Bioengineered human colon organoids with in vivo-like cellular complexity and function.

Organoids and organs-on-a-chip have emerged as powerful tools for modeling human gut physiology and disease in vitro. Although physiologically relevant, these systems often lack the environmental milieu, spatial organization, cell type diversity, and maturity necessary for mimicking human intestinal mucosa. To instead generate models closely resembling in vivo tissue, we herein integrated organoid and organ-on-a-chip technology to develop an advanced human organoid model, called "mini-colons." By employing an asymmetric stimulation with growth factors, we greatly enhanced tissue longevity and replicated in vivo-like diversity and patterning of proliferative and differentiated cell types. Mini-colons contain abundant mucus-producing goblet cells and, signifying mini-colon maturation, single-cell RNA sequencing reveals emerging mature and functional colonocytes. This methodology is expanded to generate microtissues from the small intestine and incorporate additional microenvironmental components. Finally, our bioengineered organoids provide a precise platform to systematically study human gut physiology and pathology, and a reliable preclinical model for drug safety assessment.

Profiling the heterogeneity of colorectal cancer consensus molecular subtypes using spatial transcriptomics.

The consensus molecular subtypes (CMS) of colorectal cancer (CRC) is the most widely-used gene expression-based classification and has contributed to a better understanding of disease heterogeneity and prognosis. Nevertheless, CMS intratumoral heterogeneity restricts its clinical application, stressing the necessity of further characterizing the composition and architecture of CRC. Here, we used Spatial Transcriptomics (ST) in combination with single-cell RNA sequencing (scRNA-seq) to decipher the spatially resolved cellular and molecular composition of CRC. In addition to mapping the intratumoral heterogeneity of CMS and their microenvironment, we identified cell communication events in the tumor-stroma interface of CMS2 carcinomas. This includes tumor growth-inhibiting as well as -activating signals, such as the potential regulation of the ETV4 transcriptional activity by DCN or the PLAU-PLAUR ligand-receptor interaction. Our study illustrates the potential of ST to resolve CRC molecular heterogeneity and thereby help advance personalized therapy.

Analysis of off-tumour toxicities of T-cell-engaging bispecific antibodies via donor-matched intestinal organoids and tumouroids.

Predicting the toxicity of cancer immunotherapies preclinically is challenging because models of tumours and healthy organs do not typically fully recapitulate the expression of relevant human antigens. Here we show that patient-derived intestinal organoids and tumouroids supplemented with immune cells can be used to study the on-target off-tumour toxicities of T-cell-engaging bispecific antibodies (TCBs), and to capture clinical toxicities not predicted by conventional tissue-based models as well as inter-patient variabilities in TCB responses. We analysed the mechanisms of T-cell-mediated damage of neoplastic and donor-matched healthy epithelia at a single-cell resolution using multiplexed immunofluorescence. We found that TCBs that target the epithelial cell-adhesion molecule led to apoptosis in healthy organoids in accordance with clinical observations, and that apoptosis is associated with T-cell activation, cytokine release and intra-epithelial T-cell infiltration. Conversely, tumour organoids were more resistant to damage, probably owing to a reduced efficiency of T-cell infiltration within the epithelium. Patient-derived intestinal organoids can aid the study of immune-epithelial interactions as well as the preclinical and clinical development of cancer immunotherapies.

Human immunocompetent Organ-on-Chip platforms allow safety profiling of tumor-targeted T-cell bispecific antibodies.

Traditional drug safety assessment often fails to predict complications in humans, especially when the drug targets the immune system. Here, we show the unprecedented capability of two human Organs-on-Chips to evaluate the safety profile of T-cell bispecific antibodies (TCBs) targeting tumor antigens. Although promising for cancer immunotherapy, TCBs are associated with an on-target, off-tumor risk due to low levels of expression of tumor antigens in healthy tissues. We leveraged in vivo target expression and toxicity data of TCBs targeting folate receptor 1 (FOLR1) or carcinoembryonic antigen (CEA) to design and validate human immunocompetent Organs-on-Chips safety platforms. We discovered that the Lung-Chip and Intestine-Chip could reproduce and predict target-dependent TCB safety liabilities, based on sensitivity to key determinants thereof, such as target expression and antibody affinity. These novel tools broaden the research options available for mechanistic understandings of engineered therapeutic antibodies and assessing safety in tissues susceptible to adverse events.

Neutrophilic infiltration in organ-on-a-chip model of tissue inflammation.

The multiphasic etiology of tissue inflammation and the fundamental immunological differences between species render inflammatory pathologies difficult to recapitulate in animal models, and account for the paucity of therapies that are successfully translated from rodents to humans. Here, we present a human-relevant organ-on-a-chip platform for experimental inflammatory diseases. We created an immunocompetent in vitro gut model by incorporating intestinal epithelial and immune cells into microfluidic chambers that permit cell movement across an extracellular matrix (ECM) and fluidic channels. This is the first model that integrates a mucosal barrier, a three-dimensional ECM, resident and infiltrating immune cells, and simulates a functional crosstalk that ultimately triggers cellular processes representative of inflammation. Under homeostatic conditions, enterocytes form a tight epithelium and subepithelial macrophages are non-activated. Introduction of pro-inflammatory mediators triggers macrophage activation and inflammation-induced intestinal barrier leakiness. Neutrophils in a parallel, matrix-separated non-epithelial channel are attracted by such a pro-inflammatory microenvironment and migrate through the extracellular matrix, further exacerbating tissue inflammation and damage. With this model, we provide the foundations to recapitulate and investigate the onset of tissue inflammation in a controlled, human-relevant system.

Exploration of Cell Development Pathways through High-Dimensional Single Cell Analysis in Trajectory Space.

High-dimensional single cell profiling coupled with computational modeling is emerging as a powerful tool to elucidate developmental programs directing cell lineages. We introduce tSpace, an algorithm based on the concept of "trajectory space", in which cells are defined by their distance along nearest neighbor pathways to every other cell in a population. Graphical mapping of cells in trajectory space allows unsupervised reconstruction and exploration of complex developmental sequences. Applied to flow and mass cytometry data, the method faithfully reconstructs thymic T cell development and reveals development and trafficking regulation of tonsillar B cells. Applied to the single cell transcriptome of mouse intestine and C. elegans, the method recapitulates development from intestinal stem cells to specialized epithelial phenotypes more faithfully than existing algorithms and orders C. elegans cells concordantly to the associated embryonic time. tSpace profiling of complex populations is well suited for hypothesis generation in developing cell systems.

Type I interferon signaling before hematopoietic stem cell transplantation lowers donor T cell activation via reduced allogenicity of recipient cells.

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.

RIG-I activating immunostimulatory RNA boosts the efficacy of anticancer vaccines and synergizes with immune checkpoint blockade.

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.

A Mucosal and Cutaneous Chemokine Ligand for the Lymphocyte Chemoattractant Receptor GPR15.

Chemoattractants control lymphocyte recruitment from the blood, contributing to the systemic organization of the immune system. The G protein-linked receptor GPR15 mediates lymphocyte homing to the large intestines and skin. Here we show that the 9 kDa CC-motif containing cationic polypeptide AP57/colon-derived sushi containing domain-2 binding factor (CSBF), encoded by C10orf99 in the human and 2610528A11Rik in the mouse, functions as a chemokine ligand for GPR15 (GPR15L). GPR15L binds GPR15 and attracts GPR15-expressing T cells including lymphocytes in colon-draining lymph nodes and Vγ3+ thymic precursors of dermal epithelial T cells. Patterns of GPR15L expression by epithelial cells in adult mice and humans suggest a homeostatic role for the chemokine in lymphocyte localization to the large intestines, as well as a role in homing to the epidermis during wound healing or inflammation. GPR15L is also significantly expressed in squamous mucosa of the oral cavity and esophagus with still poorly defined regulation. Identification of the chemotactic activity of GPR15L adds to its reported antibacterial and tumor cell growth regulatory functions and suggests the potential of targeting GPR15L-GPR15 interactions for modulation of mucosal and cutaneous inflammation.

RIG-I/MAVS and STING signaling promote gut integrity during irradiation- and immune-mediated tissue injury.

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.

Card9 controls Dectin-1-induced T-cell cytotoxicity and tumor growth in mice.

Activation of the C-type lectin receptor Dectin-1 by ß-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.

Vitamin D immunoregulation through dendritic cells.

Vitamin D (VD3) has been linked to immunological processes, and its supplementation may have a role in treatment or prevention of diseases with underlying autoimmune or pro-inflammatory states. As initiators of the immune responses, dendritic cells (DC) are a potential target of VD3 to dampen autoimmunity and inflammation, but the role of DC in VD3-mediated immunomodulation in vivo is not understood. In addition to being targets of VD3, DC can provide a local source of bioactive VD3 for regulation of T-cell responses. Here we review existing studies that describe the tolerogenic potential of VD3 on DC, and discuss them in the context of current understanding of DC development and function. We speculate on mechanisms that might account for the potent but poorly understood tolerogenic activities of VD3 and the role of DC as both targets and sources of this hormone.

Activity of therapeutic JAK 1/2 blockade in graft-versus-host disease.

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.

The Nlrp3 inflammasome regulates acute graft-versus-host disease.

The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome-mediated IL-1ß production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1ß or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro-IL-1ß cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1ß originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1ß were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication.

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence.

Fungal infections represent a serious threat, particularly in immunocompromised patients. Interleukin-1beta (IL-1beta) is a key pro-inflammatory factor in innate antifungal immunity. The mechanism by which the mammalian immune system regulates IL-1beta production after fungal recognition is unclear. Two signals are generally required for IL-1beta production: an NF-kappaB-dependent signal that induces the synthesis of pro-IL-1beta (p35), and a second signal that triggers proteolytic pro-IL-1beta processing to produce bioactive IL-1beta (p17) via Caspase-1-containing multiprotein complexes called inflammasomes. Here we demonstrate that the tyrosine kinase Syk, operating downstream of several immunoreceptor tyrosine-based activation motif (ITAM)-coupled fungal pattern recognition receptors, controls both pro-IL-1beta synthesis and inflammasome activation after cell stimulation with Candida albicans. Whereas Syk signalling for pro-IL-1beta synthesis selectively uses the Card9 pathway, inflammasome activation by the fungus involves reactive oxygen species production and potassium efflux. Genetic deletion or pharmalogical inhibition of Syk selectively abrogated inflammasome activation by C. albicans but not by inflammasome activators such as Salmonella typhimurium or the bacterial toxin nigericin. Nlrp3 (also known as NALP3) was identified as the critical NOD-like receptor family member that transduces the fungal recognition signal to the inflammasome adaptor Asc (Pycard) for Caspase-1 (Casp1) activation and pro-IL-1beta processing. Consistent with an essential role for Nlrp3 inflammasomes in antifungal immunity, we show that Nlrp3-deficient mice are hypersusceptible to Candida albicans infection. Thus, our results demonstrate the molecular basis for IL-1beta production after fungal infection and identify a crucial function for the Nlrp3 inflammasome in mammalian host defence in vivo.

5'-Triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma.

Genetic and epigenetic plasticity allows tumors to evade single-targeted treatments. Here we direct Bcl2-specific short interfering RNA (siRNA) with 5'-triphosphate ends (3p-siRNA) against melanoma. Recognition of 5'-triphosphate by the cytosolic antiviral helicase retinoic acid-induced protein I (Rig-I, encoded by Ddx58) activated innate immune cells such as dendritic cells and directly induced expression of interferons (IFNs) and apoptosis in tumor cells. These Rig-I-mediated activities synergized with siRNA-mediated Bcl2 silencing to provoke massive apoptosis of tumor cells in lung metastases in vivo. The therapeutic activity required natural killer cells and IFN, as well as silencing of Bcl2, as evidenced by rescue with a mutated Bcl2 target, by site-specific cleavage of Bcl2 messenger RNA in lung metastases and downregulation of Bcl-2 protein in tumor cells in vivo. Together, 3p-siRNA represents a single molecule-based approach in which Rig-I activation on both the immune- and tumor cell level corrects immune ignorance and in which gene silencing corrects key molecular events that govern tumor cell survival.

TRADD protein is an essential component of the RIG-like helicase antiviral pathway.

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-kappaB. Loss of TRADD prevented Cardif-dependent activation of IFN-beta, reduced the production of IFN-beta in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses.