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.

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.

Achieving durable clinical responses to immune checkpoint inhibitors still remains a challenge. Here we demonstrate in preclinical models 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 (Fig. 1A). 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 tumor tissue. Consistently, therapeutic targeting of RIG-I with 5'-triphosphorylated-RNA in both tumor and non-malignant host cells potently augmented the efficacy of CTLA-4 checkpoint blockade in several tumor 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 (Fig. 1B). Moreover, in melanoma patients treated with anti-CTLA-4 checkpoint blockade, high RIG-I transcriptional activity significantly associated with durable clinical responses (Fig. 1C). Our preclinical data further demonstrate that tumor cell-intrinsic RIG-I signaling is also an essential pathway for the efficacy of other immunomodulating anticancer treatments including radiotherapy or hypomethylating agents such as 5-azacytidine. We thus identify aberrant tumor cell-intrinsic RIG-I signaling to be a crucial mechanism underlying cancer resistance to checkpoint inhibitor-based and other immunotherapies. These data have immediate translational potential as a RIG-I agonist for human application has been tested in phase I/II clinical trials with local administration in solid tumors and lymphomas (NCT03065023). Intratumoral RIG-I gene expression may not only serve as a biomarker to select patients that will likely benefit from anti-CTLA-4 therapy, but clinical RIG-I targeting in patients may also increase overall response rates of checkpoint inhibitor-based immunotherapy of malignancy including lymphoma. Figure 1. (A) Wild-type (WT) mice were bilaterally challenged with either WT or RIG-I-deficient (RIG-I-/-) B16.OVA melanoma cells.Recipients were repeatedly treated with anti-CTLA-4. Some mice were additionally injected with the RIG-I ligand 3pRNA into the right-sided tumor. Overall survival of treated mice bearing WT or RIG-I-/- B16.OVA tumors. (B) Overall survival in 456 TCGA melanoma patients by expression of DDX58 (RIG-I) in tumor samples. (C)DDX58 (RIG-I) expression in tumor samples from 18 patients with durable clinical response to anti-CTLA-4 treatment versus non-responders. Date give values from individual patients + geometric mean. Figure 1 Disclosures van den Brink: Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Flagship Ventures: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Evelo: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria; Therakos: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Merck & Co, Inc.: Consultancy, Honoraria; Acute Leukemia Forum (ALF): Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Other: Licensing.