DEC-205 targeting of mDCs from melanoma patients results in cytokine release by MAGE-A3/DP4–specific CD4⁺ T cells. (A) Stimulatory capacity of DEC-205–targeted mDCs from melanoma patients. mDCs, generated from melanoma patients, were loaded for 48 hours with MAGE-A3 by DEC-205 targeting (targeting), using 1 μg/mL anti–DEC-205scFv-MAGE-A3-KKL fusion protein, or for 3 hours by peptide pulsing using the MAGE-A3/HLA-DP4 peptide. As negative control, mDCs were incubated with 1 μg/mL of the anti–MCSPscFv-MAGE-A3-KKL protein (respective control), the NY-ESO-1/HLA-DP4 peptide (respective control), medium (med), or with solvent (sc). These differently treated DCs were used to stimulate autologous MAGE-A3/HLA-DP4–specific CD4⁺ T cells, generated by TCR-RNA electroporation (“Cytokine-secretion assay”). IL-2, TNF, and IFN-γ concentrations were analyzed in the supernatants after 18 hours of coculture. Data are mean values ± SEM of 4 experiments with cells from patients 1, 2, 3, and 4. (B) Stimulatory capacity of DEC-205–targeted DCs from melanoma patients after cryopreservation. The remaining mDCs from panel A were frozen after the different treatments. After thawing, the DCs were used to stimulate allogenic MAGE-A3/HLA-DP4–specific CD4⁺ T cells generated by electroporation with RNA encoding for a TCR. Supernatants were analyzed as described in panel A. Data are mean values ± SEM of 4 experiments with cells from patients 2, 3, 4, and 5. P values were calculated with the Mann-Whitney U test. *P < .05.