Figure 4. Augmented antigen-specific proliferation, IL-10 and IFN-γ production in HSP70 immunized mice.

Mice were immunized with PBS or heat shock protein 70 (HSP70) and arthritis was induced as described in figure 1. Spleen cells were isolated on day 35 and cultured (2×10⁵/well) in the presence of recombinant microbial HSP70, proteoglycan (PG) or ovalbumin (Ova) (all at 10 µg/ml). (A) After 96 hours incubation proliferation was determined by measuring ³H incorporation. Results are expressed as mean stimulation index (CPM Antigen/CPM background)±SEM. The mean background values (responses without antigen) were for the PBS control group 8390±2135 and for the HSP70 group 10 180±2741. (B–C) For in vitro cytokine secretion the spleen cells were cultured in the presence of antigen. After 72 hours supernatants were harvested and assayed by multiplex analysis for IL-10 and IFN-γ excretion. Open bars show the PBS immunized group and filled bars the HSP70 immunized group. Values represent the mean±SEM of cytokine levels. Significant difference (p<0.05, Mann-Whitney U Test) is indicated by the asterisk. Data are representative for at least two independent experiments.