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Fig. 2 | Journal for ImmunoTherapy of Cancer

Fig. 2

From: Effective cancer immunotherapy by natural mouse conventional type-1 dendritic cells bearing dead tumor antigen

Fig. 2

TCL induces cDC1 activation and TCL-loaded cDC1s generate optimal CD8+ T cell activation in vivo. a HMGB1 content measured by ELISA in supernatants of B16-OVA cells treated with indicated agents (n = 3–4). b Quantification (left) and representative histograms (right) of CD86 expression on untreated cDC1s (Untreat.), cDC1s treated either with B16-OVA tumor cell lysate (TCL) or washed B16-OVA TCL containing only cellular components +/− 20 μg/ml poly I:C. After 1 h, cDC1s were washed and cultured for 4 h followed by flow cytometric analysis. Combined data of 4 independent experiments. MFI, mean fluorescent intensity. c Spleen cDC1s were cultured with B16-OVA TCL, poly I:C, Hiltonol and/or BO112 for 1 h, re-purified and plated with CellTrace Violet (CV)-labelled OT-I cells. Quantification (left) and representative histograms (right) of OT-I cell proliferation 3 days after. Combined data of 3 independent experiments. d cDC1s were cultured for 1 h with poly I:C, soluble OVA protein and/or B16-OVA TCL and 2 × 105 cDC1s were injected intravenously (IV) into CD45.2+ recipient mice that had received 1-2 × 105 CV-labelled CD45.1+ OT-I CD8+ T cells one-day prior. Equally treated “leftover” B16-OVA TCL served as negative control (Additional file 1: Figure S2d). Total OT-I cell number and number of IFNγ-producing OT-I cells after re-stimulation with OVA257–264 peptide was determined by flow cytometric quantification in spleen 5 days later. One representative of 2 independent experiments with n = 3 mice/group/experiment. e cDC1s were cultured for 1 h with poly I:C and B16-OVA TCL and 2 × 105 cDC1s were IV or intradermally (ID) injected into CD45.2+ recipient mice that had received 1-2 × 105 CD45.1+ OT-I CD8+ T cells one-day prior. Equally treated “leftover” B16-OVA TCL served as negative control (Additional file 1: Figure S2d). OT-I number and frequency was determined by flow cytometric quantification in spleen and draining lymph node (iLN) 5 days later. One representative of 2 independent experiments with n = 4 mice/group/experiment. *P < 0.05, **P < 0.01, ***P < 0.001 by a & c-e one-way ANOVA and Tukey post hoc test or b paired Student’s t test

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