Grayson B. Lipford

Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition

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.

Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG‐oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as “natures adjuvant” since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG‐ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen‐presenting cell (APC) function. Unfractionated murine bone marrow‐derived DC and FACS®‐fractionated MHC class IIlow (termed immature DC) or MHC class IIhigh populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG‐ODN. Similar to lipopolysaccharide, i.s. CpG‐ODN caused up‐regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL‐12, IL‐6 and TNF‐α. CpG‐ODN‐activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B‐driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG‐ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.

CpG‐DNA‐specific activation of antigen‐presenting cells requires stress kinase activity and is preceded by non‐specific endocytosis and endosomal maturation

Unmethylated CpG motifs in bacterial DNA, plasmid DNA and synthetic oligodeoxynucleotides (CpG ODN) activate dendritic cells (DC) and macrophages in a CD40‐CD40 ligand‐independent fashion. To understand the molecular mechanisms involved we focused on the cellular uptake of CpG ODN, the need for endosomal maturation and the role of the stress kinase pathway. Here we demonstrate that CpG‐DNA induces phosphorylation of Jun N‐terminal kinase kinase 1 (JNKK1/SEK/MKK4) and subsequent activation of the stress kinases JNK1/2 and p38 in murine macrophages and dendritic cells. This leads to activation of the transcription factor activating protein‐1 (AP‐1) via phosphorylation of its constituents c‐Jun and ATF2. Moreover, stress kinase activation is essential for CpG‐DNA‐induced cytokine release of tumor necrosis factor α (TNFα) and interleukin‐12 (IL‐12), as inhibition of p38 results in severe impairment of this biological response. We further demonstrate that cellular uptake via endocytosis and subsequent endosomal maturation is essential for signalling, since competition by non‐CpG‐DNA or compounds blocking endosomal maturation such as chloroquine or bafilomycin A prevent all aspects of cellular activation. The data suggest that endosomal maturation is required for translation of intraendosomal CpG ODN sequences into signalling via the stress kinase pathway, where p38 kinase activation represents an essential step in CpG‐ODN‐triggered activation of antigen‐presenting cells.

The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily.

Loxoribine (7‐allyl‐7,8‐dihydro‐8‐oxo‐guanosine) acts as synthetic adjuvant in anti‐tumor responses. Here we first demonstrate that loxoribine activates cells of the innate immune system selectively via the Toll‐like receptor (TLR) 7/MyD88‐dependent signaling pathway. TLR7‐ and MyD88‐deficient immune cells fail to proliferate or produce cytokines in response to loxoribine, and genetic complementation of TLR7‐deficient cells with murine or human TLR7 confers responsiveness. Subsequently we show that cellular activation by loxoribine and resiquimod (R‐848), a stimulus for TLR7 and TLR8, depends on acidification and maturation of endosomes and targets MyD88 to vesicular structures with lysosomal characteristics. This mode of TLR7 and TLR8 action resembles CpG‐DNA‐driven TLR9 activation. We thus conclude that TLR7, 8 and 9 form a functional subgroup within the TLR family that recognizes pathogen‐associated molecular patterns in endosomal/lysosomal compartments.

Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner

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.

Established Human Papillomavirus Type 16-Expressing Tumors Are Effectively Eradicated Following Vaccination with Long Peptides

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E743–77, containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8+ T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4+ Th cells. The level of the induced E7-specific CD8+ T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8+ T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.

Bacterial CpG-DNA Triggers Activation and Maturation of Human CD11c−, CD123+ Dendritic Cells

Human plasmacytoid precursor dendritic cells (ppDC) are a major source of type I IFN upon exposure to virus and bacteria, yet the stimulus causing their maturation into DCs is unknown. After PBMC activation with immunostimulatory bacterial DNA sequences (CpG-DNA) we found that ppDC are the primary source of IFN-α. In fact, either CpG-DNA or dsRNA (poly(I:C)) induced IFN-α from purified ppDC. Surprisingly, only CpG-DNA triggered purified ppDC survival, maturation, and production of TNF, GM-CSF, IL-6, and IL-8, but not IL-10 or IL-12. Known DC activators such as CD40 ligation triggered ppDC maturation, but only IL-8 production, while bacterial LPS was negative for all activation criteria. An additional finding was that only CpG-DNA could counteract IL-4-induced apoptosis in ppDC. Therefore, CpG-DNA represents a pathogen-associated molecular pattern for ppDC. In contrast to these finding, CpG-DNA, like LPS, caused TNF, IL-6, and IL-12 release from PBMC and purified monocytes; however, differentiation of monocytes into DCs with GM-CSF and IL-4 unexpectedly resulted in refractoriness to CpG-DNA, but not LPS. Taken together, these results suggest that within a DC subset a multiplicity of responses can be generated by distinct environmental stimuli and that responses to a given stimulus may be dissimilar between DC subsets.

TLRs Govern Neutrophil Activity in Aspergillosis

Polymorphonuclear neutrophils (PMNs) are essential in initiation and execution of the acute inflammatory response and subsequent resolution of fungal infection. PMNs, however, may act as double-edged swords, as the excessive release of oxidants and proteases may be responsible for injury to organs and fungal sepsis. To identify regulatory mechanisms that may balance PMN-dependent protection and immunopathology in fungal infections, the involvement of different TLR-activation pathways was evaluated on human PMNs exposed to the fungus Aspergillus fumigatus. Recognition of Aspergillus and activation of PMNs occurred through the involvement of distinct members of the TLR family, each likely activating specialized antifungal effector functions. By affecting the balance between fungicidal oxidative and nonoxidative mechanisms, pro- and anti-inflammatory cytokine production, and apoptosis vs necrosis, the different TLRs ultimately impacted on the quality of microbicidal activity and inflammatory pathology. Signaling through TLR2 promoted the fungicidal activity of PMNs through oxidative pathways involving extracellular release of gelatinases and proinflammatory cytokines while TLR4 favored the oxidative pathways through the participation of azurophil, myeloperoxidase-positive, granules and IL-10. This translated in vivo in the occurrence of different patterns of fungal clearance and inflammatory pathology. Both pathways were variably affected by signaling through TLR3, TLR5, TLR6, TLR7, TLR8, and TLR9. The ability of selected individual TLRs to restore antifungal functions in defective PMNs suggests that the coordinated outputs of activation of multiple TLRs may contribute to PMN function in aspergillosis.

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.

CpG-DNA aided cross-presentation of soluble antigens by dendritic cells

For cross‐presentation immature dendritic cells (DC) require enhanced antigen (Ag) uptake and a maturation signal to prime for MHC class I‐restricted CTL responses in vivo. While immunostimulatory CpG‐DNA provides, via TLR9, the maturation signal, CpG‐DNA linked to Ag augments cellular Ag uptake. In this study we show that CpG‐DNA ovalbumin (OVA) conjugates trigger in vivo peptide‐specific CTL responses at tenfold lower Ag doses compared to a mixture of CpG‐DNA plus OVA. We provide evidence that CpG‐DNA‐OVA conjugates shift OVA uptake by immature DC from the presumably inefficient fluid phase pinocytosis to efficient DNA receptor‐mediated endocytosis. Since the DNA‐binding receptor mediating endocytosis lacks any sequence specificity, cellular uptake of OVA conjugated with either stimulatory or non‐stimulatory oligonucleotides (ODN) is equally enhanced. As a consequence cross‐linking of OVA with either stimulatory or non‐stimulatory DNA yields, via enhanced OVA uptake, efficient generation and presentation of the dominant OVA‐CTL epitope SIINFEKL. However, only stimulatory CpG‐ODN cross‐linked to OVA provide the DC maturation signal required to trigger robust primary CTL responses towards the cross‐presented MHC class I complexed T cell epitope SIINFEKL. Our studies show that stimulatory CpG‐ODN linked to Ag fulfill a dual role: enhancement of Ag uptake yielding efficient Ag cross‐presentation by DC and in addition, their activation into professional DC.