Figure 6

Induction of calcium release by CRAM presented chemokines. MEC-1 cells (CRAM⁺/CCR7⁺) were loaded with fluo-3/AM and then stimulated by Nalm6 cells (CRAM⁺/CCR7^-) pre-incubated with CCL19 or CCL21 (100 ng/ml) for 30 min at 37°C and then treated as mentioned on the graph x axis. A. Calcium release was monitored by flow cytometry, allowing the discrimination between Nalm6 and MEC-1 cells, according to their SSC/FSC characteristics (Additional file 2, Figure S2). Spots represent results in arbitrary units from individual experiments and mean values of the five independent experiments are depicted as a line. Statistical analyses were done performing a T-test. B. Same experimental settings but this time changing the temperature of the initial incubation step (4°C: blue line, 37°C: pink and red lines), and the length of second incubation as indicated at various temperatures (4°C: pink line, 37°C blue and red lines). After incubations at different temperatures, the calcium release experiments were done at 37°C. C. Localisation of CCL19 in the cellular suspension used to induce the calcium release of MEC-1 cells was done using ELISA. Cells treated in the same way than for calcium experiments were used to quantify CCL19 in the supernatant (blue), at the cellular surface (pink) or internalised (green). When kept at 37°C throughout the experiments, the proportion of CCL19 internalized into the cells is higher, suggesting a possible time associated regulation of CCL19 by CRAM. D. Schematic illustration of molecular mechanisms explaining the previous results: incubation at 4°C blocks the internalisation of CRAM and hence its ability to sustain a CCL19 induced answer over time.