Single-cell multi-parametric characterization of microbiota by flow cytometry.

It has become evident, that the microbes colonizing the human body have a great impact on health and disease. Investigations of microbiota currently primarily rely on culturomics, high-throughput sequencing and metaproteomics which have considerably advanced our knowledge regarding the role of the microbiota in our environment and for our health. While single-cell phenotyping of immune cells and other somatic cells by flow cytometry has become widely used, the detailed analysis of bacterial cells such as the human microbiota on the single-cell level, is lagging behind. Here, we outline a protocol for the single-cell characterization of bacterial cells from complex microbiota samples, such as stool, by multi-parametric flow cytometry. Our protocol describes the isotype-specific detection of host-antibody coating of intestinal bacteria ex vivo, which together with quantitative DNA staining and light scatter detection comprise an individual's microbiota fingerprint. Cryoconservation and appropriate staining controls ensure reliable, reproducible data generation and analysis. We have automated the analysis of the multi-dimensional data using a segmentation approach by self-organizing map (SOM) algorithm for downstream comparative analyses. Our protocol can be adapted to integrate further phenotypic markers and uses the power of analytical cytometry for the characterization of bacteria on the single-cell level.

DSS treatment does not affect murine colonic microbiota in absence of the host.

The prevalence of inflammatory bowel disease (IBD) is rising globally; however, its etiology is still not fully understood. Patient genetics, immune system, and intestinal microbiota are considered critical factors contributing to IBD. Preclinical animal models are crucial to better understand the importance of individual contributing factors. Among these, the dextran sodium sulfate (DSS) colitis model is the most widely used. DSS treatment induces gut inflammation and dysbiosis. However, its exact mode of action remains unclear. To determine whether DSS treatment induces pathogenic changes in the microbiota, we investigated the microbiota-modulating effects of DSS on murine microbiota in vitro. For this purpose, we cultured murine microbiota from the colon in six replicate continuous bioreactors. Three bioreactors were supplemented with 1% DSS and compared with the remaining PBS-treated control bioreactors by means of microbiota taxonomy and functionality. Using metaproteomics, we did not identify significant changes in microbial taxonomy, either at the phylum or genus levels. No differences in the metabolic pathways were observed. Furthermore, the global metabolome and targeted short-chain fatty acid (SCFA) quantification did not reveal any DSS-related changes. DSS had negligible effects on microbial functionality and taxonomy in vitro in the absence of the host environment. Our results underline that the DSS colitis mouse model is a suitable model to study host-microbiota interactions, which may help to understand how intestinal inflammation modulates the microbiota at the taxonomic and functional levels.

Single-cell phenotyping of bacteria combined with deep sequencing for improved contextualization of microbiome analyses.

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.

Cross-regulation of antibody responses against the SARS-CoV-2 Spike protein and commensal microbiota via molecular mimicry.

The commensal microflora provides a repertoire of antigens that illicit mucosal antibodies. In some cases, these antibodies can cross-react with host proteins, inducing autoimmunity, or with other microbial antigens. We demonstrate that the oral microbiota can induce salivary anti-SARS-CoV-2 Spike IgG antibodies via molecular mimicry. Anti-Spike IgG antibodies in the saliva correlated with enhanced abundance of Streptococcus salivarius 1 month after anti-SARS-CoV-2 vaccination. Several human commensal bacteria, including S. salivarius, were recognized by SARS-CoV-2-neutralizing monoclonal antibodies and induced cross-reactive anti-Spike antibodies in mice, facilitating SARS-CoV-2 clearance. A specific S. salivarius protein, RSSL-01370, contains regions with homology to the Spike receptor-binding domain, and immunization of mice with RSSL-01370 elicited anti-Spike IgG antibodies in the serum. Additionally, oral S. salivarius supplementation enhanced salivary anti-Spike antibodies in vaccinated individuals. Altogether, these data show that distinct species of the human microbiota can express molecular mimics of SARS-CoV-2 Spike protein, potentially enhancing protective immunity.

IL-17A-producing CD8+ T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.

Rapid detection and online analysis of microbial changes through flow cytometry.

Short-read 16 S rRNA gene sequencing is the dominating technology to profile microbial communities in different habitats. Its uncontested taxonomic resolution paved the way for major contributions to the field. Sample measurement and analysis, that is, sequencing, is rather slow-in order of days. Alternatively, flow cytometry can be used to profile the microbiota of various sources within a few minutes per sample. To keep up with high measurement speed, we developed the open source-analyzing tool FlowSoFine. To validate the ability to distinguish microbial profiles, we examined human skin samples of three body sites (N = 3 × 54) with flow cytometry and 16 S rRNA gene amplicon sequencing. Confirmed by sequencing of the very same samples, body site was found to be significantly different by flow cytometry. For a proof-of-principle multidimensional approach, using stool samples of patients (N = 40) with/without inflammatory bowel diseases, we could discriminate the health status by their bacterial patterns. In conclusion, FlowSoFine enables the generation and comparison of cytometric fingerprints of microbial communities from different sources. The implemented interface supports the user through all analytical steps to work out the biological relevant signals from raw measurements to publication ready figures. Furthermore, we present flow cytometry as a valid method for skin microbiota analysis.

A ubiquitous bone marrow reservoir of preexisting SARS-CoV-2-reactive memory CD4+ T lymphocytes in unexposed individuals.

Circulating, blood-borne SARS-CoV-2-reactive memory T cells in persons so far unexposed to SARS-CoV-2 or the vaccines have been described in 20-100% of the adult population. They are credited with determining the efficacy of the immune response in COVID-19. Here, we demonstrate the presence of preexisting memory CD4+ T cells reacting to peptides of the spike, membrane, or nucleocapsid proteins of SARS-CoV-2 in the bone marrow of all 17 persons investigated that had previously not been exposed to SARS-CoV-2 or one of the vaccines targeting it, with only 15 of these persons also having such cells detectable circulating in the blood. The preexisting SARS-CoV-2-reactive memory CD4+ T cells of the bone marrow are abundant and polyfunctional, with the phenotype of central memory T cells. They are tissue-resident, at least in those persons who do not have such cells in the blood, and about 30% of them express CD69. Bone marrow resident SARS-CoV-2-reactive memory CD4+ memory T cells are also abundant in vaccinated persons analyzed 10-168 days after 1°-4° vaccination. Apart from securing the bone marrow, preexisting cross-reactive memory CD4+ T cells may play an important role in shaping the systemic immune response to SARS-CoV-2 and the vaccines, and contribute essentially to the rapid establishment of long-lasting immunity provided by memory plasma cells, already upon primary infection.

Flow cytometry can reliably capture gut microbial composition in healthy adults as well as dysbiosis dynamics in patients with aggressive B-cell non-Hodgkin lymphoma.

Modulation of commensal gut microbiota is increasingly recognized as a promising strategy to reduce mortality in patients with malignant diseases, but monitoring for dysbiosis is generally not routine clinical practice due to equipment, expertise and funding required for sequencing analysis. A low-threshold alternative is microbial diversity profiling by single-cell flow cytometry (FCM), which we compared to 16S rRNA sequencing in human fecal samples and employed to characterize longitudinal changes in the microbiome composition of patients with aggressive B-cell non-Hodgkin lymphoma undergoing chemoimmunotherapy. Diversity measures obtained from both methods were correlated and captured identical trends in microbial community structures, finding no difference in patients' pretreatment alpha or beta diversity compared to healthy controls and a significant and progressive loss of alpha diversity during chemoimmunotherapy. Our results highlight the potential of FCM-based microbiome profiling as a reliable and accessible diagnostic tool that can provide novel insights into cancer therapy-associated dysbiosis dynamics.

Data-Driven Mathematical Model of Apoptosis Regulation in Memory Plasma Cells.

Memory plasma cells constitutively produce copious amounts of antibodies, imposing a critical risk factor for autoimmune disease. We previously found that plasma cell survival requires secreted factors such as APRIL and direct contact to stromal cells, which act in concert to activate NF-κB- and PI3K-dependent signaling pathways to prevent cell death. However, the regulatory properties of the underlying biochemical network are confounded by the complexity of potential interaction and cross-regulation pathways. Here, based on flow-cytometric quantification of key signaling proteins in the presence or absence of the survival signals APRIL and contact to the stromal cell line ST2, we generated a quantitative model of plasma cell survival. Our model emphasizes the non-redundant nature of the two plasma cell survival signals APRIL and stromal cell contact, and highlights a requirement for differential regulation of individual caspases. The modeling approach allowed us to unify distinct data sets and derive a consistent picture of the intertwined signaling and apoptosis pathways regulating plasma cell survival.

[How T lymphocytes coordinate rheumatic inflammation]. / Wie T-Lymphozyten rheumatische Entzündung koordinieren.

Helper T (Th) cells play a decisive role in triggering and maintaining chronic rheumatic inflammation. Via secretion of proinflammatory cytokines and expression of costimulatory cell surface molecules, Th lymphocytes coordinate the recruitment and activation of effector cells, which are ultimately responsible for the immunopathology and tissue destruction. However, therapeutic approaches aimed at eliminating Th cells were unsuccessful due to their lack of selectivity. At the German Rheumatism Research Center (Deutsches Rheuma-Forschungszentrum, DRFZ), we are working to improve the understanding of the Th cells involved in chronic inflammatory reactions. Based on this understanding, our aim is to develop novel treatment strategies that selectively target the pathogenic Th lymphocytes causing rheumatic inflammation. The current article summarizes the DRFZ's research activities on this subject.

Resident memory CD4+ T lymphocytes mobilize from bone marrow to contribute to a systemic secondary immune reaction.

Resident memory T lymphocytes (TRM ) of epithelial tissues and the Bm protect their host tissue. To what extent these cells are mobilized and contribute to systemic immune reactions is less clear. Here, we show that in secondary immune reactions to the measles-mumps-rubella (MMR) vaccine, CD4+ TRM are mobilized into the blood within 16 to 48 h after immunization in humans. This mobilization of TRM is cognate: TRM recognizing other antigens are not mobilized, unless they cross-react with the vaccine. We also demonstrate through methylome analyses that TRM are mobilized from the Bm. These mobilized cells make significant contribution to the systemic immune reaction, as evidenced by their T-cell receptor Vß clonotypes represented among the newly generated circulating memory T-cells, 14 days after vaccination. Thus, TRM of the Bm confer not only local, but also systemic immune memory.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

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.

Intestinal Microbiome in Hematopoietic Stem Cell Transplantation For Autoimmune Diseases: Considerations and Perspectives on Behalf of Autoimmune Diseases Working Party (ADWP) of the EBMT.

Over the past decades, hematopoietic stem cell transplantation (HSCT) has been evolving as specific treatment for patients with severe and refractory autoimmune diseases (ADs), where mechanistic studies have provided evidence for a profound immune renewal facilitating the observed beneficial responses. The intestinal microbiome plays an important role in host physiology including shaping the immune repertoire. The relationships between intestinal microbiota composition and outcomes after HSCT for hematologic diseases have been identified, particularly for predicting the mortality from infectious and non-infectious causes. Furthermore, therapeutic manipulations of the gut microbiota, such as fecal microbiota transplant (FMT), have emerged as promising therapeutic approaches for restoring the functional and anatomical integrity of the intestinal microbiota post-transplantation. Although changes in the intestinal microbiome have been linked to various ADs, studies investigating the effect of intestinal dysbiosis on HSCT outcomes for ADs are scarce and require further attention. Herein, we describe some of the landmark microbiome studies in HSCT recipients and patients with chronic ADs, and discuss the challenges and opportunities of microbiome research for diagnostic and therapeutic purposes in the context of HSCT for ADs.

T-bet and RORα control lymph node formation by regulating embryonic innate lymphoid cell differentiation.

The generation of lymphoid tissues during embryogenesis relies on group 3 innate lymphoid cells (ILC3) displaying lymphoid tissue inducer (LTi) activity and expressing the master transcription factor RORγt. Accordingly, RORγt-deficient mice lack ILC3 and lymphoid structures, including lymph nodes (LN). Whereas T-bet affects differentiation and functions of ILC3 postnatally, the role of T-bet in regulating fetal ILC3 and LN formation remains completely unknown. Using multiple mouse models and single-cell analyses of fetal ILCs and ILC progenitors (ILCP), here we identify a key role for T-bet during embryogenesis and show that its deficiency rescues LN formation in RORγt-deficient mice. Mechanistically, T-bet deletion skews the differentiation fate of fetal ILCs and promotes the accumulation of PLZFhi ILCP expressing central LTi molecules in a RORα-dependent fashion. Our data unveil an unexpected role for T-bet and RORα during embryonic ILC function and highlight that RORγt is crucial in counteracting the suppressive effects of T-bet.

Keeping up with the stress of antibody production: BAFF and APRIL maintain memory plasma cells.

Memory plasma cells, also called long-lived plasma cells, provide 'humoral immunity' by continued secretion of protective antibodies against pathogens, which the immune system has once encountered. They are maintained mainly in the bone marrow, docking on to stromal cells individually. In those niches they can apparently persist for decades (Chang et al., 2018 [1]). Integrin-mediated contact to the stromal cell provides an essential survival signal to the plasma cell, activating the PI3K signalling pathway, downregulating FoxO1/3a and repressing the activation of caspases 3 and 7. In a redundant form, the cytokines BAFF and APRIL, ligands of the plasma cell receptors TACI and BCMA, provide a second essential survival signal, preventing activation of caspase 12, as triggered by endoplasmic reticulum stress.

Maintenance of quiescent immune memory in the bone marrow.

The adaptive immune system has the important ability to generate and maintain a memory for antigens once encountered. Recent progress in understanding the organization of immunological memory has challenged the established paradigm of maintenance of memory by restless, circulating, and "homeostatically" proliferating lymphocytes. Among other tissues, the bone marrow has emerged as a preferred resting place for memory lymphocytes providing both local and systemic long-term protection. Why the bone marrow? There, mesenchymal stromal cells provide a privileged environment for quiescent memory B and T lymphocytes, the protagonists of secondary immune reactions, and for memory plasma cells providing persistent humoral immunity. In this review, we discuss the dedicated role of the bone marrow for the maintenance of memory lymphocytes and its implications for immunological memory.

Quantifying Antigen-Specific T-Cells by Assessing Their Antigen-Induced Proliferation.

A critical property of T cells when activated by their cognate antigen-MHC complex is the initiation of cell cycle activity and clonal expansion. In this chapter, we describe how the proliferation of T cells can be assessed on the single cell level by flow cytometry and how this can be used to identify and potentially isolate antigen-reactive T cells.