Figure 5. Enriched splenic DCs do not mediate cooperation between HEL_105–120 and OVA_323–339-specific CD4 T cell populations.

BALB/c mice were seeded with 10⁵ MACS purified CD4⁺ DO11.10 T cells one day prior to injecting splenocytes cultured in the presence of HEL_105–120 alone or in the presence of both HEL_105–120 and OVA_323–339. After harvesting and washing, splenic DC populations were isolated by positive selection. Both DC depleted and DC enriched (DC+) peptide-pulsed cells were injected in proportion to the standard injection of 10⁷ (10⁷ and 2×10⁵ cells respectively). In one experiment the number of DC enriched cells was increased five-fold (10⁶ DC+). These injections were given on days 0, 3, and 6. On day 9 after the first injection, the HEL_105–120-specific cytokine-producing cells in the spleens, and draining popliteal lymph nodes (DLN), were enumerated by ELISpot, in mice given HEL_105–120-pulsed APC (H), or HEL_105–120 and OVA_323–339-pulsed APC (H+O). Each data point represents the number of specific ELISpot-forming cells in the lymphoid organ 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. The data presented are pooled values from three independent experiments.