Multiomics uncovers developing immunological lineages in human

The development of the human immune system during embryonic and fetal life has historically been difficult to research due to limited access to human tissue. Experimental animal models have been widely used to study development but cellular and molecular programmes may not be conserved across species. The advent of multiomic single-cell technologies and an increase in human developmental tissue biobank resources have facilitated single-cell multiomic studies focused on human immune development. A critical question in the near future is "How do we best reconcile scientific findings across multiple omic modalities, developmental time, and organismic space?" In this review, we discuss the application of single-cell multiomic technologies to unravel the major cellular lineages in the prenatal human immune system. We also identify key areas where the combined power of multiomics technologies can be leveraged to address specific immunological gaps in our current knowledge and explore new research horizons in human development.     

Bioinformatics for study of autoimmunity

Recent years have witnessed an explosive growth in available biological data pertaining to autoimmunity research. This includes a tremendous quantity of sequence data (biological structures, genetic and physical maps, pathways, etc.) generated by genome and proteome projects plus extensive clinical and epidemiological data. Autoimmunity research stands to greatly benefit from this data so long as appropriate strategies are available to enable full access to and utilization of this data. The quantity and complexity of this biological data necessitates use of advanced bioinformatics strategies for its efficient retrieval, analysis and interpretation. Major progress has been made in development of specialized tools for storage, analysis and modeling of immunological data, and this has led to development of a whole new field know as immunoinformatics. With advances in novel high-throughput immunology technologies immunoinformatics is transforming understanding of how the immune system functions. This paper reviews advances in the field of immunoinformatics pertinent to autoimmunity research including databases, tools in genomics and proteomics, tools for study of B- and T-cell epitopes, integrative approaches, and web servers.     

Bioinformatics for study of autoimmunity

Recent years have witnessed an explosive growth in available biological data pertaining to autoimmunity research. This includes a tremendous quantity of sequence data (biological structures, genetic and physical maps, pathways, etc.) generated by genome and proteome projects plus extensive clinical and epidemiological data. Autoimmunity research stands to greatly benefit from this data so long as appropriate strategies are available to enable full access to and utilization of this data. The quantity and complexity of this biological data necessitates use of advanced bioinformatics strategies for its efficient retrieval, analysis and interpretation. Major progress has been made in development of specialized tools for storage, analysis and modeling of immunological data, and this has led to development of a whole new field know as immunoinformatics. With advances in novel high-throughput immunology technologies immunoinformatics is transforming understanding of how the immune system functions. This paper reviews advances in the field of immunoinformatics pertinent to autoimmunity research including databases, tools in genomics and proteomics, tools for study of B- and T-cell epitopes, integrative approaches, and web servers.     

Measuring Ag-specific immune responses: understanding immunopathogenesis and improving diagnostics in infectious disease, autoimmunity and cancer

Characterization of antigen-specific immune responses at the single-cell level has been made possible by recent advancements in reagent and technology development, combined with increasing knowledge of molecular mechanisms. Fluorescently labelled MHC-peptide multimers and antigens identify directly specific T and B cells, respectively, whereas dynamic assays exploit mediator production or secretion, or the changes in surface expression of other proteins, to identify specific lymphocytes--some techniques enabling the recovery of viable cells. Meanwhile, multiparameter flow cytometry has emerged as the most versatile platform for integrating most of these methods. As the complexity of experimental data increases, so does the level of technical sophistication required for analysis and interpretation, both in terms of basic research and modern medicine, with new applications for infectious diseases, autoimmunity and cancer.     

Measuring single-cell protein secretion in immunology: Technologies,advances, and applications

The dynamics, nature, strength, and ultimately protective capabilities of an active immune response are determined by the extracellular constitution and concentration of various soluble factors. Generated effector cells secrete such mediators, including antibodies, chemo- and cytokines to achieve functionality. These secreted factors organize the individual immune cells into functional tissues, initiate, orchestrate, and regulate the immune response. Therefore, a single-cell resolved analysis of protein secretion is a valuable tool for studying the heterogeneity and functionality of immune cells. This review aims to provide a comparative overview of various methods to characterize immune reactions by measuring single-cell protein secretion. Spot-based and cytometry-based assays, such as ELISpot and flow cytometry, respectively, are well-established methods applied in basic research and clinical settings. Emerging novel technologies, such as microfluidic platforms, offer new ways to measure and exploit protein secretion in immune reactions. Further technological advances will allow the deciphering of protein secretion in immunological responses with unprecedented detail, linking secretion to functionality. Here, we summarize the development and recent advances of tools that allow the analysis of protein secretion at the single-cell level, and discuss and contrast their applications within immunology.     

基于液质联用的单细胞蛋白质组学研究进展

蛋白质是细胞功能的主要执行者,由于其无法在体外进行扩增,单细胞蛋白质组学技术相较单细胞基因组学和转录组学技术而言发展相对滞后。传统的蛋白质组学技术可获得大量细胞蛋白表达的平均值,但忽略了细胞亚型及细胞异质性等信息。单细胞水平的蛋白质分析有助于阐明细胞不同表型与异质性的分子基础。随着质谱仪的快速发展,基于质谱的方法将单细胞蛋白质组学推向新的高度。本文综述了近年来基于液质联用方法的单细胞蛋白质组学在单细胞挑选、样品前处理、同位素标签技术、肽段分离、质谱采集、数据分析等方面的研究进展,及其在生物医学研究中的应用,并对未来单细胞蛋白质组学面临的挑战和发展前景进行了展望。单细胞蛋白质组学技术的进步将为生物医学研究领域提供新的思路和解决方案,并加深我们对人类健康和疾病的理解。     

Dendritic Cells in the Context of Human Tumors: Biology and Experimental Tools

Dendritic cells (DC) are the most potent and versatile antigen-presenting cells (APC) in the immune system. DC have an exceptional ability to comprehend the immune context of a captured antigen based on molecular signals identified from its vicinity. The analyzed information is then conveyed to other immune effector cells. Such capability enables DC to play a pivotal role in mediating either an immunogenic response or immune tolerance towards an acquired antigen. This review summarizes current knowledge on DC in the context of human tumors. It covers the basics of human DC biology, elaborating on the different markers, morphology and function of the different subsets of human DC. Human blood-borne DC are comprised of at least three subsets consisting of one plasmacytoid DC (pDC) and two to three myeloid DC (mDC) subsets. Some tissues have unique DC. Each subset has a different phenotype and function and may induce pro-tumoral or anti-tumoral effects. The review also discusses two methods fundamental to the research of DC on the single-cell level: multicolor flow cytometry (FCM) and image-based cytometry (IC). These methods, along with new genomics and proteomics tools, can provide high-resolution information on specific DC subsets and on immune and tumor cells with which they interact. The different layers of collected biological data may then be integrated using Immune-Cytomics modeling approaches. Such novel integrated approaches may help unravel the complex network of cellular interactions that DC carry out within tumors, and may help harness this complex immunological information into the development of more effective treatments for cancer.     

Genetic predisposition to autoimmunity--what have we learned?

Rapid advances in genetic technologies have led to the identification of more than 85 loci that contribute to susceptibility to autoimmune diseases. These susceptibility genes are distributed throughout the innate and adaptive immune systems, indicating that dysregulations in both immune systems participate in the development of autoimmunity. A significant subset of these susceptibility genes are shared between multiple autoimmune diseases. However, the dysregulation of specific pathways, such as the pathogen recognition receptors of the innate immune system and the TNF supergene family, are significantly involved in some autoimmune diseases. Although these findings dramatically increase the details available concerning the nature of genetic predisposition to autoimmunity, a mechanistic understanding of the processes involved has not been achieved. Future studies must focus on correlating phenotypes with specific genotypes to improve our understanding of the immune processes that are dysregulated during the development of autoimmunity.     

Macrophage subsets in atherosclerosis as defined by single-cell technologies

Macrophages play a major role in the pathogenesis of atherosclerosis. Many studies have shone light on the different phenotypes and functions that macrophages can acquire upon exposure to local cues. The microenvironment of the atherosclerotic plaque contains a plethora of macrophage-controlling factors, such as cytokines, oxidised low-density lipoproteins and cell debris. Previous research has determined macrophage function within the plaque mainly by using immunohistochemistry and bulk analysis. The recent development and rapid progress of single-cell technologies, such as cytometry by time of flight and single-cell RNA sequencing, now enable comprehensive mapping of the wide range of cell types and their phenotypes present in atherosclerotic plaques. In this review we discuss recent advances applying these technologies in defining macrophage subsets residing in the atherosclerotic arterial wall of mice and men. Resulting from these studies, we describe three main macrophage subsets: resident-like, pro-inflammatory and anti-inflammatory foamy TREM2^hi macrophages, which are found in both mouse and human atherosclerotic plaques. Furthermore, we discuss macrophage subset-specific markers and functions. More insights into the characteristics and phenotype of immune cells within the atherosclerotic plaque may guide future clinical approaches to treat disease. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

FcγRIIB and autoimmunity

Autoimmune diseases are characterized by adaptive immune responses against self-antigens, including humoral responses resulting in the production of autoantibodies. Autoantibodies generate inflammation by activating complement and engaging Fcγ receptors (FcγRs). The inhibitory receptor FcγRIIB plays a central role in regulating the generation of autoantibodies and their effector functions, which include activation of innate immune cells and the cellular arm of the adaptive immune system, via effects on antigen presentation to CD4 T cells. Polymorphisms in FcγRIIB have been associated with susceptibility to autoimmunity but protection against infections in humans and mice. In the last few years, new mechanisms by which FcγRIIB controls the adaptive immune response have been described. Notably, FcγRIIB has been shown to regulate germinal center B cells and dendritic cell migration, with potential impact on the development of autoimmune diseases. Recent work has also highlighted the implication of FcγRIIB on the regulation of the innate immune system, via inhibition of Toll-like receptor- and complement receptor-mediated activation. This review will provide an update on the role of FcγRIIB in adaptive immune responses in autoimmunity, and then focus on their emerging function in innate immunity.     

Cellular stress and innate inflammation in organ-specific autoimmunity: lessons learned from vitiligo

For decades, research in autoimmunity has focused primarily on immune contributions to disease. Yet recent studies report elevated levels of reactive oxygen species and abnormal activation of the unfolded protein response in cells targeted by autoimmunity, implicating cellular stress originating from the target tissue as a contributing factor. A better understanding of this contribution may help to answer important lingering questions in organ-specific autoimmunity, as to what factors initiate disease and what directs its tissue specificity. Vitiligo, an autoimmune disease of the skin, has been the focus of translational research for over 30 years, and both melanocyte stress and immune mechanisms have been thought to be mutually exclusive explanations for pathogenesis. Chemical-induced vitiligo is a unique clinical presentation that reflects the importance of environmental influences on autoimmunity, provides insight into a new paradigm linking cell stress to the immune response, and serves as a template for other autoimmune diseases. In this review, I will discuss the evidence for cell stress contributions to a number of autoimmune diseases, the questions that remain, and how vitiligo, an underappreciated example of organ-specific autoimmunity, helps to answer them.     

Toll-like receptors: paving the path to T cell-driven autoimmunity?

The development of autoimmunity is often associated with the presence of pathogen-associated molecular patterns (PAMPs) and signaling through toll-like receptors (TLRs). Largely, the importance of PAMP-TLR ligation has been attributed to inducing the maturation of antigen-presenting cells and production of proinflammatory cytokines and chemokines. Recent evidence now shows that PAMPs can activate effector and regulatory T cells revealing a further level of complexity in the development of autoimmunity. TLR signaling on T cells acts as a form of costimulation, lowering the 'strength of signal' required for proliferation and survival. This apparent mechanism of immune homeostasis may break tolerance or anergy upon pathogen infection and promote the development of immune responses against self-antigens.     

Epigenetic regulation of the immune system in health and disease

Epigenetics comprises various mechanisms that mold chromatin structures and regulate gene expression with stability, thus defining cell identity and function and adapting cells to environmental changes. Alteration of these mechanisms contributes to the inception of various pathological conditions. Given the complexity of the immune system, one would predict that a higher-order, supragenetic regulation is indispensable for generation of its constituents and control of its functions. Here, we summarize various aspects of immune system physiology and pathology in which epigenetic pathways have been implicated. Increasing knowledge in this field, together with the development of specific tools with which to manipulate epigenetic pathways, might form a basis for new strategies of immune function modulation, both to optimize immune therapies for infections or cancer and to control immune alterations in aging or autoimmunity.     

Immunomodulation under the lens of real-time in vivo imaging

Modulation of cells and molecules of the immune system not only represents a major opportunity to treat a variety of diseases including infections, cancer, autoimmune, and inflammatory disorders but could also help understand the intricacies of immune responses. A detailed mechanistic understanding of how a specific immune intervention may provide clinical benefit is essential for the rational design of efficient immunomodulators. Visualizing the impact of immunomodulation in real-time and in vivo has emerged as an important approach to achieve this goal. In this review, we aim to illustrate how multiphoton intravital imaging has helped clarify the mode of action of immunomodulatory strategies such as antibodies or cell therapies. We also discuss how optogenetics combined with imaging will further help manipulate and precisely understand immunomodulatory pathways. Combined with other single-cell technologies, in vivo dynamic imaging has therefore a major potential for guiding preclinical development of immunomodulatory drugs.     

Yin and Yang of Biofilm Formation and Cyclic di-GMP Signaling of the Gastrointestinal Pathogen Salmonella enterica Serovar Typhimurium

Within the last 60 years, microbiological research has challenged many dogmas such as bacteria being unicellular microorganisms directed by nutrient sources; these investigations produced new dogmas such as cyclic diguanylate monophosphate (cyclic di-GMP) second messenger signaling as a ubiquitous regulator of the fundamental sessility/motility lifestyle switch on the single-cell level. Successive investigations have not yet challenged this view; however, the complexity of cyclic di-GMP as an intracellular bacterial signal, and, less explored, as an extracellular signaling molecule in combination with the conformational flexibility of the molecule, provides endless opportunities for cross-kingdom interactions. Cyclic di-GMP-directed microbial biofilms commonly stimulate the immune system on a lower level, whereas host-sensed cyclic di-GMP broadly stimulates the innate and adaptive immune responses. Furthermore, while the intracellular second messenger cyclic di-GMP signaling promotes bacterial biofilm formation and chronic infections, oppositely, Salmonella Typhimurium cellulose biofilm inside immune cells is not endorsed. These observations only touch on the complexity of the interaction of biofilm microbial cells with its host. In this review, we describe the Yin and Yang interactive concepts of biofilm formation and cyclic di-GMP signaling using S. Typhimurium as an example.     

Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: A critical review

Herein we will review the role of glycans in the immune system. Specific topics covered include: the glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode “self” identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migration pattern of immune cells; and how the combination of Fc N-glycans and Ig isotype dictate the effector function of immunoglobulins. We speculate that the latter may be responsible for the well-documented association between alterations of the serum glycome and autoimmunity. Due to technological limitations, the extent of these autoimmune-associated glycan alterations and their role in disease pathophysiology has not been fully elucidated. Thus, we also review the current technologies available for glycan analysis, placing an emphasis on Multiple Reaction Monitoring (MRM), a rapid high-throughput technology that has great potential for glycan biomarker research. Finally, we put forth The Altered Glycan Theory of Autoimmunity, which states that each autoimmune disease will have a unique glycan signature characterized by the site-specific relative abundances of individual glycan structures on immune cells and extracellular proteins, especially the site-specific glycosylation patterns of the different immunoglobulin(Ig) classes and subclasses.     

单细胞转录组学技术在肢体再生研究中的应用

蝾螈具有极强的肢体再生能力,爪蟾在发育过程中再生能力则逐渐下降,而人类和其它哺乳动物的成年个体没有肢体再生能力。借助单细胞转录组学技术可以分析细胞转录水平的优势,以蝾螈、爪蟾等肢体可再生的动物为模型,近年研究已发现并鉴定了一系列调控肢体再生的关键基因、信号通路和细胞类群,提示再生能力存在潜在的进化保守机制。单细胞转录组学技术在跨物种研究中的应用,使得免疫细胞、成纤维细胞类型和能量代谢机制对再生的重要影响得到进一步挖掘,再生组织细胞来源和异质性得以进一步明确,为解释再生相关机制奠定理论依据。单细胞多组学技术的发展和应用将有望为进一步研究有效提升哺乳动物再生能力指明方向。