Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus–1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-α and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)–deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.

Stress-inducible MICA, a distant homolog of major histocompatibility complex (MHC) class I, functions as an antigen for γδ T cells and is frequently expressed in epithelial tumors. A receptor for MICA was detected on most γδ T cells, CD8 ⁺ αβ T cells, and natural killer (NK) cells and was identified as NKG2D. Effector cells from all these subsets could be stimulated by ligation of NKG2D. Engagement of NKG2D activated cytolytic responses of γδ T cells and NK cells against transfectants and epithelial tumor cells expressing MICA. These results define an activating immunoreceptor-MHC ligand interaction that may promote antitumor NK and T cell responses.

The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins, which function as mediators of innate immunity for recognition of pathogen-derived ligands and subsequent cell activation via the Toll/IL-1R signal pathway. Here, we show that human TLR9 (hTLR9) expression in human immune cells correlates with responsiveness to bacterial deoxycytidylate-phosphate-deoxyguanylate (CpG)-DNA. Notably “gain of function” to immunostimulatory CpG-DNA is achieved by expressing TLR9 in human nonresponder cells. Transfection of either human or murine TLR9 conferred responsiveness in a CD14- and MD2-independent manner, yet required species-specific CpG-DNA motifs for initiation of the Toll/IL-1R signal pathway via MyD88. The optimal CpG motif for hTLR9 was GTCGTT, whereas the optimal murine sequence was GACGTT. Overall, these data suggest that hTLR9 conveys CpG-DNA responsiveness to human cells by directly engaging immunostimulating CpG-DNA.

Many immune receptors are composed of separate ligand-binding and signal-transducing subunits. In natural killer (NK) and T cells, DAP10 was identified as a cell surface adaptor protein in an activating receptor complex with NKG2D, a receptor for the stress-inducible and tumor-associated major histocompatibility complex molecule MICA. Within the DAP10 cytoplasmic domain, an Src homology 2 (SH2) domain–binding site was capable of recruiting the p85 subunit of the phosphatidylinositol 3-kinase (PI 3-kinase), providing for NKG2D-dependent signal transduction. Thus, NKG2D-DAP10 receptor complexes may activate NK and T cell responses against MICA-bearing tumors.

Toll‐like receptors (TLR) recognize bacterial and viral components, but direct interaction of receptor and ligand is unclear. Here, we demonstrate that TLR9 binds directly and sequence‐specifically to single‐stranded unmethylated CpG‐DNA containing a phosphodiester backbone. TLR9‐CpG‐DNA interaction occurs at the acidic pH (6.5–5.0) found in endosomes and lysosomes. By sequence comparison we identified a potential CpG‐DNA binding domain homologous to that described for methyl‐CpG‐DNA binding proteins. Amino acid substitutions in this region abrogated CpG‐DNA binding and led to loss of NF‐κB activation. Furthermore, chloroquine and quinacrine, therapeutic agents for autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, directly blocked TLR9‐CpG‐DNA interaction but not TLR2‐Pam3Cys binding. Our results demonstrate direct binding of TLR9 to CpG‐DNA and suggest that the therapeutic activity of chloroquine and quinacrine in autoimmune diseases may be due to its activity as a TLR9 antagonist and inhibitor of endosomal acidification.