Figure 2. Administration of HEL_105–120 and OVA_323–339-pulsed splenocytes to DO11.10-seeded mice results in significant cooperative enhancement of HEL_105–120-specific effector CD4 T cell generation.

One day prior to injection of pulsed splenocytes, BALB/c mice were injected with 10⁵ MACS purified CD4⁺ DO11.10 T cells. BALB/c splenocytes were cultured overnight in the presence of either HEL_105–120 alone, OVA_323–339 alone, or both peptides. Following harvest and extensive washing, 10⁷ peptide-pulsed splenocytes were injected subcutaneously into the footpad and lower leg of seeded BALB/c mice on days 0, 3, and 6. Nine days after the initial injection of pulsed splenocytes, the HEL_105–120 and OVA_323–339-specific cytokine producing cells in the spleens and draining popliteal lymph nodes (DLN) were enumerated by ELISpot in mice given only HEL_105–120-pulsed splenocytes (H), OVA_323–339-pulsed splenocytes (O), and in mice given splenocytes pulsed with both HEL_105–120 and OVA_323–339 (H+O). Each data point represents the number of peptide-specific ELISpot-forming cells in the spleen or popliteal lymph node of a single mouse. P-values indicate the probability that the means of the indicated samples are not different as assessed by unpaired, two-tailed T-tests. Results presented are pooled data from four to five independent experiments.