Generation of Dendritic Cell-like Antigen-presenting Cells in Long-term Mixed Leucocyte Culture: Phenotypic and Functional Studies
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The mechanisms contributing to the proliferation and differentiation of antigen-presenting cell (APC) precursors upon antigen stimulation or tissue injury are poorly understood. Herein, we report the induction of a population of dendritic cell-like cells (DLC) with potent antigen-presentation function from unfractionated spleen cells by means of repetitive allostimulation in long-term mixed leucocyte cultures (LT-MLC). Initially, only a few adherent DLC were observed. By 4-6 weeks, however, there were large numbers of DLC which survived persistently. Features of these DLC are closely related to dendritic cells (DC), including (1) dendritic, veiled or spiny-processed morphology; (2) expression of a wide array of leucocyte surface markers including DC-associated or restricted antigens: 33D1, NLDC-145, CD11c (N418), heat-stable antigen (HSA), CD44, B7-1 and B7-2; (3) ability to migrate to draining lymph nodes and white pulp area of spleen; (4) expression of high level of major histocompatability complex (MHC) class II molecules and (5) more potent mixed leucocyte reaction (MLR)-stimulating capacity than peritoneal macrophages and APC-enriched spleen cells. DLC-stimulated MLR was inhibited by monoclonal antibodies (mAbs) to B7-1, B7-2, intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), leucocyte-function associated antigen-1 (LFA-1) or very-late activation antigen-4 (VLA-4) by 30-55%. When maintained for more than 2 months, the DLC did not lose their MLR-stimulating activity, but many surface markers were down-regulated except for Mac-2 and VCAM-1, which remained stable or were up-regulated, respectively. In short-term culture, the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-2 enhanced proliferation of DLC, while tumour necrosis factor-alpha (TNF-alpha) and IL-4 did not. IL-4 suppressed not only 'spontaneous', but also GM-CSF-enhanced proliferation, suggesting that cytokines play a differential role in DLC proliferation. These results confirm that professional APC can proliferate in response to repetitive antigen stimulation, and their proliferation is differentially regulated by cytokines. A comparison study of DLC with typical DC is being carried out in our laboratory.