Identification of AQP4 T cell determinants
In contrast to B cells and their secreted antibodies, which frequently recognize conformational determinants, Ag-specific CD4+
T cells recognize specific Ag determinants in association with MHC II molecules expressed on the cell surface of Ag presenting cells (APCs) 
. We first identified the immunogenic regions of AQP4 by testing proliferative T cell responses to individual peptides from a library of overlapping 15-mers and 20-mers encompassing the entire AQP4 sequence. By direct immunization of C57BL/6 mice with murine AQP4 peptides, we identified five regions of AQP4 that contain T cell determinants: p21-40, p91-110, p101-120, p161-180, p231-250 and p261-280 (). Of these peptides, proliferative responses to p21-40, p91-110, p161-180 and p261-280 were more robust. AQP4 T cell determinants in SJL/J were located within p11-30, p21-40, p101-120, 126-140 and p261-280 (). T cells that proliferated to AQP4 were CD4+
and MHC II-restricted (data not shown). All AQP4 T cell determinants in C57BL/6 and SJL/J mice were found within putative transmembrane or cytoplasmic domains (
). Specifically, we did not identify T cell determinants within the three extracellular domains, the A, C, and E loops 
, which may contain B cell determinants recognized by AQP4-specific antibodies from NMO patients 
Identification of AQP4 peptides that elicit T cell responses.
Localization of identified murine AQP4 T cell epitopes.
AQP4 p21-40 is the naturally processed immunodominant determinant for recognition by CD4+ T cells
Although peptide fragments can bind cell surface MHC molecules directly and elicit T cell immune responses, presentation of native Ag by APCs to CD4+
T cells in vivo usually requires processing, which involves proteolytic degradation of protein within APCs and association of peptide cleavage products with MHC II molecules 
. Thus, in parallel with our analysis by direct immunization of AQP4 peptides, C57BL/6 and SJL/J mice were separately immunized with intact AQP4, and tested for recall to intact AQP4 and the individual overlapping AQP4 peptides. Using this approach, we could identify the naturally processed immunodominant AQP4 determinants. Intact human AQP4, which is 93% homologous to mouse AQP4 
(), was used as the immunogen. Immunization with recombinant human AQP4 elicited significant proliferation to itself in both strains (), although the magnitude was lower in comparison to the stimulation induced by the most immunogenic AQP4 peptides (). For the negative controls, no response was found to either MOG 35-55 (C57BL/6) or PLP 139-151 (SJL/J) after AQP4 immunization (data not shown). In both strains, a significant recall response was detected to mouse AQP4 p21-40 ( and ), indicating that this is a naturally processed determinant of intact AQP4. T cells that recognized AQP4 p21-40 secreted IFN-γ and IL-17, indicating that both Th1 and Th17 immune responses were elicited (). Our findings also suggest that peptides p91-110, p101-120, p166-180, p231-250 and p261-280, which elicited responses in C57BL/6 mice (), and p11-30, p101-120, p126-140 and p261-280, which elicited responses in SJL/J mice, were not efficiently processed (). To further examine this possibility, we generated T cell lines to these AQP4 peptides. In contrast to AQP4 p21-40-specific T cells, other AQP4 peptide-specific T cell lines proliferated in response to their respective AQP4 determinants, but less efficiently, or not at all, to intact AQP4 (). Collectively, our results indicate that AQP4 p21-40 contains a naturally processed immunodominant AQP4 determinant.
Identification of naturally processed AQP4 determinants.
AQP4 p21-40 is a naturally processed immunodominant determinant of intact AQP4.
T cell epitope specificity within the immunodominant AQP4 p21-40
In general, MHC II-restricted CD4+
T cells recognize 9-13 amino acid peptides 
. In order to further characterize the immunodominant AQP4 T cell epitope(s), synthetic truncated peptides corresponding to sequences within AQP4 21-40 were synthesized and tested for recognition by p21-40-specific T cells. AQP4 p21-40-specific T cells from C57BL/6 mice proliferated in response to p21-35, p23-35 and p24-35, but not to p26-40 (). Shorter AQP4 peptides, p25-35 and p23-34 also stimulated proliferation of p21-40-specific T cells, although less efficiently. These results indicate that p24-35 is the core immunodominant AQP4 T cell determinant in C57BL/6 mice.
Characterization of the minimal core T cell epitope within AQP4 p21-40.
AQP4 p21-40-specific T cells from SJL/J mice also recognized p21-35, but neither p25-38 (), nor p26-40 (data not shown). Those T cells responded nearly as efficiently to AQP4 p23-35 as to p21-35, but did not respond to p24-35 or p25-35, demonstrating that the immunodominant AQP4 T cell determinant in SJL/J mice is p23-35. Thus, our data suggest that the immunodominant T cell epitopes for C57BL/6 and SJL/J mice are located in the same 13 amino acid sequence, p23-35, although the fine specificities are not identical.
It is notable that the amino acid residues with the immunodominant determinant are important in AQP4 functions 
. Residues 24-26 are critical for the assembly of orthogonal arrays of particles (OAPs), which are thought to enhance water transport 
. AQP4 residues 23-32 also contain the site required for the interaction with the regulator glutamate receptor 5 (mGluR5) and the catalytic subunit of Na,K-ATPase in forming macromolecular microdomains that may participate in K+
. Although intriguing, T cell recognition of this same region of AQP4 is probably coincidental, since determinants recognized by CD4+
T cells are generally created through processing and association with MHC II molecules 
. Further, unlike professional APCs, including microglia and dendritic cells, astrocytes, which express AQP4, are not efficient APCs in vivo 
While mouse and human AQP4 are 93% homologous, it is recognized that small differences in amino acid sequences, e.g. single residue substitutions, in heterologous proteins can have important effects on MHC binding as well as TCR-mediated stimulation 
. However, three of the immunogenic AQP4 peptides that we identified, p91-110 and p166-180 in C57BL/6 mice (), and p126-140 in SJL/J mice (), share identical sequences between mouse and human AQP4 (). Further, we observed cross-reactivity for most immunogenic AQP4 peptides that were not entirely homologous. Mouse AQP4 p21-40, which has xenogenic substitutions at residues 21 and 39, stimulated T cell responses to both itself and human AQP4 p21-40, and mouse p21-40-specific T cell lines were also stimulated by human intact AQP4 (). Our findings clearly indicate that AQP4 21-40 is an immunodominant region of AQP4 in both strains.
In this investigation, we have identified immunogenic T cell determinants using a library of overlapping peptides encompassing the entire sequence of AQP4. While this approach may be considered labor-intensive, several different in silico
methods have recently been developed in order to efficiently identify potential MHC-restricted T cell determinants 
. Many of these programs base predictions upon existing quantitative peptide binding affinities for specific MHC molecules 
. In general, methods for predicting MHC I-restricted determinants have advanced more rapidly, in part due to the fact that MHC II has an open binding groove accommodating larger peptides. For most programs available, there is better poor resolution for I-Ab
() than for I-As
(), possibly reflecting the larger database of known I-Ab
-restricted T cell determinants of Ag. MetaSVMp, a program that utilizes relative affinities of identified determinants from the immune epitope database (IEDB) to predict MHC II binding peptides 
, identified eight distinct regions of AQP4 that contain potential determinants that bind I-Ab
for T cell recognition ( top panel). AQP4 p21-40 ranked among the top 12 percentile of predicted I-Ab
-binding AQP4 15-mers ( bottom panel). However, MetaSVMp did not predict T cell recognition of AQP4 166-180 or 261-280. MetaSVMp also predicted the AQP4 sequence 121-140 within the top one percentile, yet we did not detect proliferation to this peptide either by recall to direct peptide immunization or in response to intact AQP4 in C57BL/6 mice. Therefore, our results caution against relying solely upon using predictive algorithms for identification of T cell epitopes of proteins.
Predicted I-Ab- and I-As- binding mAQP4 epitopes for T cell recognition.
The presence of T cells within active NMO lesions 
, evidence for clonal T cell expansion in peripheral blood of NMO patients 
, and the observations that AQP4-specific antibodies from NMO patients were not pathogenic in rats in the absence of CNS inflammation provide indirect evidence suggesting that T cells may participate in the etiology of NMO. Knowing that AQP4-specific antibodies in NMO serum are T-cell dependent IgG (IgG1) points to antigen-specific CD4+
T cell-mediation of its humoral response. However, there are several questions regarding the location and nature of AQP4-specific T cells in NMO pathogenesis. Do AQP4-specific T cells direct the AQP4-specific immune response outside the CNS? Do AQP4-specific T cells participate directly in CNS inflammation? Serum NMO IgG concentrations are higher than CSF levels 
, indicating that development of AQP4-specific antibodies occurs outside the CNS and is driven by stimulation within the peripheral immune compartment. Observations that encephalitogenic MBP-specific T cells promoted the development of NMO-like lesions upon transfer of AQP4-specific antibodies provided evidence that inflammation initiated by a T cell immune response to a CNS myelin antigen was sufficient for entry of AQP4-specific antibodies, and questions whether the cellular response that initiates CNS inflammation in NMO must necessarily target AQP4.
Elevated levels of IFN-γ and IL-17 have also been detected within the CSF of NMO patients 
, supporting the existence of both Th1 and Th17 responses. Active NMO lesions are characterized by the abundance of eosinophils and neutrophils 
, which suggests that Th2 and Th17 cells may have a key role in the CNS inflammation. While it may not be feasible in clinical studies to assess whether AQP4-specific T cells participate directly in NMO pathogenesis, now that we have discovered the T cell determinants of AQP4, it should permit investigators to determine the relative contribution of these subsets of AQP4-specific T cells in the initiation of CNS inflammatory responses in models. Efforts in developing a murine AQP4-based NMO model should focus on the AQP4 T cell epitopes identified, and in particular to the N-terminal region. The identification of regions of AQP4 recognized in mice may also provide insights regarding recognition of human AQP4 by NMO patients.