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. 2020 Jan 22;11(1):48. doi: 10.1038/s41419-020-2236-3

Fig. 4. Virus-induced immunogenic cell death can activate DCs capable of stimulating antigen-specific T-cell responses.

Fig. 4

Experiments were performed using Ad, SFV4, and VV-infected HOS and A549 cells (MOI = 10) at 48 h. DAMPs: (a) Extracellular ATP (relative luminescence) was measured and represented as percentage relative to non-infected control (CT) cells. b Released HMGB1 was measured by ELISA. Calreticulin (CRT) exposure (c) and HSP90 expression (d) were measured by flow cytometry. DC phagocytosis: pp65-copGFP-expressing HOS cells (HOS-copGFP-pp65) and A549 cells (A549-copGFP-pp65) were infected with Ad, SFV4 or VV for 48 h. The infected cells were collected, washed and co-cultured with immature DCs at a ratio of 1:1 for 2 h before flow cytometry analysis. The population of CD1a+GFP+ double-positive cells were determined as the proportion of phagocytosis by DCs (e) and the correlation between fold change of phagocytosis and fold change of CRT (f). Cytokine release: (g) Supernatants from the co-cultures were collected after 24 h and Th1, Th2, pro- and anti-inflammatory cytokines were measured by Mesoscale. DC maturation: (h) After 48 h of co-culture, DC maturation was examined in terms of upregulation of the co-stimulatory molecules CD83, CD86, CD80 and CD40. T-cell priming: (i) Illustration of the experimental set-up. T-cells genetically engineered with a TCR specific for an HLA-A2-restricted epitope of pp65 (pp65-TCR T-cells) were stimulated for 6 h with autologous DCs that had been co-cultured for 48 h with Ad-, SFV4- and VV-infected HOS-copGFP-pp65 and A549-copGFP-pp65 cells, respectively. Uninfected cells were used as control (CT). j IFN-γ was measured by means of ELISA after 16 h. All data are expressed as mean ± SEM and performed with at least three different donors. Statistical analysis for normally distributed data were performed by means of one-way ANOVA with Bonferroni post hoc test and for not normally distributed data were performed by means of Kruskal–Wallis test with Dunn’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001 and **** < 0.0001).