Autoantibodies targeting the aquaporin-4 (AQP4) water channel protein and the myelin oligodendrocyte glycoprotein (MOG) are associated with a broad spectrum of human central nervous system (CNS) demyelinating diseases (1, 2). While AQP4-specific antibodies target the AQP4 water channel protein expressed on astrocyte end-feet processes causing a severe astrocytopathy called neuromyelitis optica (NMO) (3, 4), MOG-specific antibodies target the extracellular N-terminal immunoglobulin variable (IgV)-domain of MOG expressed on myelin-forming oligodendrocytes (2, 5, 6). Autoantibodies against AQP4 (AQP4-Ab) have emerged as highly sensitive and specific biomarker for the diagnosis of NMO (3). However, not all patients presenting with clinical features suggestive of an NMO-disease phenotype are positive for AQP4-Ab (7), and a significant proportion of those seronegative patients harbor antibodies to MOG. This created a diagnostic uncertainty reflected in the pathogenetically undefined category of NMO spectrum disorders (NMOSD) proposed in 2015 (1, 2, 8).

Several lines of evidence suggest that autoreactive CD4⁺ T lymphocytes are key players in the pathogenesis of Ab-associated demyelinating CNS diseases. First, passive transfer models using AQP4-specific human IgG are not considered pathogenic without T-cell induced disruption of the blood brain barrier (BBB) (9–11). The exact relevance of T-cell independent pathogenicity of a high affinity rodent monoclonal AQP4-Ab (12) remains to be determined, as serum AQP4-Ab in human NMOSD patients are polyclonal, with a wide range of affinities and often much lower antibody titers (9, 10, 13–15). The serum concentration of AQP4-Ab is many times higher than in the cerebrospinal fluid (CSF) (13, 16–18), and peripheral B cells have the capacity to produce AQP4-Ab in vitro (19, 20). Thus, it is supposed that these antibodies are produced outside the CNS and that T effector cells might initiate CNS inflammation leading to BBB disruption and entry of antibodies (6, 9–11, 21–23). Local activation of CD4⁺ T-cells in the CNS is indispensable for providing an inflammatory microenvironment that also enables the initiation of CNS inflammation orchestrating BBB breakdown, lesion location and formation and thus facilitates Ab-mediated disease propagation (6, 11, 23). Second, AQP4-Ab and MOG-Ab are class-switched complement-fixing antibodies depending on T-cell help to be generated emphasizing the pivotal role of antigen-specific T-cell responses. Finally, there is ample evidence that activated T-cells are enriched at lesion sites (11, 24, 25) and that the pathogenic effectors are CD4⁺ T-cells of either T helper (Th)-1 lineage producing pro-inflammatory interferon (IFN)-ɤ or of Th17 lineage producing pro-inflammatory interleukin (IL)-17A (26, 27). Moreover, NMOSD patients also display a higher proportion of Th17 cells or cytokines like IL-6 (28–34).

While the high diagnostic value of AQP4-Ab as hallmark serologic marker in NMOSD has been shown and AQP4-specific T-cells have been examined in NMOSD patients (31, 35–38), the role of MOG-Ab or MOG-specific T-cells is less clear. Since MOG-Ab can be found in up to 50% of AQP4-Ab seronegative NMOSD patients, it is possible that MOG-specific T-cells could play a role in NMOSD development. So far there is no published information about MOG-specific T-cells in NMOSD and related conditions. Until now all studies focused on MOG-specific T-cell responses from MS patients (39–41) or in experimental autoimmune encephalomyelitis (42–44).

Here, we aimed to analyze the T-cell reactivity in response to selected eight AQP4 and nine MOG peptides and their possible restriction to a particular human leukocyte antigen (HLA)-DQ and HLA-DR genotype, and to examine the functional phenotype of autoreactive CD4⁺ T-cells in patients with AQP4-Ab or MOG-Ab.

In this study we analyzed peripheral blood T-cell responses to AQP4 and MOG peptides in individuals with AQP4-Ab, MOG-Ab, MS, and HC. We identified significantly increased AQP4-specific CD4⁺ T-cell reactivity to AQP4 peptide 156–170 in 4 of 8 AQP4-Ab positive NMOSD patients, but in none of the other groups. In contrast, we could not detect any significant disease-specific T-cell response to other AQP4 or MOG peptides. AQP4 peptide 156–170 has already been described as a T-cell epitope in NMOSD patients (31) and is also one of the most important B-cell epitopes recognized by AQP4-Ab (14, 15, 58, 59). Three other immunodominant T-cell epitopes/peptides of the AQP4 protein have been described by Varrin-Doyer et al. (31), which were also included in our study. However, we and other authors could not confirm immunodominance for these particular determinants (38, 60). The possible reasons for this discrepancy could be explained by the different methods used (CFSE, ³H-thymidine incorporation proliferation assays, cytokine secretion) and the different genetic background, i.e., HLA associations of the study populations. Several studies have identified over-representation of HLA-DPB1^*0501, HLA-DRB1^*0301, or HLA-DRB3 in NMO patients (31, 61–63). However, only DRB1^*0301 but not any of the other HLA alleles was overrepresented in our study population, indicating differences in the genetic background. In contrast, we found an overrepresentation of HLA-DQB1^*02 and HLA-DRB1^*01 in our study.

Some AQP4-specific T-cells (particularly against AQP4 peptides 11–30, 61–80, 63–76, 91–110, 139–153, and 281–305) were also present in participants with MOG-Ab, MS, and HC, consistent with previous studies demonstrating antibody response against linear AQP4 peptides (64). The presence of these AQP4-specific T-cell responses may reflect unspecific bystander activation, i.e. T-cell receptor (TCR)-independent activation of autoreactive T-cells by pro-inflammatory cytokines during inflammation, and/or epitope spreading (65).

We found no differences in MOG-specific T-cells between the four different groups with our experimental approach. The reason for the observed results could be explained by ignorance of the immune system of the MOG protein (66). In contrast to the AQP4 protein, which is also highly expressed in the periphery (67, 68) and hence underlies highly regulated mechanisms of self-tolerance (69, 70), MOG is only expressed in the CNS at very low levels (71, 72) and therefore not subject to intense immune surveillance. This might explain why HC showed lower response to AQP4 determinants, but in vitro stimulation with MOG peptides also caused profound T cell response in some healthy subjects.

Synthetic MOG peptides used in this study may not accurately represent naturally processed antigen and MHC-presented peptides in an in vivo setting (73). One of the major pathogenic mechanisms of MOG-Ab is considered the enhanced presentation of native MOG protein to T cells via Fc receptor mediated internalization of the antigen-Ab complex (74–76). Therefore, it is possible that MOG-reactive T cells can only be detected using intact MOG protein as the antigen. Indeed, Bronge et al. were able to identify increased frequencies of IFN-γ, IL-22 and IL-17A producing MOG-specific T-cells in patients with MS using bead-bound MOG as the antigen (77).

A major limitation of our study is the small number of included participants. This number reflects the expected number for our clinical centers, since NMOSD and MOG-related disorders are rare with a worldwide prevalence of 1–4/100,000 comparatively similar in most populations. Other limitations are that most patients received immunosuppressive therapy during sample collection related to their severe clinical presentations. Most AQP4-Ab positive patients (6/8) and 4/10 MOG-Ab positive patients received immunomodulatory or immunosuppressive treatment at the time of sample collection. Although B-cell depleting therapy is known to affect T-cell responses in patients with MS (78), 3/4 AQP4-Ab patients who had increased T-cell reactivity to AQP4 peptide 156-170 were treated with rituximab and the fourth patient was under high-dose corticosteroids before the initiation of rituximab treatment.

Importantly, the detailed characterization of single peptides, i.e. known “candidate antigens” based on their encephalitogenicity in animal models and/or their immunodominance in humans is crucial for a potential use in antigen-specific tolerance induction therapies (60, 79). For future studies, the implementation of new unbiased approaches may provide additional perspectives. These new strategies differ from previous studies by using combinatorial peptide libraries, which e.g., cover the entire protein or which allow the discovery of novel “unknown” antigens (80). Discrepancies to other studies might be explained by the use of different assays. We and all other investigators in this field face the issue of very low precursor frequencies of CNS antigen-specific T cells in PBMC preparations. However, the CFSE dilution assay used here is a powerful and sensitive method for directly detecting proliferation of rare autoantigen-specific human T-cells (31, 81). Moreover, the late addition of IL-2 during a re-stimulation step (82–84) offers high sensitivity and specificity. This strategy effectively increases the sensitivity for rare antigen-specific T-cells by selectively facilitating the proliferation of T lymphocytes that express the IL-2 receptor alpha-chain CD25 following antigen recognition (82, 85). However, even though our culture conditions promote the survival of mostly proliferating T-cells, other cells might skew the cytokine response. Indeed, it is well acknowledged that the key Th17-polarizing cytokine IL-6 is produced by myeloid cells (monocytes) rather than T-cells. Furthermore, the timing of sample collection might influence the relative abundance of the different cytokines and therefore explain differences to previous studies. Finally, additional factors such as the TCR avidity or the peptide concentration in different assays may play a role for various specificities. The application of (i) a lower peptide concentration (here used 20 μg/ml per peptide is higher compared to other studies), (ii) the use of native protein antigens instead of peptides, (iii) purified memory T-cells instead of PBMC, and/or (iv) the co-culture of T-cells with autologous APC, e.g., monocytes or EBV-transformed B cells, may be critical improvements for future experiments.

To conclude, this report investigates AQP4- and MOG-specific T-cell reactivities in human individuals presenting with AQP4-Ab and MOG-Ab positive demyelinating diseases. Our in vitro data corroborates previous findings showing the involvement of AQP4-specific T-cells in AQP4-Ab positive NMOSD and confirms the AQP4 peptide 156-170 as specific T-cell epitope. In contrast, no disease-relevant MOG peptide was identified. Future confirmatory studies using an unbiased approach for epitope discovery in larger cohorts may overcome main limitations of small sample size and the use of a limited collection of synthetic peptides in this study.

All datasets generated for this study are included in the article/supplementary material.

The studies involving human participants were reviewed and approved by the local Ethics Committee of Medical University of Innsbruck, Austria (study number AN3041) and University of Zurich, Switzerland (KEK ZH 2013-0001) and all patients and controls gave written informed consent. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

LH and MRe performed experiments, analyzed and interpreted data, and drafted the manuscript. MRa performed experiments and participated in the analysis and interpretation of the data. VG and MRe designed the study and MRe and AL analyzed and interpreted data and revisited the article critically for important intellectual content. AP performed experiments and analyzed the data. KR, MS, HH, TB, and AL provided patient material and revisited the article critically for important intellectual content. All authors approved the final version of the manuscript.

The Medical University of Innsbruck and the University Hospital Innsbruck (MRe) receive payments for antibody assays (AQP4 and other anti-neuronal and anti-glial antibodies) and for AQP4 antibody validation assays organized by Euroimmun (Luebeck, Germany). HH has participated in meetings sponsored by, received speaker honoraria or travel funding from Bayer, Biogen, Merck, Novartis, Sanofi-Genzyme, Siemens, Teva, and received honoraria for acting as consultant for Biogen and Teva. KR received speaker honoraria from Merck, Novartis and served as a consultant for PARADIGM-Study, Novartis with no compensation. TB has participated in meetings sponsored by and received honoraria (lectures, advisory boards, consultations) from pharmaceutical companies marketing treatments for multiple sclerosis: Almirall, Bayer, Biogen, Biologix, Bionorica, Genzyme, MedDay, Merck, Novartis, Octapharma, Roche, Sanofi/Genzyme, TG Pharmaceuticals, TEVA-ratiopharm and UCB. His institution has received financial support in the last 12 months by unrestricted research grants (Biogen, Merck, Novartis, Roche, Sanofi/Genzyme) and for participation in clinical trials in multiple sclerosis sponsored by Biogen, Merck, Novartis, Roche, Sanofi/Genzyme, and TEVA. AL received financial compensation and/or travel support for lectures and advice from Biogen, Merck, Novartis, Teva, Genzyme, Bayer, Celgene and he is a co-founder of Cellerys and co-inventor on a patent held by the University of Zurich on the use of peptide-coupled cells for treatment of MS.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.