Figure 7. Cooperative enhancement of HEL_105–120-specific effector CD4 T cell generation results in increased HEL_105–120-specific effector cell responses following secondary immunization.

One day prior to injection of pulsed splenocytes, BALB/c mice were seeded with 10⁵ MACS purified CD4⁺ DO11.10 T cells. BALB/c splenocytes were cultured overnight in the presence of either OVA_323–339 alone, HEL_105–120 alone, or both HEL_105–120 and OVA_323–339. Following harvest, B cells were isolated by MACS negative selection and 3.5×10⁶ peptide-pulsed B cells were injected subcutaneously into the footpad and lower leg of seeded BALB/c mice on days 0, 3, and 6. Seven weeks later all mice were injected intraperitoneally with 100 ug heat-aggregated HEL protein in saline. Ten days post-challenge the HEL_105–120-specific cytokine producing cells in the spleen of mice given OVA_323–339-pulsed B cells (O), HEL_105–120-pulsed B cells (H), and OVA_323–339 and HEL_105–120-pulsed B cells (H+O) were assessed by ELISpot. Each data point represents the number of specific ELISpot-forming cells in the spleen of a single mouse. The probability that the mean number of ELISpot-forming cells in all groups is the same was assessed by one-way ANOVA. Post-hoc comparisons revealed significant differences between the means of the groups indicated. These are pooled results of three independent experiments.