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The gut microbiota is a critical factor regulating mammalian metabolism. The host immune system, in turn, can shape the microbiome, in part via immunoglobulin A (IgA) antibodies. Petersen et al. report that mice defective in T follicular helper cell development and gut IgA production show hallmarks of metabolic syndrome with age (see the Perspective by Wang and Hooper). These mice gain more weight, accumulate more fat, and show greater insulin resistance compared with controls. IgA in these mice inappropriately targets Clostridia species and allows for the outgrowth of Desulfovibrio. Clostridia suppress and Desulfovibrio enhance host lipid absorption by modulating CD36 expression. A better understanding of the microbial products that modulate lipid absorption may open the door to future therapies for obesity and metabolic disease. Science , this issue p. eaat9351 ; see also p. 316 Immune control of gut microbes maintains beneficial microbial populations that constrain lipid metabolism to prevent weight gain. The microbiota influences obesity, yet organisms that protect from disease remain unknown. During studies interrogating host-microbiota interactions, we observed the development of age-associated metabolic syndrome (MetS). Expansion of Desulfovibrio and loss of Clostridia were key features associated with obesity in this model and are present in humans with MetS. T cell–dependent events were required to prevent disease, and replacement of Clostridia rescued obesity. Inappropriate immunoglobulin A targeting of Clostridia and increased Desulfovibrio antagonized the colonization of beneficial Clostridia. Transcriptional and metabolic analysis revealed enhanced lipid absorption in the obese host. Colonization of germ-free mice with Clostridia, but not Desulfovibrio , down-regulated genes that control lipid absorption and reduced adiposity. Thus, immune control of the microbiota maintains beneficial microbial populations that constrain lipid metabolism to prevent MetS.

Background Single cell RNA sequencing (scRNAseq) has provided invaluable insights into cellular heterogeneity and functional states in health and disease. During the analysis of scRNAseq data, annotating the biological identity of cell clusters is an important step before downstream analyses and it remains technically challenging. The current solutions for annotating single cell clusters generally lack a graphical user interface, can be computationally intensive or have a limited scope. On the other hand, manually annotating single cell clusters by examining the expression of marker genes can be subjective and labor-intensive. To improve the quality and efficiency of annotating cell clusters in scRNAseq data, we present a web-based R/Shiny app and R package, Cluster Identity PRedictor (CIPR) , which provides a graphical user interface to quickly score gene expression profiles of unknown cell clusters against mouse or human references, or a custom dataset provided by the user. CIPR can be easily integrated into the current pipelines to facilitate scRNAseq data analysis. Results CIPR employs multiple approaches for calculating the identity score at the cluster level and can accept inputs generated by popular scRNAseq analysis software. CIPR provides 2 mouse and 5 human reference datasets, and its pipeline allows inter-species comparisons and the ability to upload a custom reference dataset for specialized studies. The option to filter out lowly variable genes and to exclude irrelevant reference cell subsets from the analysis can improve the discriminatory power of CIPR suggesting that it can be tailored to different experimental contexts. Benchmarking CIPR against existing functionally similar software revealed that our algorithm is less computationally demanding, it performs significantly faster and provides accurate predictions for multiple cell clusters in a scRNAseq experiment involving tumor-infiltrating immune cells. Conclusions CIPR facilitates scRNAseq data analysis by annotating unknown cell clusters in an objective and efficient manner. Platform independence owing to Shiny framework and the requirement for a minimal programming experience allows this software to be used by researchers from different backgrounds. CIPR can accurately predict the identity of a variety of cell clusters and can be used in various experimental contexts across a broad spectrum of research areas.

Tumor associated macrophage responses are regulated by distinct metabolic states that affect their function. However, the ability of specific signals in the local tumor microenvironment to program macrophage metabolism remains under investigation. Here, we identify NAMPT, the rate limiting enzyme in NAD salvage synthesis, as a target of STAT1 during cellular activation by interferon gamma, an important driver of macrophage polarization and antitumor responses. We demonstrate that STAT1 occupies a conserved element within the first intron of Nampt , termed Nampt-Regulatory Element-1 (NRE1). Through disruption of NRE1 or pharmacological inhibition, a subset of M1 genes is sensitive to NAMPT activity through its impact on glycolytic processes. scRNAseq is used to profile in vivo responses by NRE1-deficient, tumor-associated leukocytes in melanoma tumors through the creation of a unique mouse strain. Reduced Nampt and inflammatory gene expression are present in specific myeloid and APC populations; moreover, targeted ablation of NRE1 in macrophage lineages results in greater tumor burden. Finally, elevated NAMPT expression correlates with IFNγ responses and melanoma patient survival. This study identifies IFN and STAT1-inducible Nampt as an important factor that shapes the metabolic program and function of tumor associated macrophages. IFNy can polarize macrophages in the tumour microenvironment to an inflammatory state and thereby contributes to their anti-tumour function. Here the authors show an underlying mechanism in this process is IFNy-driven STAT1 occupancy and activation of NRE1, a regulatory region within the NAMPT gene, thereby implicating inducible NAD salvage synthesis in TAM functions.

Background Metastatic breast cancer (MBC) is incurable, with a 5-year survival rate of 28%. In the USA, more than 42,000 patients die from MBC every year. The most common type of breast cancer is estrogen receptor-positive (ER+), and more patients die from ER+ breast cancer than from any other subtype. ER+ tumors can be successfully treated with hormone therapy, but many tumors acquire endocrine resistance, at which point treatment options are limited. There is an urgent need for model systems that better represent human ER+ MBC in vivo, where tumors can metastasize. Patient-derived xenografts (PDX) made from MBC spontaneously metastasize, but the immunodeficient host is a caveat, given the known role of the immune system in tumor progression and response to therapy. Thus, we attempted to develop an immune-humanized PDX model of ER+ MBC. Methods NSG-SGM3 mice were immune-humanized with CD34+ hematopoietic stem cells, followed by engraftment of human ER+ endocrine resistant MBC tumor fragments. Strategies for exogenous estrogen supplementation were compared, and immune-humanization in blood, bone marrow, spleen, and tumors was assessed by flow cytometry and tissue immunostaining. Characterization of the new model includes assessment of the human tumor microenvironment performed by immunostaining. Results We describe the development of an immune-humanized PDX model of estrogen-independent endocrine resistant ER+ MBC. Importantly, our model harbors a naturally occurring ESR1 mutation, and immune-humanization recapitulates the lymphocyte-excluded and myeloid-rich tumor microenvironment of human ER+ breast tumors. Conclusion This model sets the stage for development of other clinically relevant models of human breast cancer and should allow future studies on mechanisms of endocrine resistance and tumor-immune interactions in an immune-humanized in vivo setting.

Tumour-host immune interactions lead to complex changes in the tumour microenvironment (TME), impacting progression, metastasis and response to therapy. While it is clear that cancer cells can have the capacity to alter immune landscapes, our understanding of this process is incomplete. Herein we show that endocytic trafficking at the plasma membrane, mediated by the small GTPase ARF6, enables melanoma cells to impose an immunosuppressive TME that accelerates tumour development. This ARF6-dependent TME is vulnerable to immune checkpoint blockade therapy (ICB) but in murine melanoma, loss of Arf6 causes resistance to ICB. Likewise, downregulation of ARF6 in patient tumours correlates with inferior overall survival after ICB. Mechanistically, these phenotypes are at least partially explained by ARF6-dependent recycling, which controls plasma membrane density of the interferon-gamma receptor. Collectively, our findings reveal the importance of endomembrane trafficking in outfitting tumour cells with the ability to shape their immune microenvironment and respond to immunotherapy. The small GTPase ARF6 is known to regulate endocytosis and recycling of plasma membrane proteins. Here the authors show that tumourintrinsic ARF6 promotes an immunosuppressive microenvironment that accelerates melanoma progression but that is vulnerable to immune checkpoint blockade, mechanistically linked to ARF6-dependent recycling of interferon-gamma receptors in tumour cells.

Epidemiological studies have identified a correlation between maternal helminth infections and reduced immunity to some early childhood vaccinations, but the cellular basis for this is poorly understood. Here, we investigated the effects of maternal Schistosoma mansoni infection on steady-state offspring immunity, as well as immunity induced by a commercial tetanus/diphtheria vaccine using a dual IL-4 reporter mouse model of maternal schistosomiasis. We demonstrate that offspring born to S . mansoni infected mothers have reduced circulating plasma cells and peripheral lymph node follicular dendritic cells at steady state. These reductions correlate with reduced production of IL-4 by iNKT cells, the cellular source of IL-4 in the peripheral lymph node during early life. These defects in follicular dendritic cells and IL-4 production were maintained long-term with reduced secretion of IL-4 in the germinal center and reduced generation of TFH, memory B, and memory T cells in response to immunization with tetanus/diphtheria. Using single-cell RNASeq following tetanus/diphtheria immunization of offspring, we identified a defect in cell-cycle and cell-proliferation pathways in addition to a reduction in Ebf-1, a key B-cell transcription factor, in the majority of follicular B cells. These reductions are dependent on the presence of egg antigens in the mother, as offspring born to single-sex infected mothers do not have these transcriptional defects. These data indicate that maternal schistosomiasis leads to long-term defects in antigen-induced cellular immunity, and for the first time provide key mechanistic insight into the factors regulating reduced immunity in offspring born to S . mansoni infected mothers.

Despite the success of chimeric antigen receptor T (CART) cell therapy in hematological malignancies, durable remissions remain low. Here, we report CART senescence as a potential resistance mechanism in 41BB-costimulated CART cell therapy. To mimic cancer relapse, we utilized an in vitro model with repeated CART cell activation cycles followed by rest periods. Using CD19-targeted CART cells with costimulation via 4-1BB-CD3ζ (BBζ) or CD28-CD3ζ (28ζ), we showed that CART cells undergo functional, phenotypical, and transcriptomic changes of senescence, which is more prominent in BBζ. We then utilized two additional independent strategies to induce senescence through MYC activation and irradiation. Induction of senescence impaired BBζ activity but improved 28ζ activity in preclinical studies. These findings were supported by analyses of independent patient data sets; senescence signatures in CART cell products were associated with non-response to BBζ but with improved clinical outcomes in 28ζ treatment. In summary, our study identifies senescence as a potential mechanism of failure predominantly in 41BB-costimulated CART cells. Significance We identified senescence as a cause of failure in CART cell therapy, predominantly in 4-1BB-costimulated CART cells.

Single-cell RNA-seq (scRNA-seq) is a powerful genomics technology to interrogate the cellular composition and behaviors of complex systems. While the number of scRNA-seq datasets and available computational analysis tools have grown exponentially, there are limited systematic data sharing strategies to allow rapid exploration and re-analysis of single-cell datasets, particularly in the cardiovascular field. We previously introduced PlaqView, an open-source web portal for the exploration and analysis of published atherosclerosis single-cell datasets. Now, we introduce PlaqView 2.0 (www.plaqview.com) , which provides expanded features and functionalities as well as additional cardiovascular single-cell datasets. We showcase improved PlaqView functionality, backend data processing, user-interface, and capacity. PlaqView brings new or improved tools to explore scRNA-seq data, including gene query, metadata browser, cell identity prediction, ad hoc RNA-trajectory analysis, and drug-gene interaction prediction. PlaqView serves as one of the largest central repositories for cardiovascular single-cell datasets, which now includes data from human aortic aneurysm, gene-specific mouse knockouts, and healthy references. PlaqView 2.0 brings advanced tools and high-performance computing directly to users without the need for any programming knowledge. Lastly, we outline steps to generalize and repurpose PlaqView's framework for single-cell datasets from other fields.

Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LAT G135D ) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LAT G135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LAT G135D T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LAT G135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity. Lo and colleagues provide evidence for the TCR kinetic proofreading model by LAT Gly135Asp alteration to reveal functional consequences of altered kinetics in TCR activation in thymic selection and mature T-cell responses.

Aged skeletal muscle is characterized by poor muscle recovery following disuse coinciding with an impaired muscle pro‐inflammatory macrophage response. Macrophage inflammatory status is regulated by its metabolic state, but little is understood of macrophage metabolism and its relation to macrophage inflammation in the context of muscle recovery and aging. Therefore, the purpose of this study was to thoroughly characterize macrophage metabolism and inflammation in aged muscle during early recovery following disuse atrophy using single cell transcriptomics and functional assays. Young (4–5 months) and old (20–22 months) male C57BL/6 mice underwent 14 days of hindlimb unloading followed by 4 days of ambulatory recovery. CD45+ cells were isolated from solei muscles and analyzed using 10x Genomics single cell RNA sequencing. We found that aged pro‐inflammatory macrophage clusters were characterized with an impaired inflammatory and glycolytic transcriptome, and this dysregulation was accompanied by a suppression of HIF‐1α and its immediate downstream target, Glut1. As a follow‐up, bone marrow‐derived macrophages were isolated from a separate cohort of young and old mice at 4‐d recovery and were polarized to a pro‐inflammatory phenotype and used for glycolysis stress test, phagocytosis activity assay, and targeted GC‐MS metabolomics. Aged bone marrow‐derived pro‐inflammatory macrophages were characterized with impaired glycolysis and phagocytosis function, decreased succinate and an accumulation of glycolytic metabolic intermediates overall supporting reduced glycolytic flux and macrophage function. Our results indicate that the metabolic reprograming and function of aged skeletal muscle pro‐inflammatory macrophages are dysfunctional during early recovery from disuse atrophy possibly attributing to attenuated regrowth. Summary of metabolic dysfunction in aged pro‐inflammatory macrophages after 4 days of recovery from disuse atrophy. Figure depicts aged skeletal muscle macrophages during the early recovery phase following disuse atrophy (4 days of reloading). Aged pro‐inflammatory macrophages during recovery are present with impaired inflammation and glycolytic metabolism and accompanied by lower HIF‐1α. The lack of HIF‐1α transcription in the single cell sequencing may be regulated by the presence of succinate which typical increases in normal (young) pro‐inflammatory macrophages.