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Figure 2 | Breast Cancer Research

Figure 2

From: Targeting of the non-mutated tumor antigen HER2/neu to mature dendritic cells induces an integrated immune response that protects against breast cancer in mice

Figure 2

A single dose of DEC-HER2 fusion monoclonal antibody (mAb) vaccine immunizes HER2-specific CD4+ T cells in vivo. (A) Groups of C57BL/6 mice were vaccinated intraperitoneally with 5 μg of DEC-HER2 or Ctrl Ig-HER2 in combination with poly IC (50 μg) and anti-CD40 antibody (25 μg). Additional groups received adjuvants alone or were left untreated. Two weeks after immunization, splenocytes were re-stimulated without added peptides (medium alone) or with 2 μg/mL HER2 peptide pools 1 to 7 or irrelevant HIV gag peptides. Intracellular cytokine staining was determined by fluorescence-activated cell sorting after 6 hours of in vitro stimulation. The percentage of IFNγ+CD4+ T cells is shown as mean ± standard error with three or four mice per group. The results of one of three independent experiments are shown. (B) CFSE dilution assay. Mice were immunized as in (A), and bulk splenocytes were labeled with 2.5 μM CFSE and re-stimulated with 0.2 μg of HER2 peptide pool 1-7 or HIV gag peptide mix or medium alone for 4 days. T-cell proliferation was analyzed in combination with intracellular cytokine staining of the cells re-stimulated for the last 6 hours. Percentages of proliferating (CFSElow) and IFNγ+CD4+ T cells are shown with three or four mice per group. The results of one of two independent experiments are shown. (C) Th1/Th2/Th17 cytokine enzyme-linked immunosorbent assay (ELISA). Mice were immunized as in (A). Two weeks after the boost, splenic CD4+ T cells and CD11c+ cells were isolated. CD4+ cells (3 × 105) were incubated with 1 × 105 CD11c+ cells in 96-well U-bottomed plates in the presence of 2 μg/mL HER2 peptide pool 5 or HIV gag peptides for 48 hours. Concentrations of IFNγ/IL-4/IL-10/IL-17 in culture supernatant were measured by ELISA. The results of one of two independent experiments (n = 4 mice per group) are shown. *P < 0.05, **P < 0.01, ***P < 0.001. CFSE, 5,6-carboxy fluorescein diacetate succinimidyl ester; HER, human epidermal growth factor receptor; IFNγ, interferon-gamma; Ig, immunoglobulin; IL, interleukin; NS, not statistically significant; poly IC, polyinosinic/polycytidylic acid; Th, T helper.

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