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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.

The NF-κB regulator A20 limits inflammation by providing negative feedback in myeloid cells and B cells. Functional lack of A20 has been linked to several inflammatory and autoimmune diseases. To define how A20 affects the functionality of T effector cells in a highly inflammatory environment, we performed conventional allogeneic hematopoietic stem cell transplantation (allo-HSCT) with A20-deficient CD4+ and CD8+ donor T cells in mice. Severity and mortality of graft-versus-host disease (GVHD) after allo-HSCT was drastically reduced in recipients transplanted with conventional doses of A20-deficient T cells. Consistently, we found that the A20-deficient donor T cell compartment was strongly diminished at various timepoints after allo-HSCT. However, proportionally more A20-deficient donor T cells produced IFN-γ and systemic inflammation was elevated early after allo-HSCT. Consequently, increasing the dose of transplanted A20-deficient T cells reversed the original phenotype and resulted in enhanced GVHD mortality compared to recipients that received A20+/+ T cells. Still, A20-deficient T cells, activated either through T cell receptor-dependent or -independent mechanisms, were less viable than control A20+/+ T cells, highlighting that A20 balances both, T cell activation and survival. Thus, our findings suggest that targeting A20 in T cells may allow to modulate T cell mediated inflammatory diseases like GVHD. This article is protected by copyright. All rights reserved

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.

Graft-versus-host-disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation (allo-HCT) characterized by the production of high levels of proinflammatory cytokines. Activated Janus kinases (JAKs) are required for T-effector cell responses in different inflammatory diseases, and their blockade could potently reduce acute GVHD. We observed that inhibition of JAK1/2 signaling resulted in reduced proliferation of effector T cells and suppression of proinflammatory cytokine production in response to alloantigen in mice. In vivo JAK 1/2 inhibition improved survival of mice developing acute GVHD and reduced histopathological GVHD grading, serum levels of proinflammatory cytokines, and expansion of alloreactive luc-transgenic T cells. Mechanistically, we could show that ruxolitinib impaired differentiation of CD4(+) T cells into IFN-γ- and IL17A-producing cells, and that both T-cell phenotypes are linked to GVHD. Conversely, ruxolitinib treatment in allo-HCT recipients increased FoxP3(+) regulatory T cells, which are linked to immunologic tolerance. Based on these results, we treated 6 patients with steroid-refractory GVHD with ruxolitinib. All patients responded with respect to clinical GVHD symptoms and serum levels of proinflammatory cytokines. In summary, ruxolitinib represents a novel targeted approach in GVHD by suppression of proinflammatory signaling that mediates tissue damage and by promotion of tolerogenic Treg cells.

3081Background: Strong inter-individual variation in clinical response to immune checkpoint inhibitors (ICB) including anti-CTLA-4 remains a major challenge, but the molecular pathways that modulat...

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.

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.

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.

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.

Graft-versus-host-disease (GvHD) constitutes a severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In GvHD, tissue damage is mediated by pro-inflammatory cytokines. Cytokine responses are mediated by activated Janus kinases (JAKs). We hypothesized that JAK1/2 inhibition might reduce T effector cell responses and inflammatory cytokine production in an allogeneic system, thereby ameliorating GvHD. We established an allogeneic (major mismatch) cell culture system using naive BALB/c CD4+ CD62Lhigh T cells that were co-cultured with C57BL/6 (B6) bone marrow derived dendritic cells (DC). JAK 1/2 signaling was specifically blocked using the small molecule inhibitor Ruxolitinib (INCB018424). By using a 3H Thymidine proliferation assay, we found that Ruxolitinib was able to inhibit the proliferation of allogeneic CD4+ effector T cells. In our intracellular cytokine staining experiments Ruxolitinib was able to impair the differentiation of naïve T cells into IFN-gamma and IL17A-producing T effector cells. Both cytokines – IFN-gamma and IL-17A – have been linked to aggravated courses of GvHD severity. In vivo administration of Ruxolitinib signficantly reduced lethal GvHD after allo-HSCT in a murine major mismatch model. BALB/c recipient mice were irradiated and received C57BL/6 (B6) T cell depleted bone marrow along with CD4+ and CD8+ T cells. Animals that were treated with an oral gavage of Ruxolitinib twice daily displayed significantly lower serum levels of IFN-gamma, TNF-alpha, IL-10 and IL-12p70, lower histological and clinical GvHD scores and improved overall survival compared to the control group. By using luciferase-positive (luc+) CD4+ and CD8+ T cells, we were able to visualize and quantify T cell migration after GvHD induction. The recipient mice received luc+ T cells along with conventional T cell depleted bone marrow. We were able to detect the luc+ T cell signal with a bioluminescence imaging system. At day 9 after allogeneic bone marrow transplantation, migration of donor T cells to GvHD target sites was significantly reduced in Ruxolitinib treated mice compared to control mice. In summary, our data demonstrate that suppression of inflammatory cytokine responses by JAK1/2 inhibitors modulates T effector cell responses in an allogeneic setting and improves survival in a murine major mismatch GvHD model. These observations suggest that JAK1/2 inhibition might be used for the treatment of GvHD following allo-HSCT. Disclosures: No relevant conflicts of interest to declare.