Dendritic cells (DC) are potent antigen-presenting cells and understanding their mechanisms of antigen uptake is important for loading DC with antigen for immunotherapy. The multilectin receptors, DEC-205 and macrophage mannose receptor (MMR), are potential antigen-uptake receptors; therefore, we examined their expression and FITC-dextran uptake by various human DC preparations. The RT-PCR analysis detected low levels of DEC-205 mRNA in immature blood DC, Langerhans cells (LC) and immature monocyte-derived DC (Mo-DC). Its mRNA expression increased markedly upon activation, indicating that DEC-205 is an activation-associated molecule. In Mo-DC, the expression of cell-surface DEC-205 increased markedly during maturation. In blood DC, however, the cell-surface expression of DEC-205 did not change during activation, suggesting the presence of a large intracellular pool of DEC-205 or post-transcriptional regulation. Immature Mo-DC expressed abundant MMR, but its expression diminished upon maturation. Blood DC and LC did not express detectable levels of the MMR. FITC-dextran uptake by both immature and activated blood DC was 30- to 70-fold less than that of LC, immature Mo-DC and macrophages. In contrast to immature Mo-DC, the FITC-dextran uptake by LC was not inhibited effectively by mannose, an inhibitor for MMR-mediated FITC-dextran uptake. Thus, unlike Mo-DC, blood DC and LC do not use the MMR for carbohydrate-conjugated antigen uptake and alternative receptors may yet be defined on these DC. Therefore, DEC-205 may have a different specificity as an antigen uptake receptor or contribute to an alternative DC function.

The CMRF-44 and CD83 (HB15) antigens are associated with functional maturation and activation of blood dendritic cells (DC). We describe the expression of these antigens on freshly isolated epidermal Langerhans cells and dermal DC as well as the distribution of CD83+/ CMRF-44++-activated DC within sections of normal human skin. Fresh Langerhans cells were prepared by standard techniques and large numbers of enriched (25%-55%), viable dermal DC were obtained using an improved collagenase treatment protocol with density gradient enrichment. Freshly isolated Langerhans cells and dermal DC had similar costimulator and activation antigen expression, and both stimulated moderate levels of allogeneic T lymphocyte proliferation as determined in the 7 d mixed leukocyte reaction. In situ labeling of DC within skin sections revealed a population of CD83 and CMRF-44 positive dermal cells of which most (approximately 75%) were in intimate contact with CD3+ T lymphocytes, especially in the adnexal regions. In contrast, only 25%-30% of the more numerous CD1a++ dermal DC population were directly apposed to T lymphocytes. The CMRF-44++ dermal DC population stimulated an allogeneic mixed leukocyte reaction, confirming their identity as DC. These data, plus comparative data obtained for migratory dermal DC, suggest that only a small proportion of dermal DC have been triggered to a more advanced state of differentiation or activation. The striking association of the activated dermal DC population with T lymphocytes suggests that communication between these two cell types in situ may occur early in the immune response to cutaneous antigen.

Dendritic cells (DC) originate from a bone marrow (BM) precursor and circulate via the blood to most body tissues where they fulfill a role in antigen surveillance. Little is known about DC numbers in disease, although the reported increase in tissue DC turnover due to inflammatory stimuli suggests that blood DC numbers may be altered in some clinical situations. The lack of a defined method for counting DC has limited patient studies. We therefore developed a method suitable for routine monitoring of blood DC numbers, using the CMRF44 monoclonal antibody (MoAb) and flow cytometry to identify DC. A normal range was determined from samples drawn from 103 healthy adults. The mean percentage of DC present in blood mononuclear cells (MNC) was 0.42%, and the mean absolute DC count was 10 × 106 DC/L blood. The normal ranges for DC (mean ± 1.96 standard deviation [SD]) were 0.15% to 0.70% MNC or 3 to 17 × 106 DC/L blood. This method has applications for monitoring attempts to mobilize DC into the blood to facilitate their collection for immunotherapeutic purposes and for counting blood DC in other patients. In preliminary studies, we have found a statistically significant decrease in the blood DC counts in individuals at the time of blood stem cell harvest and in patients with acute illnesses, including allogeneic bone marrow transplant (BMT) recipients with acute graft-versus-host disease (aGVHD).

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Dendritic cells (DC) are potent antigen-presenting cells (APC) with the capacity to stimulate a primary T lymphocyte immune response and are therefore of interest for potential immunotherapeutic applications. Freshly isolated DC or DC precursors may be preferable for studies of antigen uptake and the potential control of APC costimulator activity. In this report, we report that the monoclonal antibody CMRF-44 can be used to detect early DC differentiation. The majority of DC circulating in blood do not express any known DC lineage specific markers, but can be identified by CMRF-44 labeling after a brief period of in vitro culture. The sequential acquisition of DC activation antigens allows the identification of two stages of DC maturation/activation. Cytokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF ) and tumor necrosis factor (TNF )α, enhance both phases of this process, whereas CD40-ligand trimer preferentially enhances the final DC maturation to a fully mature, activated phenotype. DC positively selected using CMRF-44 possess potent allostimulatory activity and are efficient at the uptake, processing, and presentation of soluble antigens for both primary and secondary immune responses. CMRF-44+ DC are also more potent than other APC types at restimulation of a chronic myeloid leukemia peptide specific T-cell clone. The use of a purified population of freshly isolated DC may be advantageous in attempts to initiate, maintain, and direct immune responses for immunotherapeutic applications.