CD5+/Mac-1− peritoneal B cells: A novel B cell subset that exhibits characteristics of B-1 cells

Abstract Background Aging is associated with increased intrinsic B cell inflammation, decreased protective antibody responses and increased autoimmune antibody responses. The effects of aging on the metabolic phenotype of B cells and on the metabolic programs that lead to the secretion of protective versus autoimmune antibodies are not known. Methods Splenic B cells and the major splenic B cell subsets, Follicular (FO) and Age-associated B cells (ABCs), were isolated from the spleens of young and old mice and left unstimulated. The RNA was collected to measure the expression of markers associated with intrinsic inflammation and autoimmune antibody production by qPCR. B cells and B cell subsets were also stimulated with CpG and supernatants collected after 7 days to measure autoimmune IgG secretion by ELISA. Metabolic measures (oxygen consumption rate, extracellular acidification rate and glucose uptake) were performed using a Seahorse XFp extracellular flux analyzer. Results Results have identified the subset of ABCs, whose frequencies and numbers increase with age and represent the most pro-inflammatory B cell subset, as the cell type mainly if not exclusively responsible for the expression of inflammatory markers and for the secretion of autoimmune antibodies in the spleen of old mice. Hyper-inflammatory ABCs from old mice are also hyper-metabolic, as compared to those from young mice and to the subset of FO B cells, a feature needed not only to support their higher expression of RNA for inflammatory markers but also their higher autoimmune antibody secretion. Conclusions These results identify a relationship between intrinsic inflammation, metabolism and autoimmune B cells and suggest possible ways to understand cellular mechanisms that lead to the generation of pathogenic B cells, that are hyper-inflammatory and hyper-metabolic, and secrete IgG antibodies with autoimmune specificities.

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OP0186 CHANGES IN CIRCULATING B CELL LEVELS AND IMMUNOPHENOTYPE ARE ASSOCIATED WITH DEVELOPMENT OF ARTHRITIS FOLLOWING TREATMENT WITH CHECKPOINT INHIBITORS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.908 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 112.2-113

Author(s):

M. Gatto ◽

S. Bjursten ◽

C. Jonell ◽

C. Jonsson ◽

S. Mcgrath ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Adverse Events ◽

B Cell ◽

Mononuclear Cells ◽

Checkpoint Inhibitors ◽

Therapy Monitoring ◽

Cell Subset ◽

Peripheral Blood Mononuclear ◽

Transitional B Cells

Background:Inflammatory arthritis (IA) is frequent among rheumatic side effects induced by checkpoint inhibitor (CPI) therapy for metastatic malignancies1. While T cells are likely to sustain the inflammatory process2, fewer data are available concerning the role of B cells3.Objectives:To investigate the phenotype of circulating B cells in patients who develop CPI-induced IA (CPI-IA) and to compare it with features of B cells in patients not developing immune-related adverse events (irAE) upon CPI treatment.Methods:B cell subsets at baseline (before CPI initiation) and during CPI treatment were analyzed in CPI-IA patients and in patients receiving CPI but who did not develop irAE (non-irAE). Peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry and B cells were identified as CD19+ and divided into naïve (CD27-IgD+), memory (CD27+IgD+/-), double negative (CD27-IgD-) and transitional (CD10+CD24+CD38+/hi) B cells. Levels of CD21, an activation marker on transitional B cells, were also analyzed. Non-parametric tests were used for analysis of differences between groups.Results:Six CPI-IA and 7 non-irAE patients matched for age, gender and CPI treatment were included, who had received CPI treatment due to metastatic melanoma. Flow cytometry revealed a significant increase of circulating B cells (p=0.002) (Figure 1A) and especially of transitional B cells in CPI-IA patients vs. non-irAE (median %, range: 7.8 (4.5-11.4) vs. 3.2 (1.6-4.3),p=0.007) (Figure 1B), while no remarkable changes were seen across other subsets. Transitional B cell levels significantly decreased from active to quiescent CPI-IA in all patients (p=0.008). In two CPI-IA patients for whom baseline sampling was available, the increase of transitional levels occurred early after CPI treatment and before CPI-IA onset. Levels of expression of CD21 on transitional B cells were increased in CPI-IA vs. non-irAE (p=0.01).Conclusion:Transitional B cells are expanded in CPI-IA patients and seem to increase early after start of CPI therapy. Monitoring this B cell subset might lead to closer follow-up and earlier diagnosis of CPI-IA.References:[1]Ramos-Casals M, Brahmer JR, Callahan MK, et al. Immune-related adverse events of checkpoint inhibitors. Nat Rev Dis Primers 2020;6:38[2]Murray-Brown W, Wilsdon TD, Weedon H, et al. Nivolumab-induced synovitis is characterized by florid T cell infiltration and rapid resolution with synovial biopsy-guided therapy. J Immunother Cancer 2020;8:e000281[3]Das R, Bar N, Ferreira M, et al. Early B cell changes predict autoimmunity following combination immune checkpoint blockade. J Clin Invest. 2018;128:715-2Disclosure of Interests:None declared

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CD1c but neither CD1a nor CD1b molecules are expressed on normal, activated, and malignant human B cells: identification of a new B-cell subset

Blood ◽

10.1182/blood.v72.1.241.241 ◽

1988 ◽

Vol 72 (1) ◽

pp. 241-247 ◽

Cited By ~ 49

Author(s):

D Delia ◽

G Cattoretti ◽

N Polli ◽

E Fontanella ◽

A Aiello ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

De Novo ◽

Cell Subset ◽

Ultrastructural Level ◽

Cytoplasmic Staining ◽

Clear Cut ◽

Hodgkin's Lymphomas

Abstract The CD1 cluster of monoclonal antibodies (MoAbs) CD1a, CD1b, and CD1c, identifies molecules that are differentially expressed on hematopoietic and nonhematopoietic tissues. Our earlier finding that the mantle zone (MZ) but not the germinal center (GC) of normal lymph nodes (LN) is CD1c+, CD1a-, and CD1b- prompted us to further investigate the expression of these molecules on normal, activated, and malignant B cells. We report that blood and spleen contain CD1c+ B cells that account for 49% +/- 20.4% (mean +/- SD) and 50.9% +/- 4.4% of the total B cell population, respectively. CD1a- and CD1b-specific MoAbs are unreactive with both B and T cells; these latter are CD1c- as well. When CD1c+ and CD1c- B cells are activated in vitro, the CD1c molecule is upregulated in the former subset and induced de novo in the latter. Conversely, activated blood T cells remain CD1c-. Neither CD1a nor CD1b molecules are detected on activated T and B lymphocytes. At ultrastructural level, the CD1c+ B cells exhibit distinctive features, namely, condensed chromatin with or without a nucleolus and a unique cluster of cytoplasmic vesicles and organelles; the number of nucleolated cells is higher in the spleen (95%) than in the tonsil (40%) or blood (5%). These findings further confirm the similarity between blood and MZ B cells. The CD1c expression assessed on 27 B-cell chronic lymphocytic leukemias (B-CLL) and 46 B non-Hodgkin's lymphomas (B-NHL) was detected on 41% and 32% of cases, respectively; the latter comprised four follicular and 11 diffuse histotypes. The Burkitt's lymphomas were CD1c-negative. The B-cell neoplasms were all CD1a- and, except for four with a weak cytoplasmic staining, all CD1b- as well. The clear-cut CD1c distribution in normal LN (MZ+, GC-) contrasted with the evidence that some B-NHL cells of GC origin (eg, follicular with predominantly small cleaved cells) were CD1c+. Overall, the finding that CD1c expression is restricted to a fraction of B cells present in lymphoid organs and in peripheral blood indicates that CD1c is a powerful marker for the identification and dissection of B-cell subsets whose functional properties can now be evaluated.

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Proportions of B-cell subsets are altered in incomplete systemic lupus erythematosus and correlate with interferon score and IgG levels

Rheumatology ◽

10.1093/rheumatology/keaa114 ◽

2020 ◽

Vol 59 (9) ◽

pp. 2616-2624

Author(s):

Svenja Henning ◽

Wietske M Lambers ◽

Berber Doornbos-van der Meer ◽

Wayel H Abdulahad ◽

Frans G M Kroese ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Lupus Erythematosus ◽

Cell Subset ◽

Cross Sectional Study ◽

Type I ◽

Healthy Controls ◽

Sectional Study ◽

Cross Sectional ◽

B Cell Subsets

Abstract Objectives Incomplete SLE (iSLE) patients display symptoms typical for SLE but have insufficient criteria to fulfil the diagnosis. Biomarkers are needed to identify iSLE patients that will progress to SLE. IFN type I activation, B-cell-activating factor (BAFF) and B-cell subset distortions play an important role in the pathogenesis of SLE. The aim of this cross-sectional study was to investigate whether B-cell subsets are altered in iSLE patients, and whether these alterations correlate with IFN scores and BAFF levels. Methods iSLE patients (n = 34), SLE patients (n = 41) with quiescent disease (SLEDAI ≤4) and healthy controls (n = 22) were included. Proportions of B-cell subsets were measured with flow cytometry, IFN scores with RT-PCR and BAFF levels with ELISA. Results Proportions of age-associated B-cells were elevated in iSLE patients compared with healthy controls and correlated with IgG levels. In iSLE patients, IFN scores and BAFF levels were significantly increased compared with healthy controls. Also, IFN scores correlated with proportions of switched memory B-cells, plasma cells and IgG levels, and correlated negatively with complement levels in iSLE patients. Conclusion In this cross-sectional study, distortions in B-cell subsets were observed in iSLE patients and were correlated with IFN scores and IgG levels. Since these factors play an important role in the pathogenesis of SLE, iSLE patients with these distortions, high IFN scores, and high levels of IgG and BAFF may be at risk for progression to SLE.

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Antibody Production and In Vitro Behavior of CD27-Defined B-Cell Subsets: Persistent Hepatitis C Virus Infection Changes the Rules

Journal of Virology ◽

10.1128/jvi.80.8.3923-3934.2006 ◽

2006 ◽

Vol 80 (8) ◽

pp. 3923-3934 ◽

Cited By ~ 56

Author(s):

Vito Racanelli ◽

Maria Antonia Frassanito ◽

Patrizia Leone ◽

Maria Galiano ◽

Valli De Re ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

B Cells ◽

B Cell ◽

Feedback Inhibition ◽

Cell Subset ◽

Functional Program ◽

Cell Functions

ABSTRACT There is growing interest in the tendency of B cells to change their functional program in response to overwhelming antigen loading, perhaps by regulating specific parameters, such as efficiency of activation, proliferation rate, differentiation to antibody-secreting cells (ASC), and rate of cell death in culture. We show that individuals persistently infected with hepatitis C virus (HCV) carry high levels of circulating immunoglobulin G (IgG) and IgG-secreting cells (IgG-ASC). Thus, generalized polyclonal activation of B-cell functions may be supposed. While IgGs include virus-related and unrelated antibodies, IgG-ASC do not include HCV-specific plasma cells. Despite signs of widespread activation, B cells do not accumulate and memory B cells seem to be reduced in the blood of HCV-infected individuals. This apparent discrepancy may reflect the unconventional activation kinetics and functional responsiveness of the CD27+ B-cell subset in vitro. Following stimulation with T-cell-derived signals in the absence of B-cell receptor (BCR) engagement, CD27+ B cells do not expand but rapidly differentiate to secrete Ig and then undergo apoptosis. We propose that their enhanced sensitivity to BCR-independent noncognate T-cell help maintains a constant level of nonspecific serum antibodies and ASC and serves as a backup mechanism of feedback inhibition to prevent exaggerated B-cell responses that could be the cause of significant immunopathology.

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B Cell Subsets as Severity-Associated Signatures in COVID-19 Patients

Frontiers in Immunology ◽

10.3389/fimmu.2020.611004 ◽

2020 ◽

Vol 11 ◽

Author(s):

Víctor A. Sosa-Hernández ◽

Jiram Torres-Ruíz ◽

Rodrigo Cervantes-Díaz ◽

Sandra Romero-Ramírez ◽

José C. Páez-Franco ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Subset ◽

Hierarchical Cluster ◽

Specific Subset ◽

Respiratory Parameters ◽

Cell Subpopulations ◽

Severity Of The Disease ◽

B Cell Subsets ◽

Relationship Of

BackgroundSARS-CoV-2 infection represents a global health problem that has affected millions of people. The fine host immune response and its association with the disease course have not yet been fully elucidated. Consequently, we analyze circulating B cell subsets and their possible relationship with COVID-19 features and severity.MethodsUsing a multiparametric flow cytometric approach, we determined B cell subsets frequencies from 52 COVID-19 patients, grouped them by hierarchical cluster analysis, and correlated their values with clinical data.ResultsThe frequency of CD19+ B cells is increased in severe COVID-19 compared to mild cases. Specific subset frequencies such as transitional B cell subsets increase in mild/moderate cases but decrease with the severity of the disease. Memory B compartment decreased in severe and critical cases, and antibody-secreting cells are increased according to the severity of the disease. Other non-typical subsets such as double-negative B cells also showed significant changes according to disease severity. Globally, these differences allow us to identify severity-associated patient clusters with specific altered subsets. Finally, respiratory parameters, biomarkers of inflammation, and clinical scores exhibited correlations with some of these subpopulations.ConclusionsThe severity of COVID-19 is accompanied by changes in the B cell subpopulations, either immature or terminally differentiated. Furthermore, the existing relationship of B cell subset frequencies with clinical and laboratory parameters suggest that these lymphocytes could serve as potential biomarkers and even active participants in the adaptive antiviral response mounted against SARS-CoV-2.

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Belimumab alters transitional B-cell subset proportions in patients with stable systemic lupus erythematosus

Lupus ◽

10.1177/0961203319869468 ◽

2019 ◽

Vol 28 (11) ◽

pp. 1337-1343 ◽

Cited By ~ 1

Author(s):

A Benitez ◽

K Torralba ◽

M Ngo ◽

L M Salto ◽

K S Choi ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

B Cells ◽

B Cell ◽

Lupus Erythematosus ◽

Mononuclear Cells ◽

Cell Subset ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Immature B Cells

Objective We evaluated the effects of the B-cell activating factor (BAFF)-targeting antibody Belimumab on human nonmemory B-cell pools. Human B-cell pools were identified using surface markers adapted from mouse studies that specifically assessed reductions in immature B cells due to BAFF depletion. Patients with systemic lupus erythematosus (SLE) have high levels of both BAFF and immature B cells. Mechanistic mouse studies provide a framework for understanding human responses to therapies that target B cells. Methods Peripheral blood mononuclear cells were isolated from healthy donors and SLE patients on Belimumab or standard-of-care therapy (SCT). Cells were stained for flow cytometry to identify B-cell subsets based on CD21/CD24. Differences in subset proportions were determined by one-way ANOVA and Tukey’s post hoc test. Results Patients treated with Belimumab show alterations in the nonmemory B-cell pool characterized by a decrease in the Transitional 2 (T2) subset ( p = 0.002), and an increase in the proportion of Transitional 1 (T1) cells ( p = 0.005) as compared with healthy donors and SCT patients. The naïve B-cell compartment showed no significant differences between the groups ( p = 0.293). Conclusion Using a translational approach, we show that Belimumab-mediated BAFF depletion reduces the T2 subset in patients, similar to observations in mouse models with BAFF depletion.

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Unbalanced X chromosome mosaicism in B cells of mice with X-linked immunodeficiency.

Journal of Experimental Medicine ◽

10.1084/jem.158.3.920 ◽

1983 ◽

Vol 158 (3) ◽

pp. 920-931 ◽

Cited By ~ 38

Author(s):

M H Nahm ◽

J W Paslay ◽

J M Davie

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

X Chromosome ◽

Cell Subset ◽

Phosphoglycerate Kinase ◽

Mutant Gene ◽

Abnormal Development ◽

Female Mice ◽

Chromosomal Genes

The immunodeficiency in CBA/N mice is reflected by abnormal development of a subset of B lymphocytes. However, it is not clear how xid, the mutant gene in CBA/N mice, affects the development of this subset. Specifically, it is not known if the xid gene influences the development of the B cell subset directly or indirectly by providing the improper developmental milieu through effects on other cells. We investigated this question using female mice heterozygous for two x chromosomal genes, xid and Pgk-1 (phosphoglycerate kinase-1). Since females are mosaic because of x chromosome inactivation, their lymphocytes can be studied for the choice of the x chromosome, using the two PGK-1 isoenzymes as the cytological marker. We find that B lymphocytes in the spleen prefer the x chromosome without xid while the remaining splenocytes and cells from other tissues do not. This suggests that xid affects B lymphocytes directly and not through their developmental milieu. Furthermore, our data suggest that the precursors for IgG1- and IgG3-producing cells may be both few and different.

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Protein Signature of LMP1 Signaling in PTLDs Identifies and Mimics Inter/Perifollicular CD30+ EBV− B Blasts.

Blood ◽

10.1182/blood.v104.11.3251.3251 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3251-3251

Author(s):

Rita Shaknovich ◽

Katia Basso ◽

Govind Bhagat ◽

Bachir Alobeid ◽

Giorgio Cattoretti

Keyword(s):

B Cells ◽

B Cell ◽

Plasma Cells ◽

Cell Subset ◽

Cellular Transformation ◽

Cell Types ◽

Marginal Zone B Cells ◽

Latently Infected ◽

Protein Signature

Abstract EBV-associated B-cell Post-Transpant Lymphoproliferative Disorders (PTLDs) represent a diverse group of lesions morphologically, in clinical presentation and behaviour, ranging from early reversible lesions to monomorphic aggressive lymphomas. Polymorphic cases, which represent the focus of our analysis, contain a mixture of cells in various EBV latency stages, defined by EBNA1, EBNA2 and LMP1 immunostaining. LMP1 is a key viral protein for cellular transformation and, analogously to CD40, engages TNF Receptor Associated Proteins and activates NF-kB and NF-kB-responsive genes. We analyzed the protein signature of LMP1 in PTLDs and non-PTLD tonsils by double staining for LMP1, CD30, CD20, Pax5 and signaling molecules. A remarkably conserved set of proteins, associated with LMP1/CD40 signaling and NF-kB activation is expressed both in the EBV-infected lymphoid population in polymorphic PTLDs and in a normal B-cell subset(s) in reactive tonsils. These proteins include highly expressed CD30, JunB, nuclear cRel, TRAF-1, Bcl-XL, MUM1, CCL22 and downregulated BCL6 and CD10. We observed that EBV infection, possibly through LMP1 and LMP2A signaling, results in varioius degrees of differentiation within the neoplastic clone. EBER+ terminally differentiated mucosa-associated IRTA-1+ marginal zone B-cells and CD138+ plasma cells were identified in most cases, including control post-transplant tonsils with no overt disease. We document for the first time in situ, in-vivo evidence of EBV latently infected post-Germinal Center B cells of marginal and plasma cell types in PTLDs. Polymorphic PTLD cases represent EBV-induced expansion of B cells, mimicking CD40L-like activated Peri/Interfollicular CD30+ normal B-cells.

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The BCRs Expressed by Leukemia Cells from TCL1 Transgenic Mice Resemble Those of Unmutated B-CLL.

Blood ◽

10.1182/blood.v106.11.49.49 ◽

2005 ◽

Vol 106 (11) ◽

pp. 49-49

Author(s):

Xiao J. Yan ◽

Emilia Albesiano ◽

Gloria Telusma ◽

Nicola Zanesi ◽

Carlo M. Croce ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Dna Sequences ◽

Cell Subset ◽

Lymphocytic Leukemia ◽

Antigen Specificity ◽

Germline Genes ◽

Pcr Products ◽

Vh Genes ◽

V Gene

Abstract Introduction: Eμ-T cell leukemia-1 (TCL-1) transgenic (Tg) mice serve as models of human B-cell chronic lymphocytic leukemia (B-CLL). These animals develop oligoclonal expansions of CD5+ B cells, one of which transforms into a B-CLL-like cell at ~13 - 18 months of age. A major unanswered question is whether the IgV gene restrictions seen in the TCL1 Tg model resemble those identified in human B-CLL. Therefore we analyzed the DNA sequences of the expressed, rearranged VHDJH and VLJL from 11 TCL1 Tg mice for V gene use, association with specific D and J segments, and shared H and L CDR3 motifs. Methods: Total RNA was isolated from spleens and lymph nodes of mice with obvious leukemia and reverse-transcribed to cDNA. To determine the Ig VH genes used by B cell clonal expansions, consensus FR1 primers and consensus JH primers were used for PCR. For Ig VL genes, Vκ consensus primers and Cκ primers were used. PCR products were either sequenced directly or cloned into vectors and then analyzed. DNA sequences were compared to the mouse Ig V gene germline genes deposited in NCBI GenBank and IMGT V-Quest. To confirm that nucleotide differences were actual point somatic mutations and not polymorphisms of known VH germline genes or heretofore unrecognized germline genes, PCR was performed on DNA from splenocytes of non-Tg mice using primers specific for the intron upstream of FR1 and the recombination signal sequences 3′ of the gene. PCR products were cloned and up to 60 colonies were sequenced. Using this approach, three new germline genes were identified and reported to GenBank. HCDR3 motifs were used to search both nucleotide and protein databases to identify similar sequences of known antigen specificity or B-cell subset origin. Results: DNA sequences of the VHDJH and VLJL from all (n=11) TCL1 Tg mice studied were <2% different from the most similar germline counterpart. Eight of the 11 clones used VH 1 family genes and the other three used VH 3, 5 and 12 family genes. HCDR3 and LCDR3 of these sequences frequently contained charged amino acids at the V-(D)-J junctions. Database searches for sequences similar to those of the TCL1 clones revealed groups of non-B-CLL sequences with identical or very similar HCDR3 motifs; some of these groups used the same VH gene and others used different VH genes. These structurally similar antibodies were either autoantibodies or antibodies produced by B-1 cells. One anti-bacterial antibody also was included. Conclusions: The clones that eventually become leukemic in TCL1 mice resemble those of human B-CLL cases with the worst clinical outcome in that they do not exhibit significant levels of Ig V gene mutations and they are structurally similar to autoantibodies and anti-microbial antibodies. Therefore, this model will be valuable in analyzing the development and progression of B-CLL cells from normal CD5+ B cells and the role that antigen-receptor engagement by autoantigens and microbial antigens plays in this process.

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