Virus-associated activation of innate immunity induces rapid disruption of Peyer's patches in mice.

Early in the course of infection, detection of pathogen-associated molecular patterns by innate immune receptors can shape the subsequent adaptive immune response. Here we investigate the influence of virus-associated innate immune activation on lymphocyte distribution in secondary lymphoid organs. We show for the first time that virus infection of mice induces rapid disruption of the Peyer's patches but not of other secondary lymphoid organs. The observed effect was not dependent on an active infectious process, but due to innate immune activation and could be mimicked by virus-associated molecular patterns such as the synthetic double-stranded RNA poly(I:C). Profound histomorphologic changes in Peyer's patches were associated with depletion of organ cellularity, most prominent among the B-cell subset. We demonstrate that the disruption is entirely dependent on type I interferon (IFN). At the cellular level, we show that virus-associated immune activation by IFN-α blocks B-cell trafficking to the Peyer's patches by downregulating expression of the homing molecule α4ß7-integrin. In summary, our data identify a mechanism that results in type I IFN-dependent rapid but reversible disruption of intestinal lymphoid organs during systemic viral immune activation. We propose that such rerouted lymphocyte trafficking may impact the development of B-cell immunity to systemic viral pathogens.

TLR activation excludes circulating naive CD8+ T cells from gut-associated lymphoid organs in mice.

The trafficking of effector T cells is tightly regulated by the expression of site-specific sets of homing molecules. In contrast, naive T cells are generally assumed to express a uniform pattern of homing molecules and to follow a random distribution within the blood and secondary lymphoid organs. In this study, we demonstrate that systemic infection fundamentally modifies the trafficking of circulating naive CD8(+) T cells. We show that on naive CD8(+) T cells, the constitutive expression of the integrin α4ß7 that effects their entry into GALT is downregulated following infection of mice with Salmonella typhimurium. We further show that this downregulation is dependent on TLR signaling, and that the TLR-activated naive CD8(+) T cells are blocked from entering GALT. This contrasts strongly with Ag-experienced effector T cells, for which TLR costimulation in the GALT potently upregulates α4ß7 and enhances trafficking to intestinal tissues. Thus, TLR activation leads to opposite effects on migration of naive and effector CD8(+) T cells. Our data identify a mechanism that excludes noncognate CD8(+) T cells from selected immune compartments during TLR-induced systemic inflammation.

Cellular immunostimulation by CpG-sequence-coated DNA origami structures.

To investigate the potential of DNA origami constructs as programmable and noncytotoxic immunostimulants, we tested the immune responses induced by hollow 30-helix DNA origami tubes covered with up to 62 cytosine-phosphate-guanine (CpG) sequences in freshly isolated spleen cells. Unmethylated CpG sequences that are highly specific for bacterial DNA are recognized by a specialized receptor of the innate immune system localized in the endosome, the Toll-like receptor 9 (TLR9). When incubated with oligonucleotides containing CpGs, immune cells are stimulated through TLR9 to produce and secrete cytokine mediators such as interleukin-6 (IL-6) and interleukin-12p70 (IL-12p70), a process associated with the initiation of an immune response. In our studies, the DNA origami tube built from an 8634 nt long variant of the commonly used single-stranded DNA origami scaffold M13mp18 and 227 staple oligonucleotides decorated with 62 CpG-containing oligonucleotides triggered a strong immune response, characterized by cytokine production and immune cell activation, which was entirely dependent on TLR9 stimulation. Such decorated origami tubes also triggered higher immunostimulation than equal amounts of CpG oligonucleotides associated with a standard carrier system such as Lipofectamine. In the absence of CpG oligonucleotides, cytokine production induced by the origami tubes was low and was not related to TLR9 recognition. Fluorescent microscopy revealed localization of CpG-containing DNA origami structures in the endosome. The DNA constructs showed in contrast to Lipofectamine no detectable toxicity and did not affect the viability of splenocytes. We thus demonstrate that DNA origami constructs represent a delivery system for CpG oligonucleotides that is both efficient and nontoxic.

Systemic cancer therapy with a small molecule agonist of toll-like receptor 7 can be improved by circumventing TLR tolerance.

Topical application of small molecule Toll-like receptor 7 (TLR7) agonists is highly effective for the treatment of skin tumors, whereas their systemic application has been largely unsuccessful for cancer therapy. One reason may be that repeated systemic application of TLR ligands can induce a state of immune unresponsiveness, termed TLR tolerance. We show here that a single injection of the TLR7 agonist R848 in mice induces a short period of increased response to TLR stimulation followed by a state of hyporesponsiveness lasting several days. This state is characterized by inhibited secretion of the key cytokines interleukin (IL)-12p70 and IL-6 as well as by a block in IFN-α production. We show for the first time that at the cellular level, TLR7 tolerance occurs in both plasmacytoid and myeloid dendritic cells, two cell populations that play a critical role in the initiation and amplification of antitumor immune responses. We further show that TLR7 tolerance in plasmacytoid dendritic cells is accompanied by downregulation of the adaptor protein IL-1 receptor-associated kinase 1. On the basis of these findings, we have designed a novel strategy for the treatment of tumors by using cycles of repeated R848 injections separated by treatment-free intervals. We show in CT26 tumor-bearing mice that this protocol circumvents TLR7 tolerance and improves the efficacy of cancer immunotherapy.

Antigen delivery to plasmacytoid dendritic cells via BST2 induces protective T cell-mediated immunity.

Plasmacytoid dendritic cells (PDCs) are capable of presenting Ags to T cells in a tolerogenic or immunogenic manner depending on the formulation of the Ag and the mode of stimulation. It has not been investigated whether effective adaptive immune responses useful for vaccination can be induced by Ab-mediated Ag targeting to PDCs in vivo. In this study, we show that Ag delivered to murine PDCs via bone marrow stromal cell Ag 2 (BST2)/CD317 in combination with TLR agonists as adjuvants is specifically presented by PDCs in vivo and elicits strong cellular and humoral immune responses. These include IFN-γ production by CD4(+) T cells and high Ab titers with a broad range of IgG isotypes. In addition, BST2-mediated Ag delivery in the presence of polyinosinic-polycytidylic acid as adjuvant induces cytotoxic T lymphocytes that are functional in vivo. A single immunization with Ag-fused anti-BST2 Ab together with polyinosinic-polycytidylic acid as adjuvant is sufficient to trigger protective immunity against subsequent viral infection and tumor growth. We conclude that despite the potential tolerogenic properties of PDCs, Ag targeting to PDCs in combination with TLR agonists as adjuvants is an effective vaccination strategy.

CpG blocks immunosuppression by myeloid-derived suppressor cells in tumor-bearing mice.

PURPOSE: The Toll-like receptor (TLR) 9 ligand CpG has been used successfully for the immunotherapy of cancer. Chronic CpG application in tumor-free hosts leads, however, to the expansion of myeloid-derived suppressor cells (MDSC), which can cause T-cell suppression and may thus hamper the development of an effective immune response. Here, we investigated the effect of TLR9 activation on the function of MDSC in tumor-bearing mice. EXPERIMENTAL DESIGN: We investigated the effect of CpG treatment on the number, phenotype, and function of MDSC in mice bearing subcutaneous C26 tumors and in CEA424-TAg mice bearing autochthonous gastric tumors. RESULTS: CpG treatment blocks the suppressive activity of MDSC on T-cell proliferation in both tumor models. Inhibition of MDSC function by CpG was particularly pronounced for a highly suppressive Ly6G(hi) polymorphonuclear subset of MDSC. We further show that TLR9 activation by CpG promotes maturation and differentiation of MDSC and strongly decreases the proportion of Ly6G(hi) MDSC in both tumor-bearing and tumor-free mice. We demonstrate that IFN-α produced by plasmacytoid dendritic cells upon CpG stimulation is a key effector for the induction of MDSC maturation in vitro and show that treatment of mice with recombinant IFN-α is sufficient to block MDSC suppressivity. CONCLUSIONS: We show here for the first time that TLR9 activation inhibits the regulatory function of MDSC in tumor-bearing mice and define a role for the antitumoral cytokine IFN-α in this process.

Delivery of immunostimulatory RNA oligonucleotides by gelatin nanoparticles triggers an efficient antitumoral response.

RNA oligonucleotides have emerged as a new class of biologicals that can silence gene expression but also stimulate immune responses through specific pattern-recognition receptors. The development of effective delivery systems remains a major challenge for the therapeutic application of the RNA oligonucleotides. In this study, we have established a novel biodegradable carrier system that is highly effective for the delivery of immunostimulatory RNA oligonucleotides. Formulation of RNA oligonucleotides with cationized gelatin nanoparticles potentiates immune activation through the Toll-like receptor 7 (TLR7) in both myeloid and plasmacytoid dendritic cells. Further, nanoparticle-delivered RNA oligonucleotides trigger production of the antitumoral cytokines IL-12 and IFN-α. Binding to gelatin nanoparticles protects RNA oligonucleotides from degradation by nucleases, facilitates their uptake by dendritic cells, and targets these nucleic acids to the endosomal compartment in which they are recognized by TLR7. In these effects, the nanoparticles are superior to the conventional transfection reagents lipofectamine, polyethylenimine, and DOTAP. In vivo, the delivery of TLR7-activating RNA oligonucleotides by gelatin nanoparticles triggers antigen-specific CD8+ T-cell and antibody responses. Indeed, immunization with RNA-loaded nanoparticles leads to an efficient antitumoral immune response in two different mouse tumor models. Thus, gelatin-based nanoparticles represent a novel delivery system for immunostimulatory RNA oligonucleotides that is both effective and nontoxic.

Efficient eradication of subcutaneous but not of autochthonous gastric tumors by adoptive T cell transfer in an SV40 T antigen mouse model.

In stomach cancer, there is a need for new therapeutic strategies, in particular for the treatment of unresectable tumors and micrometastases. We investigated the efficacy of immunotherapy in an autochthonous model of gastric cancer, the CEA424-SV40 T Ag (TAg) transgenic mice. Treatment efficacy against both the autochthonous tumors and s.c. tumors induced by the derived cell line mGC3 were assessed. In wild-type mice, a dendritic cell vaccine loaded with irradiated tumor cells combined with CpG oligonucleotides induced efficient cytotoxic T cell and memory responses against mGC3 s.c. tumors. In contrast, neither s.c. nor autochthonous tumors responded to vaccination in CEA424-SV40 TAg mice, indicating tolerance to the SV40 TAg. To examine whether tumors in these mice were principally accessible to immunotherapy, splenocytes from immune wild-type mice were adoptively transferred into CEA424-SV40 TAg transgenic mice. Treated mice showed complete regression of the s.c. tumors associated with intratumoral infiltrates of CD8 and CD4 T cells. In contrast, the autochthonous gastric tumors in the same mice were poorly infiltrated and did not regress. Thus, even in the presence of an active anti-tumoral T cell response, autochthonous gastric tumors do not respond to immunotherapy. This is the first comparison of the efficacy of adoptive T cell transfer between transplanted s.c. tumors and autochthonous tumors in the same animals. Our results suggest that in gastric cancer patients, even a strong anti-tumor T cell response will not efficiently penetrate the tumor in the absence of additional therapeutic strategies targeting the tumor microenvironment.

Immunostimulatory RNA oligonucleotides induce an effective antitumoral NK cell response through the TLR7.

RNA oligonucleotides containing immune-activating sequences promote the development of cytotoxic T cell and B cell responses to Ag. In this study, we show for the first time that immunostimulatory RNA oligonucleotides induce a NK cell response that prevents growth of NK-sensitive tumors. Treatment of mice with immunostimulatory RNA oligonucleotides activates NK cells in a sequence-dependent manner, leading to enhanced IFN-gamma production and increased cytotoxicity. Use of gene-deficient mice showed that NK activation is entirely TLR7-dependent. We further demonstrate that NK activation is indirectly induced through IL-12 and type I IFN production by dendritic cells. Reconstitution of TLR7-deficient mice with wild-type dendritic cells restores NK activation upon treatment with immunostimulatory RNA oligonucleotides. Thus, by activating both NK cells and CTLs, RNA oligonucleotides stimulate two major cellular effectors of antitumor immunity. This dual activation may enhance the efficacy of immunotherapeutic strategies against cancer by preventing the development of tumor immune escape variants.

DNA vaccination efficiently induces antibodies to Nogo-A and does not exacerbate experimental autoimmune encephalomyelitis.

Antibodies against the neurite outgrowth inhibitor Nogo-A enhance axonal regeneration following spinal cord injury. However, antibodies directed against myelin components can also enhance CNS inflammation. The present study was designed to assess the efficacy of DNA vaccination for generating antibodies against Nogo-A and to study their pathogenic potential in a mouse model for multiple sclerosis. Mice were immunized by a single i.m. injection of a plasmid expression vector encoding either full length membrane-integral Nogo-A equipped with a signal peptide or two versions of its large N-terminal extramembrane region. The presence of serum antibodies to Nogo-A was measured 4 weeks after injection by ELISA, Western blotting and immunohistochemistry. DNA vaccination efficiently induced production of Nogo-A-specific antibodies that recognized recombinant, intracellular Nogo-A in cell culture but also stained native Nogo-A on the oligodendrocyte surface. Experimental autoimmune encephalomyelitis was induced in DNA-vaccinated mice by immunization with proteolipid peptide (a.a. 139-154). In contrast to vaccination with DNA encoding myelin oligodendrocyte glycoprotein that exacerbates this disease, Nogo-A DNA vaccination did not enhance clinical severity of disease. In summary, DNA vaccination is a simple and efficient method for generating an antibody response to Nogo-A. No pathogenicity was observed even during a full-blown inflammatory response of the central nervous system.

Novel mouse mutants with primary cellular immunodeficiencies generated by genome-wide mutagenesis.

BACKGROUND: Primary cellular immunodeficiencies are a group of genetic disorders in which 1 or more components of the cellular immune system are lacking or dysfunctional. OBJECTIVE: We sought to identify novel mouse mutants that display primary cellular immunodeficiencies. METHODS: Genome-wide N-ethyl-N-nitrosourea mutagenesis was performed in mice, followed by a phenotype screen of immunologic blood parameters. RESULTS: We identified novel mouse mutants with isolated B-cell deficiency, combined block in early B- and T-cell development, combined T-cell and natural killer cell reduction, and 3 different forms of T-cell deficiencies. One of the mutants, designated DeltaT3, displayed a combined phenotype of increased IgE, absence of peripheral T cells, and block in late thymocyte differentiation. In addition, DeltaT3 mice were unable to mount specific humoral immune responses. Chromosomal mapping and sequencing of candidate genes revealed a novel point mutation in the kinase domain of the T-cell receptor zeta chain-associated protein kinase (Zap70). In contrast to Zap70-deficient mice, DeltaT3 mutants displayed normal Zap70 mRNA and residual Zap70 protein levels. Complementation studies with Zap70-deficient mice confirmed that the point mutation found in Zap70 was causative for the DeltaT3 phenotype, including increased IgE plasma levels, a phenotype that has not been associated with altered Zap70 function in the past. CONCLUSION: Random genome-wide mutagenesis combined with a phenotype screen can be used to generate novel mouse mutants with primary cellular immunodeficiencies.

Immunostimulatory RNA oligonucleotides trigger an antigen-specific cytotoxic T-cell and IgG2a response.

Single-stranded RNA oligonucleotides containing an immunostimulatory motif (immunostimulatory RNA [isRNA]) are potent inducers of interferon-alpha via the Toll-like receptor 7. We investigated the effect of isRNA on the development of an immune response. We show that isRNA activates dendritic cells and induces production of Th1-type cytokines both in vitro and in vivo. Cytokine production led to bystander activation of T and B cells. We further demonstrate that isRNA triggers the generation of antigen-specific cytotoxic T cells and of an IgG2a-biased antibody response to antigen in a sequence-dependent manner. In summary, we provide evidence for the first time that isRNA oligonucleotides can simultaneously activate the innate and adaptive arms of the immune system.