Deleting the mitochondrial respiration negative regulator MCJ enhances the efficacy of CD8+ T cell adoptive therapies in pre-clinical studies.

Mitochondrial respiration is essential for the survival and function of T cells used in adoptive cellular therapies. However, strategies that specifically enhance mitochondrial respiration to promote T cell function remain limited. Here, we investigate methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial complex I expressed in CD8 cells, as a target for improving the efficacy of adoptive T cell therapies. We demonstrate that MCJ inhibits mitochondrial respiration in murine CD8+ CAR-T cells and that deletion of MCJ increases their in vitro and in vivo efficacy against murine B cell leukaemia. Similarly, MCJ deletion in ovalbumin (OVA)-specific CD8+ T cells also increases their efficacy against established OVA-expressing melanoma tumors in vivo. Furthermore, we show for the first time that MCJ is expressed in human CD8 cells and that the level of MCJ expression correlates with the functional activity of CD8+ CAR-T cells. Silencing MCJ expression in human CD8 CAR-T cells increases their mitochondrial metabolism and enhances their anti-tumor activity. Thus, targeting MCJ may represent a potential therapeutic strategy to increase mitochondrial metabolism and improve the efficacy of adoptive T cell therapies.

IL-4 Licenses B Cell Activation Through Cholesterol Synthesis.

Lymphocyte activation involves a transition from quiescence and associated catabolic metabolism to a metabolic state with noted similarities to cancer cells such as heavy reliance on aerobic glycolysis for energy demands and increased nutrient requirements for biomass accumulation and cell division 1-3 . Following antigen receptor ligation, lymphocytes require spatiotemporally distinct "second signals". These include costimulatory receptor or cytokine signaling, which engage discrete programs that often involve remodeling of organelles and increased nutrient uptake or synthesis to meet changing biochemical demands 4-6 . One such signaling molecule, IL-4, is a highly pleiotropic cytokine that was first identified as a B cell co-mitogen over 30 years ago 7 . However, how IL-4 signaling mechanistically supports B cell proliferation is incompletely understood. Here, using single cell RNA sequencing we find that the cholesterol biosynthetic program is transcriptionally upregulated following IL-4 signaling during the early B cell response to influenza virus infection, and is required for B cell activation in vivo . By limiting lipid availability in vitro , we determine cholesterol to be essential for B cells to expand their endoplasmic reticulum, progress through cell cycle, and proliferate. In sum, we demonstrate that the well-known ability of IL-4 to act as a B cell growth factor is through a previously unknown rewiring of specific lipid anabolic programs, relieving sensitivity of cells to environmental nutrient availability.

DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells.

Epithelial barrier dysfunction and crypt destruction are hallmarks of inflammatory bowel disease (IBD). Intestinal stem cells (ISCs) residing in the crypts play a crucial role in the continuous self-renewal and rapid recovery of intestinal epithelial cells (IECs). However, how ISCs are dysregulated in IBD remains poorly understood. Here, we observe reduced DHX9 protein levels in IBD patients, and mice with conditional DHX9 depletion in the intestinal epithelium (Dhx9ΔIEC) exhibit an increased susceptibility to experimental colitis. Notably, Dhx9ΔIEC mice display a significant reduction in the numbers of ISCs and Paneth cells. Further investigation using ISC-specific or Paneth cell-specific Dhx9-deficient mice demonstrates the involvement of ISC-expressed DHX9 in maintaining epithelial homeostasis. Mechanistically, DHX9 deficiency leads to abnormal R-loop accumulation, resulting in genomic instability and the cGAS-STING-mediated inflammatory response, which together impair ISC function and contribute to the pathogenesis of IBD. Collectively, our findings highlight R-loop-mediated genomic instability in ISCs as a risk factor in IBD.

Gene trajectory inference for single-cell data by optimal transport metrics.

Single-cell RNA sequencing has been widely used to investigate cell state transitions and gene dynamics of biological processes. Current strategies to infer the sequential dynamics of genes in a process typically rely on constructing cell pseudotime through cell trajectory inference. However, the presence of concurrent gene processes in the same group of cells and technical noise can obscure the true progression of the processes studied. To address this challenge, we present GeneTrajectory, an approach that identifies trajectories of genes rather than trajectories of cells. Specifically, optimal transport distances are calculated between gene distributions across the cell-cell graph to extract gene programs and define their gene pseudotemporal order. Here we demonstrate that GeneTrajectory accurately extracts progressive gene dynamics in myeloid lineage maturation. Moreover, we show that GeneTrajectory deconvolves key gene programs underlying mouse skin hair follicle dermal condensate differentiation that could not be resolved by cell trajectory approaches. GeneTrajectory facilitates the discovery of gene programs that control the changes and activities of biological processes.

IL-10 constrains sphingolipid metabolism to limit inflammation.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.

Bioactive signalling lipids as drivers of chronic liver diseases.

Lipids are important in multiple cellular functions, with most having structural or energy storage roles. However, a small fraction of lipids exert bioactive roles through binding to G protein-coupled receptors and induce a plethora of processes including cell proliferation, differentiation, growth, migration, apoptosis, senescence and survival. Bioactive signalling lipids are potent modulators of metabolism and energy homeostasis, inflammation, tissue repair and malignant transformation. All these events are involved in the initiation and progression of chronic liver diseases. In this review, we focus specifically on the roles of bioactive lipids derived from phospholipids (lyso-phospholipids) and poly-unsaturated fatty acids (eicosanoids, pro-resolving lipid mediators and endocannabinoids) in prevalent chronic liver diseases (alcohol-associated liver disease, non-alcoholic fatty liver disease, viral hepatitis and hepatocellular carcinoma). We discuss the balance between pathogenic and beneficial bioactive lipids as well as potential therapeutic targets related to the agonism or antagonism of their receptors.

IL-10 dampens antitumor immunity and promotes liver metastasis via PD-L1 induction.

BACKGROUND & AIMS: The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy in vivo. METHODS: To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. RESULTS: We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. CONCLUSIONS: Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. IMPACT AND IMPLICATIONS: Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.

Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection.

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.

What approaches are needed to understand human development and disease?

Researchers are leveraging what we have learned from model organisms to understand if the same principles arise in human physiology, development, and disease. In this collection of Voices, we asked researchers from different fields to discuss what tools and insights they are using to answer fundamental questions in human biology.

IL-6 trans-signaling in a humanized mouse model of scleroderma.

Fibrosis is regulated by interactions between immune and mesenchymal cells. However, the capacity of cell types to modulate human fibrosis pathology is poorly understood due to lack of a fully humanized model system. MISTRG6 mice were engineered by homologous mouse/human gene replacement to develop an immune system like humans when engrafted with human hematopoietic stem cells (HSCs). We utilized MISTRG6 mice to model scleroderma by transplantation of healthy or scleroderma skin from a patient with pansclerotic morphea to humanized mice engrafted with unmatched allogeneic HSC. We identified that scleroderma skin grafts contained both skin and bone marrow-derived human CD4 and CD8 T cells along with human endothelial cells and pericytes. Unlike healthy skin, fibroblasts in scleroderma skin were depleted and replaced by mouse fibroblasts. Furthermore, HSC engraftment alleviated multiple signatures of fibrosis, including expression of collagen and interferon genes, and proliferation and activation of human T cells. Fibrosis improvement correlated with reduced markers of T cell activation and expression of human IL-6 by mesenchymal cells. Mechanistic studies supported a model whereby IL-6 trans-signaling driven by CD4 T cell-derived soluble IL-6 receptor complexed with fibroblast-derived IL-6 promoted excess extracellular matrix gene expression. Thus, MISTRG6 mice transplanted with scleroderma skin demonstrated multiple fibrotic responses centered around human IL-6 signaling, which was improved by the presence of healthy bone marrow-derived immune cells. Our results highlight the importance of IL-6 trans-signaling in pathogenesis of scleroderma and the ability of healthy bone marrow-derived immune cells to mitigate disease.

ALKBH5 modulates hematopoietic stem and progenitor cell energy metabolism through m6A modification-mediated RNA stability control.

N6-methyladenosine (m6A) RNA modification controls numerous cellular processes. To what extent these post-transcriptional regulatory mechanisms play a role in hematopoiesis has not been fully elucidated. We here show that the m6A demethylase alkB homolog 5 (ALKBH5) controls mitochondrial ATP production and modulates hematopoietic stem and progenitor cell (HSPC) fitness in an m6A-dependent manner. Loss of ALKBH5 results in increased RNA methylation and instability of oxoglutarate-dehydrogenase (Ogdh) messenger RNA and reduction of OGDH protein levels. Limited OGDH availability slows the tricarboxylic acid (TCA) cycle with accumulation of α-ketoglutarate (α-KG) and conversion of α-KG into L-2-hydroxyglutarate (L-2-HG). L-2-HG inhibits energy production in both murine and human hematopoietic cells in vitro. Impaired mitochondrial energy production confers competitive disadvantage to HSPCs and limits clonogenicity of Mll-AF9-induced leukemia. Our study uncovers a mechanism whereby the RNA m6A demethylase ALKBH5 regulates the stability of metabolic enzyme transcripts, thereby controlling energy metabolism in hematopoiesis and leukemia.

Humanized mouse liver reveals endothelial control of essential hepatic metabolic functions.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment.

BACKGROUND: Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD: With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient's hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual's TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS: Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS: Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing.

IL-7R licenses a population of epigenetically poised memory CD8+ T cells with superior antitumor efficacy that are critical for melanoma memory.

Recurrence of advanced melanoma after therapy is a major risk factor for reduced survival, and treatment options are limited. Antitumor immune memory plays a critical role in preventing melanoma recurrence and memory T cells could be a potent cell-based therapy, but the identity, and functional properties of the required immune cells are incompletely understood. Here, we show that an IL-7Rhi tumor-specific CD8+ population is critical for antitumor memory and can be epigenetically augmented to drive powerful antitumor immune responses. Using a model of functional antimelanoma memory, we found that high IL-7R expression selectively marks a CD8+ population in lymphoid organs that plays critical roles in maintaining tumor remission after immunotherapy or surgical resection. This population has intrinsic cytotoxic activity, lacks markers of exhaustion and has superior antitumor efficacy. IL-7Rhi cells have a functionally poised epigenetic landscape regulated by DNA methylation, which can be augmented by hypomethylating agents to confer improved survival and complete melanoma clearance in naive mice. Importantly, greater than 95% of tumor-specific T cells in draining lymph nodes after therapy express high levels of IL-7R. This overlap between IL-7Rhi and antigen-specific T cells allows for enrichment of a potent functional CD8+ population without determining antigen-specificity, which we demonstrate in a melanoma model without a known antigen. We identify that IL-7R expression in human melanoma is an independent prognostic factor of improved survival. These findings advance our basic understanding of antitumor memory and suggest a cell-based therapy using high IL-7R expression to enrich for a lymph node population with superior antitumor activity that can be augmented by hypomethylating agents.

METTL3-mediated m6A methylation orchestrates mRNA stability and dsRNA contents to equilibrate γδ T1 and γδ T17 cells.

γδ T cells make key contributions to tissue physiology and immunosurveillance through two main functionally distinct subsets, γδ T1 and γδ T17. m6A methylation plays critical roles in controlling numerous aspects of mRNA metabolism that govern mRNA turnover, gene expression, and cellular functional specialization; however, its role in γδ T cells remains less well understood. Here, we find that m6A methylation controls the functional specification of γδ T17 vs. γδ T1 cells. Mechanistically, m6A methylation prevents the formation of endogenous double-stranded RNAs and promotes the degradation of Stat1 transcripts, which converge to prevent over-activation of STAT1 signaling and ensuing inhibition of γδ T17. Deleting Mettl3, the key enzyme in the m6A methyltransferases complex, in γδ T cells reduces interleukin-17 (IL-17) production and ameliorates γδ T17-mediated psoriasis. In summary, our work shows that METTL3-mediated m6A methylation orchestrates mRNA stability and double-stranded RNA (dsRNA) contents to equilibrate γδ T1 and γδ T17 cells.

Gasdermin D licenses MHCII induction to maintain food tolerance in small intestine.

The intestinal epithelial cells (IECs) constitute the primary barrier between host cells and numerous foreign antigens; it is unclear how IECs induce the protective immunity against pathogens while maintaining the immune tolerance to food. Here, we found IECs accumulate a less recognized 13-kD N-terminal fragment of GSDMD that is cleaved by caspase-3/7 in response to dietary antigens. Unlike the 30-kD GSDMD cleavage fragment that executes pyroptosis, the IEC-accumulated GSDMD cleavage fragment translocates to the nucleus and induces the transcription of CIITA and MHCII molecules, which in turn induces the Tr1 cells in upper small intestine. Mice treated with a caspase-3/7 inhibitor, mice with GSDMD mutation resistant to caspase-3/7 cleavage, mice with MHCII deficiency in IECs, and mice with Tr1 deficiency all displayed a disrupted food tolerance phenotype. Our study supports that differential cleavage of GSDMD can be understood as a regulatory hub controlling immunity versus tolerance in the small intestine.

A Treg-specific long noncoding RNA maintains immune-metabolic homeostasis in aging liver.

Regulatory T (Treg) cells modulate several aging-related liver diseases. However, the molecular mechanisms regulating Treg function in this context are unknown. Here we identified a long noncoding RNA, Altre (aging liver Treg-expressed non-protein-coding RNA), which was specifically expressed in the nucleus of Treg cells and increased with aging. Treg-specific deletion of Altre did not affect Treg homeostasis and function in young mice but caused Treg metabolic dysfunction, inflammatory liver microenvironment, liver fibrosis and liver cancer in aged mice. Depletion of Altre reduced Treg mitochondrial integrity and respiratory capacity, and induced reactive oxygen species accumulation, thus increasing intrahepatic Treg apoptosis in aged mice. Moreover, lipidomic analysis identified a specific lipid species driving Treg aging and apoptosis in the aging liver microenvironment. Mechanistically, Altre interacts with Yin Yang 1 to orchestrate its occupation on chromatin, thereby regulating the expression of a group of mitochondrial genes, and maintaining optimal mitochondrial function and Treg fitness in the liver of aged mice. In conclusion, the Treg-specific nuclear long noncoding RNA Altre maintains the immune-metabolic homeostasis of the aged liver through Yin Yang 1-regulated optimal mitochondrial function and the Treg-sustained liver immune microenvironment. Thus, Altre is a potential therapeutic target for the treatment of liver diseases affecting older adults.