Summary.A stromal cell-dependent long-term culture (LTC) system has been developed from spleen which continuously generates non-adherent cells with dendritic cell (DC) characteristics. Bioassays using factor-dependent cell lines have revealed that both spleen and thymic stromal cultures secrete interleukin (IL)-3 and IL-6-like growth factors. Conditioned medium from LTC also contains factors which appear to be unrelated to IL-3 and induces growth of stromal cells from bone marrow. Non-adherent cells generated in LTC were not T or B lymphoid cells or granulocytes, nor were mast cells detectable. Morphological and electron microscopic examination has also excluded the presence of macrophages (Mø). Cells with DC morphology have been detected by both light and electron microscopy. The majority of cells in the non-adherent population were found to have multiple membrane pseudopodia, with a small acentric nucleus. These appear to be the precursors of DC. They expressed cell surface markers detectable with DC-specific antibodies and antibody specific for major histocompatibility complex Class II molecules. A proportion of cells also expressed myeloid markers, but since this expression was not supported by histochemical staining for myeloperoxidase or non-specific esterase, it was concluded that the cells produced are not typical of the myeloid lineage. Cells generated in LTC were shown to be potent stimulators for allogeneic and syngeneic MLR and for antigen-specific T-cell proliferation.
Summary Dendritic cells (DC) are distinguishable from other antigen-presenting cells hy their potent antigen-presenting capacity. They are not only efficient at presenting peptide antigen but can also process and present soluble protein antigens to antigen-specific T cells and cloned T cell lines. They are very strong stimulators of both allogeneic and syngeneic mixed lymphocyte reactions and have a unique capacity to stimulate naive T cells. The potent functional capacity of DC is related to a high-level expression of major histocompatibility complex class IAI molecules and constitutive expression of costimulatory molecules, such as CDS0/CDS6. as well as heat stable antigen. CD4n and the leucocyte function antigen (LFA) family of adhesion molecules. Recent studies have shov^n that DC are also involved m regulation of the immune response via induction of both central and peripheral tolerance.
The study of dendritic cells (DC) has been hampered by the difficulty of isolating rare cells for analysis of their phenotype and function. Interpretation of the DC lineage has been largely influenced by studies on cell populations which can be readily isolated and amplified in the presence of cytokines. Long term cultures (LTC) from murine spleen have been shown to support continuous in vitro hematopoiesis of DC dependent on interaction with a stromal cell monolayer. LTC-DC represent a single, stable class of DC derived by constant turnover of spleen DC progenitors maintained within stroma. They represent a resident DC population in spleen. The functional characteristics of LTC-DC have been studied in terms of capacity to stimulate T cells and response to activation by environmental stimuli. LTC-DC have many morphological, phenotypic and functional properties reflecting an immature or partially mature, marginal zone-like CD4(-)CD8(-) splenic DC subset. They are highly endocytic and can process and present protein antigen to naive hen egg lysozyme (HEL)-specific MHC-II-restricted TCR-Tg CD4(+) T cells. They do not, however, induce T cell proliferation in a mixed lymphocyte reaction. LTC-DC do not respond in a typical fashion to common DC activators like LPS and CD40L. They upregulate MHC-I and CD80/CD86 but not MHC-II and CD40. They reflect an endogenous, immature DC subset in spleen with properties distinct from immature DC located in peripheral tissues.
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