T cells may represent BDC2.5-like cells.     

Complement-dependent and -independent aquaporin 4-antibody-mediated cytotoxicity in human astrocytes: Pathogenetic implications in neuromyelitis optica

BackgroundNeuromyelitis optica (NMO) is an inflammatory disease caused by the aquaporin (AQP)-4-antibody. Pathological studies on NMO have revealed extensive astrocytic damage, as evidenced by the loss of AQP4 and glial fibrillary acidic protein (GFAP), specifically in perivascular regions with immunoglobulin and complement depositions, although other pathological patterns, such as a loss of AQP4 without astrocyte destruction and clasmatodendrosis, have also been observed. Previous studies have shown that complement-dependent antibody-mediated astrocyte lysis is likely a major pathomechanism in NMO. However, there are also data to suggest antibody-mediated astrocyte dysfunction in the absence of complement. Thus, the importance of complement inhibitory proteins in complement-dependent AQP4-antibody-mediated astrocyte lysis in NMO is unclear. In most of the previous studies, the complement and target cells (astrocytes or AQP4-transfected cells) were derived from different species; however, the complement inhibitory proteins that are expressed on the cell surface cannot protect themselves against complement-dependent cytolysis unless the complements and complement inhibitory proteins are from the same species. To resolve these issues, we studied human astrocytes in primary culture treated with AQP4-antibody in the presence or absence of human complement and examined the effect of complement inhibitory proteins using small interfering RNA (siRNA).MethodsPurified IgG (10 mg/mL) was obtained from 5 patients with AQP4-antibody-positive NMO, 3 patients with multiple sclerosis (MS), and 3 healthy controls. Confluent human astrocytes transfected with Venus-M1-AQP4-cDNA were incubated with IgG (5% volume). After washing, we cultured the cells with human complements with or without heat inactivation. We observed time-lapse morphological and immunohistochemical changes using a fluorescence microscope. We also evaluated cytotoxicity using a propidium iodide (PI) kit and the lactate dehydrogenase (LDH) assay.ResultAQP4-antibody alone caused clustering and degradation followed by endocytosis of membraneous AQP4, thereby resulting in decreased cellular adherence and the shrinkage of astrocytic processes. However, these changes were partially reversed by the removal of IgG in culture. In contrast, following the application of AQP4-antibody and non-heated human complements, the cell bodies and nuclei started to swell. At 3 h, most of the astrocytes had lost mobility and adherence and were eventually destroyed or had swollen and were then destroyed. In addition, the remaining adherent cells were mostly PI-positive, indicating necrosis. Astrocyte lysis caused by rabbit complement occurred much faster than did cell lysis with human complement. However, the cell lysis was significantly enhanced by the transfection of astrocytes with siRNA against human CD55 and CD59, which are major complement inhibitory proteins on the astrocyte membrane. AQP4-antibody-negative IgG in MS or control did not induce such changes.ConclusionTaken together, these findings suggest that both complement-dependent and complement-independent AQP4-antibody-mediated astrocytopathies may operate in NMO, potentially contributing to diverse pathological patterns. Our results also suggest that the effect of complement inhibitory proteins should be considered when evaluating AQP4-antibody-mediated cytotoxicity in AQP4-expressing cells.     

Functional Fv fragment of an antibody specific for CD28: Fv-mediated co-stimulation of T cells

The most predominant co-stimulation pathway, which is critical for T cell activation and proliferation, is the CD28-B7 pathway. The anti-CD28 monoclonal antibody (mAb) also provides a co-stimulatory signal to T cells. In order to construct a functional Fv fragment (complex of VH and VL domains) of anti-CD28 antibody using a bacterial expression system, cDNA encoding the variable regions of immunoglobulin from 15E8 hybridoma cells was cloned and expressed in Escherichia coli. The Fv fragment was obtained as a soluble protein from the periplasmic fraction and showed a binding pattern similar to parental IgG. The Fv fragment induced proliferation of peripheral blood mononuclear cells in the presence of anti-CD3 or anti-CD2 mAb and enhanced anti-tumor activity of anti-MUC1x(anti)-CD3 bispecific antibody when tested with lymphokine-activated killer cells with T cell phenotype. Thus, the anti-CD28 Fv fragment will be promising not only for the study of co-stimulation, but also for cancer immunotherapy.     

Functional characterization of human T cells immortalized by oncogene transfection

We have succeeded in immortalizing human lymphocytes derived from the peripheral blood of a healthy donor and of an atopic patient, and from the lymph node of a cancer patient by oncogene transfection (Alam et al., 1996). All immortalized human lymphocytes were shown to be CD3+ and CD19–, indicating that these immortalized human lymphocytes were all T cells. We established 317, 154 and 692 individual immortalized human T cell lines derived from the healthy donor, the atopic patient and the cancer patient, respectively. The ratios of CD4+ and CD8+ subpopulations within the set containing immortalized T cells derived from the healthy donor were shown to be varied depending on the combinations of transfected oncogenes used. However, CD8+ cells were found to be the dominant subpopulation of immortalized T cells derived from the atopic patient and the cancer patient. These immortalized T cells showed different proliferative responses in the presence of exogenous human IL–2 depending on their origin, and was consistent with the surface expression of the IL–2 receptor. Furthermore, the cytokine secretion patterns of these immortalized T cells stimulated with mitogen were investigated. The results showed that the immortalized T cells from the healthy donor is able to secrete various kinds of cytokines such as IL–2, IL–10, -IFN and GM-CSF. However, immortalized T cells from the cancer patient was shown to only secrete IL–2 and GM-CSF. These results suggest that depending on the origin, the immortalized T cells came from different subsets or from cells in different activated states. Mixed lymphocytes reactions demonstrated that these immortalized T cells are able to proliferate in the presence of allogenic or xenogenic stimulator cells, suggesting that they maintain the ability to recognize specific antigens on the stimulator cells and can proliferate even after the immortalization. Furthermore, immortalized T cells derived from the healthy donor and the cancer patient strongly responded to K562 cells, suggesting that MHC-nonrestricted killer T cells were also immortalized.Abbreviations IL–2R – interleukin 2 receptor; MLR – mixed lymphocyte reaction     

Continuously proliferating T killer cells specific for H-2b targets: selection and characterization.

BALB/c (H-2d) thymus-derived lymphocytes sensitized to C57BL/6 (H-2b) alloantigens have been propagated in vitro for over 9 months. These T lymphocytes are specifically cytotoxic to H-2b target cells but are stimulated to proliferate by both H-2b and H-2k spleen cells. This indicates that for these selected cells the antigen requirements for cell proliferation are different from those for cell-mediated cytotoxicity. If not continuously stimulated with allogeneic spleen cells, the cytotoxic cultures fail to divide and rapidly lose their cytotoxic activity. Allogeneic erythrocytes do not stimulate cell proliferation in "quiescent" cell cultures and allogeneic tumor cells do so only in the presence of spleen cells. However, "quiescent" cell cultures display cytotoxicity in the presence of phytohemagglutinin A as do cell cultures which have lost their cytotoxic activity although they proliferate upon allogeneic stimulation. The significance of these findings is discussed.     

Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action.

ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes. Here we report a functional characterization of the T4 DNA ligase. One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins. An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding. All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation. From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction. The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase. The formation of an S. complex seems to be relevant during both blunt-end ligation and DNA relaxation. Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.     

Functional characterization of Fas ligand on tumor cells escaping active specific immunotherapy.

Mice transgenic for the rat HER-2/neu oncogene (rNeu-TG) developed spontaneous breast tumors that can escape a rNeu-specific immune response induced by active specific immunotherapy (ASI). The ability of these escape tumors to grow appeared to be due to upregulation of the Fas ligand (Fas-L) molecule. In an effort to develop tools for the better elucidation of the role of Fas-L and other regulatory mechanisms in tumor escape, we established cell lines derived from escape tumors. These tumor cell lines retained MHC class I, rNeu and Fas-L expression in vitro and formed tumors in vaccinated mice. Tumor growth was accompanied by permanent Fas-L expression in vivo, both in vaccinated and control vaccinated mice, indicating that these cells have acquired constitutive Fas-L expression. Moreover, these cells induced target cell apoptosis in vitro. Thus, these cells represent a unique tool to elucidate the importance of Fas-L expressed by tumors that escaped efficient systemic immune responses.     

Backward bifurcation in a model for HTLV-I infection of CD4+ T cells

Human T-cell Lymphotropic Virus Type I (HTLV-I) primarily infects CD4⁺ helper T cells. HTLV-I infection is clinically linked to the development of Adult T-cell Leukemia/Lymphoma and of HTLV-I Associated Myelopathy/Tropical Spastic Paraparesis, among other illnesses. HTLV-I transmission can be either horizontal through cell-to-cell contact, or vertical through mitotic division of infected CD4⁺ T cells. It has been observed that HTLV-I infection has a high proviral load but a low rate of proviral genetic variation. This suggests that vertical transmission through mitotic division of infected cells may play an important role.We consider and analyze a mathematical model for HTLV-I infection of CD4⁺ T cells that incorporates both horizontal and vertical transmission. Among interesting dynamical behaviors of the model is a backward bifurcation which raises many new challenges to effective infection control.     

IL-4 responsive CD4+ T cells specific for myelin basic protein: IL-2 confers a prolonged postactivation refractory phase

This study compared myelin basic protein-specific T cells from Lewis rats that were derived in the presence of either rat IL-4 or IL-2. Interleukin-4 was a maintenance factor that enabled derivation of long-term T cell lines. When activated, IL-4 dependent lines were lacking in IL-2 production capacity but maintained high levels of responsiveness to IL-2 and recognized IL-2 as a dominant growth factor. Activated IL-4 dependent T cells rapidly reverted to a quiescent phenotype in the presence of IL-4 and rapidly regained myelin basic protein reactivity. In contrast, activated IL-2 dependent T cells that were propagated in IL-2 had a more persistent blastogenic phenotype and a prolonged refractory phase. Interleukin-4 dependent lines that were propagated in IL-2 up-regulated the capacity to produce IL-2 and also acquired prolonged postactivation refractoriness. Thus, IL-2 was a dominant growth factor that conferred prolonged activation-dependent non-responsiveness. The coupling of dominant growth factor activity with prolonged postactivation refractoriness may be associated with the requisite role of IL-2 in homeostatic self-tolerance.     

Comparison of a T cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical

It has been widely assumed that T cells from TCR-transgenic (Tg) mice better represent the behavior of T cells from normal mice than do in vitro cultures of T cell clones. We have found that autoreactive T cells arising in the presumably more physiological environment of the BDC-2.5 TCR-Tg mouse, despite being apparently "naive" in surface phenotype, are highly activated functionally and do not resemble CD4(+) T cells from a spontaneously diabetic nonobese diabetic (NOD) mouse or the NOD-derived, diabetogenic CD4(+) T cell clone of origin, BDC-2.5. Our results suggest that autoreactive T cells cloned from the spontaneously diabetic NOD mouse more closely resemble effector T cells arising during the natural disease process.