Impact of deranged B cell subsets distribution in the development of HCV-related cirrhosis and HCC in type two diabetes mellitus.

Type II diabetes (T2D) may worsen the course of hepatitis C virus infection with a greater risk of liver cirrhosis (LC) and hepatocellular carcinoma (HCC). In chronic viral infections, the deranged B cell subset signifies uncontrolled disease. The study aimed to verify the relation between B cell subsets' distribution and liver disease progression in chronic hepatitis C (CHC) patients with T2D. A total of 67 CHC patients were divided into two groups; 33 non-diabetic and 34 with T2D. Each group was subdivided into CHC-without LC or HCC (N-CHC), CHC-with LC (CHC-LC), and CHC-with HCC (CHC-HCC). Twenty-seven healthy individuals also participated as controls. Flow cytometry was used to analyze CD19+ B cell subsets based on the expression of CD24 and CD38. CD19+CD24hiCD38hi Immature/transitional B cells elevated in diabetic than non-diabetic patients. In diabetic patients, while CD19+CD24+CD38- primarily memory B cells were higher in CHC-N and CHC-HCC groups than LC with a good predictive accuracy of LC, the opposite was observed for CD19+CD24-CD38- new memory B cells. Only in diabetic patients, the CD19+CD24intCD38int naïve mature B cells were high in CHC-HCC patients with good prognostic accuracy of HCC. Merely in diabetic patients, several correlations were observed between B cell subsets and liver function. Immature/transitional B cells increase remarkably in diabetic CHCpatients and might have a role in liver disease progression. Memory and Naïve B cells are good potential predictors of LC and HCCin diabetic CHCpatients, respectively. Further studies are needed to investigate the role of the CD19+CD24-CD38- new memory B cells in disease progression in CHC patients.

Differential effects of disease modifying drugs on peripheral blood B cell subsets: A cross sectional study in multiple sclerosis patients treated with interferon-ß, glatiramer acetate, dimethyl fumarate, fingolimod or natalizumab.

BACKGROUND: Several disease modifying drugs (DMDs) have been approved for the treatment of multiple sclerosis (MS), however, little is known about their differential impact on peripheral blood (PB) B cell subsets. METHODS: We performed a cross sectional study on PB B cells in MS patients treated with interferon-ß (n = 25), glatiramer acetate (n = 19), dimethyl fumarate (n = 15), fingolimod (n = 16) or natalizumab (n = 22), untreated MS patients (n = 20), and in patients with non-inflammatory neurological diseases (n = 12). Besides analyzing routine laboratory data, flow cytometry was performed to analyze naïve B cells (CD19+CD20+CD27-IgD+), non-class switched (CD19+CD20+CD27+IgD+) and class-switched memory B cells (CD19+CD20+CD27+IgD-), double negative B cells (CD19+CD20lowCD27-IgD-) and plasmablasts (CD19+CD20lowCD27+CD38++). RESULTS: Treatment associated changes were found for the overall B cell pool as well as for all B cell subsets. Natalizumab increased absolute numbers and percentage of all B cells mainly by expanding the memory B cell pool. Fingolimod decreased absolute numbers of all B cell subsets and the percentage of total B cells. Fingolimod, dimethyl fumarate and interferon-ß treatments were associated with an increase in the fraction of naïve B cells while class switched and non-class switched memory B cells showed decreased percentages. CONCLUSION: Our results highlight differential effects of DMDs on the PB B cell compartment. Across the examined treatments, a decreased percentage of memory B cells was found in dimethyl fumarate, interferon-ß and fingolimod treated patients which might contribute to the drugs' mode of action in MS. Further studies are necessary to decipher the exact role of B cell subsets during MS pathogenesis.

An Integrated Multi-omic Single-Cell Atlas of Human B Cell Identity.

B cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27- early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function.

Hepatitis B core-specific memory B cell responses associate with clinical parameters in patients with chronic HBV.

BACKGROUND & AIMS: Little is known about the frequency, phenotype and function of HBV-specific B cells during chronic infection. Here we study HBcAg and HBsAg-specific B cells in different clinical phases of a chronic HBV infection. METHODS: We included 118 treatment naïve and 34 nucleos(t)ide analogue-treated patients with chronic HBV and 23 healthy HBsAg-vaccinated controls. Global and HBV-specific B lymphocytes were examined by FACS using fluorescently labeled HBsAg and HBcAg as baits. Functional HBV-specific B cell responses were quantified in B cell ELISPOT assays. Anti-HBs and anti-HBc antibodies were measured in serum and in ELISPOT supernatant by ELISA. RESULTS: Higher HBcAg-directed B cell responses were found in HBV clinical phases with elevated vs. low serum alanine aminotransferase (ALT) levels, irrespective of the HBeAg-status. In contrast, HBsAg-directed responses were lower and did not significantly fluctuate. In individual patients a mean 17.8-fold more circulating B cells target HBcAg than HBsAg baits. These HBcAg-specific B cells present a classical memory B cell profile and have slightly higher CD69 expression levels compared to global memory B cells. Viral suppression and ALT normalization upon treatment led to a numeric and functional reduction of HBcAg-specific B cell responses, accompanied by progressive decreases in serum anti-HBc antibodies. CONCLUSION: HBcAg-specific memory B cells present a classical memory B cell phenotype, vary in number and function throughout HBV's natural history and are significantly reduced during antiviral treatment. LAY SUMMARY: In recent years, studies examining the role of B cells during chronic hepatitis B virus infection have regained interest. We show that circulating B cells more often target the hepatitis B core antigen than the hepatitis surface antigen. Moreover, these hepatitis B core-specific B cells associate with the natural history of chronic HBV, and their responses decline during effective antiviral treatment.

Classification of mouse B cell types using surfaceome proteotype maps.

System-wide quantification of the cell surface proteotype and identification of extracellular glycosylation sites is challenging when samples are limited. Here, we miniaturize and automate the previously described Cell Surface Capture (CSC) technology, increasing sensitivity, reproducibility and throughput. We use this technology, which we call autoCSC, to create population-specific surfaceome maps of developing mouse B cells and use targeted flow cytometry to uncover developmental cell subpopulations.

Double-edge Role of B Cells in Tumor Immunity: Potential Molecular Mechanism.

B cells are a heterogeneous population, which have distinct functions of antigen presentation, activating T cells, and secreting antibodies, cytokines as well as protease. It is supposed that the balance among these B cells subpopulation (resting B cells, activated B cells, Bregs, and other differentiated B cells) will determine the ultimate role of B cells in tumor immunity. There has been increasing evidence supporting opposite roles of B cells in tumor immunity, though there are no general acceptable phenotypes for them. Recent years, a new designated subset of B cells identified as Bregs has emerged from immunosuppressive and/or regulatory functions in tumor immune responses. Therefore, transferring activated B cells would be possible to become a promising strategy against tumor via conquering the immunosuppressive status of B cells in future. Understanding the potential mechanism of double-edge role of B cells will help researchers utilize activated B cells to improve their anti-tumor response. Moreover, the molecular pathways related to B cell differentiation are involved in its tumor-promoting effect, such as NF-κB, STAT3, BTK. So, we review the molecular and signaling pathway mechanisms of B cells involved in both tumor-promoting and tumor-suppressive immunity, in order to help researchers optimize B cells to fight cancer better.

Identification of cross-reactive antibodies for the detection of lymphocytes, myeloid cells and haematopoietic precursors in the naked mole rat.

The naked mole rat (Heterocephalus glaber, NMR) is a rodent with exceptional longevity, low rates of age-related diseases and spontaneous carcinogenesis. The NMR represents an attractive animal model in longevity and cancer research, but there are no NMR-specific antibodies available to study its immune system with respect to age- and cancer-related questions. Substantial homology of major NMR immune cell markers with those of Guinea pig, human and, to a lesser extent, mouse and rat origin are implicated for the existence of immunological cross-reactivity. We identified 10 antibodies recognising eight immunophenotypic markers expressed on the NMR's T and B lymphocytes, macrophages/monocytes and putative haematopoietic precursors and used them for an immunophenotyping of leukocyte subsets of peripheral blood, spleen and bone marrow samples. Overall, we found that the leukocyte composition of NMR peripheral blood is comparable to that of mice. Notably, the frequency of cytotoxic T cells was found to be lower in the NMR compared to corresponding mouse tissues and human blood. Antibodies used in the present paper are available either commercially or from the scientific community and will provide new opportunities for the NMR as a model system in ageing- and cancer-related research areas.

Innate-like B cell subsets during immune responses: Beyond antibody production.

B lymphocytes are recognized for their crucial role in the adaptive immunity since they represent the only leukocyte lineage capable of differentiating into Ab-secreting cells. However, it has been demonstrated that these lymphocytes can exert several Ab-independent functions, including engulfing and processing Ags for presentation to T cells, secreting soluble mediators, providing co-stimulatory signals, and even participating in lymphoid tissues development. Beyond that, several reports claiming the existence of multiple B cell subsets contributing directly to innate immune responses have appeared. These "innate-like" B lymphocytes, whose phenotype, development pathways, tissue distribution, and functions are in most cases notoriously different from those of conventional B cells, are crucial to early protective responses against pathogens by exerting "crossover" defensive strategies that blur the established boundaries of innate and adaptive branches of immunity. Examples of these mechanisms include the rapid secretion of the polyspecific natural Abs, increased susceptibility to innate receptors-mediated activation, cytokine secretion, downstream priming of other innate cells, usage of specific variable immunoglobulin gene-segments, and other features. As these new insights emerge, it is becoming preponderant to redefine the functionality of B cells beyond their classical adaptive-immune tasks.

Extended immunophenotyping reference values in a healthy pediatric population.

BACKGROUND: For the accurate diagnosis of immunodeficiencies is crucial to compare patients' immunology laboratory values with age-sex matched controls, yet there is a paucity of normal values for most populations. OBJECTIVES: To define appropriate reference values of extended lymphocyte subpopulations and T-cell receptor excision circle (TRECs) levels in healthy pediatric donors between 1 month and 18 years of age. METHODS: Extended immunophenotyping values were obtained by analysis of multiparameter flow cytometry panels for the following subpopulations: CD4+ and CD8+ Naive, Effector, Effector Memory and Central Memory, T helper subpopulations and their degrees of activation, T Regulatory cells, Recent Thymic Emigrants (RTE), B Lymphocyte subpopulations (Transitional, Naive, Preswitch-Memory, Switch-Memory, Plasmablasts, CD21low, and Exhausted), and subpopulations for Monocytes, NK cells and Dendritic Cells. RESULTS: Median values and the 10th and 90th percentiles were obtained for 32 lymphocyte and monocyte subpopulations, and for TRECs levels in each age group of children. Naive CD4+ and CD8+ T-cell populations tended to decrease with age, with significant difference between the groups, in parallel with the reduction in thymic function assessed by TRECs counts and the recent thymic emigrant population. Relative numbers of Th cell populations tended to increase with age. The percentage of class-switched B cell populations showed a significant increase between the youngest group and the others. CONCLUSION: This study provides essential data for interpreting extended immunophenotyping profiles in the pediatric and young adult populations, which could be of value for the diagnosis of PIDs and immune-mediated diseases, particularly those associated with subtle immunological abnormalities. © 2018 International Clinical Cytometry Society.

Human CD30+ B cells represent a unique subset related to Hodgkin lymphoma cells.

Very few B cells in germinal centers (GCs) and extrafollicular (EF) regions of lymph nodes express CD30. Their specific features and relationship to CD30-expressing Hodgkin and Reed/Sternberg (HRS) cells of Hodgkin lymphoma are unclear but highly relevant, because numerous patients with lymphoma are currently treated with an anti-CD30 immunotoxin. We performed a comprehensive analysis of human CD30+ B cells. Phenotypic and IgV gene analyses indicated that CD30+ GC B lymphocytes represent typical GC B cells, and that CD30+ EF B cells are mostly post-GC B cells. The transcriptomes of CD30+ GC and EF B cells largely overlapped, sharing a strong MYC signature, but were strikingly different from conventional GC B cells and memory B and plasma cells, respectively. CD30+ GC B cells represent MYC+ centrocytes redifferentiating into centroblasts; CD30+ EF B cells represent active, proliferating memory B cells. HRS cells shared typical transcriptome patterns with CD30+ B cells, suggesting that they originate from these lymphocytes or acquire their characteristic features during lymphomagenesis. By comparing HRS to normal CD30+ B cells we redefined aberrant and disease-specific features of HRS cells. A remarkable downregulation of genes regulating genomic stability and cytokinesis in HRS cells may explain their genomic instability and multinuclearity.

Alternative Th17 and CD4+ CD25+ FoxP3+ cell frequencies increase and correlate with worse cardiac function in Chagas cardiomyopathy.

Immune homeostasis has been suggested to play an important role in the clinical evolution of chronic Chagas disease; however, the immunopathologic factors involved have not been fully elucidated. Therefore, our study aimed to analyse the frequency of CD4+ CD25+ FoxP3+ cells, classic Th17 cells, alternative Th17 cells and IL-17+ B cells from peripheral blood of chronic cardiac patients after in vitro stimulation with Trypanosoma cruzi soluble EPI antigen. Patients were selected and classified according to clinical evaluation of cardiac involvement: mild, B1 (CARD1) (n = 20) and severe, C (CARD2) (n = 11). Patients with the indeterminate form of CD were included as the control group A (IND) (n = 17). Blood samples were collected and cultured in the presence of EPI antigen. Cells frequency and median fluorescence intensity (MFI) were obtained by flow cytometry. Our results showed that only CD4+ CD25+ FoxP3+ , CD4+ CD25high FoxP3+ , CD4+ IL-17+ IFN-γ- and CD4+ IL-17+ IFN-γ+ cells are more frequent in patients with severe cardiac disease and correlate with worse global cardiac function. However, while indeterminate patients demonstrated a positive correlation between CD4+ CD25+ FoxP3+ and CD4+ IL-17+ IFN-γ- Th17 cells, this relationship was not observed in cardiac patients. IL-17 expression by Th17 cells and B cells correlated with disease progression. Altogether our results suggest that the clinical progression of Chagas cardiomyopathy involves worsening of inflammation and impairment of immunoregulatory mechanisms.

Monitoring of dynamic changes in Keyhole Limpet Hemocyanin (KLH)-specific B cells in KLH-vaccinated cancer patients.

Keyhole limpet hemocyanin (KLH) is used as an immunogenic neo-antigen for various clinical applications and during vaccine development. For advanced monitoring of KLH-based interventions, we developed a flow cytometry-based assay for the ex vivo detection, phenotyping and isolation of KLH-specific B cells. As proof-of-principle, we analyzed 10 melanoma patients exposed to KLH during anti-cancer dendritic cell vaccination. Our assay demonstrated sensitive and specific detection of KLH-specific B cells in peripheral blood and KLH-specific B cell frequencies strongly correlated with anti-KLH serum antibody titers. Profiling of B cell subsets over the vaccination course revealed that KLH-specific B cells matured from naïve to class-switched memory B cells, confirming the prototypic B cell response to a neo-antigen. We conclude that flow-cytometric detection and in-depth phenotyping of KLH-specific B cells is specific, sensitive, and scalable. Our findings provide novel opportunities to monitor KLH-specific immune responses and serve as a blueprint for the development of new flow-cytometric protocols.

Identification of Nascent Memory CD8 T Cells and Modeling of Their Ontogeny.

Primary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector, and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify Bcl2 and Mki67 as a marker combination that enables the tracking of nascent memory cells within the effector phase. We then use a formal approach based on mathematical models describing the dynamics of population size evolution to test potential progeny links and demonstrate that most cells follow a linear naive→early effector→late effector→memory pathway. Moreover, our mathematical model allows long-term prediction of memory cell numbers from a few early experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate their genealogy and to predict the outcome of immunization regimens in terms of memory cell numbers generated.

Differential effects of Cytomegalovirus carriage on the immune phenotype of middle-aged males and females.

The elderly population is more susceptible to infections as a result of an altered immune response, commonly referred to as immunosenescence. Cytomegalovirus (CMV)-infection associated changes in blood lymphocytes are known to impact this process, but the interaction with gender remains unclear. Therefore, we analysed the effects and interaction of gender and CMV on the absolute numbers of a comprehensive set of naive and memory T- and B-cell subsets in people between 50 and 65 years of age. Enumeration and characterisation of lymphocyte subsets by flow cytometry was performed on fresh whole blood samples from 255 middle-aged persons. CMV-IgG serostatus was determined by ELISA. Gender was a major factor affecting immune cell numbers. CMV infection was mainly associated with an expansion of late-differentiated T-cell subsets. CMV+ males carried lower numbers of total CD4+, CD4+ central memory (CM) and follicular helper T-cells than females and CMV- males. Moreover, CMV+ males had significantly lower numbers of regulatory T (Treg)-cells and memory B-cells than CMV+ females. We here demonstrate an interaction between the effects of CMV infection and gender on T- and B-cells in middle-aged individuals. These differential effects on adaptive immunity between males and females may have implications for vaccination strategies at middle-age.

Decreased memory B cells and increased CD8 memory T cells in blood of breastfed children: the generation R study.

BACKGROUND: Breastfeeding provides a protective effect against infectious diseases in infancy. Still, immunological evidence for enhanced adaptive immunity in breastfed children remains inconclusive. OBJECTIVE: To determine whether breastfeeding affects B- and T-cell memory in the first years of life. METHODS: We performed immunophenotypic analysis on blood samples within a population-based prospective cohort study. Participants included children at 6 months (n=258), 14 months (n=166), 25 months (n=112) and 6 years of age (n=332) with both data on breastfeeding and blood lymphocytes. Total B- and T-cell numbers and their memory subsets were determined with 6-color flow cytometry. Mothers completed questionnaires on breastfeeding when their children were aged 2, 6, and 12 months. Multiple linear regression models with adjustments for potential confounders were performed. RESULTS: Per month continuation of breastfeeding, a 3% (95% CI -6, -1) decrease in CD27+IgM+, a 2% (95 CI % -5, -1) decrease in CD27+IgA+ and a 2% (95% CI -4, -1) decrease in CD27-IgG+ memory B cell numbers were observed at 6 months of age. CD8 T-cell numbers at 6 months of age were 20% (95% CI 3, 37) higher in breastfed than in non-breastfed infants. This was mainly found for central memory CD8 T cells and associated with exposure to breast milk, rather than duration. The same trend was observed at 14 months, but associations disappeared at older ages. CONCLUSIONS: Longer breastfeeding is associated with increased CD8 T-cell memory, but not B-cell memory numbers in the first 6 months of life. This transient skewing towards T cell memory might contribute to the protective effect against infectious diseases in infancy.

[Abnormal expression of chemokine receptor on B cells in patients with SLE].

SLE is a systemic autoimmune disease characterized by overactivation of autoreactive memory B cells. However, little is known about the mechanism of qualitative abnormality of B cells. The subset classification of T cells by expression pattern of master transcription factors and chemokine receptors has been established. The biology of T cells is useful information to assess qualitative abnormality of B cells. Therefore, we focused on the expression of chemokine receptors such as CXCR5 and CXCR3 on B cells in order to define the B cell subset classification in patients with SLE. Our results revealed that pathological B cells, which lose CXCR5 and express CXCR3, might be involved in autoantibody production through the interaction with Tfh cells, and in acquisition of effector function of memory B cells during the pathological process in SLE. In addition, the results revealed that those effector B cells still remained after improvement of disease activity by immunosuppressive therapy, indicating that the quantitative abnormality, which is not improved by current therapy, may underlie in this disease.

The majority of human memory B cells recognizing RhD and tetanus resides in IgM+ B cells.

B cell memory to T cell-dependent (TD) Ags are considered to largely reside in class-switched CD27(+) cells. However, we previously observed that anti-RhD (D) Igs cloned from two donors, hyperimmunized with D(+) erythrocytes, were predominantly of the IgM isotype. We therefore analyzed in this study the phenotype and frequency of D- and tetanus toxoid-specific B cells by culturing B cells in limiting dilution upon irradiated CD40L-expressing EL4.B5 cells and testing the culture supernatant. Most Ag-specific B cells for both TD Ags were found to reside in the IgM-expressing B cells, including CD27(-) B cells, in both hyperimmunized donors and nonhyperimmunized volunteers. Only shortly after immunization a sharp increase in Ag-specific CD27(+)IgG(+) B cells was observed. Next, B cells were enriched with D(+) erythrocyte ghosts and sorted as single cells. Sequencing of IGHV, IGLV, IGKV, and BCL6 genes from these D-specific B cell clones demonstrated that both CD27(-)IgM(+) and CD27(+)IgM(+) B cells harbored somatic mutations, documenting their Ag-selected nature. Furthermore, sequencing revealed a clonal relationship between the CD27(-)IgM(+), CD27(+)IgM(+), and CD27(+)IgG(+) B cell subsets. These data strongly support the recently described multiple layers of memory B cells to TD Ags in mice, where IgM(+) B cells represent a memory reservoir which can re-enter the germinal center and ensure replenishment of class-switched memory CD27(+) B cells from Ag-experienced precursors.