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SUMMARYSepsis is a systemic response to infection with life-threatening consequences. Our understanding of the impact of sepsis across organs of the body is rudimentary. Here, using mouse models of sepsis, we generate a dynamic, organism-wide map of the pathogenesis of the disease, revealing the spatiotemporal patterns of the effects of sepsis across tissues. These data revealed two interorgan mechanisms key in sepsis. First, we discover a simplifying principle in the systemic behavior of the cytokine network during sepsis, whereby a hierarchical cytokine circuit arising from the pairwise effects of TNF plus IL-18, IFN-γ, or IL-1β explains half of all the cellular effects of sepsis on 195 cell types across 9 organs. Second, we find that the secreted phospholipase PLA2G5 mediates hemolysis in blood, contributing to organ failure during sepsis. These results provide fundamental insights to help build a unifying mechanistic framework for the pathophysiological effects of sepsis on the body.

The immune system operates at the scale of the whole organism in mammals. We currently lack experimental approaches to systematically track and study organism-wide molecular processes in mice. Here we describe an integrated toolkit for measuring gene expression in whole tissues, PME-seq (3-prime mRNA extension sequencing), that is applicable to most mouse organs and any mouse model of interest. Further, the methods for RNA-seq described in this protocol are broadly applicable to other sample types beyond whole organs, such as tissue samples or isolated cell populations. We report procedures to collect, store and lyse a dozen of organ types using conditions compatible with the extraction of high-quality RNA. In addition, we detail protocols to perform high-throughput and low-cost RNA extraction and sequencing as well as downstream data analysis. The protocol takes 5 days to process 384 mouse organs from collecting tissues to obtaining raw sequencing data, with additional time required for data analysis and mining. The protocol is accessible to individuals with basic skills in (i) mouse perfusion and dissection for sample collection, and (ii) computation using Unix and R for data analysis. Overall, the methods presented here fill a unique gap in our toolbox for studying organism-wide processes in immunology and physiology.

Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy., Visualisation of TARP localisation is hindered by existing imaging tools. Here the authors report a labelling and imaging platform using genetic code expansion and non-canonical amino acids; they use this to fluorescently label live neurons and localise TARP proteins using super resolution microscopy.

SUMMARYThe immune system makes decisions in response to complex combinations of microbial inputs. We do not understand the combinatorial logic that governs how the interplay between higher-order combinations of microbial or adjuvant signals shape immune responses, which hampers the rational design of vaccines and immunotherapies. Here, using in vitro coculture experiments and statistical analyses, we discover a general property for the combinatorial sensing of microbial signals, whereby the effects of triplet combinations of adjuvants on immune responses can be explained by the effects of single and pairwise stimulations. Mechanistically, we find that adjuvant singles and pairs dictate the information signaled by triplets in mouse and human DCs at the levels of transcription, chromatin and protein secretion. Furthermore, we exploit this simplifying property to develop and characterize cell-based immunotherapies using adjuvant combinations with anti-tumor properties in mouse models. We conclude that the processing of complex mixtures of microbial or adjuvant inputs by immune cells is governed by pairwise effects, which will inform the rationale combination of immunomodulatory agents such as adjuvants to manipulate immunity.

The immune system is a dynamic mesh of molecules, cells and tissues spanning the entire organism. Despite a wealth of knowledge about the components of the immune system, little is known about the general rules governing the organismal circuitry of immunity. Deciphering the immune system at the scale of the whole organism is crucial to understanding fundamental problems in immunobiology and physiology, and to manipulate immunity for maintaining health and preventing disease. Here I discuss the emerging principles of inter-organ communications during immune responses by focusing on three common themes that are the regulation of the (i) composition, (ii) condition and (iii) coordination of communicating organs by molecular and cellular factors. Based on these common principles, I emphasize fundamental gaps in our knowledge of organismal immune processes and the outlook to tackle immunity at the scale of the whole organism.

The immune system makes decisions in response to combinations of multiple microbial inputs. We do not understand the combinatorial logic governing how higher-order combinations of microbial signals shape immune responses. Here, using coculture experiments and statistical analyses, we discover a general property for the combinatorial sensing of microbial signals, whereby the effects of triplet combinations of microbial signals on immune responses can be predicted by combining the effects of single and pairs. Mechanistically, we find that singles and pairs dictate the information signaled by triplets in mouse and human DCs at the levels of transcription, chromatin, and protein secretion. We exploit this simplifying property to develop cell-based immunotherapies prepared with adjuvant combinations that trigger protective responses in mouse models of cancer. We conclude that the processing of multiple input signals by innate immune cells is governed by pairwise effects, which will inform the rationale combination of adjuvants to manipulate immunity.

Building an integrated view of cellular responses to environmental cues remains a fundamental challenge due to the complexity of intracellular networks in mammalian cells. Here, we introduce an integrative biochemical and genetic framework to dissect signal transduction events using multiple data types and, in particular, to unify signaling and transcriptional networks. Using the Toll-like receptor (TLR) system as a model cellular response, we generate multifaceted datasets on physical, enzymatic, and functional interactions and integrate these data to reveal biochemical paths that connect TLR4 signaling to transcription. We define the roles of proximal TLR4 kinases, identify and functionally test two dozen candidate regulators, and demonstrate a role for Ap1ar (encoding the Gadkin protein) and its binding partner, Picalm, potentially linking vesicle transport with pro-inflammatory responses. Our study thus demonstrates how deciphering dynamic cellular responses by integrating datasets on various regulatory layers defines key components and higher-order logic underlying signaling-to-transcription pathways. Keywords: pathogen-sensing pathways; Toll-like receptors; TLRs; phosphoproteomics; protein-protein interactions; large-scale in vitro kinase assay; signaling; transcriptional network analysis, National Institutes of Health (U.S.) (Grant U54 AI057159), National Institutes of Health (U.S.) (Award DP2 OD002230), National Institutes of Health (U.S.) (Award P50 HG006193)

Follicular regulatory T cells (TFR cells) inhibit follicular helper T cell (TFH cell)-mediated antibody production. Sharpe and colleagues show that TFR cells induce a distinct suppressive state in TFH cells and B cells that can be reversed by the cytokine IL-21. Follicular regulatory T cells (TFR cells) inhibit follicular helper T cell (TFH cell)–mediated antibody production. The mechanisms by which TFR cells exert their key immunoregulatory functions are largely unknown. Here we found that TFR cells induced a distinct suppressive state in TFH cells and B cells, in which effector transcriptional signatures were maintained but key effector molecules and metabolic pathways were suppressed. The suppression of B cell antibody production and metabolism by TFR cells was durable and persisted even in the absence of TFR cells. This durable suppression was due in part to epigenetic changes. The cytokine IL-21 was able to overcome TFR cell–mediated suppression and inhibited TFR cells and stimulated B cells. By determining mechanisms of TFR cell-mediated suppression, we have identified methods for modulating the function of TFR cells and antibody production.

Type 1 interferon (IFN) is a key mediator of organismal responses to pathogens, eliciting prototypical “interferon signature genes” that encode antiviral and inflammatory mediators. For a global view of IFN signatures and regulatory pathways, we performed gene expression and chromatin analyses of the IFN-induced response across a range of immunocyte lineages. These distinguished ISGs by cell-type specificity, kinetics, and sensitivity to tonic IFN and revealed underlying changes in chromatin configuration. We combined 1,398 human and mouse datasets to computationally infer ISG modules and their regulators, validated by genetic analysis in both species. Some ISGs are controlled by Stat1/2 and Irf9 and the ISRE DNA motif, but others appeared dependent on non-canonical factors. This regulatory framework helped to interpret JAK1 blockade pharmacology, different clusters being affected under tonic or IFN-stimulated conditions, and the IFN signatures previously associated with human diseases, revealing unrecognized subtleties in disease footprints, as affected by human ancestry., Pfizer Inc.

The immune system decides upon actions in response to complex combinations of microbial inputs. Dozens of innate immune sensing pathways can collectively respond to combinations of microbial and immunomodulatory agents such as adjuvants. We currently lack a clear understanding for the combinatorial logic that governs how the interplay between higher-order combinations of microbial or adjuvant signals shape immune responses. Here, using in vitro coculture experiments and statistical analyses, we uncover an intrinsic property of pathogen-sensing pathways whereby the effects of higher-order combinations of stimuli on immune responses can be explained by single and pairwise effects. Surprisingly, this property of combinatorial innate sensing is mechanistically rooted in the molecular organization underlying pathogen-sensing pathways. Indeed, we find that the outputs of mouse and human innate immune dendritic cells upon triplet stimulations are dictated by single and pairwise effects at the levels of transcription, chromatin and protein secretion. Furthermore, we exploit this simplifying property of combinatorial pathogen sensing to develop and characterize cell-based immunotherapies with potent anti-tumor properties in mouse models using combinations of adjuvants. We conclude that the processing of complex mixtures of microbial or adjuvant inputs by immune cells is governed by pairwise effects, which suggests a path towards the rationale selection of adjuvant combinations to alter immunity.

Although PD-1 pathway inhibitors are revolutionizing cancer treatment, the mechanisms by which PD-1 regulates antitumor immunity are not fully understood. To determine PD-1 functions on different cell types, we used PD-1 conditional knockout mice. Subcutaneous transplantation of MC38 adenocarcinoma tumor cells in mice with complete deletion of PD-1 selectively on CD8+ T cells improved CD8+ T functions in the tumor microenvironment (TME). CD8+ T cells were required for the protective effects of PD-1 deletion in this model. To assess whether loss of PD-1 on all cells was necessary for improved antitumor immunity, we restricted PD-1 deletion to only half of the T-cell population. We hypothesized that a cell-intrinsic loss of PD-1 was necessary for improved T-cell fitness and effector functions. Here, deletion of PD-1 indeed led to T cell-intrinsic boosts in function. Unexpectedly, however, there was also a bystander effect that improved functions of PD-1-expressing CD8+ T cells in the TME. These data suggest that complete loss of PD-1 is not necessary for optimal tumor immunity, a finding that has important implications for applying PD-1-based immunotherapies to cancer patients. This abstract is also being presented as Poster B82. Citation Format: Kristen E. Pauken, Vikram R. Juneja, Alison Ringel, Jared H. Rowe, Kelly P. Burke, Peter T. Sage, Martin W. LaFleur, Juhi R. Kuchroo, Noga Ron-Harel, Seth Maleri, Jaclyn M. Long, Gordon J. Freeman, Nicolas Chevrier, Marcia C. Haigis, Arlene H. Sharpe. Loss of PD-1 promotes antitumor immunity by improving functions of both PD-1+ and PD-1- CD8+ T cells in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR9.

A fundamental challenge in immunology is to decipher the principles governing immune responses at the whole-organism scale. Here, using a comparative infection model, we observe immune signal propagation within and between organs to obtain a dynamic map of immune processes at the organism level. We uncover two inter-organ mechanisms of protective immunity mediated by soluble and cellular factors. First, analyzing ligand-receptor connectivity across tissues reveals that type I IFNs trigger a whole-body antiviral state, protecting the host within hours after skin vaccination. Second, combining parabiosis, single-cell analyses, and gene knockouts, we uncover a multi-organ web of tissue-resident memory T cells that functionally adapt to their environment to stop viral spread across the organism. These results have implications for manipulating tissue-resident memory T cells through vaccination and open up new lines of inquiry for the analysis of immune responses at the organism level.

Bisphosphonates are a class of drugs that are widely used to inhibit loss of bone mass in patients. We show here that the administration of clinically relevant doses of bisphosphonates in mice increases antibody responses to live and inactive viruses, proteins, haptens and existing commercial vaccine formulations. Bisphosphonates exert this adjuvant-like activity in the absence of CD4+ and γδ T cells, neutrophils or dendritic cells and their effect does not rely on local macrophage depletion nor does it depend upon Toll-like receptor signaling or the inflammasome. Rather, bisphosphonates target directly B cells and enhance B cell expansion and antibody production upon antigen encounter. These data establish bisphosphonates as a novel class of adjuvants that boost humoral immune responses.

A circuit level understanding of immune cells and hematological cancers has been severely impeded by a lack of techniques that enable intracellular perturbation without significantly altering cell viability and function. Here, we demonstrate that vertical silicon nanowires (NWs) enable gene-specific manipulation of diverse murine and human immune cells with negligible toxicity. To illustrate the power of the technique, we then apply NW-mediated gene silencing to investigate the role of the Wnt signaling pathway in chronic lymphocytic leukemia (CLL). Remarkably, CLL-B cells from different patients exhibit tremendous heterogeneity in their response to the knockdown of a single gene, LEF1. This functional heterogeneity defines three distinct patient groups not discernible by conventional CLL cytogenetic markers and provides a prognostic indicator for patients' time to first therapy. Analyses of gene expression signatures associated with these functional patient subgroups reveal unique insights into the underlying molecular basis for disease heterogeneity. Overall, our findings suggest a functional classification that can potentially guide the selection of patient-specific therapies in CLL and highlight the opportunities for nanotechnology to drive biological inquiry.

Although PD-1 pathway inhibitors are revolutionizing cancer treatment, the mechanisms by which PD-1 regulates anti-tumor immunity are not fully understood. Following subcutaneous transplantation of MC38 adenocarcinoma tumor cells into mice, we show that complete loss of PD-1 selectively on CD8+ T cells improved metabolic activity and functions in the tumor microenvironment (TME). Since clinically PD-1 inhibitors likely act on T cells post-priming, we next deleted PD-1 after initial priming and restricted deletion to roughly 50% of cells. Loss of PD-1 led to T cell-intrinsic boosts in metabolism and CD8+ T cells that lost PD-1 after priming preferentially formed anti-tumor memory cells, suggesting PD-1 antagonizes memory formation. Unexpectedly, there was also a bystander effect that improved functions of PD-1 expressing CD8+ T cells in the TME. These data suggest that complete loss of PD-1 is not necessary for optimal tumor immunity, and that enhancing the functions of a subset of CD8+ T cells can promote an antitumor microenvironment and immunologic memory. Citation Format: Kristen E. Pauken, Vikram R. Juneja, Peter T. Sage, Martin W. LaFleur, Juhi R. Kuchroo, Alison Ringel, Noga Ron-Harel, Seth P. Maleri, Gordon J. Freeman, Nicolas Chevrier, Marcia C. Haigis, Arlene H. Sharpe. PD-1 modulation promotes antitumor immunity by improving metabolic fitness of both PD-1+ and PD-1- CD8+ T cells in the tumor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A19.

How the immune system readies for battle Although gene expression is tightly controlled at both the RNA and protein levels, the quantitative contribution of each step, especially during dynamic responses, remains largely unknown. Indeed, there has been much debate whether changes in RNA level contribute substantially to protein-level regulation. Jovanovic et al. built a genome-scale model of the temporal dynamics of differential protein expression during the stimulation of immunological dendritic cells (see the Perspective by Li and Biggin). Newly stimulated functions involved the up-regulation of specific RNAs and concomitant increases in the levels of the proteins they encode, whereas housekeeping functions were regulated posttranscriptionally at the protein level. Science , this issue 10.1126/science.1259038 ; see also p. 1066

Toll-like receptors (TLRs) recognize distinct microbial components and induce innate immune responses. TLR5 is triggered by bacterial flagellin. Here we generated Tlr5-/- mice and assessed TLR5 function in vivo. Unlike other TLRs, TLR5 was not expressed on conventional dendritic cells or macrophages. In contrast, TLR5 was expressed mainly on intestinal CD11c+ lamina propria cells (LPCs). CD11c+ LPCs detected pathogenic bacteria and secreted proinflammatory cytokines in a TLR5-dependent way. However, CD11c+ LPCs do not express TLR4 and did not secrete proinflammatory cytokines after exposure to a commensal bacterium. Notably, transport of pathogenic Salmonella typhimurium from the intestinal tract to mesenteric lymph nodes was impaired in Tlr5-/- mice. These data suggest that CD11c+ LPCs, via TLR5, detect and are used by pathogenic bacteria in the intestinal lumen.

Over expression of the inhibitory receptor PD-1 commonly occurs in cancer, and PD-1 signaling suppresses protective anti-tumor immune responses. While early clinical trials with PD-1 inhibitors in cancer have been promising, the majority of patients have not shown durable clinical benefit, and the mechanisms underlying both successes and failures remain poorly defined. CD8+ T cells can directly kill tumor cells, and CD8+T cell infiltration of human tumors positively correlates with responses to PD-1 blockade. CD8+ T cells in tumors (TILs) often show high expression of PD-1; however, PD-1 is also expressed by other cell types, including CD4+ T cells, regulatory T cells (Treg), B cells, and some myeloid cells. Consequently, we hypothesized that loss of PD-1 only on CD8+ T cells may not be sufficient to control tumor burden. To address this question, we compared tumor responses in mice completely deficient for PD-1 (referred to as cKO) or selectively deficient for PD-1 on CD8+ T cells (E8i-cre PD-1f/f mice, referred to as CD8ΔPD-1). Following subcutaneous implantation of MC38 adenocarcinoma tumor cells, 100% of PD-1 cKO mice cleared the tumor. In contrast, only 70% of CD8ΔPD-1 mice controlled the tumor. Moreover, in CD8ΔPD-1 mice that controlled tumor, the average time to clearance was delayed compared to PD-1 cKO mice. In both PD-1 cKO and CD8ΔPD-1 mice, CD8+ TILs showed elevated granzyme B levels and an increased CD8 to Treg ratio, suggesting that CD8+ T cell-independent mechanisms contribute to immunity following loss of PD-1 in MC38 tumors. On-going work examining both tumor cells and infiltrating immune cells is focused on identifying these CD8+T cell-independent mechanisms of PD-1-mediated suppression.

Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene-expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.

International audience; Natural killer (NK) cell tolerance to self is partly ensured by major histocompatibility complex (MHC) class I-specific inhibitory receptors on NK cells, which dampen their reactivity when engaged. However, NK cells that do not detect self MHC class I are not autoreactive. We used dynamic fluorescence correlation spectroscopy to show that MHC class I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC class I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We propose that the confinement of activating receptors at the plasma membrane is pivotal to ensuring the self-tolerance of NK cells.

SUMMARY Deciphering the signaling networks that underlie normal and disease processes remains a major challenge. Here, we report the discovery of signaling components involved in the Toll-like receptor (TLR) response of immune dendritic cells (DCs), including a previously unkown pathway shared across mammalian antiviral responses. By combining transcriptional profiling, genetic and small-molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known regulators, involved in TLR signaling. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Our study illustrates the power of combining systematic measurements and perturbations to elucidate complex signaling circuits and discover potential therapeutic targets.

Models of mammalian regulatory networks controlling gene expression have been inferred from genomic data but have largely not been validated. We present an unbiased strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We applied this approach to derive regulatory networks that control the transcriptional response of mouse primary dendritic cells to pathogens. Our approach revealed the regulatory functions of 125 transcription factors, chromatin modifiers, and RNA binding proteins, which enabled the construction of a network model consisting of 24 core regulators and 76 fine-tuners that help to explain how pathogen-sensing pathways achieve specificity. This study establishes a broadly applicable, comprehensive, and unbiased approach to reveal the wiring and functions of a regulatory network controlling a major transcriptional response in primary mammalian cells., Burroughs Wellcome Fund (Career Award at the Scientific Interface), National Institutes of Health (U.S.) (Pioneer Award), Alfred P. Sloan Foundation

Neutralizing antibodies have been thought to be required for protection against acutely cytopathic viruses, such as the neurotropic vesicular stomatitis virus (VSV). Utilizing mice that possess B cells but lack antibodies, we show here that survival upon subcutaneous (s.c.) VSV challenge was independent of neutralizing antibody production or cell-mediated adaptive immunity. However, B cells were absolutely required to provide lymphotoxin (LT) alpha 1 beta 2, which maintained a protective subcapsular sinus (SCS) macrophage phenotype within virus draining lymph nodes (LNs). Macrophages within the SCS of B cell-deficient LNs, or of mice that lack LT alpha 1 beta 2 selectively in B cells, displayed an aberrant phenotype, failed to replicate VSV, and therefore did not produce type I interferons, which were required to prevent fatal VSV invasion of intranodal nerves. Thus, although B cells are essential for survival during VSV infection, their contribution involves the provision of innate differentiation and maintenance signals to macrophages, rather than adaptive immune mechanisms.