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. 2019 Jun 26;10:1450. doi: 10.3389/fimmu.2019.01450

Figure 1.

Figure 1

GzmA promotes DC maturation. (A) Recombinant GzmA is predominantly dimeric. Reduced and non-reduced GzmA were run on 10–12 % SDS-PAGE and stained with Coomassie Brilliant Blue. (B) Phenotypic maturation. FL-DCs were generated from BMMNCs from C57BL/6 mice in the presence of Flt3L. The expression of costimulatory molecules (CD40 and CD86) on FL-DCs (CD11c+ cells) were analyzed 24 h after stimulation with graded dose of GzmA or LPS (100 ng/ml) (n = 3). (C) Expression of CD86 on the pDC (CD11cdimB220+) of FL-DCs after stimulation with GzmA (1 μg/ml), LPS (100 ng/ml) (n = 5). (D) Cytokine production by FL-DCs stimulated with GzmA. FL-DCs were cultured in the presence of GzmA or LPS for 24 h and the supernatants were assayed for IL-12p40 and IFN-α by ELISA (mean ± SEM, n = 5). *p < 0.05, **p < 0.01 (Kruskal Wallis test). (E) IFN-α production by pDCs and cDCs. pDCs, and cDCs were sorted from FL-DCs and stimulated with GzmA (1 μg/ml), LPS (100 ng/ml), or CpG-ODN (1 μg/ml) for 24 h and the supernatants were assayed for IFN-α by ELISA (mean ± SEM, n = 4–6). *p < 0.05, **p < 0.01 (Kruskal Wallis test). (F) Effect of treatment of GzmA with nucleases or heat. GzmA was pre-treated with nothing (non), DNase, RNase or heat-inactivated and then added to FL-DCs. Supernatants from the cultures were analyzed the production for IFN-α. The data show the ratio of IFN-α production in the GzmA treated:untreated cultures, the latter set to 1 (mean ± SEM, n = 5). ***p <0.001 (Kruskal Wallis test).