Isolation, cultivation and phenotypic characterization of rat thymic dendritic cells

Rat thymic dendritic cells (TDC) were isolated from thymic cell suspension by Nycodenz gradients of different densities and osmolarities. After cultivation of these cells for 3 days in the conditioned medium (TE-R 2.5 + HT supernatant) prepared by cocultivation of a medullary thymic epithelial cell line (TE-R 2.5) and hydrocortisone resistant thymocytes, the purity (75-85%) and survival of TDC significantly increased. The supernatant contained moderate activities of IL-1 and IL-6, low levels of TNF-alpha and IL-2 and factor(s) that strongly stimulated the proliferation of a mouse macrophage cell line RAW 264.7. TDC survival in culture was significantly increased by GM-CSF and decreased by IL-6 and IFN-gamma. The phenotype of TDC was studied by flow cytometry using a panel of monoclonal antibodies (mAb) to rat cell surface markers. It was found that almost all freshly isolated TDC expressed major histocompatibility complex (MHC) class I and class II molecules as well as CD45. Most TDC were LFA-1 (CD11a)(+), CD18(+), ICAM-1 (CD54)(+) and CD53 (OX-44)(+), but only certain subsets expressed CD11b and thymocyte markers Thy1, CD2, CD4 and CD8. Upregulation in the expression of almost all the markers was observed after cultivation of TDC. In addition, cultivated, but not freshly isolated TDC expressed CD25 (IL-2R alpha) and CD45RC (OX-22) molecules. Cultivated TDC had strong accessory function in autologous thymocyte proliferation.