Additional file 1: of DECIPHER: harnessing local sequence context to improve protein multiple sequence alignment

This supplementary file contains a description of the DECIPHER algorithm, Figures S1–S6, and Tables S1–S2. (PDF 719 kb

Additional file 1: Figure S1. of Quality filtering of Illumina index reads mitigates sample cross-talk

Average quality score per-base for each read type. Tables S1 and S2: Lists of strains and primers used in this study. (DOCX 450 kb

Epithelial V-Like Antigen Mediates Efficacy of Anti-Alpha₄ Integrin Treatment in a Mouse Model of Multiple Sclerosis

<div><p>Natalizumab inhibits the transmigration of activated T lymphocytes into the brain and is highly efficacious in multiple sclerosis (MS). However, from a pharmacogenomic perspective, its efficacy and safety in specific patients remain unclear. Here our goal was to analyze the effects of epithelial V-like antigen (EVA) on anti-alpha₄ integrin (VLA4) efficacy in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EVA has been previously characterized in human CD4 T lymphocytes, mouse thymic development, and choroid plexus epithelial cells. Further analysis here demonstrated expression in B lymphocytes and an increase in EVA⁺ lymphocytes following immunization. Following active induction of EAE using the MOG^35–55 active immunization model, EVA deficient mice developed more severe EAE and white matter tissue injury as compared to wild type controls. This severe EAE phenotype did not respond to anti-VLA4 treatment. In both the control antibody and anti-VLA4 conditions, these mice demonstrated persistent CNS invasion of mature B lymphocyte (CD19⁺, CD21⁺, sIgG⁺), increased serum autoantibody levels, and extensive complement and IgG deposition within lesions containing CD5⁺IgG⁺ cells. Wild type mice treated with control antibody also demonstrated the presence of CD19⁺, CD21⁺, sIgG⁺ cells within the CNS during peak EAE disease severity and detectable serum autoantibody. In contrast, wild type mice treated with anti-VLA4 demonstrated reduced serum autoantibody levels as compared to wild type controls and EVA-knockout mice. As expected, anti-VLA4 treatment in wild type mice reduced the total numbers of all CNS mononuclear cells and markedly decreased CD4 T lymphocyte invasion. Treatment also reduced the frequency of CD19⁺, CD21⁺, sIgG⁺ cells in the CNS. These results suggest that anti-VLA4 treatment may reduce B lymphocyte associated autoimmunity in some individuals and that EVA expression is necessary for an optimal therapeutic response. We postulate that these findings could optimize the selection of treatment responders.</p></div

EVA-deficient mice demonstrate VLA4-independent CNS invasion during EAE.

<p>Flow cytometry analysis of mononuclear subsets from spinal cords of mice at onset of peak EAE (day 19) was performed. <b>A–B.</b> As predicted, total mononuclear cell counts (left y-axis) and percent CD4 invasion (right Y-axis) were reduced in wild type, litter-mate control mice treated with anti-VLA4. EVA-deficient mice demonstrated similar immune cell recruitment independent of treatment with control anti-HEL antibody or anti-VLA4. <b>C.</b> Percent differences in CD4, CD8, CD11b and CD19 populations were less marked between groups. There was a modest decrease in CD11b⁺ cells in spinal cords of EVA^−/− control treated mice as compared to the wild type control treatment group. Representative FACS plots are shown. Analysis of pooled data from multiple experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070954#pone-0070954-t002" target="_blank">Table 2</a>.</p

Analysis of EVA⁺ mononuclear cell subsets in C57BL6 mice.

*<p><i><u>P</u></i><0.05 as compared to the naive condition.</p

Increased EAE disease severity and resistance to anti-VLA4 treatment in EVA-deficient mice.

<p>EAE was induced by active immunization at 2 sites with MOG peptide^35–55 emusified in CFA (0.1 mL of emulsion/site; 1 mg/ml MOG^35–55 and 2.5 mg/ml H37Ra killed mycobacterium). Pertussis toxin (100 ng ip) was administered on days 0 and 1. Age and sex-matched control littermates were used for the wild type group. Approximately 50% of the mice used for each condition were male. Animals were observed on a daily basis for signs of clinical EAE. The animals were graded as follows: 1, limp tail; 2, partial hind limb paralysis; 3. complete hind limb paralysis; 4, hind and front limb paralysis; 5, moribund. Animals were cared for in accordance with University of Wisconsin guidelines. <b>A.</b> As compared to sex and age-matched littermate wild type controls, EVA-deficient mice developed more severe EAE as determined by analysis of daily, cumulative and peak disease scores. Cumulative disease score was 48.4±8.2 in knockout mice (n = 12) and 12.5±4.3 in wild type (n = 10; <i>P</i><0.01); peak disease score was 3.08±0.47 in knockouts and 1.30±0.30 in wild type (<i>P</i><0.01). Differences in daily disease scores were statistically significant from day 15 through day 30, the final day of observation. <b>B.</b> In separate experiments, mice were treated with 4 mg/kg of either IgG₂ isotype control anti-HEL (hen egg lysozyme) or anti-mouse VLA4 (PS/2 monoclonal antibody) intraperitoneally every 4 days for 28 days starting at day 0. As expected, wild type litter-mate controls treated with anti-VLA4 had significantly reduced disease severity as compared to anti-HEL-treated mice. Cumulative disease scores were 31.8±4.1 in the control anti-HEL treatment group versus 6.1±2.3 in the anti-VLA4 group (<i>P</i><0.01, n = 10 for both groups) and, peak disease scores were 2.50±0.22 and 0.60±0.22, respectively (<i>P</i><0.01). Differences in daily disease scores were statistically significant from day 17 through day 30. EVA-deficient mice were resistant to anti-VLA4 treatment and developed severe disease independent of treatment. Cumulative score was 49.1±7.8 in the control anti-HEL group and 41.1±6.4 in the anti-VLA4 group (<i>NS</i>; n = 9 for anti-HEL and n = 8 for anti-VLA4). Peak disease score was 3.22±0.36 in the anti-HEL control group and 3.38±0.60 in the anti-VLA4 group (<i>NS</i>).</p

Analysis of humoral responses during EAE.

<p><b>A.</b> Flow cytometry analysis was performed to examine mature B cells within spinal cord infiltrates. Cells were stained for expression of CD19 and surface IgG (sIgG) and analyzed by flow cytometry. At the onset of peak disease (day 19), anti-VLA4 treatment reduced CNS invasion in wild type, but not EVA-deficient mice. At a late stage of EAE (day 40), EVA-deficient mice demonstrated persistence of high levels of mature CD19⁺sIgG⁺ (surface IgG-positive) B lymphocytes. Representative FACS plots are shown. Analysis of pooled data from multiple experiments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070954#pone-0070954-t002" target="_blank">Tables 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070954#pone-0070954-t003" target="_blank">3</a> and the bar graphs. <b>B–C.</b> B cell subset analysis in EAE spinal cord revealed that the CD19⁺sIgG⁺ population shown in <b>A</b> expresses high levels of the maturation marker, CD21, but not the plasma cell marker, CD138. Quantitative analysis revealed persistence of mature B cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070954#pone-0070954-t003" target="_blank">Table 3</a> and bar graph) in EVA-deficient mice as compared to wild type litter- mates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070954#pone-0070954-t003" target="_blank">Table 3</a> and bar graph). At onset of peak disease, anti-VLA4 treatment reduced invasion of this population in wild type but not EVA-deficient mice. <b>D–E.</b> We also identified a population of CD19^intsIgG^int cells from EVA-deficient mice that was increased in spinal cord infiltrates as compared to both control treatment conditions at onset of peak disease (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070954#pone-0070954-t002" target="_blank">Table 2</a> and bar graph). In EVA-deficient mice, most of these cells expressed intermediate and high levels of CD5. <b>F.</b> Serum autoantibody levels (anti-MOG IgG) were measured at onset of peak disease by ELISA. EVA-deficient mice demonstrated increased autoantibody levels as compared to wild type mice from the same treatment condition. Anti-VLA4 treatment prevented the development of autoantibody in wild type mice. Although treatment appeared to reduce levels in EVA-deficient mice, autoantibody serum concentration remained higher than that seen in wild type control treated mice. At onset of peak EAE, serum IgG anti-MOG levels were 393.4±36.1 µg/ml in the knockout anti-HEL treated group, 91.6±8.7 µg/ml in knockout anti-VLA4, 52.3±19.5 µg/ml in wild type anti-HEL and 12.2±1.7 µg/ml wild type anti-VLA4.</p

Characterization of EVA⁺ lymphocyte subsets <i>in vivo.</i>

<p><b>A.</b>In naïve, unimmunized mice, approximately 3–9% of mononuclear cells express EVA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070954#pone-0070954-t001" target="_blank">Table 1</a>). A majority of these are CD19⁺ B lymphocytes. For flow cytometry analysis of lymphocyte subsets, gating was performed on the mononuclear subset as defined by FSC/SSC criteria. <b>B.</b> Following immunization with MOG^35–55 using CFA and pertussis toxin as adjuvants, the percent of EVA⁺ lymphocytes prior to disease onset (day 12) increased markedly in lymph node and peripheral blood (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070954#pone-0070954-t001" target="_blank">Table 1</a>).</p

Distinct appearance of parenchymal spinal cord lesions in EVA-deficient mice during EAE.

<p><b>A.</b> Hematoxylin and eosin stained sections of spinal cord lesions at onset of peak disease (day 19) demonstrated meningeal immune cell infiltration in all genotype and treatment conditions (longitudinal sections in lateral white matter). Scale bars, 50 µm<b>. B, C.</b> Immunofluorescence staining of longitudinal spinal cord sections in EVA-deficient conditions revealed parenchymal lesions devoid of myelin basic protein (MBP; green) associated with small numbers of CD4 and CD11b cells (red) and larger numbers of cells with blastic-appearing nuclei (DAPI nuclear stain; blue) that contained prominent nucleoli. Wild type lesions in control antibody treated mice demonstrated parenchymal invasion adjacent to meningeal infiltrates with CD4 and CD11b cells distributed throughout. A small number of meningeal inflammatory infiltrates were seen in anti-VLA4 treated mice. Scale bars, 50 µm.</p