Figure 2. sH4 exacerbates disease in CIA model.

(A) Schematic representation of the full length murine B7-H4, B7-H4V, B7-H4VC, and B7-H4Ig fragments, which were inserted into pcDNA plasmid. IgV, immunoglobulin V-like region; IgC, immunoglobulin C-like region; TM, transmembrane region; CT, cytoplasmic tail. Effect of decoy B7-H4 in incidence (B) and (C) clinical score of CIA. Mice were immunized with CII in complete Freund's adjuvant (CFA) on day 0 and day 21. The incidence and clinical score were recorded daily based on the standard described in Materials and Methods. Three groups of mice were injected with the indicated plasmids on day −1 and day 20. Each point represents results from pool of ten mice and expressed as means ± 95% confidence interval. Data represent three independent experiments. Statistical analysis was performed by repeated ANOVA method (p<0.0001). The following Tukey-Kramer test showed significant differences at each points after day 28 between the control group and other two groups (p<0.01). (D) Effect of decoy B7-H4 in the generation of autoantibodies against CII in CIA sera. CIA mice were treated with indicated plasmids and sera were sampled at day 30 after collagen immunization. Serum levels of IgG against CII were determined by specific ELISA as described Materials and Methods. Each group represents results from a pool of five mice. The top and bottom of each rectangular box denotes the 75th and 25th percentiles, respectively, with the median shown inside the box. Error bars extending from each box represent the maximum and minimum. Data represent three independent experiments. Statistical analysis was performed by ANOVA method followed by the Scheffé test. (E) Effect of decoy B7-H4 in the proliferation of T cells reactive to collagen in CIA mice. CIA mice were first treated with indicated plasmids in vivo as indicated in (B). Whole splenocytes from the mice at day 30 after collagen immunization were cultured in the presence of the indicated concentrations of CII for 72 h. The cultures were pulsed with ³HTdR 18 h before harvest, and cpm were determined by a scintillation counter. Each point represents results from three wells and data represent two independent experiments and expressed as means±95% confidence interval. (F) Effect of decoy B7-H4 in the production of cytokines from splenocytes reactive to CII in CIA mice. Supernatants in splenocyte cultures as described in (E) at 72 h were collected and assessed for IFN-γ and IL-17 by ELISA. Each group represents results from three wells and data represent three independent experiments. The top and bottom of each rectangular box denote the 75th and 25th percentiles, respectively, with the median shown inside the box. Error bars extending from each box represent the maximum and minimum. Statistical analysis was performed by ANOVA followed by the Scheffé test.