Figure 4

Cross-presentation of HER2 protein by DEC-HER2 immunization. (A) FVB/N mice were primed and boosted with DEC-HER2 or Ctrl Ig-HER2 (5 μg) in combination with poly IC (50 μg). Two weeks after the boost, spleen CD8⁺ T cells were purified by magnetic-activated cell sorting and re-stimulated with spleen CD11c⁺ DCs in the presence of medium alone or 1 μg/mL HIV gag peptide or HER2 peptide pools. IFNγ production was measured by enzyme-linked immunosorbent spot (ELISPOT) assay. (B) Proliferative capacity of HER2-specific CD8⁺ T cells. Mice were immunized as in (A), and bulk splenocytes were labeled with CFSE and re-stimulated with medium or 200 ng/mL HIV gag or HER2 peptide pool 1-7 for 4 days. Cells were re-stimulated for the last 6 hours, and IFNγ production was measured by intracellular cytokine staining. (C) HLA-A2 transgenic mice were vaccinated as in (A), and HER2-specific CD8⁺ T-cell responses were measured by ELISPOT assay. Purified CD8⁺ T cells were re-stimulated with medium alone or 1 μg/mL HIV gag peptide mix or HER2 peptide pool 1-7. All experiments were performed with at least three mice per group, and the results of one of three experiments are shown. ***P < 0.001. CFSE, 5,6-carboxy fluorescein diacetate succinimidyl ester; HER, human epidermal growth factor receptor; IFNγ, interferon-gamma; Ig, immunoglobulin; poly IC, polyinosinic/polycytidylic acid.