Identification of naturally processed AQP4 determinants.

<p>AQP4 peptides were tested for their ability to induce T cell proliferative responses in intact hAQP4-primed (A) C57BL/6 and (B) SJL/J mice. Each 20-mer AQP4 peptide is indicated by first residue. Mice were immunized subcutaneously with 100 µg recombinant intact hAQP4 in CFA. 10–12 days later, lymph node cells were cultured in vitro for recall responses to the indicated human or mouse overlapping 20-mer peptides. Data are shown as stimulation indices (SI's) of mean proliferative responses in the presence of peptide (25 µg/ml) compared to the absence of antigen (background). Standard errors (+/− SEM) are shown for proliferative responses tested in triplicate. Recall to intact hAQP4 is shown for comparison.</p

Characterization of the minimal core T cell epitope within AQP4 p21-40.

<p>AQP4 p21-40-specific T cells were restimulated with truncated peptides to determine the core of the p21-40 determinant in (A, B) p21-40 specific C57BL/6 cell lines and (C, D) p21-40 specific SJL/J primary lymph node cells. Cell lines were restimulated with irradiated syngeneic splenic APC and various concentrations of p21-40 or truncated peptides. After 48 hours (cell lines) or 72 hours (LN), cultures were pulsed with ³H-thymidine and harvested 16 hours later. Data shown represent means of triplicates +/− SEM.</p

Localization of identified murine AQP4 T cell epitopes.

<p>AQP4 peptides that elicited proliferative responses in (A) C57BL/6 and (B) SJL/J mice are located within putative transmembrane and cytoplasmic domains. (C) Sequences of human (hAQP4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015050#pone.0015050-Yang1" target="_blank">[34]</a> and murine <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015050#pone.0015050-Turtzo1" target="_blank">[17]</a> AQP4 (mAQP4). Dashes represent homologous regions between the two species.</p

Predicted I-A^b- and I-A^s- binding mAQP4 epitopes for T cell recognition.

<p>MetaSVMp, a quantitative binding affinity program that employs the immune epitope database (IEDB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015050#pone.0015050-Hu1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015050#pone.0015050-Zhang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015050#pone.0015050-Nielsen2" target="_blank">[30]</a>, was used to identify potential I-A^b- and I-A^s-binding of AQP4 determinants for T cell recognition. Inverse of 50% inhibition concentration values (1/IC₅₀) is plotted for I-A^b (A top panel) and I-A^s (B top panel). Peaks correspond to increased predicted binding affinity: strong binding, IC₅₀<500 nM; moderate binding, IC₅₀>500 nM and <5,000 nM, and non-binding, IC₅₀>5,000 nM. Peaks correspond to increased predicted binding affinity. The top 20% of all possible epitopes are shown for I-A^b (A bottom panel) and I-A^s (B bottom panel). MetaSVMp percentile ranks were acquired from the MetaMHC web-based application; tall peaks correspond to top-ranked predicted epitopes.</p

AQP4 p21-40 is a naturally processed immunodominant determinant of intact AQP4.

<p>(A) C57BL/6 (above) and SJL/J (below) mice were immunized subcutaneously with 100 µg recombinant intact hAQP4 in CFA. Lymph nodes were harvested at day 10–12 and cultured in the presence of various concentrations of either intact hAQP4 (left), or individual murine AQP4 peptides (right) for 4 days. Proliferation was measured by ³H-thymidine incorporation, and are presented as mean cpm +/− SEM for triplicates. (B) C57BL/6 (above) and SJL/J (below) m21-40 primed lymph node cells were assayed for cytokine production and proliferation in response to m21-40 peptide. Supernatants were collected after 72 hr for IFN-γ and IL-17A for ELISA. Data are presented as mean +/− SEM for triplicates. (C) Proliferation of C57BL/6 T cell lines specific to p21-40, p91-110, p166-180, and p261-280 were assayed following re-stimulation with various concentrations of intact hAQP4 (left) and self-peptides (right). Data are shown as stimulation indices (SI's) of triplicates of antigen proliferation over no antigen conditions (background).</p