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Fig. 2 | Journal of Hematology & Oncology

Fig. 2

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. 2

LA-pulsed DCs induced dual cytotoxicity of CTLs and NK cells against breast cancer in vitro. a Strategy of exploring cytotoxicity of YL/DC-CTL, LA/DC-CTL or LA/DC-NK. DCs were stimulated with 50 μg/ml peptide twice at an interval of 24 h, treated with 30 ug/mL mitomycin C for 30 min, and then cocultured with CD8+ T cells at a ratio of 1:20 on day 1 and day 6 to generate epitope/DC-CTLs. NK cells were cocultured with epitope-pulsed DCs at a ratio of 20:1 on day 1 and day 6 to generate epitope/DC-NK. b CD45RO and CD69 expression were significantly upregulated in YL/DC-CTLs or LA/DC-CTLs (n = 3), suggesting a stronger memory function. c Perforin/granzyme-B/FasL levels in YL/DC-CTLs or LA/DC-CTLs were significantly increased against MDA-MB-231 cells (HLA-A2.1+/ECM1+) using flow cytometry. d The cytotoxicity of epitope/DC-CTLs against epithelial cell MCF-10A (HLA-A2.1+/ECM1), cancer cell MCF-7 (HLA-A2.1/ECM1+), MDA-MB-231 (HLA-A2.1+/ECM1+), and BT-549 (HLA-A2.1+/ECM1+) was detected by calcein-release assay (n = 3); E/T ratio, effector/target cell ratio. e Cytotoxicity of epitope/DC-CTLs against ECM1-silenced MDA-MB-231 and HLA-A2.1-knocked-in MCF-7 cells (n = 3) f Cytotoxicity of DC-PBMCs or DC-CTLs against NK-sensitive K562 cells (n = 3). g Cytotoxicity of epitope/DC-NK cells against tumour cells (n = 3). h Representative immunohistochemical staining of ECM1. Scale bars, 50 μm. i, j Cytotoxicity of epitope/DC-CTLs or epitope/DC-NK cells on primary breast cancer cells from a HLA-A2.1+/ECM1 patient (n = 1) and HLA-A2.1+/ECM1+ patients (n = 6). k, l Cytotoxicity of epitope/DC-CTLs (n = 2) or epitope/DC-NK cells(n = 1) on breast cancer microtissue blocks from HLA-A2.1+/ECM1+ patients. The number represented the inhibition rates (IR%). NC, negative control, water. NP, negative peptide, the peptide (PPGRPSPDN) derived from ECM1 with largest predicted IC50 of affinity with HLA-A2.1. b–g, i, j P values were obtained from independent-samples t-test; error bars denote standard deviation (SD). Compared with NC, *P < 0.05, **P < 0.01, ***P < 0.001; compared with NP, #P < 0.05, ##P < 0.01, ###P < 0.001

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