In this report, we have demonstrated for the first time that peripheral blood T cells from NMO patients and HC proliferate in response to intact AQP4 and AQP4 peptides. However, the frequency and magnitude of T-cell responses to AQP4 determinants was greater in NMO patients. Expansion of those autoreactive T cells provides further evidence that AQP4 is the autoantigen in NMO. It is notable that 3 of the AQP4 T-cell determinants, p61–80, p131–150, and p211–230 are respectively located in extracellular A, C, and E loops, AQP4 domains targeted by NMO-IgG.14
Whereas antibodies that target membrane proteins frequently bind conformational determinants exposed on their extracellular surfaces,31
T cells, which are restricted by HLA-D molecules, recognize linear processed peptides that can originate from extracellular, transmembrane, or intracellular domains.21
Thus, T and B cells may recognize distinct epitopes of the same autoantigen.32, 33
Nevertheless, with regard to the B–T collaboration required for IgG production, it is intriguing that p61–80, a naturally processed immunodominant AQP4 T-cell determinant, also represents a target for pathogenic AQP4-specific antibodies.14
The frequency of AQP4 p61–80-specific Th17 cells was significantly elevated in NMO patients, a finding that suggests that these autoantigen-specific T cells are a source of IL-17 that drives immunopathogenesis in NMO. In this regard, it was recently demonstrated that Th17 cells more efficiently drive naive B cells to secrete Ig than Th1 cells.34
Although our study relates to the peripheral immune response in NMO, our finding that AQP4-specific T cells exhibit a Th17 bias may also be relevant to development of CNS inflammation in NMO. Although neutrophils and eosinophils comprise the predominant cell types within NMO lesions, T cells are also detected.6, 35
As it is recognized that NMO IgG alone does not induce CNS inflammation and Th17 cells can promote tissue accumulation of neutrophils, AQP4-specific T cells may be sentinel adaptive immune cells directing CNS inflammation in NMO. Via IL-17 secretion, Th17 cells may compromise integrity of the BBB,36, 37
promote endothelial activation, and stimulate transendothelial migration of neutrophils.38
Thus, AQP4-specific Th17 cells may participate in multiple steps of NMO pathogenesis.
Many genetic and environmental factors may contribute to the development of autoimmunity. There is increasing evidence that commensal and pathogenic gut microbiota alter susceptibility to multiple sclerosis (MS), rheumatoid arthritis, type I diabetes, and systemic lupus erythematosus.39, 40
In this report, we observed a striking sequence homology between the AQP4 T-cell epitope p63–76, which contains predicted binding motifs for 2 NMO-associated HLA-DR molecules,41
and p204–217 of a C. perfringens
ABC-TP. C. perfringens
is a ubiquitous gram-positive spore-forming bacterium found in human commensal gut flora and also includes specific strains frequently associated with enterotoxin-mediated food poisoning.42
We observed T-cell reactivity to ABC-TP p204–217 in peripheral blood of NMO patients, as well as cross-reactivity between it and AQP4 p63–76. Interestingly, we detected 60 to 70% homology between p63–76 and the expressed or predicted ABC-TP in other Clostridium
species, including the commensal bacteria C. scindens
and C. hylemonae
as well as the pathogenic strain C. sporogenes
. Thus, molecular mimicry could account for T-cell reactivity to AQP4. Besides molecular mimicry, microbes can also exploit innate mechanisms that stimulate proinflammatory or anti-inflammatory immune responses. Recently, it was observed that commensal Clostridium
-related species alter the balance between Th17 and Treg in mice, and can influence development of autoimmunity.43, 44
We hypothesize that a Clostridium
species may have dual functions in NMO pathogenesis, (1) exposing a determinant that cross-reacts with self-antigen; and (2) serving as its own proinflammatory adjuvant, promoting Th17 polarization. This demonstration of molecular mimicry may stimulate further investigation of the potential role of Clostridium
species in NMO pathogenesis.
Based upon the presumption that the AQP4-specific antibodies of NMO IgG are pathogenic, approaches that reduce humoral immunity, including plasmapheresis, intravenous IgG, and CD20 B-cell depletion, are commonly used in NMO therapy. Although favorable responses to those treatments have been reported, they are often incomplete.45
Recognition that AQP4-specific antibodies are T cell-dependent, and alone are not pathogenic in the absence of CNS inflammation, suggests that therapies directed against the cellular arm of NMO pathogenesis could be beneficial. Interestingly, IFN-β, an approved MS therapy that alters cellular immune responses and may influence proinflammatory Th17 activity,46
has provoked NMO exacerbations.47–49
Our observation that T cells specific for the immunodominant AQP4 epitope exhibit Th17 polarization support testing of agents that target the IL-17 axis in NMO.
Collectively, our data provide a foundation to address the potential role of AQP4-specific T cells in driving adaptive humoral and cellular immune responses in NMO pathogenesis. Our observations provide a possible connection between gastrointestinal microbiota, Th17 polarization, and molecular mimicry in the development of CNS autoimmunity.