SummaryCD34 § cells in human cord blood and marrow are known to give rise to dendritic cells (DC), as well as to other myeloid lineages. CD34 § cells are rare in adult blood, however, making it difficult to use CD34 + ceils to ascertain if DC progenitors are present in the circulation and if blood can be a starting point to obtain large numbers of these immunostimulatory antigenpresenting cells for clinical studies. A systematic search for DC progenitors was therefore carried out in several contexts. In each case, we looked initially for the distinctive proliferating aggregates that were described previously in mice. In cord blood, it was only necessary to deplete erythroid progenitors, and add granulocyte/macrophage colony-stimulating factor (GM-CSF) together with tumor necrosis factor (TNF), to observe many aggregates and the production of typical DC progeny. In adult blood from patients receiving CSFs after chemotherapy for malignancy, GM-CSF and TNF likewise generated characteristic DCs from HLA-DR negative precursors. However, in adult blood from healthy donors, the above approaches only generated small DC aggregates which then seemed to become monocytes. When interleukin 4 was used to suppress monocyte development (Jansen, J. H., G.-J. H. M. Wientjens, W. E. Fibbe, K. Willemze, and H. C. Kluin-Nelemans. 1989. J. Exp. Med. 170:577.), the addition of GM-CSF led to the formation of large proliferating DC aggregates and within 5-7 d, many nonproliferating progeny, about 3-8 million cells per 40 ml of blood. The progeny had a characteristic morphology and surface composition (e.g., abundant HLA-DK and accessory molecules for cell-mediated immunity) and were potent stimulators of quiescent T cells. Therefore, large numbers of DCs can be mobilized by specific cytokines from progenitors in the blood stream. These relatively large numbers of DC progeny should facilitate future studies of their FceRI and CD4 receptors, and their use in stimulating T cell-mediated resistance to viruses and tumors.

Dendritic cells (DCs) are considered to be promising adjuvants for inducing immunity to cancer. We used mature, monocyte-derived DCs to elicit resistance to malignant melanoma. The DCs were pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin. 11 far advanced stage IV melanoma patients, who were progressive despite standard chemotherapy, received five DC vaccinations at 14-d intervals. The first three vaccinations were administered into the skin, 3 × 106 DCs each subcutaneously and intradermally, followed by two intravenous injections of 6 × 106 and 12 × 106 DCs, respectively. Only minor (less than or equal to grade II) side effects were observed. Immunity to the recall antigen was boosted. Significant expansions of Mage-3A1–specific CD8+ cytotoxic T lymphocyte (CTL) precursors were induced in 8/11 patients. Curiously, these immune responses often declined after the intravenous vaccinations. Regressions of individual metastases (skin, lymph node, lung, and liver) were evident in 6/11 patients. Resolution of skin metastases in two of the patients was accompanied by erythema and CD8+ T cell infiltration, whereas nonregressing lesions lacked CD8+ T cells as well as Mage-3 mRNA expression. This study proves the principle that DC “vaccines” can frequently expand tumor-specific CTLs and elicit regressions even in advanced cancer and, in addition, provides evidence for an active CD8+ CTL–tumor cell interaction in situ as well as escape by lack of tumor antigen expression.

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SummaryWe have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR. and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels ofbioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 • 106 DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison oflL-12 production, and blocking experiments employing an anti-CD40 ligand n'LAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Thl type cell-mediated immune responses. IL-12, a 70-kD heterodimeric cytokine composed of co-.valentty linked p35 and p40 chains has emerged as a central cytokine in the immune response (1). IL-12 stimulates NK cells, mediates Thl development, and fosters CTL development. It can be produced by monocytes and macrophages in response to intracellular pathogens, bacteria (e.g., staphylococci) and bacterial products. Recent reports indicate that dendritic cells (DC) also release bioactive IL-12. One report described that anti-IL-12 blocks the capacity of murine DC to skew the response of naive transgenic T cells to the Thl phenotype (2), and another shows induction of IL-12 p40/p35 mRNA in bone-marrow derived murine DC upon uptake ofmicroparticle-absorbed protein antigen (3). Human epidermal Langerhans cells are also a source of IL-12 (4). We have recently used several criteria for demonstration of IL-12 p40 and p35 mRNA as well as IL-12 p40 and bioactive p70 proteins, to show that murine and human DC release IL-12 upon conventional stimuli such as staphylococcus aureus (5). We also found that DC produced bioactive IL-12 upon interaction with T cells without standard stir " ~-,~h as bacterial products. Here, we describe the regulation oflL-12 in DC.

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