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Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine (CpG) dinucleotides (CpG ODN) mimic the immunostimulatory activity of bacterial DNA and are recognized by the Toll-like receptor 9 (TLR9). CpG ODN of the B-Class stimulate strong B cell and NK cell activation and cytokine production. The highest degrees of NK stimulation as well as IFN- § secretion by plasmacytoid DC were found to occur only with A-Class ODN. A third class of CpG ODN combines the immune effects of A-and B-Class CpG ODN. C-Class ODN strongly stimulate B cell or NK cell activation and IFN- § production. In contrast to the AClass, the C-Class is wholly phosphorothioate, has no poly-G stretches, but has palindromic sequences combined with stimulatory CpG motifs. All classes stimulate TLR9-dependent signaling, but with strikingly different dose-response relationships that are quite in contrast to those observed for IFN- § . Effects similar to those on human cells were observed on mouse splenocytes. In contrast, splenocytes from TLR9-deficient mice did not show any response to the three CpG ODN classes. In vivo studies demonstrate that C-Class ODN are very potent Th1 adjuvants. C-Class ODN may represent new therapeutic drugs that combine the effects of A-and B-Class ODN for broad applications in infectious disease or cancer therapy.

Oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides mimic the immune stimulatory activity of bacterial DNA in vertebrates and are recognized by Toll-like receptor 9 (TLR9). It is also possible to detect immune activation with certain phosphorothioate sequences that lack CpG motifs. These ODN are less potent than CpG ODN and the mechanism by which they stimulate mammalian leucocytes is not understood. We here provide several lines of evidence demonstrating that the effects induced by non-CpG ODN are mediated by TLR9. First, non-CpG ODN could not stimulate cytokine secretion from the splenocytes of TLR9-deficient (TLR9(-/-)) mice. Second, immunization of TLR9(+/+) but not TLR9(-/-) mice with non-CpG ODN enhanced antigen-specific antibody responses, although these were T helper type 2 (Th2)-biased. Third, reactivity to non-CpG ODN could be reconstituted by transfection of human TLR9 into non-responsive cells. In addition, we define a new efficient immune stimulatory motif aside from the CpG dinucleotide that consists of a 5'-TC dinucleotide in a thymidine-rich background. Non-CpG ODN containing this motif induced activation of human B cells, but lacked stimulation of Th1-like cytokines and chemokines. Our study indicates that TLR9 can mediate either efficient Th1- or Th2-dominated effects depending on whether it is stimulated by CpG or certain non-CpG ODN.

Toll-like receptors (TLR) 7 and 8 are closely related members of the TLR family of pathogen-associated molecular pattern recognition receptors and have an important function in activation of innate immune responses upon viral infection. TLR7 can be activated selectively by the guanosine analogue loxoribine, whereas the imidazoquinoline derivative Resiquimod (R-848) activates both TLR7 and TLR8. We demonstrate that co-incubation of R-848 with thymidine homopolymer oligodeoxynucleotides (ODN) significantly increased activity of R-848 on TLR8-expressing HEK 293 cells, but abolished TLR7-mediated signaling. Similarly, the combination of loxoribine and thymidine ODN redirected the stimulatory effect of loxoribine away from TLR7, and toward TLR8. This alteration in ligand specificity was demonstrated both in TLRtransfected HEK cells, and also in human PBMC, with a corresponding change in cytokine production away from IFN-a secretion by TLR7-expressing plasmacytoid DC and toward IL-12, TNF-a and IFN-c secretion by TLR8-expressing monocytes and NK cells. These results demonstrate an unexpected plasticity in the ligand specificities of TLR7 and TLR8, and suggest a novel sequence-selective interaction between these receptors and synthetic phosphorothioate ODN. IntroductionComprehension of the immune systems responses to pathogens has advanced dramatically since the discovery of the family of Toll-like receptors (TLR). TLR belong to the IL-1 receptor (IL-1R) superfamily and recognize so-called pathogen-associated molecular patterns (PAMP) thereby activating innate immune responses to bacterial, viral or fungal threats. In mammals, 11 TLR have been described so far. Natural pathogenderived ligands have been identified for many TLR: lipopolysaccharide (TLR4), triacyl lipopeptides (TLR1), lipopeptides and glycolipids (TLR2), flagellin (TLR5) and zymosan (TLR2 and TLR6) are some of these bacterial and fungal components. Non-methylated, CpG-containing DNA of bacterial or viral origin has been found to activate TLR9, whereas double-stranded viral RNA was demonstrated to target TLR3 (for review see [1]). Most recently, several groups identified singlestranded RNA as well as natural ligands for TLR7 and TLR8 [2][3][4]. In addition, certain short interfering RNA (siRNA) sequences have been identified as potential TLR7 ligands [5].TLR are type I membrane receptors with TLR 1, 2, 4, 5, and 6 located on the cell surface and TLR 7, 8, and 9 located within the cell [6][7][8][9]. TLR3 was found to be expressed intracellularly and at the cell surface in certain cell types, e.g., fibroblasts [10] or epithelial cells [11,12]. For TLR9, localization in the endoplasmic reticulum and recruitment to the endosomal compart- As activation of TLR is important for stimulating immune responses, the TLR family has been a target for pharmaceutical drug development. Synthetic ligands for several TLR have been developed and some are being tested in clinical trials. CpG-containing oligodeoxynucleotides (ODN), e.g., PF-3512676 (formerly known as CPG 7909),...

The retroviral oncogene p3k (v-p3k) of avian sarcoma virus 16 (ASV16) codes for the catalytic subunit of phosphoinositide (PI) 3-kinase, p110␣. The v-P3k protein is oncogenic in vivo and in vitro; its cellular counterpart, c-P3k, lacks oncogenicity. Fusion of viral Gag sequences to the amino terminus of c-P3k activates the transforming potential. Activation can also be achieved by the addition of a myristylation signal to the amino terminus or of a farnesylation signal to the carboxyl terminus of c-P3k. A mutated myristylation signal was equally effective; it also caused a strong increase in the kinase activity of P3k. Mutations that inactivate lipid kinase activity abolish oncogenicity. The transforming activity of P3k is correlated with the ability to induce activating phosphorylation in Akt. Point mutations and amino-terminal deletions recorded in v-P3k were shown to be irrelevant to the activation of oncogenic potential. Interactions of P3k with the regulatory subunit of PI 3-kinase, p85, or with Ras are not required for transformation. These results support the conclusion that the oncogenicity of P3k depends on constitutive lipid kinase activity. Akt is an important and probably essential downstream component of the oncogenic signal from P3k.