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Although group 3 innate lymphoid cells (ILC3s) are efficient inducers of T cell responses in the spleen, they fail to induce CD4 + T cell proliferation in the gut. The signals regulating ILC3-T cell responses remain unknown. Here, we show that transcripts associated with MHC II antigen presentation are down-modulated in intestinal natural cytotoxicity receptor (NCR) − ILC3s. Further data implicate microbiota-induced IL-23 as a crucial signal for reversible silencing of MHC II in ILC3s, thereby reducing the capacity of ILC3s to present antigen to T cells in the intestinal mucosa. Moreover, IL-23-mediated MHC II suppression is dependent on mTORC1 and STAT3 phosphorylation in NCR − ILC3s. By contrast, splenic interferon-γ induces MHC II expression and CD4 + T cell stimulation by NCR − ILC3s. Our results thus identify biological circuits for tissue-specific regulation of ILC3-dependent T cell responses. These pathways may have implications for inducing or silencing T cell responses in human diseases. Group 3 innate lymphoid cells (ILC3s) promote T cell activation in the spleen but suppress it in the gut. Here, the authors show that this distinct regulation is mediated by gut microbiota-induced IL-23 and IFN-γ, respectively, and, along with the article by Rao et al, this work elucidates how cytokines set context specificity of ILC-T cell crosstalk by regulating ILC antigen presentation.

Obesity and associated diseases, such as diabetes, have reached epidemic proportions globally. In this era of “diabesity”, white adipose tissue (WAT) has become a target of high interest for therapeutic strategies. To gain insights into mechanisms of adipose (patho‐)physiology, researchers traditionally relied on animal models. Leveraging Organ‐on‐Chip technology, a microphysiological in vitro model of human WAT is introduced: a tailored microfluidic platform featuring vasculature‐like perfusion that integrates 3D tissues comprising all major WAT‐associated cellular components (mature adipocytes, organotypic endothelial barriers, stromovascular cells including adipose tissue macrophages) in an autologous manner and recapitulates pivotal WAT functions, such as energy storage and mobilization as well as endocrine and immunomodulatory activities. A precisely controllable bottom‐up approach enables the generation of a multitude of replicates per donor circumventing inter‐donor variability issues and paving the way for personalized medicine. Moreover, it allows to adjust the model's degree of complexity via a flexible mix‐and‐match approach. This WAT‐on‐Chip system constitutes the first human‐based, autologous, and immunocompetent in vitro adipose tissue model that recapitulates almost full tissue heterogeneity and can become a powerful tool for human‐relevant research in the field of metabolism and its associated diseases as well as for compound testing and personalized‐ and precision medicine applications. In the era of “diabesity”, human adipose tissue research has become more important than ever. However, a lack of predictive model systems has traditionally impaired progress in this field. A human, patient‐specific, immunocompetent white adipose tissue (WAT)‐on‐chip system is developed, which integrates virtually all WAT‐associated cell types and reliably reflects in vivo WAT functions in vitro.

Background Pancreatic ductal adenocarcinomas (PDACs) are highly lethal and aggressive with oncogenic KRAS being the main oncogenic driver of the disease. PDACs have been extensively profiled at advanced stages, and in advanced disease the tumor microenvironment is a major determinant that critically shapes patient outcomes. Since the molecular events occurring prior to invasive growth remain poorly understood, we aimed to investigate changes in the precancerous epithelium and its surrounding niche. Methods We acquired time-resolved, single-cell transcriptomic (scRNAseq), and accessible-chromatin data from human pluripotent stem cell-derived pancreatic duct-like organoids (PDLO) inducibly expressing KRAS G12D and from various niche cells. Results Analysis of the pure epithelium already revealed key signatures of matrix remodeling and inflammation-related signaling upon few days of KRAS G12D expression. Machine learning captured KRAS G12D -dependent transcriptomic classifiers with high prediction accuracy and niche preparatory relevance. Various co-culture approaches followed by scRNAseq and functional validation, including T-cell microfluidics, demonstrated that the KRAS G12D -induced PDLO-secretome activates pancreatic stellate cells (PaSCs) and protects precancerous organoids from T cell infiltration. Additional, in silico  approaches reconstructed a virtual pancreatic (pre)cancerous space to profile cell–cell interactions between PDLOs and niche cells. TNFα emerged as a top-ranked ligand and was functionally validated to mediate T-cell shielding and PaSC activation. Cyst fluid from 80 prospectively sampled Intraductal Papillary Mucinous Neoplasm (IPMNs) –well-known cystic PDAC precursor lesions– showed a stepwise TNFα rise across LGD (low-grade), HGD (high-grade), and IC (invasive cancer). Conclusion Our study reveals that oncogenic KRAS orchestrates niche-preparatory programs that precede PDAC formation and highlight a T cell exclusion program governed by epithelial-derived TNFα.

Innate lymphoid cells (ILCs) have a protective immune function at mucosal tissues but can also contribute to immunopathology. Previous work has shown that the serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1) is involved in generating protective ILC3 cytokine responses during bacterial infection. However, whether mTORC1 also regulates IFN-γ-mediated immunopathology has not been investigated. In addition, the role of mTORC2 in ILC3s is unknown. Using mice specifically defective for either mTORC1 or mTORC2 in ILC3s, we show that both mTOR complexes regulate the maintenance of ILC3s at steady state and pathological immune response during colitis. mTORC1 and to a lesser extend mTORC2 promote the proliferation of ILC3s in the small intestine. Upon activation, intestinal ILC3s produce less IFN-γ in the absence of mTOR signaling. During colitis, loss of both mTOR complexes in colonic ILC3s results in the reduced production of inflammatory mediators, recruitment of neutrophils and immunopathology. Similarly, treatment with rapamycin after colitis induction ameliorates the disease. Collectively, our data show a critical role for both mTOR complexes in controlling ILC3 cell numbers and ILC3-driven inflammation in the intestine.

Pancreatic ductal adenocarcinomas (PDACs) are highly lethal and aggressive with oncogenic KRAS being the main oncogenic driver of the disease. PDACs have been extensively profiled at advanced stages, and in advanced disease the tumor microenvironment is a major determinant that critically shapes patient outcomes. Since the molecular events occurring prior to invasive growth remain poorly understood, we aimed to investigate changes in the precancerous epithelium and its surrounding niche. We acquired time-resolved, single-cell transcriptomic (scRNAseq), and accessible-chromatin data from human pluripotent stem cell-derived pancreatic duct-like organoids (PDLO) inducibly expressing KRAS.sup.G12D and from various niche cells. Analysis of the pure epithelium already revealed key signatures of matrix remodeling and inflammation-related signaling upon few days of KRAS.sup.G12D expression. Machine learning captured KRAS.sup.G12D-dependent transcriptomic classifiers with high prediction accuracy and niche preparatory relevance. Various co-culture approaches followed by scRNAseq and functional validation, including T-cell microfluidics, demonstrated that the KRAS.sup.G12D-induced PDLO-secretome activates pancreatic stellate cells (PaSCs) and protects precancerous organoids from T cell infiltration. Additional, in silico approaches reconstructed a virtual pancreatic (pre)cancerous space to profile cell-cell interactions between PDLOs and niche cells. TNF[alpha] emerged as a top-ranked ligand and was functionally validated to mediate T-cell shielding and PaSC activation. Cyst fluid from 80 prospectively sampled Intraductal Papillary Mucinous Neoplasm (IPMNs) -well-known cystic PDAC precursor lesions- showed a stepwise TNF[alpha] rise across LGD (low-grade), HGD (high-grade), and IC (invasive cancer). Our study reveals that oncogenic KRAS orchestrates niche-preparatory programs that precede PDAC formation and highlight a T cell exclusion program governed by epithelial-derived TNF[alpha].

Reliable modeling of human adaptive immune responses is a prerequisite to understand processes leading to vaccine‐induced protective immunization, to overcome the poor predictive value of non‐clinical in vivo and in vitro models and to drive informed decisions in vaccine development pipelines. Here, we present a centrifugal microfluidics‐based organ‐on‐chip approach to generate an organotypic high density lymphoid‐tissue‐on‐chip. The model enables long‐term culture of lymphoid tissue while preventing autoactivation and shows raised antigen‐specific antibody responses against influenza vaccines for up to 4 weeks on‐chip. Antibody response of different magnitude and quality could be induced both by direct antigen exposure as well as by recruitment of peripheral antigen‐presenting cells. The model represents an attractive approach to evaluate the impact of the mode of antigen delivery on adaptive immune responses. Beyond applications in vaccine development, the lymphoid‐tissue‐on‐chip provides a platform to study cellular interactions during homeostasis, immune responses, and drug treatment over several weeks.

In the past decades, vaccine development has made great strides. Nevertheless, more often than not, vaccine candidates fail in advanced stages of development and clinical trials. A key reason is the poor predictive value of non-clinical in vivo and in vitro models, due to either species-specific differences in the immune response or insufficient reflection of physiological vaccine mechanisms. Reliable modeling of human adaptive immune responses is a prerequisite to understand processes leading to vaccine-induced protective immunization and to drive informed decisions in vaccine development pipelines. Here, we present a centrifugal microfluidics based organ-on-chip approach to generate an organotypic high density lymphoid tissue on-chip. The model enables long-term culture of lymphoid tissue and raised antigen-specific antibody responses against influenza vaccines even after four weeks on-chip. Antibody response of different magnitude and quality could be induced both by direct antigen exposure as well as by recruitment of antigen-presenting cells from the periphery. The model represents an attractive approach to evaluate the impact of the mode of antigen delivery on adaptive immune responses. Beyond applications in vaccine development, the lymphoid-tissue-on-chip provides a platform to study cellular interactions during homeostasis, immune responses and long-term impact of immunomodulators over several weeks.

The non-clinical assessment of CAR-T cells demands innovative models that are capable of predicting safety and efficacy in the clinical setting. Here, we present a novel solid tumor-on-chip model that allows CAR-T cell perfusion and integrates the vasculature and tumor lesions to recapitulate key events of CAR-T cell performance including extravasation, tumor infiltration and cytokine release. We assessed CAR-T cells targeting the ROR1 antigen against tumor aggregates that were derived from a breast cancer cell line and primary breast cancer organoids. The data show the temporal kinetic of ROR1 CAR-T cell migration and expansion, lytic activity and cytokine production over the course of 8 days, and reveal a correlation between anti-tumor efficacy and ROR1 antigen density on tumor cells. CAR-modified T cells extravasated faster, infiltrated tumor lesions stronger, persisted longer and in higher numbers than non-CAR modified T cells. Intriguingly, we detected cytokine release levels and kinetics typically observed in patients who developed cytokine release syndrome, and administered dasatinib as a pharmacologic OFF switch to control this inflammatory response. The data illustrate the ability of this tumor-on-chip platform to assess parameters associated withherapeutic outcome and the potential to aid in patient stratification and monitoring of CAR-T cell therapy.

Obesity and associated diseases, such as diabetes, have reached epidemic proportions globally. In the era of ‘diabesity’ and due to its central role for metabolic and endocrine processes, adipose tissue (specifically white adipose tissue; WAT) has become a target of high interest for therapeutic strategies. To gain insights in cellular and molecular mechanisms of adipose (patho-)physiology, researchers traditionally relied on animal models since in vitro studies on human WAT are challenging due to the large size, buoyancy, and fragility of mature white adipocytes. Leveraging the Organ-on-Chip technology, we introduce a next-generation microphysiological in vitro model of human WAT based on a tailored microfluidic platform featuring vasculature-like perfusion. The platform integrates a 3D tissue comprising all major WAT-associated cellular components in an autologous manner, including not only mature adipocytes but also organotypic endothelial barriers and stromovascular cells featuring tissue-resident innate immune cells, specifically adipose tissue macrophages. This microphysiological tissue model recapitulates pivotal WAT functions, such as energy storage and mobilization as well as endocrine and immunomodulatory activities. The combination of all individual cell types with extra cellular matrix-like hydrogels in a precisely controllable bottom-up approach enables the generation of a multitude of replicates from the same donors circumventing issues of inter-donor variability and paving the way for personalized medicine. Moreover, it allows to adjust the model’s degree of complexity to fit a specific purpose via a flexible mix- and-match approach with different cell component modules. This novel WAT-on-chip system constitutes a human-based, autologous and immunocompetent in vitro model of adipose tissue that recapitulates almost full tissue heterogeneity. In the future, the new WAT-on-chip model can become a powerful tool for human-relevant research in the field of metabolism and its associated diseases as well as for compound testing and personalized- and precision medicine applications.

Gadolinium ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) is a paramagnetic hepatobiliary magnetic resonance (MR) contrast agent. Due to its OATP1B1/B3-dependent hepatocyte-specific uptake and paramagnetic properties increasing evidence has emerged to suggest that Gd-EOB-DTPA-enhanced MRI can be potentially used for evaluation of liver function. In this paper we compare the diagnostic performance of Gd-EOB-DTPA-enhanced relaxometry-based and commonly used signal-intensity (SI)-based indices, including the hepatocellular uptake index (HUI) and SI-based indices corrected by spleen or muscle, for evaluation of liver function, determined using the Indocyanin green clearance (ICG) test. Simple linear regression model showed a significant correlation of the plasma disappearance rate of ICG (ICG-PDR) with all Gd-EOB-DTPA-enhanced MRI-based liver function indices with a significantly better correlation of relaxometry-based indices on ICG-PDR compared to SI-based indices. Among SI-based indices, HUI achieved best correlation on ICG-PDR and no significant difference of respective correlations on ICG-PDR could be shown. Assessment of liver volume and consecutive evaluation of multiple linear regression model revealed a stronger correlation of ICG-PDR with both (SI)-based and T1 relaxometry-based indices. Thus, liver function can be estimated quantitatively from Gd-EOB-DTPA–enhanced MRI-based indices. Here, indices derived from T1 relaxometry are superior to SI-based indices, and all indices benefit from taking into account respective liver volumes.