A human homologue of the Drosophila Toll protein signals activation of adaptive immunity

. Like Drosophila Toll, human Toll is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. Both Drosophila Toll and the IL-1 receptor are known to signal through the NF-kB pathway 5-7 . We show that a constitutively active mutant of human Toll transfected into human cell lines can induce the activation of NF-kB and the expression of NF-kB-controlled genes for the inflammatory cyto- kines IL-1, IL-6 and IL-8, as well as the expression of the co- stimulatory molecule B7.1, which is required for the activation of naive T cells. The Toll protein controls dorsal-ventral patterning in Drosophila embryos and activates the transcription factor Dorsal upon binding to its ligand Spatzle 8 . In adult Drosophila, the Toll/Dorsal signalling pathway participates in an anti-fungal immune response 2 . Signal- ling through Toll parallels the signalling pathway induced by the IL- 1 receptor (IL-1R) in mammalian cells: IL-1R signals through the NF-kB pathway, and Dorsal and its inhibitor Cactus are homo- logous to NF-kB and I-kB proteins, respectively 5,6 . Moreover, the cytoplasmic domain of Drosophila Toll is homologous to the cytoplasmic domain of IL-1R (ref. 9). Remarkably, the tobacco- virus-resistance gene that encodes N-protein is also similar to Toll in that it contains both a Toll signalling domain and an LRR domain 10 . It thus appears that the immune-response system mediated by Toll represents an ancient host defence mechanism 6 (Fig. 1). To inves- tigate the possibility that this pathway has been retained in the immune system of vertebrates, we used sequence and pattern searches 11 of the expressed-sequence tag (EST) database at the fragment was used to probe northern blots containing poly(A) + RNA from several organs. Most organs expressed two mRNA species: one of ,5 kilobases (kb) was predominant in most tissues except peripheral blood leukocytes (PBL), and corresponded to the length of the cDNA that we cloned. The lower band was ,4 kb long and this band was predominant in the PBL. The 4-kb band was not detectable in kidney, and liver did not contain any mRNA at all (Fig. 3). We also tested different mouse and human cell lines for expression of hToll mRNA by using PCR with reverse transcription (RT-PCR). We found mRNA for hToll in monocytes, macrophages, dendritic cells, g/d T cells, Th1 and Th2 a/b T cells, a small intestinal epithelial cell line, and a B-cell line (data not shown). The hToll gene is expressed most strongly in spleen and PBL (Fig. 3); its expression in other tissues may be due to the presence of macrophages and dendritic cells, in which it could act as an early-warning system for infection. Alternatively, hToll may be widely expressed because hToll signals through the conserved NF-kB pathway (see below) and NF- kB is a ubiquitous transcription factor. To characterize hToll functions and see whether it can induce transcription of immune response genes like dToll, we generated a dominant-positive mutant of hToll because the natural ligand of hToll is unknown. To produce a constitutively active mutant of hToll, we made use of genetic information from dToll: analysis of ventra- lizing mutants in Drosophila embryos had identified the function of the ectodomain C-flanking cysteine-rich region in dToll 16 as control- ling the activity of dToll in signal transduction. In three dominant