Evelyn Hartmann

B-Cell Lymphomas Differ in their Responsiveness to CpG Oligodeoxynucleotides

Human B cells detect CpG motifs within microbial DNA via TLR9. Synthetic CpG oligodeoxynucleotides are currently being tested in clinical trials for the therapy of different types of B cell non-Hodgkins lymphoma. However, there is only limited information on the CpG oligodeoxynucleotide sensitivity of primary malignant B cells of different non-Hodgkins lymphoma entities. Here we found that most B-cell malignancies except plasmacytoma respond to CpG oligodeoxynucleotides by up-regulating expression of costimulatory and antigen-presenting molecules, by increasing expression of CD20, and by proliferation. In an in vitro analysis of 41 individual patient-derived primary tumor samples, B-cell chronic lymphocytic leukemia (B-CLL) and marginal zone lymphoma showed the strongest activation upon stimulation with CpG oligodeoxynucleotides. Small lymphocytic lymphoma, follicular lymphoma, mantle cell lymphoma, and large cell lymphoma showed an intermediate response. Consistent with CpG oligodeoxynucleotides sensitivity, TLR9 mRNA was present in B-CLL but absent in plasmacytoma. Although CpG oligodeoxynucleotides induced proliferation in all CpG oligodeoxynucleotide–sensitive types of B-cell malignancies, proliferation was weaker than in normal B cells and at least for B-CLL was followed by increased apoptosis. In conclusion, B-cell malignancies show significant differences in their responsiveness to CpG oligodeoxynucleotides. Focusing clinical studies on patients with highly CpG oligodeoxynucleotide–sensitive B-cell malignancies may improve the clinical outcome of such trials.

Activation of Endothelial Toll-Like Receptor 3 Impairs Endothelial Function

Rationale: Endothelial dysfunction and atherosclerosis are chronic inflammatory diseases characterized by activation of the innate and acquired immune system. Specialized protein receptors of the innate immune system recognize products of microorganisms and endogenous ligands such as nucleic acids. Toll-like receptor 3 (TLR3), for example, detects long double-stranded RNA and is abundantly expressed in endothelial cells. Whether innate immunity contributes to atherogenic mechanisms in endothelial cells is poorly understood. Objective: We sought to determine the effects of TLR3 activation in endothelial cells. Methods and Results: We first investigated whether stimulation of TLR3 influences endothelial biology in mice. Intravenous injection of polyinosine polycytidylic acid, a synthetic double-stranded RNA analog and TLR3 ligand, impaired endothelium-dependent vasodilation, increased vascular production of reactive oxygen species, and reduced reendothelialization after carotid artery injury in wild-type mice compared with controls but had no effect in TLR3−/− animals. TLR3 stimulation not only induced endothelial dysfunction but also enhanced the formation of atherosclerotic plaques in apolipoprotein E–deficient mice. In vitro incubation of endothelial cells with polyinosine polycytidylic acid induced production of the proinflammatory cytokines interleukin-8 and interferon-&ggr;–induced protein 10, increased formation of reactive oxygen species, diminished proliferation, and increased apoptosis, which suggests that endothelial cells are able to directly detect and respond to TLR3 ligands. Neutralization of interleukin-8 and interferon-&ggr;–induced protein 10 antagonizes the observed negative effects of polyinosine polycytidylic acid. We found elevated levels of circulating endothelial progenitor cells in polyinosine polycytidylic acid–treated mice, although they displayed increased endothelial dysfunction. Stimulation of TLR3 in cultured endothelial progenitor cells, however, led to increased formation of reactive oxygen species, increased apoptosis, and reduced migration. Injection of endothelial progenitor cells that had been incubated with polyinosine polycytidylic acid ex vivo hindered reendothelialization after carotid artery injury. Therefore, endothelial progenitor cell function was affected by TLR3 stimulation. Finally, apolipoprotein E–deficient/TLR3-deficient mice exhibited improved endothelial function compared with apolipoprotein E–deficient/TLR3+/+ littermates. Conclusions: Immunorecognition of long double-stranded RNA by endothelial cells may be an important mechanism involved in endothelial cell activation and development of endothelial dysfunction.

Tumour-derived prostaglandin E2 and transforming growth factor-β synergize to inhibit plasmacytoid dendritic cell-derived interferon-α

In previous studies we reported that plasmacytoid dendritic cells (PDC) infiltrating head and neck cancer tissue are functionally impaired, but the molecular basis for the functional deficiency remained unclear. Here we demonstrate that tumour‐derived prostaglandin E2 (PGE2) and transforming growth factor‐β (TGF‐β) increase interleukin‐8 (IL‐8) but synergistically inhibit interferon‐α (IFN‐α) and tumour necrosis factor (TNF) production of Toll‐like receptor 7 (TLR7)‐ and Toll‐like receptor 9 (TLR9)‐stimulated PDC. The inhibitory effect of PGE2 could be mimicked by the induction of cyclic AMP (cAMP) and by inhibitors of cyclooxygenase. The contribution of tumour‐derived TGF‐β was confirmed by the TGF‐β antagonist SB‐431542. Suppression of tumour‐derived PGE2 and TGF‐β restored TLR‐induced IFN‐α production of PDC. Additionally, PGE2‐ and TGF‐β‐treated PDC display a ‘tolerogenic’ phenotype because of a downregulation of CD40 accompanied by an upregulation of CD86. Finally, in TLR‐stimulated PDC, PGE2 and TGF‐β reduce the CCR7 : CXCR4 ratio, suggesting that PDC are impaired in their ability to migrate to tumour‐draining lymph nodes but are retained in stromal cell‐derived factor 1 (SDF‐1)‐expressing tissues. Based on these data, cyclooxygenase inhibitors and TGF‐β antagonists may improve TLR7‐ and TLR9‐based tumour immunotherapy.

Targeted activation of RNA helicase retinoic acid-inducible gene-I induces proimmunogenic apoptosis of human ovarian cancer cells.

Most malignant cells are poorly immunogenic and fail to elicit an effective antitumor immune response. In contrast, viral infections of cells are promptly detected and eliminated by the immune system. Viral recognition critically hinges on cytosolic nucleic acid receptors that include the proinflammatory RNA helicase retinoic acid-inducible gene-I (RIG-I). Here, we show that targeted delivery of RIG-I agonists induced ovarian cancer cells to upregulate HLA class I and to secrete the proinflammatory cytokines CXCL10, CCL5, interleukin-6, tumor necrosis factor-alpha, and IFN-beta. Ovarian cancer cells stimulated via RIG-I became apoptotic and were readily phagocytosed by monocytes and monocyte-derived dendritic cells, which in turn upregulated HLA class I/II and costimulatory molecules and released CXCL10 and IFN-alpha. Our findings offer proof of principle that mimicking viral infection in ovarian cancer cells triggers an immunogenic form of tumor cell apoptosis that may enhance immunotherapy of ovarian cancer.

Higher activation of TLR9 in plasmacytoid dendritic cells by microbial DNA compared with self-DNA based on CpG-specific recognition of phosphodiester DNA

TLR9 detects DNA in endolysosomal compartments of human B cells and PDC. Recently, the concept of the CpG motif specificity of TLR9‐mediated detection, specifically of natural phosphodiester DNA, has been challenged. Unlike in human B cells, CpG specificity of natural phosphodiester DNA recognition in human PDC has not been analyzed in the literature. Here, we found that the induction of IFN‐α and TNF‐α in human PDC by phosphodiester ODNs containing one or two CG dinucleotides was reduced to a lower level when the CG dinucleotides were methylated and was abolished if the CGs were switched to GCs. Consistent with a high frequency of unmethylated CG dinucleotides, bacterial DNA induced high levels of IFN‐α in PDC; IFN‐α was reduced but not abolished upon methylation of bacterial DNA. Mammalian DNA containing low numbers of CG dinucleotides, which are frequently methylated, induced IFN‐α in PDC consistently but on a much lower level than bacterial DNA. For activation of PDC, phosphodiester ODNs and genomic DNA strictly required complexation with cationic molecules such as the keratinocyte‐derived antimicrobial peptide LL37 or a scrambled derivative. In conclusion, we demonstrate that self‐DNA complexed to cationic molecules activate PDC and thus, indeed, may function as DAMPs; nevertheless, the preference of PDC for CpG containing DNA provides the basis for the discrimination of microbial from self‐DNA even if DNA is presented in the condensed form of a complex.

Delivery with polycations extends the immunostimulant Ribomunyl into a potent antiviral Toll-like receptor 7/8 agonist.

BACKGROUND Upper respiratory tract infection is a frequent cause of morbidity worldwide. Although the course of infection is usually mild, it is responsible for enormous social and economic costs. Immunostimulation with bacterial extracts consisting of ribosomal RNA and proteoglycans, such as Ribomunyl, was introduced into the clinic in the 1980s as a new treatment concept, but did not achieve widespread application. Ribomunyl has been proposed to activate innate immunity, but the contribution of its RNA content as well as its antiviral potential has not been studied. METHODS Peripheral blood mononuclear cells from healthy donors and immune cells from adenoids were incubated with Ribomunyl either by itself or formulated in a complex with cationic polypeptides such as poly-l-arginine or protamine, and induction of cytokines was quantified by ELISA. RESULTS Ribomunyl in complex with either poly-l-arginine or protamine, but not on its own, was able to strongly induce interferon-α (P<0.01) and interleukin-12 (P<0.01) in peripheral blood mononuclear cells, whereas induced tumour necrosis factor-α and interleukin-6 levels were independent of the formulation. Comparable results were obtained in immune cells from adenoids, suggesting efficacy also in virus-affected tissue. Cell sorting, RNase digests and selective receptor expression show that the RNA in Ribomunyl acts as an agonist of Toll-like receptor (TLR)7 and TLR8. CONCLUSIONS Ribomunyl is, in principle, able to potently induce antiviral interferon-α and interleukin-12 via TLR7 and TLR8, respectively, but only when formulated in a complex with cationic polypeptides.