Figure 4

Interleukin-17/interferon-gamma (IL-17/IFNγ) ratio increases with disease severity and dependence on Fas ligand. (a) Single-cell suspensions of splenocytes from individual mice were cultured in the presence of 10 μg/mL of cII_259-273 peptide. Culture supernatants were collected on days 2 and 5 and tested for IFNγ and IL-17, respectively, by enzyme-linked immunosorbent assay (ELISA) as described in Materials and methods. The ratio of antigen-stimulated IL-17 to IFNγ was determined for each mouse and plotted against the disease score for that mouse. Linear regression was performed using GraphPad Prism software and showed a significant correlation between a higher IL-17/IFNγ ratio as disease severity increased (R-squared = 0.2539, F = 5.444, P = 0.033). (b-c) Splenic CD19⁺ B cells were magnetic bead-purified from naïve DBA/1 mice and cultured overnight in the presence or absence of plate-bound anti-mouse CD40 antibody. After 24 hours of culture, cII peptide was added to indicated samples, followed by the addition of splenocytes from naïve cII T-cell receptor transgenic mice that had been depleted of CD19⁺ cells by magnetic bead separation. Supernatants were collected 5 days after the addition of B-depleted cells, and IL-17 (b) and IFNγ (c) levels were determined by ELISA. Data shown are mean ± standard deviation of triplicate samples. Ag, antigen; cII, type II collagen; FasL, Fas ligand.