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Immunogenic cancer therapies, including radiation and hypomethylating agents, such as 5-azacytidine, rely on tumor cell-intrinsic activation of the RNA receptor RIG-I for their synergism with immune checkpoint inhibitors. Possible RIG-I ligands are small nuclear RNA (snRNA) and endogenous retroviral elements (ERV) leaking from the nucleus during programmed cell death.

Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Such inflammatory diseases are fostered by damage to the intestinal barrier after transplantation. Consequently, the integrity and regeneration of the intestinal barrier is a key factor in the prevention of GVHD. On one side the main driver for regeneration of damaged gut epithelium are intestinal stem cells (ISC), but on the other side these cells are themselves primary targets of donor-derived T cells. One known mechanism of T cell mediated damage to the stem cell compartment is through IFN-γ dependent ISC toxicity. Yet, little is known about how T cells are contributing to the regeneration of damaged tissue after allo-HSCT and GVHD. Methods: To address this, we used preclinical models for allo-HSCT and GVHD including transplantation of recipient mice with escalating doses of Wildtype or IFN-γ-deficient allogeneic T cells and in the presence or absence of the JAK-1/2 inhibitor ruxolitinib. Intestinal regeneration was assessed by RNA-seq, flow cytometry and a newly established ex vivo organoid recovery assay. GVHD outcome was assessed by clinical scoring, histology and survival. Additionally, we established an allogeneic co-culture system of murine or human intestinal organoids with CD4+ conventional T cells or T regs -/+ Ruxolitinib. Effects on organoid growth and cell death were assessed by size measurements and manual counting after passaging. Results: We here demonstrate that recipient mice with increasingly dense intestinal infiltration by allogeneic T cells not only developed more severe GVHD (Fig. 1A), but also showed augmented recovery potential early after allo-HSCT (Fig. 1B). This was associated with intestinal gene signatures related to epithelial regeneration and protection from GVHD. Utilizing ex vivo cultures of intestinal organoids generated from murine allo-HSCT recipients, we found that development of GVHD but also regenerative capacity of ISCs were dependent on interferon (IFN)-γ-producing T cells in the intestine (Fig. 2A-B). Mice with fulminant GVHD and enhanced organoid recovery showed accumulation of intestinal regulatory T cells (Tregs) (Fig. 2C). Ex vivo, T regs nurtured growth of intestinal organoids in an IFN-γ dependent manner (Fig. 2D-E). This effect was diminished in intestinal organoids lacking IFNγR signaling, but was independent of T reg intrinsic IFNγR signaling (Fig. 2E-F). Intriguingly, treatment of murine allo-HSCT recipients with the JAK-1/2 inhibitor ruxolitinib enhanced epithelial organoid regeneration and numbers of intestinal Tregs (Fig. 3A-B). Similarily, growth of human intestinal organoids co-cultured with allogeneic T cells could be augmented by ruxolitinib treatment (Fig. 3C). We thus propose that the level and differentiation of infiltrating intestinal T cells determines both ISC damage and epithelial regeneration during immune-mediated tissue injury, leading to a sensitive equilibrium that can be modulated by therapeutic intervention. We also provide evidence that ruxolitinib improves ISC regeneration via IFNγ-producing Treg cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Maintaining immune tolerance requires the production of Foxp3-expressing regulatory T (Treg) cells in the thymus. Activation of NF-κB transcription factors is critically required for Treg cell development, partly via initiating Foxp3 expression. NF-κB activation is controlled by a negative feedback regulation through the ubiquitin editing enzyme A20, which reduces proinflammatory signaling in myeloid cells and B cells. In naive CD4+ T cells, A20 prevents kinase RIPK3-dependent necroptosis. Using mice deficient for A20 in T lineage cells, we show that thymic and peripheral Treg cell compartments are quantitatively enlarged because of a cell-intrinsic developmental advantage of A20-deficient thymic Treg differentiation. A20-deficient thymic Treg cells exhibit reduced dependence on IL-2 but unchanged rates of proliferation and apoptosis. Activation of the NF-κB transcription factor RelA was enhanced, whereas nuclear translocation of c-Rel was decreased in A20-deficient thymic Treg cells. Furthermore, we found that the increase in Treg cells in T cell–specific A20-deficient mice was already observed in CD4+ single-positive CD25+ GITR+ Foxp3− thymic Treg cell progenitors. Treg cell precursors expressed high levels of the tumor necrosis factor receptor superfamily molecule GITR, whose stimulation is closely linked to thymic Treg cell development. A20-deficient Treg cells efficiently suppressed effector T cell–mediated graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, suggesting normal suppressive function. Holding thymic production of natural Treg cells in check, A20 thus integrates Treg cell activity and increased effector T cell survival into an efficient CD4+ T cell response.

Caspases have important functions beyond their established role in driving inflammation and apoptosis. In this issue of Immunity, Wang et al. (2017) demonstrate that inflammasome-triggered caspases cleave and inactivate the DNA sensor cGAS, thus restricting the type I interferon response to cytosolic DNA.

Innate immune recognition of RNA is key for the initiation of immunity in response to viral infection. Although the factors controlling the detection of viral RNA by innate immune receptors in host cells are increasingly well understood, little is known about the dynamic changes in signaling after the initial triggering of these receptors. In this study, we report that preconditioning with the synthetic dsRNA polyinosinic-polycytidylic acid [poly(I:C)], a mimetic of viral RNA, rapidly reprograms murine APCs by simultaneously augmenting sensitivity of endosomal TLRs and inhibiting activation of RIG-I–like receptors (RLRs) in an IFN-β–dependent manner. These changes in receptor sensitivity were also seen in vivo after treatment of mice with poly(I:C). Mechanistically, the increased sensitivity of the TLR pathway was associated with elevated MAPK and NF-κB activity. The RLR response was inhibited downstream of TANK-binding kinase-1, resulting in decreased IFN regulatory factor 3 phosphorylation. Reprogramming of pattern-recognition receptor signaling also occurred after viral infection, because infection of host cells with Sendai virus or their exposure to supernatant from virus-infected cells induced the same changes in TLR and RLR sensitivity as poly(I:C). Thus, innate recognition of viral infection critically modifies responses to pattern-recognition receptor stimulation. These dynamic adaptations to infection may reinforce antiviral immunity and at the same time serve to limit pathological inflammation.

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 g (RegIIIg). 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. 2017

Activation of the C-type lectin receptor Dectin-1 by β-glucans triggers multiple signals within DCs that result in activation of innate immunity. While these mechanisms can potently prime CD8+ cytotoxic T-cell (CTL) responses without additional adjuvants, the Dectin-1 effector pathways that control CTL induction remain unclear. Here we demonstrate that Dectin-1-induced CTL cross-priming in mice does not require inflammasome activation but strictly depends on the adapter protein Card9 in vitro. In vivo, Dectin-1-mediated Card9 activation after vaccination drives both expansion and activation of Ag-specific CTLs, resulting in long-lasting CTL responses that are sufficient to protect mice from tumor challenge. This Dectin-1-induced antitumor immune response was independent of NK cell function and completely abrogated in Card9-deficient mice. Thus, our results demonstrate that Dectin-1-triggered Card9 signaling but not inflammasome activation can potently cross-prime Ag-specific CTLs, suggesting that this pathway would be a candidate for immunotherapy and vaccine development.

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 α4β7-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.

Introduction: In lung cancer, interleukin-22 (IL-22) expression within primary tissue has been demonstrated, but the frequency and the functional consequence of IL-22 signaling have not been addressed. This study aims at analyzing the cellular effects of IL-22 on lung carcinoma cell lines and the prognostic impact of IL-22 tis- sue expression in lung cancer patients. Methods: Biological effects of IL-22 signaling were investigated in seven lung cancer cell lines by Western blot, flow cytometry, real- time polymerase chain reaction, and proliferation assays. Tumor tissue specimens of two cohorts with a total of 2300 lung cancer patients were tested for IL-22 expression by immunohistochemistry. IL-22 serum concentrations were analyzed in 103 additional patients by enzyme-linked immunosorbent assay. Results: We found the IL-22 receptor 1 (IL-22-R1) to be expressed in six of seven lung cancer cell lines. However IL-22 signaling was functional in only four cell lines, where IL-22 induced signal trans- ducer activator of transcription 3 phosphorylation and increased cell proliferation. Furthermore, IL-22 induced the expression of anti- apoptotic B-cell lymphoma 2, but did not rescue tumor cells from carboplatin-induced apoptosis. Cisplatin-resistant cell lines showed a significant up-regulation of IL-22-R1 along with a stronger prolifera- tive response to IL-22 stimulation. IL-22 was preferentially expressed in small- and large-cell lung carcinoma (58% and 46% of cases, respectively). However, no correlation between IL-22 expression by immunohistochemistry and prognosis was observed. Conclusion: 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.

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 α4β7 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 α4β7 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.

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. Cancer Res; 71(15); 5123–33. ©2011 AACR.

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.

Strong inter-individual variation in clinical response to checkpoint inhibitors such as anti-CTLA-4 remains a major challenge. In a retrospective analysis, expression of viral defense genes in human melanomas including the cytosolic RNA receptor RIG-I (DDx58) has been associated with clinical benefit to CTLA-4 blockade. However, possible interconnections of these two distinct pathways remain unknown. Using CRISPR/Cas9 technology to generate B16 melanoma cells lines that lack nucleic acid receptors or downstream signaling molecules (RIG-I, STING, IRF3/7) together with available genetically deficient mouse models, we addressed the importance of nucleic acid receptor signaling in both tumor and host cells for the efficacy of anti-CTLA-4 immunotherapy. We here provide experimental proof that anti-CTLA-4 immunotherapy relies on tumor cell-intrinsic RIG-I but not STING signaling. Following anti-CTLA-4 treatment, tumor-intrinsic RIG-I signaling critically impacts on cross-presentation of tumor-associated antigen by CD103+ dendritic cells, the expansion of tumor antigen-specific CD8+ T cells and ultimately the accumulation of CD8+ T cells within tumor tissue. Consistently, therapeutic targeting of RIG-I with 5'-phosphorylated-RNA in both tumor and non-malignant cells potently augmented the efficacy of CTLA-4 checkpoint blockade. These processes were additionally dependent on host STING, MAVS and type I interferon signaling and were closely linked to RIG-I-mediated tumor cell death. In summary, our study identifies both tumor- and host-intrinsic RIG-I signaling as fundamental requirements for anti-CTLA-4-mediated antitumor immunity. Our data predict that targeting RIG-I may serve as a basis for the development of new combination strategies to increase the response rate of checkpoint inhibitor-based immunotherapy of malignancy including lymphoma, particularly in individuals that do not have a sufficient spontaneous antitumor T-cell immune response. Disclosures van den Brink: Jazz Pharmaceuticals: Consultancy; Seres: Research Funding; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.

Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

Introduction: Targeting inhibitory T-cell receptors such as CTLA-4 has shown great promise in cancer therapy. However, strong inter-individual variation in clinical response to checkpoint inhibitors remains a major challenge. Expression of viral defense genes in human melanomas including the cytosolic RNA receptor RIG-I (DDx58) has recently been associated with clinical benefit to CTLA-4 blockade. Methods: Using CRISPR/Cas9 technology to generate B16 melanoma cells lines that lack nucleic acid receptors or downstream signaling molecules (RIG-I, STING, IRF3/7) together with available genetically deficient mouse models, we address the importance of nucleic acid receptor signaling for the efficacy of anti-CTLA-4 immunotherapy. Results: We provide experimental proof that anti-CTLA-4 immunotherapy indeed relies on tumor cell-intrinsic RIG-I/MAVS but not cGAS/STING signaling. Consistently, therapeutic targeting of RIG-I with the specific ligand 5'‑triphosphate-RNA (3pRNA) potently augments the efficacy of CTLA-4 checkpoint blockade. In situ vaccination by intratumoral 3pRNA injection enhanced cross-presentation of tumor-associated antigens by CD103+ migratory DCs in local draining lymph nodes. Subsequent expansion of tumor-specific CD8+ T cells resulted in control of local and distant tumors, otherwise resistant to anti-CTLA‑4 monotherapy. These processes were critically dependent on tumor cell-intrinsic RIG-I-mediated immunogenic cell death (ICD), but not tumor-derived type I IFN release. In such, the synergistic therapeutic effect of RIG-I activation and anti-CTLA-4 was restricted to tumor models that showed susceptibility to RIG‑I-mediated ICD. Furthermore, systemic antitumor immunity following anti-CTLA-4 +/- 3pRNA treatment required RIG-I signaling in both tumor and non-malignant host cells. Conclusion: Our study identifies a hitherto unrecognized role of RIG-I signaling in tumors and their microenvironment as a crucial component for checkpoint inhibitor-mediated immunotherapy of cancer. Therapeutic targeting of RIG-I using specific agonists strongly augments the efficacy of anti-CTLA-4 blockade and may thus serve as the basis for new combinatorial approaches. Citation Format: Simon Heidegger, Alexander Wintges, Diana Kreppel, Sarah Bek, Michael Bscheider, Martina Schmickl, Marcel R.M. van den Brink, Christian Peschel, Tobias Haas, Hendrik Poeck. Tumor- and host-intrinsic RIG-I signaling promote anticancer immunity by CTLA-4 blockade [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A005.

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.

Antibody-mediated targeting of regulatory T cell receptors such as CTLA-4 has been shown to enhance anti-tumor immune responses against several cancer entities including malignant melanoma. Yet, therapeutic success in patients remains variable underscoring the need for novel combinatorial approaches. Here we established a vaccination protocol that combines selective engagement of the nucleic acid-sensing pattern recognition receptor RIG-I, antigen and CTLA-4-blockade. We found that vaccination together with RIG-I ligation strongly synergized with CTLA-4 blockade to induce expansion and activation of antigen-specific CD8+ T cells and potent anti-tumor immunity. Cross-priming of cytotoxic T cells as well as anti-tumor immunity required the adapter protein MAVS and type I interferon (IFN) signaling and were mediated by dendritic cells. In addition, the benefit of the combined immunization with anti-CTLA-4 was reduced by systemic antibiotics pointing to the requisite of an intact commensal microbiota in this context. Together, our findings describe a novel combinatorial strategy that may form the basis for the design of new type I IFN-based regimens that enhance antigen-specific T cell reactivity against cancer. Disclosures No relevant conflicts of interest to declare.

Introduction: Although the role of Type I IFNs in initiating host defense against pathogens is well established, recent work highlights the regenerative function of this cytokine family. Yet, despite its involvement in tissue repair, the cellular targets and mechanisms of action by which Type I IFNs act during tissue regeneration are poorly understood. Results: Here, we describe a hitherto unrecognized regenerative function for the RIG-I/MAVS/IFN-I pathway through direct effects on epithelial regeneration. Mice deficient in the RIG-I adaptor MAVS were more sensitive to intestinal barrier damage after total body irradiation (TBI) and, like RIG-I deficient mice, developed worse graft-versus-host disease (GVHD) in a preclinical model for allogeneic hematopoietic stem cell transplantation (allo-HSCT). This phenotype was not associated with changes in the intestinal microbiota, but with a defect in epithelial regeneration. Moreover, in contrast to previous reports in steady-state conditions and after viral challenge, we found that interferon-α/β receptor (IFNAR) signaling in non-hematopoietic epithelial cells was crucial for tissue regeneration after acute damage. Importantly, we could demonstrate that this pathway could be therapeutically targeted with RIG-I agonists, which promoted barrier integrity and prevented GVHD. Mechanistically, Type I IFNs (either RIG-I-induced or recombinant) could promote intestinal stem cell (ISC) growth in crypt organoid cultures, suggesting that stimulation of the ISC compartment led to epithelial regeneration. Conclusion: Our findings suggest that activation of RIG-I and IFN-I can promote regeneration of intestinal epithelial cells and thus offers an innovative therapeutic strategy for the management of acute intestinal injury. Disclosures van den Brink: Merck: Honoraria; Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Regeneron: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tobira Therapeutics: Other: Advisory board attendee.