ABSTRACT
Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19low-BMPC are derived from follicular, while CD19high-BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19low- and CD19high-BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19high-BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observable in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.
Subject(s)
Antigens, CD19 , Bone Marrow , Interleukins , Plasma Cells , Adult , Female , Humans , Male , Middle Aged , Antibody-Producing Cells/immunology , Antigens, CD19/immunology , Antigens, CD19/metabolism , B-Lymphocytes/immunology , Bone Marrow/immunology , Bone Marrow Cells/immunology , Bone Marrow Cells/cytology , COVID-19/immunology , COVID-19/virology , Diphtheria-Tetanus-Pertussis Vaccine/immunology , Immunity, Humoral/immunology , Interleukins/immunology , Interleukins/metabolism , Plasma Cells/immunology , SARS-CoV-2/immunology , Single-Cell Analysis , VaccinationABSTRACT
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.
Subject(s)
Flow Cytometry , Single-Cell Analysis , Flow Cytometry/methods , Humans , Single-Cell Analysis/methods , Microbiota/genetics , Bacteria/genetics , Gastrointestinal Microbiome , Feces/microbiologyABSTRACT
Unlike Fc receptors for switched immunoglobulin (Ig) isotypes, Fc receptor for IgM (FcµR) is selectively expressed by lymphocytes. The ablation of the FcµR gene in mice impairs B cell tolerance as evidenced by concomitant production of autoantibodies of IgM and IgG isotypes. In this essay, we reiterate the autoimmune phenotypes observed in mutant mice, ie IgM homeostasis, dysregulated humoral immune responses including autoantibodies, and Mott cell formation. We also propose the potential phenotypes in individuals with FCMR deficiency and the model for FcµR-mediated regulation of self-reactive B cells.
Subject(s)
Autoimmunity , Receptors, Fc , Mice , Animals , Receptors, Fc/genetics , Autoantibodies , Immunoglobulin MABSTRACT
Autoimmunity, including that involved in chronic inflammatory rheumatic diseases, seems to be the price we have to pay for our efficient immune system. It has the ability to precisely recognize pathogens and tumor cells, to efficiently fight them, to adapt to their alterations and provide specific immunity for a lifetime. "Inoculation", and more specifically "vaccination" takes advantage of this, either by transfer of protective antibodies (passive vaccination) or by using attenuated pathogens or parts of them by which a specific protective immunity is induced (active vaccination). The idea to use vaccination to reduce undesired (auto)immunity and chronic inflammation is nothing new in rheumatology. Many biologicals are antibodies, which specifically block the mediators of inflammation and in the broader sense are similar to a passive vaccination. The active vaccination with autoantigens using the recent mRNA/liposome technology, has shown in experimental animal models that they can prevent the formation of chronic inflammatory immune reactions, in that they strengthen the physiological tolerance and deviate the immune system to noninflammatory immune reactions against the antigen; however, there is still a long way to go to achieve the actual goals of a permanent suppression of established undesired immune reactions and the regeneration of immunological tolerance.
Subject(s)
Autoimmunity , Immune Tolerance , Animals , Autoantigens , Vaccination , RegenerationABSTRACT
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.
Subject(s)
COVID-19 , Microbiota , Humans , Animals , Mice , Spike Glycoprotein, Coronavirus , Antibody Formation , Molecular Mimicry , SARS-CoV-2 , Antibodies, Monoclonal , Antibodies, Viral , Immunoglobulin A, Secretory , Immunoglobulin G , Antibodies, NeutralizingABSTRACT
For targeted intervention in coronavirus disease 2019 (COVID-19), there is a high medical need for biomarkers that predict disease progression and severity in the first days after symptom onset. This study assessed the utility of early transforming growth factor ß (TGF-ß) serum levels in COVID-19 patients to predict disease severity, fatality, and response to dexamethasone therapy. Patients with severe COVID-19 had significantly higher TGF-ß levels (416 pg/mL) as compared to patients with mild (165 pg/mL, p < 0.0001) or moderate COVID-19 (241 pg/mL; p < 0.0001). Receiver operating characteristics area under the curve values were 0.92 (95% confidence interval [CI] 0.85-0.99, cut-off: 255 pg/mL) for mild versus severe COVID-19, and 0.83 (95% CI 0.65-1.0, cut-off: 202 pg/mL) for moderate versus severe COVID-19. Patients who died of severe COVID-19 had significantly higher TGF-ß levels (453 pg/mL) as compared to convalescent patients (344 pg/mL), and TGF-ß levels predicted fatality (area under the curve: 0.75, 95% CI 0.53-0.96). TGF-ß was significantly reduced in severely ill patients treated with dexamethasone (301 pg/mL) as compared to untreated patients (416 pg/mL; p < 0.05). Early TGF-ß serum levels in COVID-19 patients predict, with high accuracy, disease severity, and fatality. In addition, TGF-ß serves as a specific biomarker to assess response to dexamethasone treatment.
Subject(s)
COVID-19 , Humans , Biomarkers , Dexamethasone/therapeutic use , Disease Progression , Transforming Growth Factor betaABSTRACT
In previous studies, Mott cells, an unusual form of plasma cells containing Ig-inclusion bodies, were frequently observed in peripheral lymphoid tissues in our IgM Fc receptor (FcµR)-deficient (KO) mouse strain. Because of discrepancies in the reported phenotypes of different Fcmr KO mouse strains, we here examined two additional available mutant strains and confirmed that such enhanced Mott-cell formation was a general phenomenon associated with FcµR deficiency. Splenic B cells from Fcmr KO mice clearly generated more Mott cells than those from WT mice when stimulated in vitro with LPS alone or a B-1, but not B-2, activation cocktail. Nucleotide sequence analysis of the Ig variable regions of a single IgMλ+ Mott-hybridoma clone developed from splenic B-1 B cells of Fcmr KO mice revealed the near (VH) or complete (Vλ) identity with the corresponding germline gene segments and the addition of six or five nucleotides at the VH/DH and DH/JH junctions, respectively. Transduction of an FcµR cDNA into the Mott hybridoma significantly reduced cells containing IgM-inclusion bodies with a concomitant increase in IgM secretion, leading to secreted IgM binding to FcµR expressed on Mott transductants. These findings suggest a regulatory role of FcµR in the formation of Mott cells and IgM-inclusion bodies.
Subject(s)
B-Lymphocytes , Receptors, Fc , Animals , Mice , Receptors, Fc/genetics , B-Lymphocytes/metabolism , Plasma Cells/metabolism , Immunoglobulin M/genetics , Immunoglobulin M/metabolismABSTRACT
IgM is the first antibody to emerge during phylogeny, ontogeny, and immune responses and serves as a first line of defense. Effector proteins interacting with the Fc portion of IgM, such as complement and its receptors, have been extensively studied for their functions. IgM Fc receptor (FcµR), identified in 2009, is the newest member of the FcR family and is intriguingly expressed by lymphocytes only, suggesting the existence of distinct functions as compared to the FcRs for switched Ig isotypes, which are expressed by various immune and non-hematopoietic cells as central mediators of antibody-triggered responses by coupling the adaptive and innate immune responses. Results from FcµR-deficient mice suggest a regulatory function of FcµR in B cell tolerance, as evidenced by their propensity to produce autoantibodies of both IgM and IgG isotypes. In this article, we discuss conflicting views about the cellular distribution and potential functions of FcµR. The signaling function of the Ig-tail tyrosine-like motif in the FcµR cytoplasmic domain is now formally shown by substitutional experiments with the IgG2 B cell receptor. The potential adaptor protein associating with FcµR and the potential cleavage of its C-terminal cytoplasmic tail after IgM binding are still enigmatic. Critical amino acid residues in the Ig-like domain of FcµR for interacting with the IgM Cµ4 domain and the mode of interaction are now defined by crystallographic and cryo-electron microscopic analyses. Some discrepancies on these interactions are discussed. Finally, elevated levels of a soluble FcµR isoform in serum samples are described as the consequence of persistent B cell receptor stimulation, as seen in chronic lymphocytic leukemia and probably in antibody-mediated autoimmune disorders.
Subject(s)
Receptors, Antigen, B-Cell , Receptors, Fc , Animals , Mice , Immunoglobulin M , Receptors, Fc/metabolism , Protein IsoformsABSTRACT
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.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Bone Marrow , CD4-Positive T-Lymphocytes , Nucleocapsid ProteinsSubject(s)
Collagen Diseases , Rheumatic Diseases , Berlin , Humans , Rheumatic Diseases/diagnosis , Rheumatic Diseases/therapyABSTRACT
The FcR for IgM (FcµR) is the newest member of the FcR family, selectively expressed by lymphocytes, and distinct from FcRs for switched Ig isotypes that are expressed by various immune cell types and non-hematopoietic cells. From studies of Fcmr-ablated mice, FcµR was shown to have a regulatory function in B-cell tolerance, as evidenced by high serum titers of autoantibodies of the IgM and IgG isotypes in mutant mice. In our previous studies, both cell-surface and serum FcµR levels were elevated in patients with chronic lymphocytic leukemia (CLL), where antigen-independent self-ligation of BCR is a hallmark of the neoplastic B cells. This was assessed by sandwich ELISA using two different ectodomain-specific mAbs. To determine whether the serum FcµR is derived from cleavage of its cell-surface receptor (shedding) or its alternative splicing to skip the transmembrane exon resulting in a 70-aa unique hydrophilic C-terminus (soluble), we developed a new mouse IgG1κ mAb specific for human soluble FcµR (solFcµR) by taking advantages of the unique nature of transductant stably producing His-tagged solFcµR and of an in vivo differential immunization. His-tagged solFcµR attached to exosomes and plasma membranes, allowing immunization and initial hybridoma screening without purification of solFcµR. Differential immunization with tolerogen (membrane FcµR) and immunogen (solFcµR) also facilitated to generate solFcµR-specific hybridomas. The resultant solFcµR-specific mAb reacted with serum FcµR in subsets of CLL patients. This mAb, along with another ectodomain-specific mAb, will be used for verifying the hypothesis that the production of solFcµR is the consequence of chronic stimulation of BCR.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , Receptors, Fc , Animals , Antibodies, Monoclonal , Antigens , B-Lymphocytes , Immunoglobulin M , Immunosuppressive Agents , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , MiceABSTRACT
Recently, mass cytometry has enabled quantification of up to 50 parameters for millions of cells per sample. It remains a challenge to analyze such high-dimensional data to exploit the richness of the inherent information, even though many valuable new analysis tools have already been developed. We propose a novel algorithm "pattern recognition of immune cells (PRI)" to tackle these high-dimensional protein combinations in the data. PRI is a tool for the analysis and visualization of cytometry data based on a three or more-parametric binning approach, feature engineering of bin properties of multivariate cell data, and a pseudo-multiparametric visualization. Using a publicly available mass cytometry dataset, we proved that reproducible feature engineering and intuitive understanding of the generated bin plots are helpful hallmarks for re-analysis with PRI. In the CD4+T cell population analyzed, PRI revealed two bin-plot patterns (CD90/CD44/CD86 and CD90/CD44/CD27) and 20 bin plot features for threshold-independent classification of mice concerning ineffective and effective tumor treatment. In addition, PRI mapped cell subsets regarding co-expression of the proliferation marker Ki67 with two major transcription factors and further delineated a specific Th1 cell subset. All these results demonstrate the added insights that can be obtained using the non-cluster-based tool PRI for re-analyses of high-dimensional cytometric data.
Subject(s)
Neoplasms , Algorithms , Animals , Mice , Neoplasms/therapy , Transcription FactorsABSTRACT
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.
Subject(s)
Phosphatidylinositol 3-Kinases , Plasma Cells , Apoptosis/physiology , Caspases/metabolism , Models, Theoretical , Plasma Cells/metabolismABSTRACT
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.