SAg incorporation in DCs and cell activation markers.

<p>Bone marrow DCs were pulsed 1 h with SEG-FITC, washed, and immunolabeled with anti CD11c-PE. (<b>A</b>) Percentage of DCs incorporating different concentration of SEG-FITC at 37°C and 4°C <i>vs.</i> non-pulsed DCs as control. (<b>B</b>) Percentage of DCs showing inhibition of SEG-FITC internalization by wortmannin (WT) and 5-ethyl-N-isopropyl amiloride (EIPA) added at final concentrations of 100 nM and 50 µM, respectively, 15 min before SAg-FITC at 50 µg/ml. (<b>C</b>) Representative histograms of the phenotype of DCs in the presence of SAgs. (<b>D</b>) Cell surface MHC-II molecules on DCs treated with SSA, SEG, SEI or LPS for 24 h, compared to non-treated basal control, and expressed in MFI. (<b>E</b>) CD80 on SAgs- or LPS-treated DCs compared to non-treated. (<b>F</b>) Endocytosis of OVA-FITC by SAg or LPS pre-treated DCs. (<b>G</b>) DCs were incubated for 1 h with SEG at 37°C or 4°C as control, washed, and cultured for additional periods from 0 to 24 h. At each endpoint, cells were washed and disrupted in order to isolate the endosomal/lysosomal compartment. These fractions were incubated with mouse splenocytes to assess proliferative activity by ³H-Thy incorporation in triplicate. The proliferation index (PI) was calculated as cpm/basal cpm. Basal represent the proliferation of T cells stimulates by DCs incubated in the absence of SAg. (<b>H</b>) SEG-pulsed DCs cultured for additional periods of 0 to 24 h. At each endpoint, cells were washed and immunolabeled with mouse anti-SEG polyclonal antibodies and anti-mouse-FITC, as second antibody to evaluate the presence of SEG on the cell surface. Figures show a representative experiment of 3–5. *<i>p</i><0.05, **<i>p</i><0.01.