Langerhans cells (LC)' are leukocytes that are distributed in the suprabasal region of squamous epithelia, particularly skin. LC undergo marked changes when epidermal cell suspensions are prepared by a standard trypsinization protocol and placed in culture for 1-3 d. Several cell surface markers (Fc receptors, the F4/80 antigen, membrane ATPase, and nonspecific esterase) decrease substantially (1, 2), while others (class I and II products of the MHC) increase (3). When accessory function for primary T-dependent immune responses is quantitated, the activity of the LC population increases some 10-30-fold (1, 4). Therefore LC seem to mature immunologically in bulk epidermal culture and acquire most of the features of lymphoid dendritic cells.Because LC maturation leads to the development of accessory function for primary immune responses, this process may contribute to the sensitization phase of cell-mediated immunity . Here, we asked whether maturation is autonomous, or ifexogenous cells and factors are required . We developed a panning technique to enrich the trace LC population from freshly dissociated mouse epidermis. When this was done, the viability and function of cultured LC was dependent upon factors that were found in the medium of keratinocyte cultures, as well as stimulated macrophages and T cells . We will describe these experiments and the identification of granulocyte/macrophage colony-stimulating factor (GM-CSF) as the principal if not exclusive mediator for the production of functioning LC.

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Two recent studies (1, 2) have identified cells in human blood that fully resemble the dendritic cells described previously in mice and rats (3). Among other similarities, the human equivalent is Ia positive and Fc receptor negative, occurs in trace numbers (<1% of blood mononuclear cells), and acts as a potent stimulator of T cell proliferation in vitro. For example, preparations enriched in dendritic cells are 10-100 times more active than monocytes or lymphocytes in stimulating the syngeneic and allogeneic mixed leukocyte reactions (MLR) ~ as well as oxidative mitogenesis--the proliferation of periodate-modified T cells (1, 2). Therefore the prevailing concept that monocytes are the principal accessory cells in man must be reexamined.Monoclonal antibodies that distinguish macrophages from dendritic cells provide new probes for accessory or stimulator cells in the immune response. Selective depletion of murine dendritic cells, with specific antibody and complement, decreases accessory function dramatically (4-6). In man, antidendritic cell antibodies are not available, but alternative and useful reagents have been obtained. For example, 3C10 and 1 D9 are related antimacrophage antibodies that do not react with dendritic cells, while 9.3F10 is an anti-HLA class II reagent (7) that reacts with both cell types. In this paper, we use these monoclonals to study the requirements for T cell proliferation. T cell growth is severely reduced when accessory cells are depleted with 9.3F10 and complement, or when an Fab fragment of 9.3F10 is added to the culture. Positive and negative monocyte selection experiments, with the fluorescence-activated cell sorter and with complement-mediated cytolysis, indicate that monocytes contribute little if at all to accessory function. In contrast, highly enriched and monocyte-depleted dendritic cells are potent stimulators of the syngeneic and allogeneic MLR and the response to soluble tetanus toxoid. Monocytes and dendritic cells express similar levels of Ia antigens, however, indicating that class II products need to be expressed on dendritic cells to induce several T cell-proliferative responses in man.

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Flow cytometers are instruments that can determine multiparameter data simultaneously and have a great potential in providing unique information about cells. The FACSCount System is designed as the first dedicated flow cytometer for the clinical laboratory. Its current configuration provides CD4, CD8, and CD3 absolute counts from 100 μl of whole blood. Adapting the FACSCount System to the clinical setting are minimal sample handling, lysis free cell preparation, automated fluorescence gating, built‐in calibrated reference beads, and appropriate error code reporting. Quality control checks ensure that reported CD4 counts, important for clinical follow‐up and patient management, are accurate, precise, and reproducible across instruments over time. © 1996 Wiley‐Liss, Inc.