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. 2017 Oct 16;7:13301. doi: 10.1038/s41598-017-13530-w

Figure 5.

Figure 5

Pretargeting approach for tumour-localized DC maturation using scFvFITC:sCD40L (A) Schematic representation of scFvFITC:sCD40L-mediated DC maturation in the presence of cancer cells pretargeted with FITC-labelled anti-tumour MAbs. (B) iDC were treated with fresh medium or medium contained scFvFITC:sCD40L (0.5 µg/ml) for 2 days. Subsequently, iDC were harvested and stained with anti-CD83-PE, anti-CD86-FITC, anti-HLA-DR-PE and appropriate isotype controls for assessment of DC maturation. (C) Flow cytometric analysis of scFvFITC:sCD40L binding to RTX-FITC-pretargeted Jurkat.CD20 cells in which binding was abrogated by addition of molar excess amounts of Fluo-A. (D) iDC were co-cultured with an increasing dose of scFvFITC:sCD40L in the presence or absence of pretargeted or non-targeted FaDu cells for 24 h. DC maturation was assessed by determining IL-12/23 levels in the culture supernatant. (E) Representative light microscopic photos of iDC clusters in co-culture with FaDu cells pretargeted with anti-EpCAM-FITC upon scFvFITC:sTRAIL treatment. (F) and (G) iDC were co-cultured with anti-EpCAM-FITC-pretargeted FaDu or anti-CD33-FITC-pretargeted FaDu cells in the presence or absence of scFvFITC:sCD40L, non- FITC fluorescein or LPS for 24 h. DC maturation was assessed by determining cellular surface CD83 expression and IL-12/23 level in the culture supernatant. (H) iDCs were co-cultured with the indicated pretargeted tumour cell lines in the presence or absence of scFvFITC:sCD40L with or without blocking reagent non- FITC fluorescein. iDC maturation was assessed by determination IL-12/23 levels in the culture supernatant. Apoptosis in all experiments was determined by Annexin V/PI staining. Statistical analysis was performed using two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant).