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.

Height is a complex human phenotype that is influenced by variations in a high number of genes. Recently, a single nucleotide polymorphism (SNP) within IL11 (rs4252548) has been described to be associated with height in adults of European ancestry. This coding SNP leads to the exchange of Arg-112 to His-112 within the cytokine Interleukin-11 (IL-11), which has a well-established role in osteoclast development and bone turnover. The functional consequences of the R112H mutation are unknown so far. In this study, we show by molecular replacement that Arg-112 does not participate in binding of IL-11 to its receptors IL-11R and glycoprotein 130 (gp130). Recombinant IL-11 R112H expressed in E. coli displays a correct four-helix-bundle folding topology, and binds with similar affinity to IL-11R and the IL-11/IL-11R/gp130 complex. IL-11 R112H induces cell proliferation and phosphorylation of the downstream transcription factor STAT3 indistinguishable from IL-11. However, IL-11 R112H fails to support the survival of osteoclast progenitor cells and is less thermally stable, which is caused by the loss of the positive charge on the protein surface since protonation of the histidine side chain recovers stability.

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Recent studies highlight immunoregulatory functions of type I interferons (IFN-I) during the pathogenesis of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that selective activation of IFN-I pathways including RIG-I/MAVS and cGAS/STING prior to allo-HSCT conditioning therapy can ameliorate the course of GVHD. However, direct effects of IFN-Is on immune cells remain ill characterized. We applied RIG-I agonists (3pRNA) to stimulate IFN-I production in murine models of conditioning therapy with total body irradiation (TBI) and GVHD. Using IFN-I receptor-deficient donor T cells and hematopoietic cells, we found that endogenous and RIG-I-induced IFN-Is do not reduce GVHD by acting on these cell types. However, 3pRNA applied before conditioning therapy reduced the ability of CD11c+ recipient cells to stimulate proliferation and interferon gamma expression of allogeneic T cells. Consistently, RIG-I activation before TBI reduced the proliferation of transplanted allogeneic T-cells. The reduced allogenicity of CD11c+ recipient cells was dependent on IFN-I signaling. Notably, this immunosuppressive function of DCs was restricted to a scenario where tissue damage occurs. Our findings uncover a context (damage by TBI) and IFN-I dependent modulation of T cells by DCs and extend the understanding about the cellular targets of IFN-I during allo-HSCT and GVHD.

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Background: Recent studies highlight immunoregulatory functions of type I interferons (IFN-I) during the pathogenesis of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that selective activation of IFN-I pathways including RIG-I/MAVS and cGAS/STING prior to allo-HSCT conditioning therapy can ameliorate the course of GVHD. However, direct effects of IFN-Is on immune cells remain ill characterised. Methods: We applied selective RIG-I agonists (3pRNA) to stimulate IFN-I production in murine models of conditioning therapy with total body irradiation (TBI) and GVHD. Results: Using IFNAR1-deficient donor T and hematopoietic donor cells, we found that endogenous and RIG-I-induced IFN-Is do not reduce GVHD by acting on these respective cell types. However, 3pRNA applied before conditioning therapy reduced the ability of CD11c+ recipient cells to stimulate proliferation and interferon gamma expression of allogeneic T cells. Consistently, RIG-I activation before TBI reduced the proliferation of transplanted T-cells after allo-HSCT. The reduced allogenicity of CD11c+ recipient cells was dependent on IFN-I signalling. Notably, this immunosuppressive function of DCs was restricted to a scenario of genotoxic tissue damage as neither RIG-I activation and IFN-I induction in naive (non-irradiated) mice altered allogeneic T cell activation. Conclusion: Our findings uncover a hitherto unknown IFN-I- and context dependent immunosuppressive function of dendritic cells. This needs to be considered in the development of IFN-I based therapeutic approaches to modulate donor T cell activation after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

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.

Background: Graft-versus-host disease (GVHD) is a dreaded complication after stem-cell transplantation (SCT). Standard treatment relies on immunosuppressants but is associated with an increased risk of infection and relapse. Up 50% of patients develop steroid-refractory GVHD, with dismal impact on SCT outcomes. New studies in mice suggest that induction of type-I interferon (IFN-I) signalling or activation of IFN-I inducing pathways such as cGAS/STING or RIG-I/MAVS can promote gut barrier integrity and limit GVHD. However, the endogenous ligands that drive this "protective" IFN-I response are poorly defined. Recent data in mice and humans suggest that microbial-derived metabolites such as small-chain fatty acids or indoles can decrease GHVD mortality. Here, we describe IFN-I inducing metabolites that improve outcomes in mouse models of gut epithelial damage and acute GVHD via a mechanism dependent on STING signalling. Methods: To investigate which cell types mediate protection and how, we generated intestinal organoids and antigen presenting cells (APC) from bone marrow of WT or genetically deficient mice (MAVS-/-, STING-/-, IFNAR-/-) under steady state conditions versus chemotherapy, total body irradiation and after allogeneic SCT in the presence or absence of microbial metabolites. Analysis was performed by microscopy, immunoblotting, qPCR, ELISA and flow cytometry. Outcomes of gut-injured mice were assessed by clinical scoring, histopathology, flow cytometry and organoid recovery. Results: Metabolite treatment promoted regeneration of intestinal organoids as assayed by organoid numbers as well as proliferation. These effects were dependent on IFN-I and STING signalling. In addition, we found activated pro-inflammatory NFkB signalling and decreased apoptosis as evidenced by reduced caspase-3 cleavage. In APCs, IFN-I responses were enhanced in the presence of metabolites including increased IFN-β production and upregulation of IFN stimulated genes. Metabolite-treated mice showed improved recovery of intestinal stem cells following gut injury by chemotherapy and irradiation and increased survival in acute GVHD. Conclusions: Our findings uncover a mechanism by which microbial metabolites amplify IFN-I signals, promote tissue regeneration and thereby prevent allo-activation and GVHD. We show for the first time that these IFN-I inducing metabolites are protective in diverse models of gut damage -- from chemotherapy and radiation to immune-mediated damage -- via a STING-dependent mechanism. Perhaps the poor prognosis of GVHD patients exhibiting a loss of microbiota diversity can be explained in part by an absence of "protective" metabolites able to amplify critical IFN signals. We are currently studying whether metabolite levels correlate with severity and outcome of GVHD in humans. Figure Disclosures No relevant conflicts of interest to declare.