Identification of critical residues of human (h)AQP4_281-300 for presentation in the context of <i>HLA-DRB1*03</i>:<i>01</i> and recognition by the B.10 T cell receptor (TCR).

<p>(A) First, the ability of hAQP4_281-300-reactive lymph node cells to recognize the alanine screening peptides was determined by ELISpot. 5.0x10⁵ cells/well lymph node cells taken ten days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with hAQP4_281-300 were restimulated with hAQP4 alanine scanning peptides (2 5 μg/mL) for 48 hours in IFNγ and IL-17 ELISpot plates (* = P-value < 0.05 and ** = P-value < 0.01). (B) Alanine screening peptides that not result in an increased frequency of IFNγ and IL-17 secreting lymph node cells were identified as the key residue peptides, and were subsequently tested in a MHC binding assay. Splenocytes taken from <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice were incubated for 12 hours in the presence of biotinylated hAQP4 alanine scanning peptides. Post incubation, cells were stained utilizing FITC-Avidin, and antigen positive cells were quantified by flow cytometry (* = P-value < 0.05 and ** = P-value < 0.01). (C) There was no Ig isotype class switch in mice immunized with mAQP4_284-299 with regard to antibody responses against whole-length AQP4 protein. (D) Critical <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, and B.10 TCR contact amino acids are specified. E₂₈₈ and L₂₉₄ are required as <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, while T₂₈₉, D₂₉₀, D₂₉₁, and I₂₉₃ are critical B.10 TCR interacting residues.</p

Immunization with human (h)AQP4_281-300 leads to an expansion of antigen-specific CD4⁺ T cells <i>in vivo</i>, and an Ig isotype switch in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice.

<p>(A) Following immunization with human (h)AQP4_281-300, an expansion of antigen-specific CD4⁺ T helper cells was detected by tetramer staining of lymph node cells. The fluorescent signal of <i>HLA-DRB1*03</i>:<i>01</i>-loaded tetramers minus the fluorescent signal of empty <i>HLA-DRB1*03</i>:<i>01</i> tetramers is shown. CD4⁺ T helper cells provide soluble mediators that drive B cell differentiation immunoglobulin (Ig) class switching. To determine whether hAQP4_281-300-reactive CD4⁺ T cells are capable of causing IgM to IgG isotype switching in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice, the concentration of Ig against hAQP4_281-300, mAQP4284-299, or with whole-length hAQP4 protein in serum of immunized mice was quantified longitudinally. Since the NMO-IgG is a human IgG1 isotype, both, the murine IgG2a and IgG2b isotype were examined as they have similar properties with regard to complement binding and the Fcγ receptor. A switch from IgM to IgG2b was detected in mice immunized with hAQP4_281-300 peptide with regard to (B) antibody responses against hAQP4_281-300 and (C) whole-length AQP4 protein. An Ig isotype switch from IgM to IgG2b was also detectable in mice immunized with whole-length AQP4 protein with regard to (D) antibody responses against hAQP4_281-300 and (E) whole-length AQP4 protein.</p

<i>HLA-DRB1*03</i>:<i>01</i> transgenic mice are disease resistant to active immunization with human aquaporin 4 (hAQP4), and adoptive transfer of hAQP4-specific T cells.

<p>(A) <i>HLA-DRB1*03</i>:<i>01</i> mice were actively immunized with proteolipid protein (PLP)_91-110 (100 μg/100 μl/mouse; positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152720#pone.0152720.ref025" target="_blank">25</a>]), or varying AQP4 antigens*(whole-length hAQP4 protein, hAQP4_281-300, murine (m)AQP4_281-300, hAQP4_281-300 with a Quil-A Incomplete Freund Adjuvant (IFA) booster on day 14 post-immunization, mAQP4_281-300 with a Quil-A IFA booster on day 14 post immunization, and hAQP4_281-300 plus mAQP4_281-300) emulsified in Complete Freund Adjuvant (CFA). Immunization with a positive control proteolipid protein (PLP)_91-110, a dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> led to typical EAE. (B) Lymph node cells taken from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 or mAQP4_281-300 were restimulated for three days and passively transferred into <i>HLA-DRB1*03</i>:<i>01</i> mice. None of these experimental approaches resulted in clinical disease. (C) Paraffin sections were stained with haematoxlin eosin (H&E) and luxol fast blue (LFB). Representative sections of the spinal cords from PLP_91-110 and hAQP4_281-300 immunized mice are shown. On histopathological examination there were no visible signs of cellular infiltration, inflammation, or demyelination within the brain and spinal cord in any experimental paradigms other than in active immunization with PLP_91-110, the dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> that led to typical EAE (spinal cord shown; inflammatory infiltrates and areas of demyelination are indicated by black arrows). (D) Fifteen days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with PLP_91-110 or hAQP4_281-300, pupillary reflex was measured via a mouse pupillometry. Mice actively immunized with hAQP4_281-300 and the control antigen PLP_91-110 did not show altered pupillary responses.</p

Human (h)AQP4_284-299 Alanine Scanning Peptides.

<p>The immunogenic region of hAQP4_281-300, hAQP4_284-299, was utilized to generate alanine scanning peptides at which each peptide sequence has a single alanine residue mutation.</p

Human (h)AQP4_281-300-specific T cells do not cross-react with murine (m) AQP4_281-300.

<p>(A) There is that a single amino acid substitution from aspartic acid in hAQP4 to glutamic acid in murine (m)AQP4 at position 290. (B) In lymph node cells of <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 there was a significant proliferation of CD4⁺ T cells when hAQP4_281-300 was used as the recall antigen (* = P-value = 0.01). Only a higher recall antigen dose of 25 μg/ml resulted in a significant increase in proliferation, whereas as a dose of 5 μg/ml did not. (C) There was no proliferative response to mAQP4_281-300 at either dose_. (D) There is a significantly increased frequency of IFNγ and IL-17 producing lymph nodes cells from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 by ELISpot assay when hAQP4_281-300, and hAQP4_281-299 are used as recall antigens. However, we were unable to detect antigen specific IFNγ and IL-17 producing lymph nodes cells when mAQP4_281-300, or the negative control hAQP4_66-79 were used as recall antigens (** = P-value < 0.01). (E) IFNγ and IL-17 producing lymph nodes cells from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with mAQP4_281-300 were undetectable with any of the recall antigens.</p

B and T cell frequency in therapeutic model of Rituximab administration.

<p>Cells from each organ were pooled from at least three mice per group and counted. Cell counts were then normalized by dividing the total counts by the numbers of mice in each group and then multiplied by the percentage of each cell type as identified by flow cytometry. Total leukocytes were identified by CD45+ events within FSC and SSC gates. B cells were identified using gates for CD19 and B220. T cells were identified by expression of CD3 and either CD4 or CD8. Numbers in parenthesis indicate the percent change in cell counts in the hCD20Tg mice as compared to WTLM controls.</p

Disruption of EAE pathogenesis by B cell depletion.

<p><b>Panel A</b>. EAE severity is similar in WTLM and hCD20Tg mice. EAE onset and severity were monitored using a 5-point scale on WTLM and hCD20Tg mice immunized with MOG_1–125. These results are representative of at least two independent experiments. <b>Panels B–D</b>. Rituximab administration prevents the induction of EAE. WTLM or hCD20Tg mice were either left untreated or were injected with 100 µg Rituximab daily for three days (Day -3,-2,-1). On Day 0, EAE was induced by immunization with MOG_1–125. <b>Panel B</b>. Disease course of WTLM and hCD20Tg mice, EAE onset and severity was monitored using a 5-point scale. Shown are the mean clinical score +/− SEM. <b>Panel C</b>. B cell depletion results in reduced levels of anti-MOG IgG in the serum. Serum was harvested on day 21 post-immunization and MOG-specific IgG levels were determined by ELISA. Results shown are the mean IgG concentration +/− SEM. Asterix indicates significant decrease as compared to Rituximab-treated WTLM mice. <b>Panel D</b>. Rituximab administration results in rapid depletion of B cells in the peripheral blood. Blood was taken from WTLM or hCD20Tg mice 3 days following the final dose of Rituximab (day 2 post-immunization). B cells were identified by flow cytometry using gates to identify lymphocytes and CD19 expressing cells. Results shown are the mean percentages of CD19+ B cells +/−SEM (*, p<0.01). <b>Panels E/F</b>. Treatment with Rituximab reduces EAE severity. EAE was initiated in WTLM and hCD20Tg mice on Day 0. Upon the appearance of clinical signs of EAE, Rituximab (100 µg) was administered daily for three treatments. <b>Panel E</b>. Disease course of WTLM and hCD20Tg mice, EAE onset and severity was monitored using a 5-point scale. Shown are the mean clinical score +/− SEM. <b>Panel F</b>. B cell depletion in peripheral blood on day 20. Significant differences were determined using an unpaired t-test (*, p<0.05; **, p<0.01). These results are representative of at least two independent experiments with Rituximab and two experiments using the 1F5 anti-human CD20 mAb (data not shown).</p

Rituximab administration alters MOG-specific recall responses.

<p><b>Panels A–E</b>. WTLM and hCD20Tg mice were treated with Rituximab daily for 3 days (Day -3,-2,-1) followed by immunization with MOG_1–125 on Day 0. On day 20 post-immunization, bulk draining lymph node cells (LNC) were isolated and recall response determined. <b>Panels A and B</b>. Identification of MOG-reactive T cells by tetramer staining. Bulk LNC were cultured for 3 days in the presence of MOG_1–125 prior to labeling with antibodies to CD3, CD4 and I-Ab tetramers to either human (A) CLIP_103–117 or (B) MOG_38–48. Numbers above boxes indicate percentages of T cells in the tetramer positive gate. <b>Panel C</b>. Secondary T cell proliferative responses were determined by CFSE dilution assay. LNC were labeled with CFSE and placed in culture with 20 µg/ml MOG_1–125 and proliferation determined by flow cytometry on day 6 of culture. Results shown are gated on CD4+ events. Numbers indicate the percentage of total cells that diluted CFSE from WTLM and hCD20Tg mice. <b>Panels D and E</b>. 48-hour supernatants from the Panel C experiments were examined for the presence of IL-17 (D) or IFNγ (E) by ELISA. Asterices indicate a significant decrease in IL-17 production (p<0.05). Results are representative of at least 2 independent experiments.</p

B and T cell dynamics following Rituximab treatment.

<p><b>Panels A and E</b>. Expression of human CD20 by hCD20Tg B cells and hCD20Tg T cells. Splenocytes from WTLM and hCD20Tg mice were stained with antibodies to human CD20, CD4 and CD19 and flow cytometry performed. Results shown are gated on CD19+CD4− events to identify B cells or gated on CD19−CD4+ events to identify T cells. <b>Panels B/C/D/F/G</b>. WTLM and hCD20Tg mice received three daily injections of Rituximab (100 µg) beginning on day 0. At 144 hours after Rituximab treatment was initiated, tissues were harvested for flow cytometry analysis. <b>Panel B</b>. Peripheral B cells are rapidly depleted following Rituximab treatment. <b>Panel C</b>. Splenic B cells are depleted following Rituximab treatment. <b>Panel D</b>. B cells in the LN (Axilary, Brachial and Inguinal) are depleted following Rituximab treatment. <b>Panel E</b>. CD4 T cells do not express human CD20. <b>Panel F</b>. Splenic CD4 T cells are reduced following Rituximab treatment. <b>Panel G</b>. CD4 T cells in the LN (Axilary, Brachial and Inguinal) are reduced following Rituximab treatment. <b>Panel H</b>. Rituximab administration does not prevent priming of inflammatory T-effector cells. WTLM and hCD20Tg mice were treated with Rituximab daily for 3 days (Day -3,-2,-1), followed by immunization with MOG_1–125 on Day 0. On day 10 post-immunization, DTH responses were elicited by subcutaneous injection of MOG_1–125 (10 µg) in the ear. The net ear swelling responses were determined at 24 hours. Results shown indicate the mean ear swelling in mmX10E-3 (background subtracted) +/− SEM. Significant differences were detected by unpaired t-test (*, p<0.05; **, p<0.01). These results are representative of at least two independent experiments.</p