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Highly effective vaccines elicit specific, robust, and durable adaptive immune responses. To advance informed vaccine design, it is critical that we understand the cellular dynamics underlying responses to different antigen formats. Here, we sought to understand how antigen-specific B and T cells were activated and participated in adaptive immune responses within the mucosal site. Using a human tonsil organoid model, we tracked the differentiation and kinetics of the adaptive immune response to influenza vaccine and virus modalities. Each antigen format elicited distinct B and T cell responses, including differences in their magnitude, diversity, phenotype, function, and breadth. These differences culminated in substantial changes in the corresponding antibody response. A major source of antigen format-related variability was the ability to recruit naive vs. memory B and T cells to the response. These findings have important implications for vaccine design and the generation of protective immune responses in the upper respiratory tract.

Impressive advances have been made to replicate human physiology in vitro over the last few years due to the growth of the organ-on-chip (OoC) field in both industrial and academic settings. OoCs are a type of micro physiological system (MPS) that imitates functional and dynamic aspects of native human organ biology on a microfluidic device. Organoids and organotypic models, ranging in their complexity from simple single-cell to complex multi-cell type constructs, are being incorporated into OoC microfluidic devices to better mimic human physiology. OoC technology has now progressed to the stage at which it has received official recognition by the Food and Drug Administration (FDA) for use as an alternative to standard procedures in drug development, such as animal studies and traditional in vitro assays. However, an area that is still lagging behind is the incorporation of the immune system, which is a critical element required to investigate human health and disease. In this review, we summaries the progress made to integrate human immunology into various OoC systems, specifically focusing on models related to organ barriers and lymphoid organs. These models utilize microfluidic devices that are either commercially available or custom made. This review explores the difference between the use of innate and adaptive immune cells and their role for modelling organ-specific diseases in OoCs. Immunocompetent multi-OoC models are also highlighted and the extent to which they recapitulate systemic physiology is discussed. Together, the aim of this review is to describe the current state of immune-OoCs, the limitations and the future perspectives needed to improve the field. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to almost seven million deaths worldwide. SARS-CoV-2 causes infection through respiratory transmission and can occur either without any symptoms or with clinical manifestations which can be mild, severe or, in some cases, even fatal. Innate immunity provides the initial defense against the virus by sensing pathogen-associated molecular patterns and triggering signaling pathways that activate the antiviral and inflammatory responses, which limit viral replication and help the identification and removal of infected cells. However, temporally dysregulated and excessive activation of the innate immune response is deleterious for the host and associates with severe COVID-19. In addition to its defensive role, innate immunity is pivotal in priming the adaptive immune response and polarizing its effector function. This capacity is relevant in the context of both SARS-CoV-2 natural infection and COVID-19 vaccination. Here, we provide an overview of the current knowledge of the innate immune responses to SARS-CoV-2 infection and vaccination. [ABSTRACT FROM AUTHOR]

Impressive advances have been made to replicate human physiology in vitro over the last few years due to the growth of the organ-on-chip (OoC) field in both industrial and academic settings. OoCs are a type of microphysiological system (MPS) that imitates functional and dynamic aspects of native human organ biology on a microfluidic device. Organoids and organotypic models, ranging in their complexity from simple single-cell to complex multi-cell type constructs, are being incorporated into OoC microfluidic devices to better mimic human physiology. OoC technology has now progressed to the stage at which it has received official recognition by the Food and Drug Administration (FDA) for use as an alternative to standard procedures in drug development, such as animal studies and traditional in vitro assays. However, an area that is still lagging behind is the incorporation of the immune system, which is a critical element required to investigate human health and disease. In this review, we summarise the progress made to integrate human immunology into various OoC systems, specifically focusing on models related to organ barriers and lymphoid organs. These models utilise microfluidic devices that are either commercially available or custom-made. This review explores the difference between the use of innate and adaptive immune cells and their role for modelling organ-specific diseases in OoCs. Immunocompetent multi-OoC models are also highlighted and the extent to which they recapitulate systemic physiology is discussed. Together, the aim of this review is to describe the current state of immune-OoCs, the limitations and the future perspectives needed to improve the field.

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

Immune responses in human tissues rely on the concerted action of different cell types. Inter-cellular communication shapes both the function of the multicellular interaction networks and the fate of the individual cells that comprise them. With the advent of new methods to profile and experimentally perturb primary human tissues, we are now in a position to systematically identify and mechanistically dissect these cell-cell interactions and their modulators. Here, we introduce the concept of multicellular hubs, functional modules of immune responses in tissues. We outline a roadmap to discover multicellular hubs in human tissues and discuss how emerging technologies may further accelerate progress in this field.

Spontaneous control of HIV infection has been repeatedly linked to antiviral CD8+ T cells but is not always permanent. To address mechanisms of durable and aborted control of viremia, we evaluated immunologic and virologic parameters longitudinally among 34 HIV-infected subjects with differential outcomes. Despite sustained recognition of autologous virus, HIV-specific proliferative and cytolytic T cell effector functions became selectively and intrinsically impaired prior to aborted control. Longitudinal transcriptomic profiling of functionally impaired HIV-specific CD8+ T cells revealed altered expression of genes related to activation, cytokine-mediated signaling, and cell cycle regulation, including increased expression of the antiproliferative transcription factor KLF2 but not of genes associated with canonical exhaustion. Lymphoid HIV-specific CD8+ T cells also exhibited poor functionality during aborted control relative to durable control. Our results identify selective functional impairment of HIV-specific CD8+ T cells as prognostic of impending aborted HIV control, with implications for clinical monitoring and immunotherapeutic strategies.

The human immune system is a distributed system of specialized cell populations with unique functions that collectively give rise to immune responses to infections and during immune-mediated diseases. Cell composition, plasma proteins, and functional responses vary among individuals, making the system difficult to interpret, but this variation is nonrandom. With careful analyses using novel experimental and computational tools, human immune system composition and function carry interpretable information. Here, we propose that systems-level analyses offer an opportunity to make human immune responses more interpretable in the future, and we discuss herein important considerations and lessons learned to this end. Predictable human immunology holds implications for better diagnostic and curative precision in patients with infectious and immune-associated diseases. Systems immunology is the study of cell–cell interactions in the immune system, their regulatory functions, and the emergent properties of immune responses. We posit that, despite large interindividual variation, cell composition is nonrandom, and understanding its rules will make human immune systems more predictable in the future. A growing number of computational tools exists for the inference of cell–cell relationships, but no 'gold standard' methods exist, and statistical power is often limiting. Minimizing the technical sources of variance by optimal experimental design and sampling procedures allows for more biological 'signals' to be discovered. A systems approach is necessary to comprehend the complexity of an organism's immune system since there is an underlying shared mechanism of cell–cell relationships. Better inferences of cell–cell interactions are essential to increase our understanding of immune responses for various health conditions. A well-planned longitudinal sampling of whole blood is vital in translating in vitro findings to in vivo biology, allowing the systematic tracking of immune system responses over time, and facilitating the development of more accurate models with improved predictability for immune system behaviors. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to almost seven million deaths worldwide. SARS-CoV-2 causes infection through respiratory transmission and can occur either without any symptoms or with clinical manifestations which can be mild, severe or, in some cases, even fatal. Innate immunity provides the initial defense against the virus by sensing pathogen-associated molecular patterns and triggering signaling pathways that activate the antiviral and inflammatory responses, which limit viral replication and help the identification and removal of infected cells. However, temporally dysregulated and excessive activation of the innate immune response is deleterious for the host and associates with severe COVID-19. In addition to its defensive role, innate immunity is pivotal in priming the adaptive immune response and polarizing its effector function. This capacity is relevant in the context of both SARS-CoV-2 natural infection and COVID-19 vaccination. Here, we provide an overview of the current knowledge of the innate immune responses to SARS-CoV-2 infection and vaccination.

Summary: Tissue‐resident memory T cells (TRM) represent a dedicated layer of localized immune memory in virtually every organ throughout the human body. By virtue of their long‐term residence in disparate tissues, TRM are shaped by a myriad of site‐specific influences and exhibit remarkable heterogeneity in form and function. Here, we review the major axes by which TRM vary, including their surface phenotypes, transcriptional programming, and the tissue‐specific adaptations that accrue over their tenancy. We discuss how localization within distinct anatomic niches both within and across major organ systems shapes TRM identity and examine mechanisms and prevailing models for TRM generation. Understanding the drivers of differentiation, function and maintenance of the various subpopulations that together define the TRM lineage may hold the key to unlocking the full potential of TRM to promote localized and protective tissue immunity throughout the body. [ABSTRACT FROM AUTHOR]

The lymph node is a highly structured organ that mediates the body’s adaptive immune response to antigens and other foreign particles. Central to its function is the distinct spatial assortment of lymphocytes and stromal cells, as well as chemokines that drive the signaling cascades which underpin immune responses. Investigations of lymph node biology were historically explored in vivo in animal models, using technologies that were breakthroughs in their time such as immunofluorescence with monoclonal antibodies, genetic reporters, in vivo two-photon imaging, and, more recently spatial biology techniques. However, new approaches are needed to enable tests of cell behavior and spatiotemporal dynamics under well controlled experimental perturbation, particularly for human immunity. This review presents a suite of technologies, comprising in vitro, ex vivo and in silico models, developed to study the lymph node or its components. We discuss the use of these tools to model cell behaviors in increasing order of complexity, from cell motility, to cell-cell interactions, to organ-level functions such as vaccination. Next, we identify current challenges regarding cell sourcing and culture, real time measurements of lymph node behavior in vivo and tool development for analysis and control of engineered cultures. Finally, we propose new research directions and offer our perspective on the future of this rapidly growing field. We anticipate that this review will be especially beneficial to immunologists looking to expand their toolkit for probing lymph node structure and function. [ABSTRACT FROM AUTHOR]

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT.

Gut microbiota has extensive and tremendous impacts on human physiology and pathology. The regulation of microbiota is therefore a cardinal problem for the mutualistic relationship, as both microbial overgrowth and excessive immune reactions toward them could potentially be detrimental to host homeostasis. Growing evidence suggests that IgA, the most dominant secretory immunoglobulin in the intestine, regulates the colonization of commensal microbiota, and consequently, the microbiota-mediated intestinal and extra-intestinal diseases. In this review, we discuss the interactions between IgA and gut microbiota particularly relevant to human pathophysiology. We review current knowledge about how IgA regulates gut microbiota in humans and about the molecular mechanisms behind this interaction. We further discuss the potential role of IgA in regulating human diseases by extrapolating experimental findings, suggesting that IgA can be a future therapeutic strategy that functionally modulates gut microbiota. [ABSTRACT FROM AUTHOR]

While meta-analysis has demonstrated increased statistical power and more robust estimations in studies, the application of this commonly accepted methodology to cytometry data has been challenging. Different cytometry studies often involve diverse sets of markers. Moreover, the detected values of the same marker are inconsistent between studies due to different experimental designs and cytometer configurations. As a result, the cell subsets identified by existing auto-gating methods cannot be directly compared across studies. We developed MetaCyto for automated meta-analysis of both flow and mass cytometry (CyTOF) data. By combining clustering methods with a silhouette scanning method, MetaCyto is able to identify commonly labeled cell subsets across studies, thus enabling meta-analysis. Applying MetaCyto across a set of ten heterogeneous cytometry studies totaling 2,926 samples enabled us to identify multiple cell populations exhibiting differences in abundance between demographic groups. Software is released to the public through Bioconductor (http://bioconductor.org/packages/release/bioc/html/MetaCyto.html).

While inbred mice have informed most of what we know about the immune system in the modern era, they have clear limitations with respect to their ability to be informative regarding genetic heterogeneity or microbial influences. They have also not been very predictive as models of human disease or vaccination results. Although there are concerted attempts to compensate for these flaws, the rapid rise of human studies, driven by both technical and conceptual advances, promises to fill in these gaps, as well as provide direct information about human diseases and vaccination responses. Work on human immunity has already provided important additional perspectives on basic immunology such as the importance of clonal deletion to self-tolerance, and while many challenges remain, it seems inevitable that "the human model" will continue to inform us about the immune system and even allow for the discovery of new mechanisms. A consideration of mouse human differences, with an emphasis on how emerging data on human immune responses, will enrich our knowledge of immunology generally and may reveal entirely new mechanisms. [ABSTRACT FROM AUTHOR]

Inborn errors of immunity (IEIs) are a group of more than 450 monogenic disorders that impair immune development and function. A subset of IEIs blend increased susceptibility to infection, autoimmunity, and malignancy and are known collectively as primary immune regulatory disorders (PIRDs). While many aspects of immune function are altered in PIRDs, one key impact is on T-cell function. By their nature, PIRDs provide unique insights into human T-cell signaling; alterations in individual signaling molecules tune downstream signaling pathways and effector function. Quantifying T-cell dysfunction in PIRDs and the underlying causative mechanisms is critical to identifying existing therapies and potential novel therapeutic targets to treat our rare patients and gain deeper insight into the basic mechanisms of T-cell function. Though there are many types of T-cell dysfunction, here we will focus on T-cell exhaustion, a key pathophysiological state. Exhaustion has been described in both human and mouse models of disease, where the chronic presence of antigen and inflammation (e.g., chronic infection or malignancy) induces a state of altered immune profile, transcriptional and epigenetic states, as well as impaired T-cell function. Since a subset of PIRDs amplify T-cell receptor (TCR) signaling and/or inflammatory cytokine signaling cascades, it is possible that they could induce T-cell exhaustion by genetically mimicking chronic infection. Here, we review the fundamentals of T-cell exhaustion and its possible role in IEIs in which genetic mutations mimic prolonged or amplified T-cell receptor and/or cytokine signaling. Given the potential insight from the many forms of PIRDs in understanding T-cell function and the challenges in obtaining primary cells from these rare disorders, we also discuss advances in CRISPR-Cas9 genome-editing technologies and potential applications to edit healthy donor T cells that could facilitate further study of mechanisms of immune dysfunctions in PIRDs. Editing T cells to match PIRD patient genetic variants will allow investigations into the mechanisms underpinning states of dysregulated T-cell function, including T-cell exhaustion.

Butyrate is a short‐chain fatty acid that is produced by commensal microbes within the intestinal microbiome through fermentation of dietary fibre. Microbial‐derived butyrate has been shown to promote immunologic and metabolic homeostasis, in part through its beneficial effects on mitochondrial function, and thus has been proposed as a possible anti‐inflammatory therapy. We tested the hypothesis that butyrate could mitigate the decrease in mitochondrial respiration in immune cells under septic conditions as a preliminary step towards better understanding the potential for butyrate as a novel therapy in sepsis. Mitochondrial respiration and content (measured as citrate synthase activity) were compared within four Epstein–Barr virus‐transformed lymphoblast (LB) cell lines exposed to either control media or lipopolysaccharide (LPS) 100 ng/ml. Both co‐incubation of LBs with LPS + butyrate and treatment with butyrate after LPS stimulation reversed the decrease in mitochondrial respiration observed in LBs exposed to LPS without butyrate. Neither LPS nor butyrate led to significant changes in citrate synthase activity. The preliminary findings support further investigation of a potential mitochondrial‐based mechanism through which butyrate may help to mitigate the immuno‐inflammatory response in sepsis. [ABSTRACT FROM AUTHOR]

Immune responses in human tissues rely on the concerted action of different cell types. Inter-cellular communication shapes both the function of the multicellular interaction networks and the fate of the individual cells that comprise them. With the advent of new methods to profile and experimentally perturb primary human tissues, we are now in a position to systematically identify and mechanistically dissect these cell-cell interactions and their modulators. Here, we introduce the concept of multicellular hubs, functional modules of immune responses in tissues. We outline a roadmap to discover multicellular hubs in human tissues and discuss how emerging technologies may further accelerate progress in this field. [ABSTRACT FROM AUTHOR]

The humanized bone marrow-liver-thymus (BLT) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain human leukocyte antigen (HLA) haplotypes or monogene deficiencies. To circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ-/-) recipient mice, we were able to expand one primary BLT mouse into a colony of 4-5 proBLT (propagated BLT) mice in 6-8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to those of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2-restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable preclinical research tool.

Immune responses in human tissues rely on the concerted action of different cell types. Inter-cellular communication shapes both the function of the multicellular interaction networks and the fate of the individual cells that comprise them. With the advent of new methods to profile and experimentally perturb primary human tissues, we are now in a position to systematically identify and mechanistically dissect these cell-cell interactions and their modulators. Here, we introduce the concept of multicellular hubs, functional modules of immune responses in tissues. We outline a roadmap to discover multicellular hubs in human tissues and discuss how emerging technologies may further accelerate progress in this field.

Multimodal single-cell profiling methods can capture immune cell variations unfolding over time at the molecular, cellular, and population levels. Transforming these data into biological insights remains challenging. Here, we introduce a framework to integrate variations at the human population and single-cell levels in vaccination responses. Comparing responses following AS03-adjuvanted versus unadjuvanted influenza vaccines with CITE-seq revealed AS03-specific early (day 1) response phenotypes, including a B cell signature of elevated germinal center competition. A correlated network of cell-type-specific transcriptional states defined the baseline immune status associated with high antibody responders to the unadjuvanted vaccine. Certain innate subsets in the network appeared "naturally adjuvanted," with transcriptional states resembling those induced uniquely by AS03-adjuvanted vaccination. Consistently, CD14+ monocytes from high responders at baseline had elevated phospho-signaling responses to lipopolysaccharide stimulation. Our findings link baseline immune setpoints to early vaccine responses, with positive implications for adjuvant development and immune response engineering. [Display omitted] • A framework for integrating human population and single-cell immune response variations • AS03 induces unique B cell survival and innate-sensing and activation states • Baseline immune setpoints are encoded as correlated networks of cell states • A high-responder baseline setpoint phenocopies cell states induced by AS03 Mulè et al. provide a framework for integrating human population and single-cell variations to study vaccine responses with and without the adjuvant AS03. They uncover AS03-specific B cell survival and innate-sensing signatures 1 day after vaccination. Furthermore, some AS03-induced response cell states are already present at baseline in "naturally adjuvanted" individuals who respond more robustly serologically to the unadjuvanted vaccine. [ABSTRACT FROM AUTHOR]

Background: CARD9 deficiency is an autosomal recessive primary immunodeficiency underlying increased susceptibility to fungal infection primarily presenting as invasive CNS Candida and/or cutaneous/invasive dermatophyte infections. More recently, a rare heterozygous dominant negative CARD9 variant c.1434 + 1G > C was reported to be protective from inflammatory bowel disease. Objective: We studied two siblings carrying homozygous CARD9 variants (c.1434 + 1G > C) and born to heterozygous asymptomatic parents. One sibling was asymptomatic and the other presented with candida esophagitis, upper respiratory infections, hypogammaglobulinemia, and low class-switched memory B cells. Methods and Results: The CARD9 c.1434 + 1G > C variant generated two mutant transcripts confirmed by mRNA and protein expression: an out-of-frame c.1358–1434 deletion/ ~ 55 kDa protein (CARD9Δex.11) and an in-frame c.1417–1434 deletion/ ~ 61 kDa protein (CARD9Δ18 nt.). Neither transcript was able to form a complete/functional CBM complex, which includes TRIM62. Based on the index patient's CVID-like phenotype, CARD9 expression was tested and detected in lymphocytes and monocytes from humans and mice. The functional impact of different CARD9 mutations and gene dosage conditions was evaluated in heterozygous and homozygous c.1434 + 1 G > C members of the index family, and in WT (two WT alleles), haploinsufficiency (one WT, one null allele), and null (two null alleles) individuals. CARD9 gene dosage impacted lymphocyte and monocyte functions including cytokine generation, MAPK activation, T-helper commitment, transcription, plasmablast differentiation, and immunoglobulin production in a differential manner. Conclusions: CARD9 exon 11 integrity is critical to CBM complex function. CARD9 is expressed and affects particular T and B cell functions in a gene dosage-dependent manner, which in turn may contribute to the phenotype of CARD9 deficiency. [ABSTRACT FROM AUTHOR]

Abstract Background Broadly, much of variance in immune system phenotype has been linked to the influence of non-heritable factors rather than genetics. In particular, two non-heritable factors: aging and human cytolomegavirus (CMV) infection, have been known to account for significant inter-individual immune variance. However, many specific relationships between them and immune composition remain unclear, especially between individuals over narrower age ranges. Further exploration of these relationships may be useful for informing personalized intervention development. Results To address this need, we evaluated 41 different cell type frequencies by mass cytometry and identified their relationships with aging and CMV seropositivity. Analyses were done using 60 healthy individuals, including 23 monozygotic twin pairs, categorized into young (12–31 years) and middle-aged (42–59 years). Aging and CMV discordance were associated with increased immune diversity between monozygotic twins overall, and particularly strongly in various T cell populations. Notably, we identified 17 and 11 cell subset frequencies as relatively influenced and uninfluenced by non-heritable factors, respectively, with results that largely matched those from studies on older-aged cohorts. Next, CD4+ T cell frequency was shown to diverge with age in twins, but with lower slope than in demographically similar non-twins, suggesting that much inter-individual variance in this cell type can be attributed to interactions between genetic and environmental factors. Several cell frequencies previously associated with memory inflation, such as CD27- CD8+ T cells and CD161+ CD4+ T cells, were positively correlated with CMV seropositivity, supporting findings that CMV infection may incur rapid aging of the immune system. Conclusions Our study confirms previous findings that aging, even within a relatively small age range and by mid-adulthood, and CMV seropositivity, both contribute significantly to inter-individual immune diversity. Notably, we identify several key immune cell subsets that vary considerably with aging, as well as others associated with memory inflation which correlate with CMV seropositivity.

Although small numbers of immune-mediated diseases are inherited due to rare genetic mutations, most are multifactorial diseases caused by multiple elements including genetic and environmental factors. In the case of autoimmune diseases, many disease-susceptibility genes, including several in the major histocompatibility gene complex, have been reported, and over the past 10 years, genome-wide association studies (GWAS) have been used to analyze disease-susceptibility loci in representative diseases. Furthermore, many disease-susceptibility variants have been found to be related to gene expression levels. The expression of genes involved in disease pathogenesis is often cell-type-specific, and this is closely related to epigenome alterations. Genomic information is present even before the onset of a disease and has a clear causal relationship to the disease (i.e. the outcome). Therefore, it is important to establish functional genetics in human immunology to understand the pathogenesis of diseases using these pieces of information. We can then apply these results to drug discovery. Here, we will review these issues, especially focusing on autoimmune diseases, and discuss current and future directions of human immune system research. [ABSTRACT FROM AUTHOR]

Professional antigen‐presenting cells (APCs), which include dendritic cells (DCs) and monocytes are essential for inducing and steering adaptive T‐cell responses. Recent technological developments in single‐cell analysis have significantly advanced our understanding of APC subset heterogeneity. To accurately resolve this functional diversity and to account for tissue‐specific adaptation, novel phenotyping markers have been described more recently. While some of these largely overlap with traditionally used markers, more fine‐grained phenotyping might be essential during inflammatory settings, where the traditional distinction between monocytes and dendritic cells has become blurred. Within this phenotype report, we provide a concise overview of traditional and recently described markers for the phenotyping of DCs and monocytes in the human system. [ABSTRACT FROM AUTHOR]

Background: Broadly, much of variance in immune system phenotype has been linked to the influence of non-heritable factors rather than genetics. In particular, two non-heritable factors: aging and human cytolomegavirus (CMV) infection, have been known to account for significant inter-individual immune variance. However, many specific relationships between them and immune composition remain unclear, especially between individuals over narrower age ranges. Further exploration of these relationships may be useful for informing personalized intervention development. Results: To address this need, we evaluated 41 different cell type frequencies by mass cytometry and identified their relationships with aging and CMV seropositivity. Analyses were done using 60 healthy individuals, including 23 monozygotic twin pairs, categorized into young (12–31 years) and middle-aged (42–59 years). Aging and CMV discordance were associated with increased immune diversity between monozygotic twins overall, and particularly strongly in various T cell populations. Notably, we identified 17 and 11 cell subset frequencies as relatively influenced and uninfluenced by non-heritable factors, respectively, with results that largely matched those from studies on older-aged cohorts. Next, CD4+ T cell frequency was shown to diverge with age in twins, but with lower slope than in demographically similar non-twins, suggesting that much inter-individual variance in this cell type can be attributed to interactions between genetic and environmental factors. Several cell frequencies previously associated with memory inflation, such as CD27- CD8+ T cells and CD161+ CD4+ T cells, were positively correlated with CMV seropositivity, supporting findings that CMV infection may incur rapid aging of the immune system. Conclusions: Our study confirms previous findings that aging, even within a relatively small age range and by mid-adulthood, and CMV seropositivity, both contribute significantly to inter-individual immune diversity. Notably, we identify several key immune cell subsets that vary considerably with aging, as well as others associated with memory inflation which correlate with CMV seropositivity. [ABSTRACT FROM AUTHOR]

Summary: Severe disease of SARS-CoV-2 is characterized by vigorous inflammatory responses in the lung, often with a sudden onset after 5–7 days of stable disease. Efforts to modulate this hyperinflammation and the associated acute respiratory distress syndrome rely on the unraveling of the immune cell interactions and cytokines that drive such responses. Given that every patient is captured at different stages of infection, longitudinal monitoring of the immune response is critical and systems-level analyses are required to capture cellular interactions. Here, we report on a systems-level blood immunomonitoring study of 37 adult patients diagnosed with COVID-19 and followed with up to 14 blood samples from acute to recovery phases of the disease. We describe an IFNγ-eosinophil axis activated before lung hyperinflammation and changes in cell-cell co-regulation during different stages of the disease. We also map an immune trajectory during recovery that is shared among patients with severe COVID-19.

The ability to genetically modify CD8 T cells using viral gene delivery has facilitated the development of next generation of cancer immunotherapies such as chimeric Ag receptor (CAR) T cells engineered to specifically kill tumor cells. Development of immunotherapies targeting NK cells have stalled in part by their resistance to traditional viral gene delivery systems. Here, an efficient approach is described to genetically edit human NK cells by electroporation and CRISPR‐Cas9 ribonucleoprotein (RNP) complexes. Electroporation pulse codes and buffer optimization for protein uptake by human NK cells and viability, and the efficiency of this approach over other methods are detailed. To highlight the transformative step this technique will have for NK cell immunotherapy drug discovery, NCR1 and CISH are deleted in primary human NK cells and murine findings are validated on their key roles in regulating NK cell antitumor function. [ABSTRACT FROM AUTHOR]

Purpose and Appropriate Sample Types: This 14‐color flow cytometry panel was designed to identify newly described subpopulations within human group 2 innate lymphoid cells (ILC2s) and other ILC subsets. This panel also allowed to identify recently reported subpopulations of peripheral blood CRTH2− c‐Kit+ ILCs. We validated this panel mostly in human peripheral blood but also confirmed that the same panel and gating strategy works well in human tonsillar cells. The panel contains a few markers indicating the activation status of ILCs. In addition, phycoerythrin (PE) channel is available for the markers of interest in each study. In the validation studies described here, PE channel was used to test the expression of some markers. These features make this panel applicable for immunophenotyping of ILCs in various disease states. © 2020 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Tissue-resident memory T cells (TRMs) in mice mediate optimal protective immunity to infection and vaccination, while in humans, the existence and properties of TRMs remain unclear. Here, we use a unique human tissue resource to determine whether human tissue memory T cells constitute a distinct subset in diverse mucosal and lymphoid tissues. We identify a core transcriptional profile within the CD69+ subset of memory CD4+ and CD8+ T cells in lung and spleen that is distinct from that of CD69− TEM cells in tissues and circulation and defines human TRMs based on homology to the transcriptional profile of mouse CD8+ TRMs. Human TRMs in diverse sites exhibit increased expression of adhesion and inhibitory molecules, produce both pro-inflammatory and regulatory cytokines, and have reduced turnover compared with circulating TEM, suggesting unique adaptations for in situ immunity. Together, our results provide a unifying signature for human TRM and a blueprint for designing tissue-targeted immunotherapies.

We provide in this paper a detailed characterization of the human peripheral CD4+ CD127lowCD25+ regulatory T cell (Treg) compartment, with a particular emphasis in defining the population expressing higher levels of the IL-6 receptor (IL-6R). We provide a description of the phenotype of this population by assessing both the surface expression by flow cytometry as well as their transcriptional profile and functional features. In addition, we also present functional data describing the responsiveness of these subsets to IL-6 signalling in vitro and to IL-2 in vivo. The data presented in this paper support the research article “Human IL-6RhiTIGIT− CD4+CD127lowCD25+ T cells display potent in vitro suppressive capacity and a distinct Th17 profile” (Ferreira RC et al., 2017; doi: 10.1016/j.clim.2017.03.002) [1].

Cytometry by Time-Of-Flight (CyTOF) uses antibodies conjugated to isotopically pure metals to identify and quantify a large number of cellular features with single-cell resolution. A barcoding approach allows for 20 unique samples to be pooled and processed together in one tube, reducing the intra-barcode technical variability. However, with only 20 samples per barcode, multiple barcode sets (batches) are required to address questions in robustly powered study designs. A batch adjustment procedure is required to reduce variability across batches and to facilitate direct comparison of runs performed across multiple barcodes run over weeks, months, or years. We describe a method using technical replicates that are included in each run to determine and apply an appropriate adjustment per batch without manual intervention. The use of technical replicate samples (i.e., anchors or reference samples) avoids assumptions of sample homogeneity among batches, and allows direct estimation of batch effects and appropriate adjustment parameters applicable to all samples within a batch. Quantification of cell subpopulations and mean signal intensity pre- and post-adjustment using both manual gating and unsupervised clustering demonstrate substantial mitigation of batch effects in the anchor samples used for this adjustment calculation, and in a second validation set of technical replicates.

The degree and type of T cell infiltration influence rectal cancer prognosis regardless of classical tumor staging. We asked whether clonal expansion and tumor infiltration are restricted to selected-phenotype T cells; which clones are accessible in peripheral blood; and what the spatial distribution of their target antigens is. From five rectal cancer patients, we isolated paired tumor-infiltrating T cells (TILs) and T cells from unaffected rectum mucosa (TUM) using 13-parameter FACS single cell index sorting. TCRαβ sequences, cytokine, and transcription factor expression were determined with single cell sequencing. TILs and TUM occupied distinct phenotype compartments and clonal expansion predominantly occurred within CD8+ T cells. Expanded TIL clones identified by paired TCRαβ sequencing and exclusively detectable in the tumor showed characteristic PD-1 and TIM-3 expression. TCRβ repertoire sequencing identified 49 out of 149 expanded TIL clones circulating in peripheral blood and 41 (84%) of these were PD-1− TIM-3−. To determine whether clonal expansion of predominantly tumor-infiltrating T cell clones was driven by antigens uniquely presented in tumor tissue, selected TCRs were reconstructed and incubated with cells isolated from corresponding tumor or unaffected mucosa. The majority of clones exclusively detected in the tumor recognized antigen at both sites. In summary, rectal cancer is infiltrated with expanded distinct-phenotype T cell clones that either i) predominantly infiltrate the tumor, ii) predominantly infiltrate the unaffected mucosa, or iii) overlap between tumor, unaffected mucosa, and peripheral blood. However, the target antigens of predominantly tumor-infiltrating TIL clones do not appear to be restricted to tumor tissue.

Immunity to influenza is unique among pathogens, in that immune memory is established both via intermittent lung localized infections with highly variable influenza virus strains and by intramuscular vaccinations with inactivated protein-based vaccines. Studies in the past decades have suggested that the B cell responses to influenza infection and vaccination are highly biased by an individual's early history of influenza infection. This reactivity likely reflects both the competitive advantage that memory B cells have in an immune response and the relatively limited diversity of epitopes in influenza hemagglutinin that are recognized by B cells. In contrast, CD4 T cells recognize a wide array of epitopes, with specificities that are heavily influenced by the diversity of influenza antigens available, and a multiplicity of functions that are determined by both priming events and subsequent confrontations with antigens. Here, we consider the events that prime and remodel the influenza-specific CD4 T cell response in humans that have highly diverse immune histories and how the CD4 repertoire may be edited in terms of functional potential and viral epitope specificity. We discuss the consequences that imprinting and remodeling may have on the potential of different human hosts to rapidly respond with protective cellular immunity to infection. Finally, these issues are discussed in the context of future avenues of investigation and vaccine strategies.

In this report, we describe a novel T437N STAT1 mutation found in a mother and 3 of her 4 children which we demonstrate yields gain-of-function. All of the four patients with the T437N STAT1 mutation experienced lymphadenopathy. However, two of the children developed Nodular Lymphocyte Predominant Hodgkin Lymphoma (NHLPL) and have responded to chemotherapeutic regimens. The fourth sibling had neither the STAT1 variant nor lymphadenopathy or malignancy. To our knowledge this is the first description of a potential association between STAT1 GOF mutations and lymphoma development.

Tuberculosis (TB) is a major infectious disease worldwide, and is associated with several challenges for control and eradication. First, more accurate diagnostic tools that better represent the spectrum of infection states are required; in particular, identify the latent TB infected individuals with high risk of developing active TB. Second, we need to better understand, from a mechanistic point of view, why the immune system is unsuccessful in some cases for control and elimination of the pathogen. Host transcriptomics is a powerful approach to identify both diagnostic and mechanistic immune signatures of diseases. We have recently reported that optimal study design for these two purposes should be guided by different sets of criteria. Here, based on already published transcriptomics signatures of tuberculosis, we further develop these guidelines and identify additional factors to consider for obtaining diagnostic vs. mechanistic signatures in terms of cohorts, samples, data generation and analysis. Diagnostic studies should aim to identify small disease signatures with high discriminatory power across all affected populations, and against similar pathologies to TB. Specific focus should be made on improving the diagnosis of infected individuals at risk of developing active disease. Conversely, mechanistic studies should focus on tissues biopsies, immune relevant cell subsets, state of the art transcriptomic techniques and bioinformatics tools to understand the biological meaning of identified gene signatures that could facilitate therapeutic interventions. Finally, investigators should ensure their data are made publicly available along with complete annotations to facilitate metadata and cross-study analyses.

ABSTRACT Although Toxoplasma virulence mechanisms targeting gamma interferon (IFN-γ)-induced cell-autonomous antiparasitic immunity have been extensively characterized in mice, the virulence mechanisms in humans remain uncertain, partly because cell-autonomous immune responses against Toxoplasma differ markedly between mice and humans. Despite the identification of inducible nitric oxide synthase (iNOS) as an anti-Toxoplasma host factor in mice, here we show that iNOS in humans is a pro-Toxoplasma host factor that promotes the growth of the parasite. The GRA15 Toxoplasma effector-dependent disarmament of IFN-γ-induced parasite growth inhibition was evident when parasite-infected monocytes were cocultured with hepatocytes. Interleukin-1β (IL-1β), produced from monocytes in a manner dependent on GRA15 and the host’s NLRP3 inflammasome, combined with IFN-γ to strongly stimulate iNOS expression in hepatocytes; this dramatically reduced the levels of indole 2,3-dioxygenase 1 (IDO1), a critically important IFN-γ-inducible anti-Toxoplasma protein in humans, thus allowing parasite growth. Taking the data together, Toxoplasma utilizes human iNOS to antagonize IFN-γ-induced IDO1-mediated cell-autonomous immunity via its GRA15 virulence factor. IMPORTANCE Toxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Gamma interferon (IFN-γ) is produced in the host to inhibit the proliferation of this parasite and eventually cause its death. Unlike mouse disease models, which involve well-characterized virulence strategies that are used by Toxoplasma to suppress IFN-γ-dependent immunity, the strategies used by Toxoplasma in humans remain unclear. Here, we show that GRA15, a Toxoplasma effector protein, suppresses the IFN-γ-induced indole-2,3-dioxygenase 1-dependent antiparasite immune response in human cells. Because NLRP3-dependent production of IL-1β and nitric oxide (NO) in Toxoplasma-infected human cells is involved in the GRA15-dependent virulence mechanism, blocking NO or IL-1β production in the host could represent a novel therapeutic approach for treating human toxoplasmosis.

Cytometry by Time-Of-Flight (CyTOF) uses antibodies conjugated to isotopically pure metals to identify and quantify a large number of cellular features with single-cell resolution. A barcoding approach allows for 20 unique samples to be pooled and processed together in one tube, reducing the intra-barcode technical variability. However, with only 20 samples per barcode, multiple barcode sets (batches) are required to address questions in robustly powered study designs. A batch adjustment procedure is required to reduce variability across batches and to facilitate direct comparison of runs performed across multiple barcodes run over weeks, months, or years. We describe a method using technical replicates that are included in each run to determine and apply an appropriate adjustment per batch without manual intervention. The use of technical replicate samples (i.e., anchors or reference samples) avoids assumptions of sample homogeneity among batches, and allows direct estimation of batch effects and appropriate adjustment parameters applicable to all samples within a batch. Quantification of cell subpopulations and mean signal intensity pre- and post-adjustment using both manual gating and unsupervised clustering demonstrate substantial mitigation of batch effects in the anchor samples used for this adjustment calculation, and in a second validation set of technical replicates. [ABSTRACT FROM AUTHOR]

Summary: Regulatory T (Treg) cells represent an essential component of peripheral tolerance. Given their potently immunosuppressive functions that is orchestrated by the lineage‐defining transcription factor forkhead box protein 3 (FoxP3), clinical modulation of these cells in autoimmunity and cancer is a promising therapeutic target. However, recent evidence in mice and humans indicates that Treg cells represent a phenotypically and functionally heterogeneic population. Indeed, both suppressive and non‐suppressive Treg cells exist in human blood that are otherwise indistinguishable from one another using classical Treg cell markers such as CD25 and FoxP3. Moreover, murine Treg cells display a degree of plasticity through which they acquire the trafficking pathways needed to home to tissues containing target effector T (Teff) cells. However, this plasticity can also result in Treg cell lineage instability and acquisition of proinflammatory Teff cell functions. Consequently, these dysfunctional CD4+FoxP3+ T cells in human and mouse may fail to maintain peripheral tolerance and instead support immunopathology. The mechanisms driving human Treg cell dysfunction are largely undefined, and obscured by the scarcity of reliable immunophenotypical markers and the disregard paid to Treg cell antigen‐specificity in functional assays. Here, we review the mechanisms controlling the stability of the FoxP3+ Treg cell lineage phenotype. Particular attention will be paid to the developmental and functional heterogeneity of human Treg cells, and how abrogating these mechanisms can lead to lineage instability and Treg cell dysfunction in diseases like immunodysregulation polyendocrinopathy enteropathy X‐linked (IPEX) syndrome, type 1 diabetes, rheumatoid arthritis and cancer. [ABSTRACT FROM AUTHOR]

γδ T lymphocytes represent ~1% of human peripheral blood mononuclear cells and even more cells in most tissues of vertebrates. Although they have important anticancer functions, most current single-cell RNA sequencing (scRNA-seq) studies do not identify γδ T lymphocytes because their transcriptomes at the single-cell level are unknown. Here we show that high-resolution clustering of large scRNA-seq datasets and a combination of gene signatures allow the specific detection of human γδ T lymphocytes and identification of their T cell receptor (TCR)Vδ1 and TCRVδ2 subsets in large datasets from complex cellmixtures. In t-distributed stochastic neighbor embedding plots from blood and tumor samples, the few γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1 and TCRVδ2 subsets formclose yet distinct subclusters, respectively neighboring NK and CD8 T cells because of expression of shared and distinct cytotoxic maturation genes. Similar pseudotime maturation trajectories of TCRVδ1 and TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets an unattended pool of terminally differentiated effector memory cells with preserved proliferative capacity, a finding confirmed by in vitro proliferation assays. Overall, the single-cell transcriptomes of thousands of individual γδ T lymphocytes from different CMV+ and CMV- donors reflect cytotoxic maturation stages driven by the immunological history of donors. This landmark study establishes the rationale for identification, subtyping, and deep characterization of human γδ T lymphocytes in further scRNA-seq studies of complex tissues in physiological and disease conditions. [ABSTRACT FROM AUTHOR]

Macrophages are heterogeneous and play critical roles in development and disease, but their diversity, function, and specification remain inadequately understood during human development. We generated a single-cell RNA sequencing map of the dynamics of human macrophage specification from PCW 4–26 across 19 tissues. We identified a microglia-like population and a proangiogenic population in 15 macrophage subtypes. Microglia-like cells, molecularly and morphologically similar to microglia in the CNS, are present in the fetal epidermis, testicle, and heart. They are the major immune population in the early epidermis, exhibit a polarized distribution along the dorsal-lateral-ventral axis, and interact with neural crest cells, modulating their differentiation along the melanocyte lineage. Through spatial and differentiation trajectory analysis, we also showed that proangiogenic macrophages are perivascular across fetal organs and likely yolk-sac-derived as microglia. Our study provides a comprehensive map of the heterogeneity and developmental dynamics of human macrophages and unravels their diverse functions during development. [Display omitted] • A spatial and temporal molecular roadmap of human prenatal macrophages • Extensive and distinctive distribution of microglia-like cells outside the CNS • Microglia-like cells in the skin interact with and regulate neural crest cells • A proangiogenic macrophage population that is perivascular across fetal organs A single-cell RNA sequencing map of human immune cells from postconceptional weeks 4–26 across 19 tissues is provided with a focus on macrophage specification. Of note, a microglia-like cell population widely distributed outside the CNS as well as a proangiogenic population were found among the 15 macrophage subtypes identified. [ABSTRACT FROM AUTHOR]

Infancy and childhood are critical life stages for generating immune memory to protect against pathogens; however, the timing, location, and pathways for memory development in humans remain elusive. Here, we investigated T cells in mucosal sites, lymphoid tissues, and blood from 96 pediatric donors aged 0–10 years using phenotypic, functional, and transcriptomic profiling. Our results revealed that memory T cells preferentially localized in the intestines and lungs during infancy and accumulated more rapidly in mucosal sites compared with blood and lymphoid organs, consistent with site-specific antigen exposure. Early life mucosal memory T cells exhibit distinct functional capacities and stem-like transcriptional profiles. In later childhood, they progressively adopt proinflammatory functions and tissue-resident signatures, coincident with increased T cell receptor (TCR) clonal expansion in mucosal and lymphoid sites. Together, our findings identify staged development of memory T cells targeted to tissues during the formative years, informing how we might promote and monitor immunity in children. [Display omitted] • Memory T cells preferentially accumulate in human mucosal tissues during early life • T RM cells undergo functional maturation over childhood • T RM cells exhibit distinct transcriptional programs and site-specific expansion over age Immune memory generation during early childhood is critical for long-term protective immunity. Connors et al. analyze the development of human memory T cells in lymphoid organs, mucosal sites, and blood over infancy and childhood. Memory T cells accumulate rapidly in the intestines and lungs and display age- and tissue-driven maturation stages. [ABSTRACT FROM AUTHOR]

The process of immune system regeneration after allogeneic stem cell transplantation is slow, complex, and insufficiently understood. An entire immune system with all of its cell populations must regenerate from infused donor hematopoietic stem cells over the course of weeks and months post-transplantation. Both innate and adaptive arms of the immune system differ in their capacity and speed to reconstitiute in the recipient, which contributes to inadequacy in global immunity during the delayed reconstitution period. Systems-level analyses of immune systems in human patients have been made possible by high-throughput and high-dimensional, state-of-the-art, single-cell methodologies such as mass cytometry. Mass cytometry has revolutionized our ability to comprehensively profile all immune cell populations simultaneously in blood or tissue samples, providing signatures of differentially regulated cells in a range of clinical conditions. Such kind of systems immunology analyses promise not only for more accurate descriptions of variation between patients but also within individual patients over time, inter-dependencies between cell populations and the inference of developmental trajectories for specific cell populations. Here, we took advantage of a recently performed longitudinal mass cytometry analysis in 26 patients with hematological malignancies followed during the first 12 months following allogeneic stem cell transplantation. We present a proof-of-principle analysis to understand the evolution of individual immune cell populations. By applying non-linear dimensionality reduction and feauture extraction algorithms, we infer trajectories for individual immune cell populations, and map continuous marker expression changes occuring during immune cell regeneration that add novel information about this developmental process.

CD4+ follicular helper T (Tfh) cells constitute a subset of effector T cells that participate in the generation of high-affinity humoral responses. They express the chemokine receptor CXCR5 and produce the cytokine IL-21, both of which are required for their contribution to germinal center formation. Uncontrolled expansion of Tfh cells is observed in various mouse models of systemic autoimmune diseases and in patients with these diseases. In particular, the frequency of circulating Tfh is correlated with disease activity and anti-DNA antibody titer in patients with systemic lupus erythematosus. Recent studies reveal functional diversity within the Tfh population in both humans and mice. We will summarize here the molecular mechanisms for Tfh cell generation, survival and function in both humans and mice, and the relationship between Tfh cells and autoimmune disease in animal models and in patients.