American Society of Hematology

Figure 3

$Figure 3. IL-2 secretion by MAGE-A3/HLA-DP4–specific CD4+ T cells after stimulation with differently loaded DCs. (A) Ag loading and maturation of DCs during this study. Depicted is a scheme indicating at which time point and at which stage DCs were loaded with the constructs and at which time point the DCs were matured. The plastic-adherent fraction of PBMCs from healthy donors was cultured in medium supplemented with granulocyte-macrophage colony-stimulating factor and IL-4. After 6 days of culture, cells were matured (black bar) for 24 hours with IL-1β, IL-6, PGE2, and TNF (mDCs), or were left immature (iDCs). iDCs and mDCs were incubated for 48 hours with the anti–DEC-205scFv-MAGE-A3-KKL or anti–MCSPscFv-MAGE-A3-KKL constructs (▨). After 24 hours of incubation with the constructs, a fraction of the iDCs was matured with the maturation cocktail (■) for 24 hours (iDCm). This ultimately leads to 3 different Ag-loaded DC populations, which were used in subsequent experiments. (B) Comparison of the stimulatory capacity of DCs loaded with MAGE-A3 by DEC-205 targeting, RNA electroporation, or direct peptide pulsing. DCs were either matured (mDCs) or were left immature (iDCs) and were incubated with 1 μg/mL of the anti–DEC-205scFv-MAGE-A3-KKL or the anti–MCSPscFv-MAGE-A3-KKL construct (as respective control) for 48 hours. A fraction of the iDCs was matured during the incubation (iDCm). In addition, mDCs were electroporated with MAGE-A3-DCLAMP RNA or MAGE-A3 RNA (as respective control) and pulsed with MAGE-A3/HLA-DP4 or NY-ESO-1/HLA-DP4 (as respective control) peptides. All these differently loaded DC populations were used to stimulate MAGE-A3/HLA-DP4–specific autologous CD4+ T cells, which were generated by TCR-RNA electroporation (“Cytokine-secretion assays”). IL-2 secretion by specific T cells was analyzed after 18 hours of coculture. Furthermore, the ratio between IL-10 to IL-2 secretion after activation of specific T cells with DEC-205–targeted iDCs, iDCm, and mDCs was analyzed (right panel). P values were calculated with the Mann-Whitney U test. *P < .05. **P < .005. ***P < .001. (C) Dose-dependent IL-2 secretion by MAGE-A3/HLA-DP4–specific CD4+ T cells after stimulation with DEC-205–loaded iDCm, generated as described in panel A, except that they were loaded with different concentrations of anti–DEC-205scFv-MAGE-A3-KKL fusion protein (as indicated). As negative control, heat-inactivated anti–DEC-205scFv-MAGE-A3-KKL protein in the same concentrations, 10 μg/mL and 1 μg/mL of anti–MCSPscFv-MAGE-A3-KKL protein, medium, and solvent control (generated by similar protein-purification protocol from mock-transfected 293T cells) were used. These DCs were used to stimulate MAGE-A3/HLA-DP4–specific autologous CD4+ T cells; and after 18 hours of coincubation, IL-2 concentrations in the supernatants were measured. Data are mean values ± SD of 4 independent experiments. DK indicates anti–DEC-205scFv-MAGE-A3-KKL fusion protein; MK, anti–MCSPscFv-MAGE-A3-KKL fusion protein; and EP, electroporation.$

IL-2 secretion by MAGE-A3/HLA-DP4–specific CD4⁺ T cells after stimulation with differently loaded DCs. (A) Ag loading and maturation of DCs during this study. Depicted is a scheme indicating at which time point and at which stage DCs were loaded with the constructs and at which time point the DCs were matured. The plastic-adherent fraction of PBMCs from healthy donors was cultured in medium supplemented with granulocyte-macrophage colony-stimulating factor and IL-4. After 6 days of culture, cells were matured (black bar) for 24 hours with IL-1β, IL-6, PGE₂, and TNF (mDCs), or were left immature (iDCs). iDCs and mDCs were incubated for 48 hours with the anti–DEC-205scFv-MAGE-A3-KKL or anti–MCSPscFv-MAGE-A3-KKL constructs (▨). After 24 hours of incubation with the constructs, a fraction of the iDCs was matured with the maturation cocktail (■) for 24 hours (iDCm). This ultimately leads to 3 different Ag-loaded DC populations, which were used in subsequent experiments. (B) Comparison of the stimulatory capacity of DCs loaded with MAGE-A3 by DEC-205 targeting, RNA electroporation, or direct peptide pulsing. DCs were either matured (mDCs) or were left immature (iDCs) and were incubated with 1 μg/mL of the anti–DEC-205scFv-MAGE-A3-KKL or the anti–MCSPscFv-MAGE-A3-KKL construct (as respective control) for 48 hours. A fraction of the iDCs was matured during the incubation (iDCm). In addition, mDCs were electroporated with MAGE-A3-DCLAMP RNA or MAGE-A3 RNA (as respective control) and pulsed with MAGE-A3/HLA-DP4 or NY-ESO-1/HLA-DP4 (as respective control) peptides. All these differently loaded DC populations were used to stimulate MAGE-A3/HLA-DP4–specific autologous CD4⁺ T cells, which were generated by TCR-RNA electroporation (“Cytokine-secretion assays”). IL-2 secretion by specific T cells was analyzed after 18 hours of coculture. Furthermore, the ratio between IL-10 to IL-2 secretion after activation of specific T cells with DEC-205–targeted iDCs, iDCm, and mDCs was analyzed (right panel). P values were calculated with the Mann-Whitney U test. *P < .05. **P < .005. ***P < .001. (C) Dose-dependent IL-2 secretion by MAGE-A3/HLA-DP4–specific CD4⁺ T cells after stimulation with DEC-205–loaded iDCm, generated as described in panel A, except that they were loaded with different concentrations of anti–DEC-205scFv-MAGE-A3-KKL fusion protein (as indicated). As negative control, heat-inactivated anti–DEC-205scFv-MAGE-A3-KKL protein in the same concentrations, 10 μg/mL and 1 μg/mL of anti–MCSPscFv-MAGE-A3-KKL protein, medium, and solvent control (generated by similar protein-purification protocol from mock-transfected 293T cells) were used. These DCs were used to stimulate MAGE-A3/HLA-DP4–specific autologous CD4⁺ T cells; and after 18 hours of coincubation, IL-2 concentrations in the supernatants were measured. Data are mean values ± SD of 4 independent experiments. DK indicates anti–DEC-205scFv-MAGE-A3-KKL fusion protein; MK, anti–MCSPscFv-MAGE-A3-KKL fusion protein; and EP, electroporation.

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