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Fig. 3 | Journal for ImmunoTherapy of Cancer

Fig. 3

From: EnanDIM - a novel family of L-nucleotide-protected TLR9 agonists for cancer immunotherapy

Fig. 3

TLR9-specific effects of EnanDIM-C and EnanDIM-A in vitro. a, effect on pDC isolated from human PBMC: incubation with EnanDIM-A and EnanDIM-C at a final concentration of 3 μM for 48 h, analysis for cytokine / chemokine production and presence of activation markers, n = 5. b, c, CpG-dependency of EnanDIM-C effect: b, incubation of donor PBMC with EnanDIM-C or CG-depleted variant EnanDIM-C(-CG) at a final concentration of 3 μM for 48 h, analysis for cytokine / chemokine production and activation of cell surface markers on monocytes and NK cells, n = 6. c, incubation of ELAM41 reporter cells with the indicated concentrations of EnanDIM-C or EnanDIM-C(-CG) for 7 h and determination of the amount of fluorescent protein via flow cytometry. The expression level of eGFP is represented by the product of the relative amount of fluorescent cells and the fluorescence intensity (AU ± SEM) from 3 independent experiments. d, type-I interferon dependency of EnanDIM-C effect via IFN-alpha capture by B18R protein (vaccinia virus-encoded receptor with specificity to type I interferons): incubation of donor PBMC with EnanDIM-C at a final concentration of 3 μM ± B18R protein (final concentration 0.5 μg/ml) for 48 h, analysis for IP-10 production and activation of cell surface markers on monocytes, NK cells and B cells, n = 6. e, cytotoxic effect of EnanDIM-C in human PBMC against Jurkat cells: incubation of donor PBMC (effector cells) with EnanDIM-C at a final concentration of 3 μM for 48 h, co-culture of PBMC with Jurkat cells as target cells for 4 h, use of different target:effector ratios, quantification of target cell death by flow cytometry - shown is one representative donor (left) as well as mean values of 8 different donors ±SEM at a target:effector ratio of 10:1 (right), ***p < 0.001; One way ANOVA, Dunnett’s multiple comparisons test. f, proposed immunomodulatory mode-of-action of the TLR9 agonist EnanDIM®

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