Sensitivity of Dendritic Cells to Microenvironment Signals

Figure 2

Leukemic cell microvesicles influence on dendritic cell differentiation. Mononuclear cells were obtained after gradient density from buffy coats of healthy individuals. After 2 h of adhesion, lymphocytes were removed and monocyte cultures were stimulated with IL-4 and GM-CSF (50 ng/mL) to induce dendritic cell differentiation. K562 supernatants (SN K562) were obtained after 3 days of cell culture in RPMI plus 10% of fetal bovine serum (FBS) followed by filtering (0.22 μm). Part of the tumor supernatant was centrifuged twice at 100000 g for 1 h to purify microvesicles. After this process, two supernatants were obtained: K562 supernatant only with microvesicles resuspended in the same original volume of medium (MV K562) and K562 supernatant with all the other products except microvesicles (free MV K562). Supernatants were added (10% of final volume) at the beginning of monocyte culture and remained until the end (5 days). Afterward, cells were stained with anti-CD14 FITC and anti-CD1a PE and data were acquired in a FACS Calibur. Graphs show the percentage of CD14⁺ (a) and CD1a⁺ (b) cells. Each individual analyzed in different conditions is represented by one symbol. ; ; . .

(a)

(b)