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.

The molecular pathways that regulate the tissue repair function of type I interferon (IFN-I) during acute tissue damage are poorly understood. We describe a protective role for IFN-I and the RIG-I/MAVS signaling pathway during acute tissue damage in mice. Mice lacking mitochondrial antiviral-signaling protein (MAVS) were more sensitive to total body irradiation– and chemotherapy-induced intestinal barrier damage. These mice developed worse graft-versus-host disease (GVHD) in a preclinical model of allogeneic hematopoietic stem cell transplantation (allo-HSCT) than did wild-type mice. This phenotype was not associated with changes in the intestinal microbiota but was associated with reduced gut epithelial integrity. Conversely, targeted activation of the RIG-I pathway during tissue injury promoted gut barrier integrity and reduced GVHD. Recombinant IFN-I or IFN-I expression induced by RIG-I promoted growth of intestinal organoids in vitro and production of the antimicrobial peptide regenerating islet–derived protein 3 γ (RegIIIγ). Our findings were not confined to RIG-I/MAVS signaling because targeted engagement of the STING (stimulator of interferon genes) pathway also protected gut barrier function and reduced GVHD. Consistent with this, STING-deficient mice suffered worse GVHD after allo-HSCT than did wild-type mice. Overall, our data suggest that activation of either RIG-I/MAVS or STING pathways during acute intestinal tissue injury in mice resulted in IFN-I signaling that maintained gut epithelial barrier integrity and reduced GVHD severity. Targeting these pathways may help to prevent acute intestinal injury and GVHD during allogeneic transplantation.

Achieving durable clinical responses to immune checkpoint inhibitors remains a challenge. Here, we demonstrate that immunotherapy with anti–CTLA-4 and its combination with anti–PD-1 rely on tumor cell–intrinsic activation of the cytosolic RNA receptor RIG-I. Mechanistically, tumor cell–intrinsic RIG-I signaling induced caspase-3–mediated tumor cell death, cross-presentation of tumor-associated antigen by CD103+ dendritic cells, subsequent expansion of tumor antigen–specific CD8+ T cells, and their accumulation within the tumor tissue. Consistently, therapeutic targeting of RIG-I with 5′– triphosphorylated RNA in both tumor and nonmalignant host cells potently augmented the efficacy of CTLA-4 checkpoint blockade in several preclinical cancer models. In humans, transcriptome analysis of primary melanoma samples revealed a strong association between high expression of DDX58 (the gene encoding RIG-I), T cell receptor and antigen presentation pathway activity, and prolonged overall survival. Moreover, in patients with melanoma treated with anti–CTLA-4 checkpoint blockade, high DDX58 RIG-I transcriptional activity significantly associated with durable clinical responses. Our data thus identify activation of RIG-I signaling in tumors and their microenvironment as a crucial component for checkpoint inhibitor–mediated immunotherapy of cancer.

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-a 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. Cancer Res; 71(15); 5123-33. Ó2011 AACR.

IL-22 is frequently expressed in lung cancer tissue. Enhanced IL-22-R1 expression and signaling in chemotherapy-refractory cell lines are indicative of a protumorigenic function of IL-22 and may contribute to a more aggressive phenotype.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only treatment with curative potential for certain aggressive hematopoietic malignancies. Its success is limited by acute graft-versus-host disease (GVHD), a life-threatening complication that occurs when allo-reactive donor T cells attack recipient organs. There is growing evidence that microbes and innate pattern-recognition receptors (PRRs) such as toll-like receptors (TLR) and nod-like receptors (NLR) are critically involved in the pathogenesis of acute GVHD. Currently, a widely accepted model postulates that intensive chemotherapy and/or total-body irradiation during pre-transplant conditioning results in tissue damage and a loss of epithelial barrier function. Subsequent translocation of bacterial components as well as release of endogenous danger molecules stimulate PRRs of host antigen-presenting cells to trigger the production of pro-inflammatory cytokines (cytokine storm) that modulate T cell allo-reactivity against host tissues, but eventually also the beneficial graft-versus-leukemia (GVL) effect. Given the limitations of existing immunosuppressive therapies, a better understanding of the molecular mechanisms that govern GVHD versus GVL is urgently needed. This may ultimately allow to design modulators, which protect from GvHD but preserve donor T-cell attack on hematologic malignancies. Here, we will briefly summarize current knowledge about the role of innate immunity in the pathogenesis of GVHD and GVL following allo-HSCT.

Multifunctional mesoporous silica nanoparticles (MSN) have attracted substantial attention with regard to their high potential for targeted drug delivery. For future clinical applications it is crucial to address safety concerns and understand the potential immunotoxicity of these nanoparticles. In this study, we assess the biocompatibility and functionality of multifunctional MSN in freshly isolated, primary murine immune cells. We show that the functionalized silica nanoparticles are rapidly and efficiently taken up into the endosomal compartment by specialized antigen-presenting cells such as dendritic cells. The silica nanoparticles showed a favorable toxicity profile and did not affect the viability of primary immune cells from the spleen in relevant concentrations. Cargo-free MSN induced only very low immune responses in primary cells as determined by surface expression of activation markers and release of pro-inflammatory cytokines such as Interleukin-6, -12 and -1β. In contrast, when surface-functionalized MSN with a pH-responsive polymer capping were loaded with an immune-activating drug, the synthetic Toll-like receptor 7 agonist R848, a strong immune response was provoked. We thus demonstrate that MSN represent an efficient drug delivery vehicle to primary immune cells that is both non-toxic and non-inflammagenic, which is a prerequisite for the use of these particles in biomedical applications.

Background Antibody-mediated targeting of regulatory T cell receptors such as CTLA-4 enhances antitumor immune responses against several cancer entities including malignant melanoma. Yet, therapeutic success in patients remains variable underscoring the need for novel combinatorial approaches. Methods Here we established a vaccination strategy that combines engagement of the nucleic acid-sensing pattern recognition receptor RIG-I, antigen and CTLA-4 blockade. We used in vitro transcribed 5′-triphosphorylated RNA (3pRNA) to therapeutically target the RIG-I pathway. We performed in vitro functional analysis in bone-marrow derived dendritic cells and investigated RIG-I-enhanced vaccines in different murine melanoma models. Findings We found that protein vaccination together with RIG-I ligation via 3pRNA strongly synergizes with CTLA-4 blockade to induce expansion and activation of antigen-specific CD8 + T cells that translates into potent antitumor immunity. RIG-I-induced cross-priming of cytotoxic T cells as well as antitumor immunity were dependent on the host adapter protein MAVS and type I interferon (IFN-I) signaling and were mediated by dendritic cells. Interpretation Overall, our data demonstrate the potency of a novel combinatorial vaccination strategy combining RIG-I-driven immunization with CTLA-4 blockade to prevent and treat experimental melanoma. Fund German Research Foundation (SFB 1335, SFB 1371), EMBO, Else Kröner-Fresenius-Foundation, German Cancer Aid, European Hematology Association, DKMS Foundation for Giving Life, Dres. Carl Maximilian and Carl Manfred Bayer-Foundation.