Characterization of antigen-processing machinery in human monocyte-derived immature and mature dendritic cells Free

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Efficient antigen processing and presentation are the major function of dendritic cells (DC) and are clearly dependent on the integrity of the entire antigen processing machinery (AMP) pathway in these cells. The panel of monoclonal antibodies developed for recognition of APM components in fixed paraffin-embedded tissues was used to evaluate expression of each of the components in human immature (iDC) and matured (mDC) dendritic cells. A flow cytometry-based method was developed for quantitative detection (MESF units) of individual APM components, and DC generated from peripheral blood monocytes of normal donors (ND; n = 12) and patients with melanoma and head neck cancer (n = 6) were compared. We also compared two methods for cell permeabilization: the microwave treatment and the incubation of cells in the presence of 0.25% saponin solution. Co-incubation of tumor cell lines with iDC were performed in microwells or transwell system to evaluate effects of tumor cells on the APM component expression. Our results show that the microwave treatment of DC was necessary to achieve optimal staining, as the percentage of positive cells was consistently higher in microwave-treated DC than saponin-permeabilized DC in all experiments performed. A considerable variability was observed among ND in expression of the APM components in DC. i DC were found to have lower (p<0.01) expression of LMP7, LMP10, TAP-1, MB1 and tapasin, but maturation in the presence of a cytokine cocktail up-regulated the expression of these proteins in DC obtained from ND and patients alike. Expression of HLA class I and HLA class II molecules, β2 microglobulin, calnexin, and calreticulin was seen in 100% of the cells in both iDC and mDC of ND. Co-incubation of iDC obtained from ND in the presence of tumor cells led to significantly decreased expression of APM components. However, subsequent maturation of these DC in the presence of cytokines reversed the tumor-induced effects and restored high levels of APM component expression characteristic of activated mDC. Exposure of iDC to the cytokine cocktail for 24 hours was accompanied by significant up-regulation of expression of the APM components in DC of patients with cancer. The largest increases were observed in expression of the HLA class I molecules, HLA class II molecules, LMP2, calnexin, DELTA, and TAP1. The data indicate that the DC generated ex vivo from monocytes of patients with cancer and matured in cytokine cocktail normalize expression of AMP components and presumably are able to process and present antigen to T cells as effectively as the DC obtained from ND.