Monophosphoryl Lipid A Activates Both Human Dendritic Cells and T Cells 1 Jamila Ismaili, 2 * Joe ¨lle Rennesson,* Ezra Aksoy,* Johan Vekemans,* Benoit Vincart,* Zoulikha Amraoui,* Francois Van Laethem, † Michel Goldman,* and Patrice M. Dubois ‡ The induction of dendritic cell (DC) maturation is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. In this study, we have investigated the effects of monophosphoryl lipid A (MPL) on human monocyte-derived DC as well as peripheral blood T cells. Calcium mobilization, mitogen-activated protein kinase activation, and the NF-B transcription factor were induced after MPL stimulation of DC and required high doses of MPL (100 g/ml). Maturation parameters such as production of IL-12 and increases in cell surface expression of HLA-DR, CD80, CD86, CD40, and CD83 were observed following DC treatment with MPL. However, lower levels of IL-12 were induced by MPL when compared with lipopolysaccharide. This is likely to be related to differences in the kinetics of extracellular signal-related kinase 1/2 and p-38 phosphorylation induced by both molecules. Although maturation induced by MPL was weaker when compared with lipopolysaccharide, it appeared to be sufficient to support optimal activation of allogeneic naive CD45RA  T cell and anti-tetanus toxoid CD4 T cells. MPL at low doses (5 g/ml) had no impact on DC maturation, while its addition to DC-T cell cocultures induced full T cell activation. The observed effect was related to the fact that MPL also acts directly on T cells, likely through their Toll-like receptors, by increasing their intracellular calcium and up-regulating their CD40 ligand expression. Together, these data support a model where MPL enhances T cell responses by having an impact on DC and T cells. The Journal of Immunology, 2002, 168: 926 –932. D endritic cells (DC) 3 are the most potent APCs. Immature DC reside in peripheral tissues and effectively capture and process native protein Ag and migrate to peripheral lymphoid organs. A maturation process, characterized by IL-12 production and the up-regulation of MHC and costimulatory mol- ecules, is critical for initiation of primary T cell response (1–3). This maturation is driven by inflammatory cytokines, such as TNF-, or bacterial products, such as lipopolysaccharide (LPS), encountered in peripheral organs (4). LPS, a constituent of the outer membrane of the cell wall of Gram-negative bacteria, is a complex glycolipid composed of a hydrophilic polysaccharide por- tion and a hydrophobic domain known as lipid A (5). Recent stud- ies (6 –11) showed that CD14 associates with Toll-like receptor (TLR)2 and TLR4, which are the signaling component of LPS, and consequently triggers its cellular transduction, leading to NF-B activation and DC maturation. The adjuvant activity of bacterial products is important not only for antibacterial responses induced by peripheral DC but also for vaccine development. However, LPS is excluded because of its high toxicity, as it is one of the main causes of septic shock in humans. The adjuvant activity of LPS resides in its lipid moiety, thus lipid A derivatives and analogs have been developed. The removal of an acid labile phosphate group and normal fatty acid groups from diphosphoryl lipid A dramatically reduced the toxicity and pyrogenicity (12). The gen- erated monophosphoryl lipid A (MPL) has been shown to activate APC and to enhance the generation of both Th1- and Th2-specific immune response in mice (13, 14). Its adjuvant effect may be tempting for vaccine development in humans. We tested its effect on maturation and activation of immature DC generated in vitro from adherent monocytes in the presence of GM-CSF and IL-4. Their maturation (surface molecule up-regulation and IL-12 pro- duction), their activation (calcium mobilization, mitogen-activated protein kinase (MAPK) activation, and NF-B translocation to the nucleus), and their ability to induce T cell responses have been assessed. We also explored the direct effect of MPL on T cells. Materials and Methods Reagents and medium LPS from Escherichia coli serotype (0128:B12) or from Salmonella mine- sota were purchased from Sigma-Aldrich (Bornem, Belgium). The S. mine- sota LPS derivative MPL (GlaxoSmithKline, Rixensart, Belgium) was pre- pared as previously described (15). Of note, LPS preparations are unlikely to contain MPL, because extrac- tion of this compound requires both alkaline and acid treatments following organic solvent extraction out of bacterial outer membranes. Cells were grown in RPMI 1640 (Life Technologies, Merelbeke, Bel- gium) supplemented with 50 M ME, 20 g/ml gentamicin, 2 mM L- glutamine, 1% nonessential amino acids (Life Technologies), and 10% FBS (Perbio, Aalst, Belgium). DC generation and stimulation PBMC from healthy volunteers were isolated by density centrifugation of heparinized blood on Lymphoprep (Nycomed, Oslo, Norway), washed with HBSS, resuspended in culture medium, and allowed to adhere in *Laboratory of Experimental Immunology, Faculte ´ de Medecine, Universite Libre de Bruxelles, Brussels, Belgium; † Laboratory of Animal Physiology, Institut de Biologie et Medecine Moleculaire, Gosselies, Belgium; ‡ GlaxoSmithKline, Rixensart, Belgium Received for publication January 16, 2001. Accepted for publication November 6, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work has been supported by grants from La Re ´gion Wallone, Formation et Impulsion a la Recherche Scientifique et Technologique, and GlaxoSmithKline. 2 Address correspondence and reprint requests to Dr. Jamila Ismaili at the current address: M.R.C. Laboratories, PO Box 273, Fajara, WA, Gambia. E-mail address: jismaili@mrc.gm 3 Abbreviations used in this paper: DC, dendritic cell; MPL, monophosphoryl lipid A; CD40L, CD40 ligand; TLR, Toll-like receptor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; LPS, lipopolysaccharide; MKK, MAPK kinase. Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00