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.

Dendritic cells and thymic epithelial cells perform important immunoregulatory functions by presenting antigens in the form of peptides bound to cell-surface major histocompatibility complex (MHC) molecules to T cells. Whereas B cells are known to present specific antigens efficiently through their surface immunoglobins, a comparable mechanism for the capture and efficient presentation of diverse antigens by dendritic cells and thymic epithelial cells has not previously been described. We show here that their antigen-presentation function is associated with the high-level expression of DEC-205, an integral membrane protein homologous to the macrophage mannose receptor and related receptors which are able to bind carbohydrates and mediate endocytosis. DEC-205 is rapidly taken up by means of coated pits and vesicles, and is delivered to a multivesicular endosomal compartment that resembles the MHC class II-containing vesicles implicated in antigen presentation. Rabbit antibodies that bind DEC-205 are presented to reactive T-cell hybridomas 100-fold more efficiently than rabbit antibodies that do not bind DEC-205. Thus DEC-205 is a novel endocytic receptor that can be used by dendritic cells and thymic epithelial cells to direct captured antigens from the extracellular space to a specialized antigen-processing compartment.

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-gamma (IFN-gamma), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-gamma bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 by murine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-gamma production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.

Freshly isolated, murine epidermal Langerhans cells (LC) are weak accessory cells for primary T cell-dependent immune responses, but increase their stimulatory capacity at least 20-fold progressively over a 3-d culture with keratinocytes. We have studied the mediators of LC maturation. LC enriched from 12-h epidermal cultures by negative selection do not survive when cultured for 60 h in standard medium. LC survive and show increased stimulatory capacity for oxidative mitogenesis and the primary MLR when 30% keratinocyte-conditioned medium is included. Of the three cytokines that are known to be produced by keratinocytes, only granulocyte/macrophage CSF (GM-CSF) maintains viability and increases stimulatory capacity. IL-1 alone does not keep LC alive, but further enhances the stimulatory activity when combined with GM-CSF. IL-3 has no effect. The increase in LC stimulatory capacity is not due to increased Ia antigen expression, which does not change between 12 and 60 h. Function is not simply due to improved viability, as GM-CSF does not enhance the function of 12-h LC when added to the short-term oxidative mitogenesis assay. By generating LC with strong stimulating activity for resting T cells, GM-CSF and IL-1 may be critical in the sensitization phase of T cell-mediated immunity.