Multimodal profiling reveals tissue-directed signatures of human immune cells altered with age.

The immune system comprises multiple cell lineages and heterogeneous subsets found in blood and tissues throughout the body. While human immune responses differ between sites and over age, the underlying sources of variation remain unclear as most studies are limited to peripheral blood. Here, we took a systems approach to comprehensively profile RNA and surface protein expression of over 1.25 million immune cells isolated from blood, lymphoid organs, and mucosal tissues of 24 organ donors aged 20-75 years. We applied a multimodal classifier to annotate the major immune cell lineages (T cells, B cells, innate lymphoid cells, and myeloid cells) and their corresponding subsets across the body, leveraging probabilistic modeling to define bases for immune variations across donors, tissue, and age. We identified dominant tissue-specific effects on immune cell composition and function across lineages for lymphoid sites, intestines, and blood-rich tissues. Age-associated effects were intrinsic to both lineage and site as manifested by macrophages in mucosal sites, B cells in lymphoid organs, and T and NK cells in blood-rich sites. Our results reveal tissue-specific signatures of immune homeostasis throughout the body and across different ages. This information provides a basis for defining the transcriptional underpinnings of immune variation and potential associations with disease-associated immune pathologies across the human lifespan.

Tr1 Cells Emerge and Suppress Effector Th17 Cells in Glomerulonephritis.

T regulatory type 1 (Tr1) cells, which are defined by their regulatory function, lack of Foxp3, and high expression of IL-10, CD49b, and LAG-3, are known to be able to suppress Th1 and Th17 in the intestine. Th1 and Th17 cells are also the main drivers of crescentic glomerulonephritis (GN), the most severe form of renal autoimmune disease. However, whether Tr1 cells emerge in renal inflammation and, moreover, whether they exhibit regulatory function during GN have not been thoroughly investigated yet. To address these questions, we used a mouse model of experimental crescentic GN and double Foxp3mRFP IL-10eGFP reporter mice. We found that Foxp3neg IL-10-producing CD4+ T cells infiltrate the kidneys during GN progression. Using single-cell RNA sequencing, we could show that these cells express the core transcriptional factors characteristic of Tr1 cells. In line with this, Tr1 cells showed a strong suppressive activity ex vivo and were protective in experimental crescentic GN in vivo. Finally, we could also identify Tr1 cells in the kidneys of patients with antineutrophil cytoplasmic autoantibody-associated GN and define their transcriptional profile. Tr1 cells are currently used in several immune-mediated inflammatory diseases, such as T-cell therapy. Thus, our study provides proof of concept for Tr1 cell-based therapies in experimental GN.

DISCERN: deep single-cell expression reconstruction for improved cell clustering and cell subtype and state detection.

BACKGROUND: Single-cell sequencing provides detailed insights into biological processes including cell differentiation and identity. While providing deep cell-specific information, the method suffers from technical constraints, most notably a limited number of expressed genes per cell, which leads to suboptimal clustering and cell type identification. RESULTS: Here, we present DISCERN, a novel deep generative network that precisely reconstructs missing single-cell gene expression using a reference dataset. DISCERN outperforms competing algorithms in expression inference resulting in greatly improved cell clustering, cell type and activity detection, and insights into the cellular regulation of disease. We show that DISCERN is robust against differences between batches and is able to keep biological differences between batches, which is a common problem for imputation and batch correction algorithms. We use DISCERN to detect two unseen COVID-19-associated T cell types, cytotoxic CD4+ and CD8+ Tc2 T helper cells, with a potential role in adverse disease outcome. We utilize T cell fraction information of patient blood to classify mild or severe COVID-19 with an AUROC of 80% that can serve as a biomarker of disease stage. DISCERN can be easily integrated into existing single-cell sequencing workflow. CONCLUSIONS: Thus, DISCERN is a flexible tool for reconstructing missing single-cell gene expression using a reference dataset and can easily be applied to a variety of data sets yielding novel insights, e.g., into disease mechanisms.

Single-cell multiomic analysis of thymocyte development reveals drivers of CD4+ T cell and CD8+ T cell lineage commitment.

The development of CD4+ T cells and CD8+ T cells in the thymus is critical to adaptive immunity and is widely studied as a model of lineage commitment. Recognition of self-peptide major histocompatibility complex (MHC) class I or II by the T cell antigen receptor (TCR) determines the CD8+ or CD4+ T cell lineage choice, respectively, but how distinct TCR signals drive transcriptional programs of lineage commitment remains largely unknown. Here we applied CITE-seq to measure RNA and surface proteins in thymocytes from wild-type and T cell lineage-restricted mice to generate a comprehensive timeline of cell states for each T cell lineage. These analyses identified a sequential process whereby all thymocytes initiate CD4+ T cell lineage differentiation during a first wave of TCR signaling, followed by a second TCR signaling wave that coincides with CD8+ T cell lineage specification. CITE-seq and pharmaceutical inhibition experiments implicated a TCR-calcineurin-NFAT-GATA3 axis in driving the CD4+ T cell fate. Our data provide a resource for understanding cell fate decisions and implicate a sequential selection process in guiding lineage choice.

DestVI identifies continuums of cell types in spatial transcriptomics data.

Most spatial transcriptomics technologies are limited by their resolution, with spot sizes larger than that of a single cell. Although joint analysis with single-cell RNA sequencing can alleviate this problem, current methods are limited to assessing discrete cell types, revealing the proportion of cell types inside each spot. To identify continuous variation of the transcriptome within cells of the same type, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI). Using simulations, we demonstrate that DestVI outperforms existing methods for estimating gene expression for every cell type inside every spot. Applied to a study of infected lymph nodes and of a mouse tumor model, DestVI provides high-resolution, accurate spatial characterization of the cellular organization of these tissues and identifies cell-type-specific changes in gene expression between different tissue regions or between conditions. DestVI is available as part of the open-source software package scvi-tools ( https://scvi-tools.org ).

Transjugular Intrahepatic Portosystemic Shunt: A Possible Risk Factor for Direct-Acting Antiviral Treatment Failure in Patients With Hepatitis C?

Direct-acting antiviral (DAA) therapies have revolutionized the treatment of chronic hepatitis C virus infection, achieving sustained virological response (SVR) rates of >90% even in patients with advanced liver cirrhosis. Having observed an unusual case of repeated DAA therapy failures in a patient with a transjugular intrahepatic portosystemic shunt (TIPS), we assessed a possible association between prior TIPS placement and DAA failure. A structured search of our clinical database revealed 10 patients who had received DAA therapy after TIPS placement. At the time of therapy, most patients (8; 80%) presented with a Child-Pugh score B, and the following DAA regimens were used: sofosbuvir/ledipasvir ± ribavirin (5 patients), sofosbuvir/daclatasvir ± ribavirin (3), sofosbuvir/velpatasvir (2), and sofosbuvir/velpatasvir/voxilaprevir (1). In total, 5 patients (50%) achieved an SVR, whereas a virological relapse occurred in the other half of the cases, including 2 patients with multiple relapses. In this patient cohort, SVR rates were unusually low for all regimens: sofosbuvir/ledipasvir ± ribavirin, 3/5 (60%); sofosbuvir/daclatasvir ± ribavirin, 2/3 (66%); sofosbuvir/velpatasvir, 0/2 (0%); and sofosbuvir/velpatasvir/voxilaprevir, 0/1 (0%), and patients with a TIPS made up a relevant proportion of DAA failures in patients with cirrhosis at our center: sofosbuvir/ledipasvir, 2/18 (11%); sofosbuvir/daclatasvir, 1/4 (25%); sofosbuvir/velpatasvir, 2/3 (66%); and sofosbuvir/velpatasvir/voxilaprevir, 1/1 (100%). Conclusion: We observed a high rate of virological relapse in patients with a TIPS who received DAA treatment and therefore postulate that TIPS placement may be a possible risk factor for DAA failure due to the profound hemodynamic changes evoked by the intervention. Longer treatment duration or addition of ribavirin might be warranted in these patients.

Liver fibrosis affects the targeting properties of drug delivery systems to macrophage subsets in vivo.

Myeloid immune cells promote inflammation and fibrosis in chronic liver diseases. Drug delivery systems, such as polymers, liposomes and microbubbles, efficiently target myeloid cells in healthy liver, but their targeting properties in hepatic fibrosis remain elusive. We therefore studied the biodistribution of three intravenously injected carrier material, i.e. 10 nm poly(N-(2-hydroxypropyl)methacrylamide) polymers, 100 nm PEGylated liposomes and 2000 nm poly(butyl cyanoacrylate) microbubbles, in two fibrosis models in immunocompetent mice. While whole-body imaging confirmed preferential hepatic uptake even after induction of liver fibrosis, flow cytometry and immunofluorescence analysis revealed markedly decreased carrier uptake by liver macrophage subsets in fibrosis, particularly for microbubbles and polymers. Importantly, carrier uptake co-localized with immune infiltrates in fibrotic livers, corroborating the intrinsic ability of the carriers to target myeloid cells in areas of inflammation. Of the tested carrier systems liposomes had the highest uptake efficiency among hepatic myeloid cells, but the lowest specificity for cellular subsets. Hepatic fibrosis affected carrier uptake in liver and partially in spleen, but not in other tissues (blood, bone marrow, lung, kidney). In conclusion, while drug carrier systems target distinct myeloid cell populations in diseased and healthy livers, hepatic fibrosis profoundly affects their targeting efficiency, supporting the need to adapt nanomedicine-based approaches in chronic liver disease.

The CCR2+ Macrophage Subset Promotes Pathogenic Angiogenesis for Tumor Vascularization in Fibrotic Livers.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) typically arises in fibrotic or cirrhotic livers, which are characterized by pathogenic angiogenesis. Myeloid immune cells, specifically tumor-associated macrophages (TAMs), may represent potential novel therapeutic targets in HCC, complementing current ablative or immune therapies. However, the detailed functions of TAM subsets in hepatocarcinogenesis have remained obscure. METHODS: TAM subsets were analyzed in-depth in human HCC samples and a combined fibrosis-HCC mouse model, established by i.p. injection with diethylnitrosamine after birth and repetitive carbon tetrachloride (CCl4) treatment for 16 weeks. Based on comprehensively phenotyping TAM subsets (fluorescence-activated cell sorter, transcriptomics) in mice, the function of CCR2+ TAM was assessed by a pharmacologic chemokine inhibitor. Angiogenesis was evaluated by contrast-enhanced micro-computed tomography and histology. RESULTS: We show that human CCR2+ TAM accumulate at the highly vascularized HCC border and express the inflammatory marker S100A9, whereas CD163+ immune-suppressive TAM accrue in the HCC center. In the fibrosis-cancer mouse model, we identified 3 major hepatic myeloid cell populations with distinct messenger RNA profiles, of which CCR2+ TAM particularly showed activated inflammatory and angiogenic pathways. Inhibiting CCR2+ TAM infiltration using a pharmacologic chemokine CCL2 antagonist in the fibrosis-HCC model significantly reduced pathogenic vascularization and hepatic blood volume, alongside attenuated tumor volume. CONCLUSIONS: The HCC microenvironment in human patients and mice is characterized by functionally distinct macrophage populations, of which the CCR2+ inflammatory TAM subset has pro-angiogenic properties. Understanding the functional differentiation of myeloid cell subsets in chronically inflamed liver may provide novel opportunities for modulating hepatic macrophages to inhibit tumor-promoting pathogenic angiogenesis.

Molecular and functional heterogeneity of IL-10-producing CD4+ T cells.

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4+ T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3neg CD4+ T cells that displays regulatory activity unlike other IL-10-producing CD4+ T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4+ T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3neg CD4+ T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.

Targeting and Modulation of Liver Myeloid Immune Cells by Hard-Shell Microbubbles.

Poly n-butylcyanoacrylate (PBCA)-based hard-shell microbubbles (MB) have manifold biomedical applications, including targeted drug delivery or contrast agents for ultrasound (US)-based liver imaging. MB and their fragments accumulate in phagocytes, especially in the liver, but it is unclear if MB affect the function of these immune cells. We herein show that human primary monocytes internalize different PBCA-MB by phagocytosis, which transiently inhibits monocyte migration in vertical chemotaxis assays and renders monocytes susceptible to cytotoxic effects of MB during US-guided destruction. Conversely, human macrophage viability and function, including cytokine release and polarization, remain unaffected after MB uptake. After i.v. injection in mice, MB predominantly accumulate in liver, especially in hepatic phagocytes (monocytes and Kupffer cells). Despite efficiently targeting myeloid immune cells in liver, MB or MB after US-elicited burst do not cause overt hepatotoxicity or inflammation. Furthermore, MB application with or without US-guided burst does not aggravate the course of experimental liver injury in mice or the inflammatory response to liver injury in vivo. In conclusion, PBCA-MB have immunomodulatory effects on primary human myeloid cells in vitro, but do not provoke hepatotoxicity, inflammation or altered response to liver injury in vivo, suggesting the safety of these MB for diagnostic and therapeutic purposes.

Targeting distinct myeloid cell populations in vivo using polymers, liposomes and microbubbles.

Identifying intended or accidental cellular targets for drug delivery systems is highly relevant for evaluating therapeutic and toxic effects. However, limited knowledge exists on the distribution of nano- and micrometer-sized carrier systems at the cellular level in different organs. We hypothesized that clinically relevant carrier materials, differing in composition and size, are able to target distinct myeloid cell subsets that control inflammatory processes, such as macrophages, neutrophils, monocytes and dendritic cells. Therefore, we analyzed the biodistribution and in vivo cellular uptake of intravenously injected poly(N-(2-hydroxypropyl) methacrylamide) polymers, PEGylated liposomes and poly(butyl cyanoacrylate) microbubbles in mice, using whole-body imaging (computed tomography - fluorescence-mediated tomography), intra-organ imaging (intravital multi-photon microscopy) and cellular analysis (flow cytometry of blood, liver, spleen, lung and kidney). While the three carrier materials shared accumulation in tissue macrophages in liver and spleen, they notably differed in uptake by other myeloid subsets. Kupffer cells and splenic red pulp macrophages rapidly take up microbubbles. Liposomes efficiently reach dendritic cells in liver, lung and kidney. Polymers exhibit the longest circulation half-life and target endothelial cells in the liver, neutrophils and alveolar macrophages. The identification of such previously unrecognized target cell populations might open up new avenues for more efficient drug delivery.

Chemokine (C-C motif) receptor 2-positive monocytes aggravate the early phase of acetaminophen-induced acute liver injury.

Acetaminophen (APAP, paracetamol) poisoning is a leading cause of acute liver failure (ALF) in humans and induces hepatocyte necrosis, followed by activation of the innate immune system, further aggravating liver injury. The role of infiltrating monocytes during the early phase of ALF is still ambiguous. Upon experimental APAP overdose in mice, monocyte-derived macrophages (MoMFs) massively accumulated in injured liver within 12-24 hours, whereas the number of tissue-resident macrophages (Kupffer cells) decreased. Influx of MoMFs is dependent on the chemokine receptor, chemokine (C-C motif) receptor 2 (CCR2), given that Ccr2-/- mice display reduced infiltration of monocytes and attenuated liver injury post-APAP overdose at early time points. As evidenced by intravital multiphoton microscopy of Ccr2 reporter mice, CCR2+ monocytes infiltrate liver as early as 8-12 hours post-APAP overdose and form dense cellular clusters around necrotic areas. CCR2+ MoMFs express a distinct pattern of inflammatory, but also repair-associated, genes in injured livers. Adoptive transfer experiments revealed that MoMFs primarily exert proinflammatory functions early post-APAP, thereby aggravating liver injury. Consequently, early pharmacological inhibition of either chemokine (C-C motif) ligand (CCL2; by the inhibitor, mNOX-E36) or CCR2 (by the orally available dual CCR2/CCR5 inhibitor, cenicriviroc) reduces monocyte infiltration and APAP-induced liver injury (AILI) in mice. Importantly, neither the early nor continuous inhibition of CCR2 hinder repair processes during resolution from injury. In line with this, human livers of ALF patients requiring liver transplantation reveal increased CD68+ hepatic macrophage numbers with massive infiltrates of periportal CCR2+ macrophages that display a proinflammatory polarization. CONCLUSION: Infiltrating monocyte-derived macrophages aggravate APAP hepatotoxicity, and the pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential by reducing the inflammatory reaction during the early phase of AILI. (Hepatology 2016;64:1667-1682).

M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.

Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring. The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange. To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4). CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Liver inflammation abrogates immunological tolerance induced by Kupffer cells.

UNLABELLED: The liver is essential for inducing immunological tolerance toward harmless antigens to maintain immune system homeostasis. However, the precise cellular mechanisms of tolerance induction against particle-bound antigens, the role of the local hepatic microenvironment, and implications for therapeutic targets in immune-mediated diseases are currently unclear. In order to elucidate cellular mechanisms of tolerance induction in healthy and injured liver, we developed a novel in vivo system combining the systemic delivery of low-dose peptide antigens coupled to inert particles, immunological readouts, and sophisticated intravital multiphoton microscopy-based imaging of liver in mice. We show that liver resident macrophages, Kupffer cells (KCs), but not hepatic monocyte-derived macrophages or dendritic cells (DCs), are the central cellular scavenger for circulating particle-associated antigens in homeostasis. KC-associated antigen presentation induces CD4 T-cell arrest, expansion of naturally occurring Foxp3(+) CD25(+) interleukin-10-producing antigen-specific regulatory T cells (Tregs) and tolerogenic immunity. Particle-associated tolerance induction in the liver protected mice from kidney inflammation in T-cell-mediated glomerulonephritis, indicating therapeutic potential of targeting KC for immune-mediated extrahepatic disorders. Liver inflammation in two independent experimental models of chronic liver injury and fibrosis abrogated tolerance induction and led to an immunogenic reprogramming of antigen-specific CD4 T cells. In injured liver, infiltrating monocyte-derived macrophages largely augment the hepatic phagocyte compartment, resulting in antigen redistribution between myeloid cell populations and, simultaneously, KCs lose signature markers of their tolerogenic phenotype. CONCLUSIONS: Hepatic induction of tissue-protective immunological tolerance against particulate antigens is dependent on KCs as well as on a noninflamed liver microenvironment, thereby providing mechanistic explanations for the clinical observation of immune dysfunction and tolerance break in patients with advanced liver diseases.