Figure 4. Induction of iNKT cell mediated IL-12 production by splenic adherent cells.

Sixty days LD infected animals were treated with GSPL as described in the legend of Figure 1. (A) Animals were sacrificed 15 days after the last treatment and iNKT cells isolated from individual experimental animals (WT BALB/c and C3H/HeJ mice) were mixed with 1∶10 of autologous splenic adherent cells (1×10⁶ adherent cells) and were cultured in 24-well culture plates containing 100 µg/mL GSPL for 24 h. Culture supernatants were assayed for the concentration of IL-12p70 and IL-12p40 in ELISA. The results are representative of three independent experiments and data shown are means ± SD; *p<0.0001 versus corresponding infected control; Student's t-test. (B) mRNA expression of IL-12p40, IL-12p35, and IL-23p19 in the spleen of each GSPL treated LD infected animals was evaluated individually by real-time PCR. The fold up-regulation of mRNA post-GSPL treatment was calculated by normalizing the amount of cytokine mRNA with the housekeeping gene GAPDH, and comparing results from treated to infected; *p<0.0001; paired two-tailed Student's t-test. (means ± SD (n=3) of one from three independent experiments are shown).