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. 2018 Mar 19;8:4804. doi: 10.1038/s41598-018-22931-4

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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DP4 constitutively presents MAGE-A3_243–258 peptide derived from chimeric Ii^MAGE-A3 regardless of the presence of Ii and HLA-DM. (a-h) DP4/MAGE-A3-specific CD4⁺ T cells were stimulated using the indicated K562-based aAPCs, and IFN-γ responses were measured by ELISPOT analysis. (a) K562 cells stably expressing either DP4 or DP4^84DEAV87 as indicated were transiently transfected with either Ii^MAGE-A3 linked with IRES-EGFP, or empty vector (control) encoding IRES-EGFP alone, and used to stimulate T cells. (b) Immunogenicity of stimulator cells was evaluated by stimulating responder T cells with the indicated K562-based aAPCs following pulse with 10 µM of either Tetanus Toxin TT_947–967 peptide (control) or MAGE-A3_243–258 peptide. (c,d) K562 cells stably expressing DP4, either in the presence or absence of stably expressed Ii and/or HLA-DM as indicated, were transiently transfected with native MAGE-A3 (c), Ii^MAGE-A3 (d), or empty vector (control) linked with IRES-EGFP, and used to stimulate T cells. (e) Immunogenicity of stimulator cells was evaluated by stimulating responder T cells with the indicated K562-based aAPCs following pulse with 10 µM of either Tetanus Toxin TT_947–967 peptide (control) or MAGE-A3_243–258 peptide. (f,g) K562 cells stably expressing DP4^84DEAV87, either in the presence or absence of stably expressed Ii and/or HLA-DM as indicated, were transiently transfected with native MAGE-A3 (f), Ii^MAGE-A3 (g), or empty vector (control) linked with IRES-EGFP and used to stimulate T cells. (h) Immunogenicity of stimulator cells was evaluated by stimulating responder T cells with the indicated K562-based aAPCs following pulse with 10 µM of either Tetanus Toxin TT_947–967 peptide (control) or MAGE-A3_243–258 peptide. Transient transfection efficiencies were normalized by EGFP expression as measured by flow cytometry (Supplementary Figure 3). The data shown represent the mean ± SD of experiments performed in triplicate. Results are representative of at least three independent experiments. ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired, two-tailed Welch’s t-test.