Figure 6. OX40L blockade significantly impairs the generation of peptide-specific cytokine producing effector CD4 T cells following injection of peptide-pulsed purified B cells.

One day prior to injection of pulsed B cells, BALB/c mice were injected with 10⁵ MACS purified CD4⁺ DO11.10 T cells. BALB/c splenocytes were cultured overnight in the presence of HEL_105–120 alone, or both HEL_105–120 and OVA_323–339. Following splenocyte 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 DO11.10-seeded BALB/c mice on days 0, 3, and 6. With each B cell injection, mice were injected intravenously with either 50 µg of an isotype-matched control antibody (iso. cont.) or with an OX40 blocking antibody (OX40 blocker). 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 for mice given only HEL_105–120-pulsed B cells (H), and in mice injected with B cells pulsed with both HEL_105–120 and OVA_323–339 (H+O). Each data point represents the number of specific ELISpot-forming cells in the spleen and 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. Data presented are pooled values from three independent experiments.