GENERATION OF DENDRITIC CELL-LIKE ANTIGEN-PRESENTING CELLS IN LONG-TERM MIXED LEUKOCYTE-CULTURE - PHENOTYPIC AND FUNCTIONAL-STUDIES

The mechanisms contributing to the proliferation and differentiation o f 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 re petitive allostimulation in long-term mixed leucocyte cultures (LT-MLC ), Initially, only a few adherent DLC were observed. By 4-6 weeks, how ever, there were large numbers of DLC which survived persistently. Fea tures of these DLC are closely related to dendritic cells (DC), includ ing: (1) dendritic, veiled or spiny-processed morphology; (2) expressi on of a wide array of leucocyte surface markers including DC-associate d or restricted antigens: 33D1, NLDC-145, CD11c (N418), heat-stable an tigen (HSA), CD44, B7-1 and B7-2; (3) ability to migrate to draining l ymph 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 p eritoneal macrophages and APC-enriched spleen cells. DLC-stimulated ML R was inhibited by monoclonal antibodies (mAbs) to B7-1, B7-2, intrace llular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), leucocyte-function associated antigen-1 (LFA-1) or very-lat e activation antigen-4 (VLA-4) by 30-55%. When maintained for more tha n 2 months, the DLC did not lose their MLR-stimulating activity, but m any surface markers were downregulated except for Mac-2 and VCAM-1, wh ich remained stable or were up-regulated, respectively. In short-term culture, the addition of granulocyte-macrophage colony-stimulating fac tor (GM-CSF) or interleukin (IL)-2 enhanced proliferation of DLC, whil e tumour necrosis factor-alpha (TNF-alpha) and IL-4 did not. IL-4 supp ressed not only 'spontaneous', but also GM-CSF-enhanced proliferation, suggesting that cytokines play a differential role in DLC proliferati on. These results confirm that professional APC can proliferate in res ponse to repetitive antigen stimulation, and their proliferation is di fferentially regula ed by cytokines. A comparison study of DLC with ty pical DC is being carried out in our laboratory.