Figure 5
Mage-b-specific immune responses in vitro. BALB/c mice were immunised with the LM-LLO-Mage-b/2nd vaccine strain and challenged with 4T1 tumour cells as outlined in Figure 1, or not challenged with 4T1 tumour cells. Two weeks after tumour challenge, mice were euthanized and spleens and draining (inguinal) lymph nodes (LNs) were analysed for in vitro immune responses upon restimulation with Mage-b. For this purpose, the number of IFNγ-producing cells in spleens of mice without (A) and with (B) 4T1 tumours and metastases were compared. Again, the number of IFNγ-producing cells were determined in spleens (C) but now compared with the number of IFNγ-producing cells in the LNs (D) of mice bearing 4T1 tumours and metastases were compared. Lymph nodes and spleens were from the same mice, and tested in the same experiment. In panel C, spleen cells depleted for CD8 T cells are shown as well. All restimulation assays were performed with bone marrow (BM) cells transfected with pcDNA3.1-Mage-b and pCMV1-GM-CSF plasmid DNA. Two days later, Mage-b-specific immune responses were analysed by ELISPOT. Controls such as BM cells transfected with pcDNA3.1-Mage-b, or with pCMV-GM-CSF, or non-transfected BM cells did not produce IFN (data not shown). The LM-LLO-Mage-b/2nd vaccine was tested in three independent experiments. Results were averaged and subjected to statistical analysis using Mann–Whitney test (n=5 mice per group in each experiment). The error bars represent the s.e.m.
