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Figure 3


Fig. 3. RyR activation induces transcription of genes involved in DC maturation and potentiates allospecific T-cell stimulation. (A) In vitro-derived iDCs were cultured for 18 hours in the presence of the indicated concentrations of LPS and with 10 mM caffeine and 20 µM dantrolene, as indicated. Total RNA was extracted and CD83, IFN{alpha}, IL12B and IL23A gene expression was evaluated by quantitative real-time PCR. Gene expression results are expressed as fold-increase as compared with values obtained in iDCs treated with medium + 1 ng/ml LPS. One representative experiment out of four is shown. (B) Experiment as in A except that in vitro-derived iDCs were cultured for 18 hours in the presence of 1 ng/ml LPS + 100 µM ATP (left panel) or in the presence of 10 mM caffeine and 20 µM dantrolene, as indicated (right panel). Gene expression results are expressed as fold-increase as compared with values obtained in iDCs treated with medium + 1 ng/ml LPS (left panel; ATP experiments) or as fold-increase as compared with values obtained in iDCs treated with medium alone (right panel). One representative experiment out of four is shown. (C) Experiment as in A except that in vitro-derived iDCs were cultured for 18 hours in the presence of 1 µM thapsigargin, or the indicated concentration of caffeine or 1 µM thapsigargin + 1 ng/ml LPS. Total RNA was extracted and IL23A gene expression was evaluated by quantitative real-time PCR. One representative experiment out of four is shown. (D) Immature DCs were harvested on day 5 of differentiation and stimulated with 1 ng/ml LPS (grey box), with 10 mM caffeine + 1 ng/ml LPS (hatched box), with 20 µM dantrolene + 10 mM caffeine + 1 ng/ml LPS (diagonal lines) or with 100 µM ATP + 1 ng/ml LPS (horizontal lines), or left untreated (empty box). DCs were then washed and added to allogenic PBMC at a ratio of 10:1 for 5 days. [3H] thymidine was then added and cells were cultured for another day. The bars indicate the mean value of c.p.m. (± s.e.m.) from triplicate samples from one donor. The experiment was repeated with similar results at least four times with different donors.





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