Ulrike Samulowitz

Identification of RNA Sequence Motifs Stimulating Sequence-Specific TLR8-Dependent Immune Responses

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 lacking CpG dinucleotides mediate Toll-like receptor 9 dependent T helper type 2 biased immune stimulation

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.

CpG oligodeoxynucleotides stimulate IFN-γ-inducible protein-10 production in human B cells

Several classes of CpG oligodeoxynucleotides (ODNs) with different immune stimulatory profiles were recently identified: the A-, B- and C-classes. In this study, we investigated the CpG-dependent stimulation of IFN-γ-inducible protein 10 (IP-10 or CXCL10) in different human immune cell types. CpG ODNs induced IP-10 in monocytes, pDCs and in B cells. Purified B cells as well as RPMI 8226 cells responded to CpG stimulation by IP-10 production. Treatment with exogenous IFN-α2b sensitized PBMCs, purified B cells as well as RPMI 8226 cells to respond more efficiently to stimulation with CpG ODNs by IP-10 production. IP-10 signaling could be directly stimulated via TLR9 in CpG-unresponsive HEK293 cells transfected with human TLR9 and an IP-10 reporter construct. Therefore, CpG-mediated IP-10 production is stimulated through IFN-α in cells that express the IFN-α receptor, a second pathway for IP-10 induction exists in TLR9-expressing B cells and pDCs where IP-10 is stimulated directly upon CpG-mediated TLR9 signaling. Our data provide a better understanding of the mechanisms through which CpG ODNs induce efficient Th1 responses.

A Novel Class of Immune-Stimulatory CpG Oligodeoxynucleotides Unifies High Potency in Type I Interferon Induction with Preferred Structural Properties

Unmethylated deoxycytidyl-deoxyguanosin dinucleotide (CpG)-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists for Toll-like receptor 9. We here describe a new class of CpG ODNs, the so-called P-Class, which combines preferred properties of known CpG ODN classes. This P-Class contains two palindromic sequences, enabling it to form concatamers, multimeric units, where each molecule is bound via Watson-Crick basepairing to a second and a third palindrome. The type I interferon-inducing potency and efficacy of the double-palindromic P-Class ODN is substantially higher than that of previously described C-Class ODNs, and they stimulate superior cytokine production upon in vivo application. The multimeric structures of the P-Class can be resolved to monomers and dimers by formulation in low-salt buffer, retaining the strong and potent immune effects. Taken together, we have discovered a novel class of CpG ODNs, the P-Class, with promising superior activity for disease application.

Immunopharmacology of CpG Oligodeoxynucleotides and Ribavirin

ABSTRACT To investigate their potential mechanisms of action, the nucleoside analogue ribavirin and a TLR9 agonist were compared. The CpG oligodeoxynucleotides (ODN) demonstrated strong TLR9-related Th1-type effects, and ribavirin appeared only to mediate signaling in TLR-transfected cells. CpG ODN represent a promising new type of therapeutic drug for hepatitis C or other infectious diseases.