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Fig. 1 | Journal of Hematology & Oncology

Fig. 1

From: HLA-A2.1-restricted ECM1-derived epitope LA through DC cross-activation priming CD8+ T and NK cells: a novel therapeutic tumour vaccine

Fig. 1

Identification of HLA-A2.1-restricted ECM1-derived epitopes with a double activation of CD8+ T and NK cells. a Strategy of identifying CTL epitopes. b ECM1 expression increased significantly in various tumour tissues compared with tumour-adjacent tissues by immunohistochemistry. c The combination between peptides and HLA-A2.1 by MOE. The number represents the S value, the free-energy between docking molecules. d T2 binding assay. Left, HLA-A2.1-binding affinity of epitopes (n = 3). T2 cells were cultured with peptide (50 μg/mL) and β2-microglobulin (3 μg/ml) for 18 h. Fluorescence index (FI) > 1.5, high affinity. Right, HLA-A2-binding stability of epitopes (n = 3). T2 cells were stimulated with peptide for 18 h and then treated with brefeldin A (10 μg/mL) for 0, 2, 4, 6 and 18 h, respectively. DC50 > 6 h, high stability. DC50, the time point when the mean FI value at 0 h was reduced to 50%. e After stimulation with peptides (50 μg/mL), IFN-γ secretion by rhECM1-sensitized (10 μg/mL) HLA-A2.1+ PBMC was detected by ELISPOT (n = 5). f Frequency of CD8+ T cells in HLA-A2.1+ PBMC (DC-) stimulated with peptide-pulsed DCs (n = 3). IFN-γ secretion and proliferative effect of epitope/DC-CTLs were detected by ELISPOT and CFSE assays (n = 3). g Frequency of NK cells (CD3CD16+CD56+) in HLA-A2.1+ PBMC (DC-) stimulated with peptide-pulsed DCs (n = 3). IFN-γ secretion and proliferative effect of epitope/DC-NK cells were detected by ELISPOT and CFSE assays (n = 3). CFSE, carboxyfluorescein succinimidyl ester; NC, negative control, water; NP, negative peptide, the peptide (PPGRPSPDN) derived from ECM1 with the largest predicted IC50. b P values were obtained with Wilcoxon signed-rank test; error bars denote medians and interquartile ranges; *P < 0.05, ***P < 0.001. e–g P values were obtained with independent-samples t-test; error bars denote standard deviation (SD); e Compared with NP, *P < 0.05; f, g Compared with NC, *P < 0.05, **P < 0.01; compared with NP, #P < 0.05, ##P < 0.01

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