Highly encephalitogenic aquaporin 4-specific T cells and NMO-IgG jointly orchestrate lesion location and tissue damage in the CNS
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In neuromyelitis optica (NMO), astrocytes become targets for pathogenic aquaporin 4 (AQP4)-specific antibodies which gain access to the central nervous system (CNS) in the course of inflammatory processes. Since these antibodies belong to a T cell-dependent subgroup of immunoglobulins, and since NMO lesions contain activated CD4+ T cells, the question arose whether AQP4-specific T cells might not only provide T cell help for antibody production, but also play an important role in the induction of NMO lesions. We show here that highly pathogenic, AQP4-peptide-specific T cells exist in Lewis rats, which recognize AQP4268–285 as their specific antigen and cause severe panencephalitis. These T cells are re-activated behind the blood–brain barrier and deeply infiltrate the CNS parenchyma of the optic nerves, the brain, and the spinal cord, while T cells with other AQP4-peptide specificities are essentially confined to the meninges. Although AQP4268–285-specific T cells are found throughout the entire neuraxis, they have NMO-typical “hotspots” for infiltration, i.e. periventricular and periaqueductal regions, hypothalamus, medulla, the dorsal horns of spinal cord, and the optic nerves. Most remarkably, together with NMO-IgG, they initiate large astrocyte-destructive lesions which are located predominantly in spinal cord gray matter. We conclude that the processing of AQP4 by antigen presenting cells in Lewis rats produces a highly encephalitogenic AQP4 epitope (AQP4268–285), that T cells specific for this epitope are found in the immune repertoire of normal Lewis rats and can be readily expanded, and that AQP4268–285-specific T cells produce NMO-like lesions in the presence of NMO-IgG.
KeywordsCNS inflammation Neuromyelitis optica T cells Aquaporin 4 ENMO
Materials and methods
Lewis rats (7–8 weeks old) were obtained from Charles River Wiga (Sulzfeld, Germany). They were housed in the Decentral Facilities of the Institute for Biomedical Research (Medical University Vienna) under standardized conditions. The experiments were approved by the Ethic Commission of the Medical University Vienna and performed with the license of the Austrian Ministry for Science and Research.
Characterization of the immunoglobulins used in transfer experiments
The NMO-IgG preparations containing pathogenic AQP4-specific antibodies derived from therapeutic plasmapheresates/sera of two different patients (“NMO-IgG9” and “pt1”; both NMO-IgGs worked equally well). The NMO-IgGs were essentially prepared and purified as described , and adjusted to an IgG concentration of 10 mg/ml. The use of it for research was approved by the Ethics Committee of Tohoku University School of Medicine (No. 2007-327) and by the Regional and National Ethical Committee of Hungary (3893.316-12464/KK4/2010 and 42341-2/2013/EKU). The normal human IgG preparation used as a negative control was commercially available (Subcuvia™, Baxter, Vienna), and was also diluted with phosphate-buffered saline (PBS) to an IgG concentration of 10 mg/ml prior to use.
Peptide and epitope sequences
Amino acid sequence
AQP4 278–323 a
For specificity tests (see below), also full-length human M23 AQP4 (gene bank accession number: NP-004019) was used, which has 100 % identity to the rat epitopes contained in AQP4207–232 (PAVIMGNWE) and AQP4268–285 (QQTKGSYME and TKGSYMEVE), and contains the human sequence of AQP4 recognized by AQP4278–323-specific T cells (GVVHVIDVD and HVIDVRGE, Table 1). For the preparation of this protein, HEK293A cells were transiently transfected with pcDNA3.1(M23)AQP4, allowing the production of AQP4 as a 6-HIS-tagged protein. 72 h later, the cells were washed with sterile phosphate-buffered saline (PBS) and exposed to lysis buffer (10 mM Tris buffer pH7.5, 100 mM NaCl, 1 mM EDTA, 1 % Triton X-100 and complete protease inhibitor cocktail tablet) for 1 h at 4 °C. The lysate was thoroughly mixed by pipetting, subjected to repeated rounds of freezing and thawing, sonicated using a Sonopuls GM70 (Bandelin, Berlin, Germany), and finally passaged through a 23 gauge needle. Ni NTA-Agarose Superflow (Qiagen) was then used for the purification of AQP4 following the instructions of the manufacturer. Briefly, Ni NTA beads were gently applied to a column, washed with 5 volumes of wash buffer (20 mM Tris/125 mM NaCl) prior to applying the lysate mix diluted 1:1 in wash buffer containing 1 % Triton X-100. Following washing steps with 10 volumes of wash buffer containing 1 % Triton X-100, we were washing the column with 10 volumes of washing buffer containing 0.1 % Triton X-100. Subsequently, we eluted AQP4 in 20 mM Tris/125 mM NaCl/0.1 % Triton X-100/600 mM Imidiazole. Following dialyses against PBS we used AQP4 in a concentration of 1 mg/ml in PBS/0.1 % Triton X-100. The eluted AQP4 protein was confirmed by Western blot (data not shown). In specificity tests, also recombinant human MOG1–125 was used, which was essentially produced and purified as described . The MOG35–55-specific T cells used were raised against rat/mouse MOG35–55 (Sigma).
Immunization and T cell line preparation
The animals were subcutaneously immunized with 100 µl of a 1:1 mixture of the relevant antigen (stock 2 mg/ml) in Freund’s incomplete adjuvans supplemented with 4 mg/ml mycobacterium tuberculosis H37Ra. 9–11 days after the immunization, the animals were killed. At this point, they were all clinically healthy and did not show any evidence for inflammation of the CNS or of peripheral organs. The lymph nodes draining the immunization site were removed, and peptide-specific T cell lines were established as described [32, 33].
Isolation of naïve T cells
The naïve T cells tested derived from the spleen of an adult Lewis rat housed under specific pathogen-free conditions. The spleen was processed to a single cell suspension, and contaminating erythrocytes were removed by incubation of the cells for 5 min in hypotonic salt solution (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA) pH 7.4.
Preparation of T cells for immunocytochemistry
T cells were embedded in HistoGel (Thermoscientific, Cheshire, UK) according to the manufacturer’s instructions and subsequently fixed with 4 % paraformaldehyde for 24 h. The HistoGel blocks were then processed for immunohistochemical analysis as detailed [2, 7].
Characterization of T cell lines
Specificity was determined in T cell proliferation assays, using 96 well plates. 5 × 105 AQP4-peptide-specific T cells were cocultured in triplicates with 1 × 106 thymic antigen presenting cells in the absence of externally added antigen or in the presence of irrelevant CNS antigens (i.e. myelin basic protein or unrelated AQP4-peptides; 10 µg/ml final) as negative controls, of the peptide against which the cell line was established as specific antigen (10 µg/ml final), or of concanavalin A (2.5 µg/ml final) as positive control [9, 11].
The human M23 AQP4 preparation had a protein concentration of 1 mg/ml in phoshate-buffered saline/0.1 % Triton X-100. To avoid the toxic effects of Triton X-100, we diluted the antigen preparation 1:100 with PBS and coated it in 100 µl aliquots over night at 4 °C onto flat-bottom 96-well plates. On the next morning, the coating solution was drained from the plates, and each well was washed gently and briefly with 100 µl culture medium containing 1 % rat serum. Then, 1 × 106 thymic antigen presenting cells in 100 µl medium were cultured in these plates for 8 h at 37 °C prior to the addition of T cells. Exactly the same procedure was applied to recombinant human MOG1–125.
The cells were cultured for 48–72 h. For the final 18–24 h in culture, [3H]-thymidine was added to reveal de novo DNA synthesis during the S-phase of the cell cycle of activated T cells.
Analysis of surface marker expression by flow cytometry
For staining, the cells were incubated for 30 min at room temperature with antibodies against rat CD4 (W3/25, mouse monoclonal, Serotec) or mouse IgG1 (Dako, Glostrup, Denmark; isotype matched control antibody). After washing, the cells were incubated for 30 min at room temperature for 30 min with polyclonal Alexa488-labeled goat anti-mouse IgG (Jackson ImmunoResearch). For staining of the αβ-T cell receptor (TCR), PE-labeled mouse anti-αβTCR (eBioscience, San Diego, CA) was used. All antibodies were used in a dilution of 1:100 in stain buffer (PBS/10 % fetal calf serum/1 mM EDTA).
Quantitative real-time polymerase chain reaction (qPCR) for the detection of transcripts for IFN-γ and IL-17
RNA was purified from freshly activated T cell blasts using the RNeasy Plus Mini Kit (Qiagen GMbH, Hilden, Germany). Genomic DNA was removed and the RNA transcribed to cDNA as described . qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions, using a 10 µl reaction mixture [5 µl SsoAdvanced Universal SYBR Green Supermix, 0.2 µl forward primer (10 pmol/µl), 0.2 µl reverse primer (10 pmol/µl), 3.6 µl double-distilled H2O, 1 µl DNA template] in a StepOnePlus system (applied biosystems). The following primers were used: IL-17 forward: 5′-TACCAGCTGATCAGGACGAG-3′; IL-17 reverse: 5′-CATCAGGCACATGGATGGAA-3′; IFN-γ forward: 5′-ATTCATGAGCATCGCCAAGTTC-3′; IFN-γ reverse: 5′-TGACAGCTGGTGAATCACTCTGAT-3′; GAPDH forward: 5′-CCGAGGGCCCACTAAAGG-3′; GAPDH reverse: 5′-ATGGGAGTTGCTGTTGAAGTCA-3′. For qPCR, an initial denaturation step (95 °C, 30 s) was followed by 40 cycles of denaturation (95 °C, 15 s) and annealing/extension (60 °C, 1 min). The absence of unspecific amplification was determined by melt curve analysis. All reactions were run in triplicates.
Induction of experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuromyelitis optica (ENMO)
Antigen injections into the cisterna magna
On day 5 after EAE induction by T cell transfer, the animals were anesthetized with Ketanest S/Rompun. Afterwards, the neck of the animals was flexed, and the skin was cut open to expose the atlanto-occipital membrane, which was then punctured with a thin glass capillary. When the entry of clear cerebrospinal fluid into the capillary indicated the accuracy of the injection site, 5 µl of AQP4 peptides (dissolved in a concentration of 2 mg/ml in RPMI), or of RPMI alone were injected slowly. Afterwards, the capillary was withdrawn and the skin was closed with wound clips. 24 h later, the animals were killed with CO2 and perfused with 4 % PFA. Brains and spinal cords were processed for immunohistochemical analysis as detailed [2, 7].
All stainings were done essentially as described [2, 7] using the mouse monoclonal antibody ED1 (to stain macrophages and activated microglia; Serotec, Germany), rabbit polyclonal antibodies against CD3 (to stain T cells; NeoMarkers, Fremont, USA), rabbit polyclonal antibodies against AQP4 (to stain astrocytes; Sigma, Germany), rabbit polyclonal or mouse monoclonal antibodies against glial fibrillary acidic protein (GFAP; from Dako, Denmark, or NeoMarkers, respectively), anti-human immunoglobulin (biotinylated donkey; polyclonal; Amersham, UK), anti-rat immunoglobulin (biotinylated donkey; polyclonal; Jackson ImmunoResearch), and anti-complement C9 (rabbit polyclonal ).
For double immunostainings of proliferating cell nuclear antigen (PCNA, mouse, clone PC10, DAKO) and CD3 (rabbit polyclonal; NeoMarkers, Fremont, USA), sections were steamed in citrate buffer for 30 min. After incubation with mouse anti-proliferating cell nuclear antigen (PCNA, clone PC10, DAKO, 1:10 000) over night at 4 °C, the sections were incubated with alkaline-phosphatase-labeled anti-mouse antibodies (Jackson ImmunoResearch, West Grove PA, USA, 1:200) for 1 h at RT and developed with Fast Blue (FB, Sigma, Germany) substrate. Then the sections were incubated with rabbit anti-CD3 (polyclonal; NeoMarkers, Fremont, USA, 1:2000) over night at 4 °C. After incubation with biotinylated anti-rabbit antibodies (1:2000) for 1 h at RT and enhancement with CSA (1:1000) for 20 min, avidin-peroxidase (Sigma) was applied and the sections were developed with aminoethyl carbazole (AEC, Sigma).
Quantitative evaluation of immunostained sections
Quantification was done by using a morphometric grid. To determine the extent of T cell infiltration, the number of CD3+ cells was determined in 3 different areas: within in the meninges, within the superficial parenchyma (<100 µm distance from the meninges) and within the deep parenchyma (>100 µm distance from the meninges).
Statistics were calculated with the IBM SPSS Statistics 21. The Mann–Whitney (Wilcoxon) W test (comparison of medians) was used in all cases. For multiple comparisons, Bonferroni correction was used.
All AQP4 epitopes suitable for binding to Lewis rat MHC class II (RT1.BL) give rise to antigen-specific T cell responses
We used peptides spanning AQP4 epitopes previously identified as potential binders to RT1.BL of Lewis rats  for the immunization of Lewis rats. Although all animals remained clinically healthy upon immunization, they mounted T cell responses against all of the peptides used. Consequently, different peptide-specific T cell lines could be established by alternating cycles of antigen-specific T cell activation and IL-2-driven T cell propagation (Table 1) which constrains the generation of TH17 cells . These cells were CD4+, and expressed the αβ T cell receptor (suppl. fig 1). All of these cell lines showed a dominant expression of IFN-γ over IL-17 (suppl. fig 2), and therefore belonged to the TH1 subset of cells. They were all responsive to their specific peptides, but the AQP427–69- and AQP4278–232-specific T cell lines did not reach a stimulation index >2 (suppl fig. 3).
We tested the encephalitogenic potential of these cells, i.e. their ability to induce CNS inflammation, by their transfer into naïve Lewis rats.
AQP4-peptide-specific T cell lines vary in encephalitogenicity and ability for parenchymal infiltration
The different AQP4-peptide-specific CD4+ T cells varied in their ability to induce CNS inflammation and could be grouped into weak, medium, and strongly encephalitogenic lines. Weak encephalitogenicity was observed upon transfer of T cells with specificity for AQP4171–190, AQP427–69, AQP4278–323, and AQP4237–277. Even after transfer of as many as 10 × 107 T cells/animal, these lines were just able to yield an average of less than 2 lesions per rat. These lesions were essentially located within the meninges, and only very few T cells entered the CNS parenchyma (Fig. 2). The recipient animals did not show any symptoms of clinical disease, in line with this weak histological evidence of CNS inflammation. Medium encephalitogenicity was observed with T cells specific for AQP4207–232 containing the epitope PAVIMGNWE. On average, transfer of these cells lead to ~10 lesions per animal, distributed along the entire neuraxis. However, these cells only barely infiltrated the CNS parenchyma. Instead, they piled up in the meninges, resulting in up to 10 layers of T cells on top of each other (Fig. 2). Although there was more histological evidence of CNS inflammation than seen with the AQP4 peptide-specific T cells described above, we still did not see signs of clinical disease. Strong encephalitogenicity was observed in T cells specific for AQP4268–285.
AQP4268–285-specific T cells are highly encephalitogenic
AQP4207–232- and AQP4268–285-specific T cells differ in their extent of activation within the CNS
The amount of available AQP4207–232-peptide limits CNS infiltration by AQP4207–232-peptide-specific T cells
Hence, T cell activation and subsequent parenchymal infiltration by AQP4207–232-peptide-specific T cells can be increased by injections of AQP4207–232 into the cisterna magna.
In the presence of NMO-IgG, the numbers of pathogenic T cells determine location and size of astrocyte-destructive lesions
Hence, in the presence of NMO-IgG, low numbers of AQP4268–285-specific T cells initiate astrocyte-destructive lesions almost exclusively in spinal cord gray matter, while higher numbers of AQP4268–285-specific T cells trigger additional lesions with AQP4 and GFAP loss in the brain.
Since the discoveries of T cells in NMO lesions , and of NMO-IgG containing pathogenic AQP4-specific antibodies with the T cell-dependent immunoglobulin subclass IgG1 [19, 20], the question arose whether AQP4 specific T cells are only responsible for T cell help in antibody formation, or whether they can also participate in directing lesions to NMO-typical sites, and induce there astrocyte-destructive lesions in the presence of NMO-IgG. And indeed, all AQP4-specific T cells established so far from wildtype animals were only weakly encephalitogenic, targeted the wrong sites, and were essentially confined to the meninges, but only poorly infiltrated the CNS parenchyma . To finally answer these questions, we used one model organism—Lewis rats—to raise T cell lines against all AQP4 epitopes predicted to bind to the MHC class II products (RT1.BL) of these animals , and to test the encephalitogenic potential of these cells in vivo. While all of these peptides could serve as antigens to produce stable CD4+ TH1 cell lines and were fully able to activate T cells in vitro, the vast majority of the resulting AQP4 peptide-specific T cells were only weakly or moderately encephalitogenic. Such cells had to be transferred in very high numbers into naïve animals, did not yield any clinical symptoms, and barely infiltrated the CNS parenchyma. However, one of the peptides used, i.e. AQP4268–285, was clearly different. It contains two overlapping epitopes (AQP4271–279 with the amino acid sequence QQTKGSYME, and AQP4273–281 with the amino acid sequence TKGSYMEVE), is the autoantigen of highly encephalitogenic T cell responses in Lewis rats, and gives rise to T cells which induce clinical symptoms, deeply immigrate the CNS parenchyma, and initiate large astrocyte-destructive lesions in the presence of NMO-IgG.
AQP4268–285-specific CD4+ T cells are found in the normal healthy immune repertoire, can be readily activated upon immunization, and induce severe panencephalitis upon injection into naïve rats. Hence, AQP4268–285 is a true self-antigen in Lewis rats.
AQP4268–285-specific T cells can immigrate into the CNS parenchyma throughout the entire neuraxis, but are particularly frequent at sites described to be typical for NMO : AQP4268–285-specific T cells cause myelitis with a strong involvement of the dorsal horns and central gray matter, optic neuritis, and encephalitis with profound infiltration around the 3rd and 4th ventricle and in the hippocampus, in the periaqueductal gray, in the cerebellum and in the medulla. These sites are also lesion sites in NMO patients with brain involvement [30, 43], which has initially been ascribed to the high AQP4 expression at these sites . However, since we also see inflammation at these sites, when animals have been challenged with other CNS-antigen-specific T cells, this distribution of brain lesions is more likely to reflect the local action of other regulatory mechanisms for T cells and antibody-mediated processes.
AQP4268–285-specific T cells yield inflammatory lesions in which ~44 % of the deeply infiltrating T cells express PCNA as a sign of recent activation. Again, this is a crucial point, since T cell activation within the CNS is an important prerequisite for the formation of astrocyte-destructive lesions in the presence of NMO-IgG lesions , and since activated CD4+ T cells are found in NMO lesions . It is tempting to speculate that this high level of PCNA expression might be due to reactivation within the CNS and not due to activation in vitro before transfer. A supportive, but certainly not definitive evidence for this speculation is the low number of proliferating AQP4207–232-specific T cells in the CNS, which is massively increased by additional injection of the respective peptide into the CSF. Unfortunately, however, we cannot formally prove whether or not antigen presenting cells in the CNS process antigens in a similar way as their counterparts in vitro do, and whether full-length AQP4 is cleaved by these cells to AQP4207–232 as efficiently in the CNS as it is in vitro.
High numbers of AQP4268–285-specific T cells target astrocyte-destructive lesions throughout the entire spinal cord, and also to the brain in NMO-IgG seropositive hosts. However, when present in low numbers, AQP4268–285-specific T cells target 98 % of all astrocyte-destructive lesions to cervical/thoracic spinal cord gray matter in NMO-IgG seropositive hosts. The preference of spinal cord is important, since NMO often presents with episodes of myelitis. Targeting of this site could result from higher levels of expression of AQP4 mRNA, protein, and large supramolecular aggregates in spinal cord and optic nerve relative to other regions of the brain , and in gray relative to white matter cord . Higher antigen concentrations might then translate to better binding of NMO-IgG, to an enhanced availability of this antigen for local antigen presenting cells and subsequent T cell activation , and to an increased astrocytotoxicity of microglia/macrophages via complement- and antibody-mediated cellular mechanisms [33, 34].
In combination with NMO-IgG, AQP4268–285-specific T cells also show a predilection for cervical/thoracic spinal cord, which are sites most often affected in NMO patients. Since this area is also most frequently targeted in the spinal cords of Lewis (LEW), LEW.1 N and LEW.1A rats with EAE provoked by the action of myelin oligodendrocyte glycoprotein (MOG)-specific antibodies and T cells , this might point to a gateway for autoreactive TH1 cells and antibodies to cross the blood–brain barrier at this site, possibly defined by regional neural activation .
We do not know yet why in our current NMO model the optic nerves are spared from astrocyte-destructive lesions, although they contain numerous inflammatory T cells and activated microglia/macrophages. Most trivially, the formation of NMO-like lesions in the optic nerve could just simply be a rare event in ENMO provoked by AQP4268–285-specific T cells and NMO-IgG, and could become visible when higher numbers of animals are examined. Alternatively, also the genetic background of our animals might play a role, since there is, again, a striking resemblance to the MOG-induced EAE model described above. In the MOG-model, demyelinating spinal cord lesions formed in LEW.1 N, LEW.1A, LEW.1AV1, BN, and DA rats, but additionally in the optic nerves only in BN and DA rats . This contribution of genetic background to disease phenotype could find its human correlate in the different, ethnicity-dependent frequencies of longitudinally extensive transverse myelitis seen at onset attack in 53 % of Caucasian vs. 33 % of Afro-Caribbean patients in an UK cohort of NMO patients .
Also low numbers of AQP4268–285-specific T cells can initiate large lesions with AQP4 loss. These findings recapitulate observations which have been made earlier in an EAE model using myelin basic protein-specific T cells with demyelinating anti-MOG antibodies , and suggest that the large, astrocyte-destructive lesions in NMO-IgG seropositive NMO patients could be provoked by the action of very few AQP4-specific T cells.
In Lewis rats, AQP4268–285 is highly encephalitogenic. For the time being, we do not know yet whether intracellular AQP4 fragments also contain highly encephalitogenic antigens in humans, since their MHC could select different epitopes. Does this mean that AQP4-specific T cells recognizing weakly encephalitogenic AQP4 epitopes are irrelevant for NMO in Lewis rats or NMO patients? Probably not. While certain antigenic fragments might not be present in sufficient amounts to warrant T cell infiltration into the intact CNS, i.e. to trigger the very first NMO lesion, they might play a crucial role in the propagation of relapses, for example when antigens are released from necrotic, astrocyte-destructive lesions. Then, the liberated antigens might become available for local antigen presenting cells [13, 26], and provide the basis for the activation of naive T cells within the CNS in a process called epitope spreading . At least in EAE and MS, this process underlies the shift of autoreactivity from primary initiating self-determinants, which invariably regress with time and might even become undetectable during periods of disease progression, to sustained secondary autoreactivity . Considering the fact that AQP4 contains a large number of potential T cell epitopes, not only in mice and rats [12, 25, 32], but also in humans [6, 23] (Fig. 1), it is tempting to speculate that this might be a strong argument in favor of very early T cell vaccination, and a strong counter-argument for later T cell vaccination as a therapeutic option for NMO patients.
This work was supported by the Austrian Science Fund [Grant Numbers P25240-B24 to MB and I916-B13 (International Programme, Eugène Devic European Network) to HL and MR], by the Else Kröner-Fresenius-Stiftung (Grant Number 2013_A283 to MB), by the Austrian Ministery of Science, Research and Economy (BIGWIG-MS to HL and MR), and by Grants-in-aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology of Japan to KF and TM. We thank Dr. Dagoberto Callegaro for sample collection, Marianne Leisser, Ulrike Köck and Angela Kury for excellent technical assistance, Verena Berg and Madhura Modak for assistance at the β-counter, and the Core Facility Flow Cytometry, Medical University of Vienna.
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