Search

Great effort was made to characterize the bacterial communities inhabiting the human body as a factor in disease, resulting in the realization that a wide spectrum of diseases is associated with an altered composition of the microbiome. However, the identification of disease‐relevant bacteria has been hindered by the high cross‐sectional diversity of individual microbiomes, and in most cases, it remains unclear whether the observed alterations are cause or consequence of disease. Hence, innovative analysis approaches are required that enable inquiries of the microbiome beyond mere taxonomic cataloging. This review highlights the utility of microbiota flow cytometry, a single‐cell analysis platform to directly interrogate cellular interactions, cell conditions, and crosstalk with the host's immune system within the microbiome to take into consideration the role of microbes as critical interaction partners of the host and the spectrum of microbiome alterations, beyond compositional changes. In conjunction with advanced sequencing approaches it could reveal the genetic potential of target bacteria and advance our understanding of taxonomic diversity and gene usage in the context of the microenvironment. Single‐cell bacterial phenotyping has the potential to change our perspective on the human microbiome and empower microbiome research for the development of microbiome‐based therapy approaches and personalized medicine. [ABSTRACT FROM AUTHOR]

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state‐of‐the‐art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human DC from lymphoid organs, and various non‐lymphoid tissues. Within this chapter, detailed protocols are presented that allow for the generation of single‐cell suspensions from mouse lymphohematopoietic tissues including spleen, peripheral lymph nodes, and thymus, with a focus on the subsequent analysis of DC by flow cytometry. However, prepared single‐cell suspensions can be subjected to other applications including sorting and cellular enrichment procedures, RNA sequencing, Western blotting, and many more. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer‐reviewed by leading experts and approved by all co‐authors, making it an essential resource for basic and clinical DC immunologists. [ABSTRACT FROM AUTHOR]

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state‐of‐the‐art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non‐lymphoid tissues. Within this article, detailed protocols are presented that allow for the generation of single cell suspensions from human lymphohematopoietic tissues including blood, spleen, thymus, and tonsils with a focus on the subsequent analysis of DC via flow cytometry, as well as flow cytometric cell sorting of primary human DC. Further, prepared single cell suspensions as well as cell sorter‐purified DC can be subjected to other applications including cellular enrichment procedures, RNA sequencing, functional assays, and many more. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer‐reviewed by leading experts and approved by all co‐authors, making it an essential resource for basic and clinical DC immunologists. [ABSTRACT FROM AUTHOR]

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state‐of‐the‐art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single‐cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in‐depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer‐reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists. [ABSTRACT FROM AUTHOR]

Growing interest surrounds adoptive cellular therapies utilizing Natural Killer (NK) cells, which can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding NK cell receptor expression and diversity in such cellular sources will guide future therapeutic designs. We used a 20‐color flow cytometry panel to compare unstimulated and cytokine‐activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD‐1, TIGIT, and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and dimensionality reduction analyses revealed enrichment in CD56neg as well as mature NKp46neg and CD56+CD16+ NK cell populations in UCB whereas CD57+ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following stimulation with IL‐12, IL‐15, and IL‐18. Cytokine stimulation was associated with the downregulation of TIGIT and CD16 on multiple NK cell subsets in UCB and APB. Among UCB CD16− NK cell populations, TIGIT+ NK cells produced more IFN‐γ than their TIGIT− counterparts. Our data demonstrate higher immune checkpoint expression on UCB NK cells compared to APB. However, the expression of TIGIT immune checkpoint is not indicative of NK cell exhaustion. [ABSTRACT FROM AUTHOR]

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

(B) CD3intCD4+ T cells were identified, while NK1.1+ NK cells, CD8+ T cells, CD19+ B cells, and Ly-6G+neutrophils are absent from healthy skin. Keywords: myeloid cells; dendritic cells; immune phenotyping; mouse lymph node; mouse skin EN myeloid cells dendritic cells immune phenotyping mouse lymph node mouse skin 2006 2009 4 12/13/22 20221201 NES 221201 This novel 26-color flow cytometry panel allows the detailed immune phenotyping of the complex network of myeloid cells in murine lymph nodes and skin. Mono...monocytes, Neutro...neutrophils, mig DC...migratory DC, res DC...resident DC, DN-DC...double-negative DC. GLO:BVO/01dec22:eji5356-gra-0001.jpg PHOTO (COLOR): . gl Abbreviations DC dendritic cells LN lymph nodes cDC conventional dendritic cells pDC plasmacytoid dendritic cells LC Langerhans cells FMO Fluorescence minus one We here describe the establishment and optimization of a 26-color flow cytometry panel to dissect the complex network of myeloid cells in mouse lymphoid and non-lymphoid tissue with a specific focus on dendritic cells (DC). [Extracted from the article]

Our cover features images related to flow cytometry techniques widely used for analysis of function and phenotypes of major human and murine immune cell subsets, superimposed on a multidimensional immune cell population scatter plot. These images are taken from the third edition of EJI's Flow Cytometry Guidelines by Cossarizza et al., a comprehensive resource prepared by flow cytometry and immunology research experts from around the world.. [Extracted from the article]

Our cover features images related to flow cytometry techniques widely used for analysis of function and phenotypes of major human and murine immune cell subsets, superimposed on a multidimensional immune cell population scatter plot. These images are taken from the third edition of EJI's Flow Cytometry Guidelines by Cossarizza et al., a comprehensive resource prepared by flow cytometry and immunology research experts from around the world.. [Extracted from the article]

Severe coronavirus disease 19 (COVID‐19) manifests with systemic immediate proinflammatory innate immune activation and altered iron turnover. Iron homeostasis, differentiation, and function of myeloid leukocytes are interconnected. Therefore, we characterized the cellularity, surface marker expression, and iron transporter phenotype of neutrophils and monocyte subsets in COVID‐19 patients within 72 h from hospital admission, and analyzed how these parameters relate to infection severity. Between March and November 2020, blood leukocyte samples from hospitalized COVID‐19 patients (n = 48) and healthy individuals (n = 7) were analyzed by flow cytometry enabling comparative analysis of 40 features. Inflammation‐driven neutrophil expansion, depletion of CD16+ nonclassical monocytes, and changes in surface expression of neutrophil and monocyte CD64 and CD86 were associated with COVID‐19 severity. By unsupervised self‐organizing map clustering, four patterns of innate myeloid response were identified and linked to varying levels of systemic inflammation, altered cellular iron trafficking and the severity of disease. These alterations of the myeloid leukocyte compartment during acute COVID‐19 may be hallmarks of inefficient viral control and immune hyperactivation and may help at risk prediction and treatment optimization. [ABSTRACT FROM AUTHOR]

Conformational change of the β2 integrin lymphocyte function‐associated antigen 1 (LFA‐1) is an early marker of T cell activation. A protocol using the mAb clone m24 recognizing the active, extended high‐affinity conformation has been previously described for the assessment of functional CD4+ and CD8+ T cells in response to MHC‐peptide stimulation. We investigated the applicability of the m24 mAb to detect the activation of γδ T cells in response to different soluble and immobilized stimuli. m24 mAb staining was associated with the expression of cytokines and was detectable as early as 10 min after stimulation, but with different kinetics depending on the nature of the stimulus. Hence, we conclude that this assay is suitable for the detection of functional γδ T cells and allows the assessment of activation more rapidly than alternative methods such as cytokine detection. Intracellular staining, protein trafficking inhibitors, or prior knowledge of the stimulating moiety recognized are no longer required for monitoring γδ T cell activation. [ABSTRACT FROM AUTHOR]

Our cover features images related to flow cytometry techniques widely used for analysis of function and phenotypes of major human and murine immune cell subsets, superimposed on a multidimensional immune cell population scatter plot. These images are taken from the third edition of EJI's Flow Cytometry Guidelines by Cossarizza et al., a comprehensive resource prepared by flow cytometry and immunology research experts from around the world.. [Extracted from the article]

Our cover features images related to flow cytometry techniques widely used for analysis of function and phenotypes of major human and murine immune cell subsets, superimposed on a multidimensional immune cell population scatter plot. These images are taken from the third edition of EJI's Flow Cytometry Guidelines by Cossarizza et al., a comprehensive resource prepared by flow cytometry and immunology research experts from around the world.. [Extracted from the article]

Cytometric immunophenotyping is a powerful tool to discover and implement T‐cell biomarkers of type 1 diabetes (T1D) progression and response to clinical therapy. Although many discovery‐based T‐cell biomarkers have been described, to date, no such markers have been widely adopted in standard practice. The heterogeneous nature of T1D and lack of standardized assays and experimental design across studies is a major barrier to the broader adoption of T‐cell immunophenotyping assays. There is an unmet need to harmonize the design of immunophenotyping assays, including those that measure antigen‐agnostic cell populations, such that data collected from different clinical trial sites and T1D cohorts are comparable, yet account for cohort‐specific features and different drug mechanisms of action. In these Guidelines, we aim to provide expert advice on how to unify aspects of study design and practice. We provide recommendations for defining cohorts, method implementation, as well as tools for data analysis and reporting by highlighting and building on selected successes. Harmonization of cytometry‐based T‐cell assays will allow researchers to better integrate findings across trials, ultimately enabling the identification and validation of biomarkers of disease progression and treatment response in T1D. [ABSTRACT FROM AUTHOR]

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers. [ABSTRACT FROM AUTHOR]

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen‐based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS‐CoV‐2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine‐like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory‐confirmed patients no SARS‐CoV‐2‐specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS‐CoV‐2 virus might provide insight into the wide array of clinical presentations of COVID‐19. [ABSTRACT FROM AUTHOR]

The dynamics of T‐cell receptor (TCR)selection in chronic HIV‐1 infection, and its association with clinical outcome, is well documented for an array of MHC‐peptide complexes and disease stages. However, the factors that may contribute to the selection and expansion of CD8+ T‐cells in chronic HIV‐2 infection, especially at the clonal level remain unclear. To address this question, we undertook a detailed molecular characterization of the clonotypic architecture of an HLA‐B*3501 restricted Gag‐specific CD8+ T‐cell response in donors chronically infected with HIV‐2 using a combination of flow cytometry, tetramer‐specific CD8+ TCR clonotyping, and in vitro assays. We show that the response to the NY9 epitope is hierarchical and narrow in terms of T‐cell receptor‐alpha (TCRA) and ‐beta (TCRB) gene usage yet clonotypically diverse. Furthermore, clonotypic dominance in shared origin CTL clones was associated with a greater magnitude of cytokine production and antigen sensitivity at limiting antigen dilution as well as enhanced cross‐reactivity for known HIV‐2 variants. Hence, our data suggest that effector mobilization and expansion in human chronic HIV‐2 infection may be linked to the qualitative features of specific CD8+ T‐cell clonotypes, which could have implications for viral control and disease outcome. [ABSTRACT FROM AUTHOR]

The phenotype of infused cells is a major determinant of Adoptive T‐cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state‐of‐the‐art and innovative culture conditions to generate stem‐like memory cells (TSCM) suitable for ACT. Noticeably, the combination of IL‐7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit‐TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T‐cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID‐19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring. [ABSTRACT FROM AUTHOR]

Myeloid‐derived suppressor cells (MDSCs) are important regulators of immune processes during sepsis in mice. However, confirming these observations in humans has been challenging due to the lack of defined preparation protocols and phenotyping schemes for MDSC subsets. Thus, it remains unclear how MDSCs are involved in acute sepsis and whether they have a role in the long‐term complications seen in survivors. Here, we combined comprehensive flow cytometry phenotyping with unsupervised clustering using self‐organizing maps to identify the three recently defined human MDSC subsets in blood from severe sepsis patients, long‐term sepsis survivors, and age‐matched controls. We demonstrated the expansion of monocytic M‐MDSCs and polymorphonuclear PMN‐MDSCs, but not early‐stage (e)‐MDSCs during acute sepsis. High levels of PMN‐MDSCs were also present in long‐term survivors many months after discharge, suggesting a possible role in sepsis‐related complications. Altogether, by employing unsupervised clustering of flow cytometric data we have confirmed the likely involvement of human MDSC subsets in acute sepsis, and revealed their expansion in sepsis survivors at late time points. The application of this strategy in future studies and in the clinical/diagnostic context would enable rapid progress toward a full understanding of the roles of MDSC in sepsis and other inflammatory conditions. [ABSTRACT FROM AUTHOR]

CD4(+) T cells and B cells are both essential for acquired immunity to Salmonella infection. It is well established that Salmonella inhibit host CD4(+) T-cell responses, but a corresponding inhibitory effect on B cells is less well defined. Here, we utilize an Ag tetramer and pull-down enrichment strategy to directly visualize OVA-specific B cells in mice, as they respond to infection with Salmonella-OVA. Surprisingly, OVA-specific B-cell expansion and germinal center formation was not detected until bacteria were cleared from the host. Furthermore, Salmonella infection also actively inhibited both B- and T-cell responses to the same coinjected Ag but this did not require the presence of iNOS. The Salmonella Pathogenicity Island 2 (SPI2) locus has been shown to be responsible for inhibition of Salmonella-specific CD4(+) T-cell responses, and an examination of SPI2-deficient bacteria demonstrated a recovery in B-cell expansion in infected mice. Together, these data suggest that Salmonella can simultaneously inhibit host B- and T-cell responses using SPI2-dependent mechanisms.

The Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively determined GPA33 expression patterns on human blood leukocyte subsets, using mass and flow cytometry. We found that GPA33 was expressed on fractions of B, dendritic, natural killer and innate lymphoid cells. Most prominent expression was found in the CD4+ T cell compartment. Naïve and CXCR5+ regulatory T cells were GPA33high, and naïve conventional CD4+ T cells expressed intermediate GPA33 levels. The expression pattern of GPA33 identified functional heterogeneity within the CD4+ central memory T cell (Tcm) population. GPA33+ CD4+ Tcm cells were fully undifferentiated, bona fide Tcm cells that lack immediate effector function, whereas GPA33– Tcm cells exhibited rapid effector functions and may represent an early stage of differentiation into effector/effector memory T cells before loss of CD62L. Expression of GPA33 in conventional CD4+ T cells suggests a role in localization and/or preservation of an undifferentiated state. These results form a basis to study the function of GPA33 and show it to be a useful marker to discriminate between different cellular subsets, especially in the CD4+ T cell lineage. [ABSTRACT FROM AUTHOR]

Our cover features images related to flow cytometry techniques widely used for analysis of function and phenotypes of major human and murine immune cell subsets, superimposed on a multidimensional immune cell population scatter plot. These images are taken from the third edition of EJI's Flow Cytometry Guidelines (Cossarizza et al. https://onlinelibrary.wiley.com/doi/10.1002/eji.202170126), a comprehensive resource prepared by flow cytometry and immunology research experts from around the world.. [Extracted from the article]

Invariant natural killer T (iNKT) cells are integral components of immune responses during many chronic diseases, yet their surface phenotypes, subset distribution, and polyfunctional capacity in this environment are largely unknown. Therefore, using flow cytometry, we determined iNKT cell phenotypic and functional characteristics in subjects with chronic inflammatory disease sarcoidosis and matched controls. We found that sarcoidosis subjects displayed lower iNKT-cell frequencies, which correlated with lung fibrosis, C-reactive protein levels, and other measures of clinical disease. The CD4(-) CD8(-) (double negative, DN) iNKT-cell population was selectively lower in diseased individuals and the remaining DN iNKT cells exhibited higher frequencies of the activation markers CD69 and CD56. Functionally, both total IFN-γ(+) and the dual-functional IFN-γ(+) TNF-α(+) iNKT cells were decreased in sarcoidosis subjects and these functional defects correlated with total iNKT-cell circulating frequencies. As the loss of polyfunctionality can reflect functional exhaustion, we measured the surface antigens programmed death-1 receptor and CD57 and found that levels inversely correlated with dual-functional iNKT-cell percentages. These findings reveal that, similar to traditional T cells, iNKT cells may also undergo functional exhaustion, and that circulating iNKT-cell frequencies reflect these defects. Programmed death-1 receptor antagonists may therefore be attractive therapeutic candidates for sarcoidosis and other iNKT-cell-mediated chronic diseases.

Non‐Hodgkin orbital lymphoma (NHOL) and idiopathic orbital inflammation (IOI) are common orbital conditions with largely unknown pathophysiology. To investigate the immune cell composition of these diseases, we performed standardized 29 parameter flow cytometry phenotyping in peripheral blood mononuclear cells of 18 NHOL patients, 21 IOI patients, and 41 unaffected controls. Automatic gating by FlowSOM revealed decreased abundance of meta‐clusters containing dendritic cells in patients, which we confirmed by manual gating. A decreased percentage of (HLA‐DR+CD303+CD123+) plasmacytoid dendritic cells (pDC) in the circulation of IOI patients and decreased (HLA‐DR+CD11c+CD1c+) conventional dendritic cells (cDC) type‐2 for IOI patients were replicated in an independent cohort of patients and controls. Meta‐analysis of both cohorts demonstrated that pDCs are also decreased in blood of NHOL patients and highlighted that the decrease in blood cDC type‐2 was specific for IOI patients compared to NHOL or controls. Deconvolution‐based estimation of immune cells in transcriptomic data of 48 orbital biopsies revealed a decrease in the abundance of pDC and cDC populations within the orbital microenvironment of IOI patients. Collectively, these data suggest a previously underappreciated role for dendritic cells in orbital disorders. [ABSTRACT FROM AUTHOR]

Celiac disease (CD) is characterized by a spectrum of intestinal inflammatory lesions. Most patients have villous atrophy (overt‐CD), while others have a morphologically normal mucosa, despite the presence of CD‐specific autoantibodies (potential‐CD). As the mechanism responsible for villous atrophy is not completely elucidated, we investigated biomarkers specific for the different celiac lesions. Phenotype and cytokine production of intestinal mucosa cells were analyzed by flow cytometry in gut biopsies of children with overt‐ or potential‐CD and in healthy controls. Density of TCRγδ+ T cells was found markedly enhanced in intestinal mucosa of children with overt‐CD compared to potential‐CD or controls. By contrast, very few IL4+ T cells infiltrated the mucosa with villous atrophy compared to morphologically normal mucosa. IL4+ T cells were classical CD4+ T‐helper cells (CD161−), producing or not IFN‐γ, and negative for IL17A. Our study demonstrated that the transition to villous atrophy in CD patients is characterized by increased density of TCRγδ+ T cells, and concomitant disappearance of IL4+ cells. These findings suggest that immunomodulatory mechanisms are active in potential‐CD to counteract the inflammatory cascade responsible of villous atrophy. Further studies are required to validate the use of IL4+ and TCRγδ+ T cells as biomarkers of the different CD forms. [ABSTRACT FROM AUTHOR]

Patients with primary progressive (PP) and secondary progressive (SP) forms of multiple sclerosis (MS) exhibit a sustained increase in the number of Th1, T cytotoxic type‐1 and Th17 cells in peripheral blood, suggesting that the progressive phase is characterized by a permanent peripheral immune activation. As T cell functionality and activation are strictly connected to their metabolic profile, we investigated the mitochondrial functionality and metabolic changes of T cell subpopulations in a cohort of progressive MS patients. T cells from progressive patients were characterized by low proliferation and increase of terminally differentiated/exhausted cells. T cells from PP patients showed lower Oxygen Consumption Rate and Extracellular Acidification Rate, lower mitochondrial mass, membrane potential and respiration than those of SP patients, a downregulation of transcription factors supporting respiration and higher tendency to shift towards glycolysis upon stimulation. Furthermore, PP effector memory T cells were characterized by higher Glucose transporter ‐1 levels and a higher expression of glycolytic‐supporting genes if compared to SP patients. Overall, our data suggest that profound differences exist in the phenotypic and metabolic features of T cells from PP and SP patients, even though the two clinical phenotypes are considered part of the same disease spectrum. [ABSTRACT FROM AUTHOR]

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Regulatory T cells (Treg) maintain immune homeostasis due to their anti-inflammatory functions. They can be generated either centrally in the thymus or in peripheral organs. Metabolites such as short-chain fatty acids produced by intestinal microbiota can induce peripheral Treg differentiation, by activating G-protein-coupled-receptors like GPR109A. In this study, we identified a novel role for GPR109A in thymic Treg development. We found that Gpr109a -/- mice had increased Treg under basal conditions in multiple organs compared with WT mice. GPR109A was not expressed on T cells but on medullary thymic epithelial cells (mTECs), as revealed by single-cell RNA sequencing in both mice and humans and confirmed by flow cytometry in mice. mTECs isolated from Gpr109a -/- mice had higher expression of autoimmune regulator (AIRE), the key regulator of Treg development, while the subset of mTECs that did not express Gpr109a in the WT displayed increased Aire expression and also enhanced signaling related to mTEC functionality. Increased thymic Treg in Gpr109a -/- mice was associated with protection from experimental autoimmune encephalomyelitis, with ameliorated clinical signs and reduced inflammation. This work identifies a novel role for GPR109A and possibly the gut microbiota, on thymic Treg development via its regulation of mTECs., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

High‐dimensional single‐cell (HDcyto) technologies, such as mass cytometry (CyTOF) and flow cytometry, are the key techniques that hold a great promise for deciphering complex biological processes. During the last decade, we witnessed an exponential increase of novel HDcyto technologies that are able to deliver an in‐depth profiling in different settings, such as various autoimmune diseases and cancer. The concurrent advance of custom data‐mining algorithms has provided a rich substrate for the development of novel tools in translational medicine research. HDcyto technologies have been successfully used to investigate cellular cues driving pathophysiological conditions, and to identify disease‐specific signatures that may serve as diagnostic biomarkers or therapeutic targets. These technologies now also offer the possibility to describe a complete cellular environment, providing unanticipated insights into human biology. In this review, we present an update on the current cutting‐edge HDcyto technologies and their applications, which are going to be fundamental in providing further insights into human immunology and pathophysiology of various diseases. Importantly, we further provide an overview of the main algorithms currently available for data mining, together with the conceptual workflow for high‐dimensional cytometric data handling and analysis. Overall, this review aims to be a handy overview for immunologists on how to design, develop and read HDcyto data. The technological advancement in high‐dimensional single cell cytometry (HDcyto) has improved our ability to investigate cellular heterogeneity in humans. With adequate automated data mining algorithms and appropriate computational power, HDcyto provides new tools for the identification of stratification biomarkers, fundamental for precision medicine. [ABSTRACT FROM AUTHOR]

Microbial flow cytometry is a powerful emerging technology with a broad range of applications including the study of complex microbial communities. Immunologists are increasingly using this technology to study antibody responses against pathogenic and commensal microbes. We employed microbial flow cytometry to quantify the proportion of fecal microbes bound by six different Ig isotypes: IgA, IgM, IgG1, IgG2b, IgG2c, and IgG3. In healthy mammals, secretory IgA (sIgA) binds to a subset of commensal microbes in the gut whereas IgG is not typically found in the intestinal tract of healthy mammals. Unexpectedly, fecal microbes isolated from SPF C57BL/6 mice housed in the Hill facility and imported from the vendors The Jackson Laboratory and Taconic Biosciences showed a strong signal in the Brilliant Violet 711 (BV711) channel. Unstained fecal samples from these mice demonstrated that the BV711 signal was due to bacterial autofluorescence. We found that murine diets containing alfalfa induce ex vivo microbial autofluorescence in the far red spectrum, likely due to chlorophyll. Analysis of unstained intestinal microbes is an important step in microbial flow cytometry to identify diet‐induced autofluorescence. We recommend fluorophores with emission spectra below 650 nm (e.g. BV421, PE). [ABSTRACT FROM AUTHOR]

Integrins regulate leukocyte trafficking during homeostasis and inflammatory conditions. However, the role of α4 and β7 integrins in guiding eosinophil transmigration into the lungs during filarial manifestation of Tropical Pulmonary Eosinophilia (TPE) has not been explored. In this study, mice exhibiting TPE manifestations were administered with in vivo neutralizing antibodies against integrins α4 and β7 or their combination and immuno-pathological parameters were evaluated. Results show an intact lung barrier, significantly lower lung inflammation and reduced eosinophil counts in the Bronchoalveolar lavage fluid and lungs of mice receiving anti-α4+β7 treatment. Reduced eosinophil peroxidase and β-hexosaminidase activity, downregulation of inflammatory genes, lower production of inflammatory lipid intermediates like prostaglandins E2 and D2, leukotriene B4 and cysteinyl leukotrienes were also noted in anti-α4+β7 treated mice. Reduced accumulation of central memory, effector memory, regulatory T cells and lower production of IL-4, IL-5, and TGF-β were other cardinal features of anti-α4+β7 treated mice lungs. Flow cytometry-sorted lung eosinophils from anti-α4+β7 treated mice showed higher apoptotic potential, downregulated anti-apoptotic gene Bcl-2, and exhibited reduced F-actin polymerization and calcium influx as compared to IgG controls. In summary, neutralization of α4+β7 integrins impairs the transmigration, activation and survival of eosinophils and reduces TPE induced pathology in mice lungs. [ABSTRACT FROM AUTHOR]

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross-platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal-labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome-conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR® labelling protocol. After labelling with 141Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4+ T-cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19-VioBlue-142Nd, CD20-VioGreen-147Sm, CD27-Cy5-167Er and CD38-Alexa488-143Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets. [ABSTRACT FROM AUTHOR]

We provide a robust four‐color fluorescence‐based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice. [ABSTRACT FROM AUTHOR]

Being the sole source of antibody, plasmablasts and plasma cells are essential for protective immunity. Due to their relative rarity, heterogeneity and the loss of many canonical B-cell markers, antibody-secreting cells (ASCs) have often been problematic to identify and further characterize. In the mouse, the combination of the expression of CD138 and BLIMP-1, has led to many insights into ASC biology, although this approach requires the use of a GFP reporter strain. In the current issue of the European Journal of Immunology, two independent studies by Wilmore et al. and Pracht et al. provide alternative approaches to identify all murine ASCs using antibodies against the cell surface proteins, Sca-1 and TACI, respectively. Here we will discuss the advantages of these new approaches to identify ASCs in the context of our emerging knowledge of the cell surface phenotype and gene expression program of various ASC subsets in the murine and human systems. [ABSTRACT FROM AUTHOR]