No abstract

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.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies to certain cellular macromolecules, such as the small nuclear ribonucleoprotein particles (snRNPs), which had been considered to be passive targets of the autoimmune response. SLE is also characterized by the increased expression of type I interferon (IFN), which appears to be associated with the development and severity of disease. Here, we show that specific, highly conserved RNA sequences within snRNPs can stimulate Toll-like receptors (TLRs) 7 and 8 as well as activate innate immune cells, such as plasmacytoid dendritic cells (pDCs), which respond by secreting high levels of type I IFN. SLE patient sera containing autoantibodies to snRNPs form immune complexes that are taken up through the Fc receptor γRII and efficiently stimulate pDCs to secrete type I IFNs. These results demonstrate that a prototype autoantigen, the snRNP, can directly stimulate innate immunity and suggest that autoantibodies against snRNP may initiate SLE by stimulating TLR7/8.

The TLRs 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. U-rich RNA sequences were recently discovered that stimulate human TLR7/8-mediated or murine TLR7-mediated immune effects. In this study we identified single-stranded RNA sequences containing defined sequence motifs that either preferentially activate human TLR8-mediated as opposed to TLR7- or TLR7/8-mediated immune responses. The identified TLR8 RNA motifs signal via TLR8 and fail to induce IFN-α from TLR7-expressing plasmacytoid dendritic cells but induce the secretion of Th1-like and proinflammatory cytokines from TLR8-expressing immune cells such as monocytes or myeloid dendritic cells. In contrast, RNA sequences containing the TLR7/8 motif signal via TLR7 and TLR8 and stimulate cytokine secretion from both TLR7- and TLR8-positive immunocytes. The TLR8-specific RNA sequences are able to trigger cytokine responses from human and bovine but not from mouse, rat, and porcine immune cells, suggesting that these species lack the capability to respond properly to TLR8 RNA ligands. In summary, we describe two classes of single-stranded TLR7/8 and TLR8 RNA agonists with diverse target cell and species specificities and immune response profiles.

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.