SummaryA new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage-specific monoclonal antibodies (mAbs) and removal with antiimmunoglobulin-coupled magnetic beads. This enriched population (~80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCK)-CD3 complex, and having TCK/3 and 3" genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T ceU markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8ce mRNA. Thymic DC presented both the CD8 c~ and/3 chains on the cell surface (Ly-2+3+), although the ce chain was in excess; the splenic DC expressed only the CD8 ol chain (Ly-2 + 3-). h is suggested that the expression of CD8 could endow certain antigen-presenting DC with a veto function. D endritic cells (DC), 1 first described by Steinman et al.(1, 2), are a minor population of irregularly shaped cells in lymphoid organs, distinguishable from both lymphocytes and macrophages. DC constitutively express high levels of both class I and class II MHC antigens (1, 2). They are highly efficient at presentation of antigen and stimulation of T lymphocytes (1-7). Those within the thymus are believed to effect deletion of developing T cells with self-reacting potential (8, 9). The surface antigenic pattern of thymic DC has been shown to differ somewhat from that of splenic DC (10-12), but it is not dear whether this implies the cells have a different origin, or a different function, or whether they simply represent different development states of the same functional lineage. There is evidence that it is the developmental state of the T cell that determines whether the T cell-DC interaction leads to T cell proliferation or to T cell death (13). As part of a study of the interaction between developing T cells in the thymus and thymic stromal elements (14), we have isolated thymic DC and compared them with those isolated from the spleen. The usual procedure for isolating DC involves enrichment in a low buoyant density fraction by centrifugation, followed by selection as cells that show an initial adherence but then release from the vessel surface when cultured overnight (10).We have used such a procedure to isolate DC from mouse thymus (15). However, we were con...

The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8α or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC development from these thymic precursors did not include granulocyte/macrophage colony stimulating factor (GM-CSF), usually required for DC development in culture. The addition of anti–GM-CSF antibody or the use of precursors from GM-CSF–deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 3 and IL-7 were present, although some stimulation by GM-CSF was noted in their absence. In contrast, DC development was enhanced by addition of the Flt3/Flk2 ligand, in line with the effects of the administration of this cytokine in vivo. The results indicate that the development of a particular lineage of DC, probably those of lymphoid precursor origin, may be independent of the myeloid hormone GM-CSF.

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