scholarly journals B Lymphocytes Producing Demyelinating Autoantibodies: Development and Function in Gene-targeted Transgenic Mice

1998 ◽  
Vol 188 (1) ◽  
pp. 169-180 ◽  
Author(s):  
Tobias Litzenburger ◽  
Reinhard Fässler ◽  
Jan Bauer ◽  
Hans Lassmann ◽  
Christopher Linington ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Cell Population ◽  
Plasma Cells ◽  
Autoimmune Encephalitis ◽  
Normal Numbers ◽  
Self Tolerance ◽  
Normal Differentiation ◽  
And Function

We studied the cellular basis of self tolerance of B cells specific for brain autoantigens using transgenic mice engineered to produce high titers of autoantibodies against the myelin oligodendrocyte glycoprotein (MOG), a surface component of central nervous system myelin. We generated “knock-in” mice by replacing the germline JH locus with the rearranged immunoglobulin (Ig) H chain variable (V) gene of a pathogenic MOG-specific monoclonal antibody. In the transgenic mice, conventional B cells reach normal numbers in bone marrow and periphery and express exclusively transgenic H chains, resulting in high titers of MOG-specific serum Igs. Additionally, about one third of transgenic B cells bind MOG, thus demonstrating the absence of active tolerization. Furthermore, peritoneal B-1 lymphocytes are strongly depleted. Upon immunization with MOG, the mature transgenic B cell population undergoes normal differentiation to plasma cells secreting MOG-specific IgG antibodies, during which both Ig isotype switching and somatic mutation occur. In naive transgenic mice, the presence of this substantial autoreactive B cell population is benign, and the mice fail to develop either spontaneous neurological disease or pathological evidence of demyelination. However, the presence of the transgene both accelerates and exacerbates experimental autoimmune encephalitis, irrespective of the identity of the initial autoimmune insult.

scholarly journals NR4A Expression by Human Marginal Zone B-Cells

Antibodies ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 50
Author(s):  
Kim Doyon-Laliberté ◽  
Josiane Chagnon-Choquet ◽  
Michelle Byrns ◽  
Matheus Aranguren ◽  
Meriam Memmi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Human Blood ◽  
Marginal Zone ◽  
Ex Vivo ◽  
Transcriptome Profiling ◽  
Marginal Zone B Cells ◽  
Healthy Donors ◽  
And Function

We have previously characterized a human blood CD19+CD1c+IgM+CD27+CD21loCD10+ innate-like B-cell population, which presents features shared by both transitional immature and marginal zone (MZ) B-cells, named herein “precursor-like” MZ B-cells. B-cells with similar attributes have been associated with regulatory potential (Breg). In order to clarify this issue and better characterize this population, we have proceeded to RNA-Seq transcriptome profiling of mature MZ and precursor-like MZ B-cells taken from the blood of healthy donors. We report that ex vivo mature MZ and precursor-like MZ B-cells express transcripts for the immunoregulatory marker CD83 and nuclear receptors NR4A1, 2, and 3, known to be associated with T-cell regulatory (Treg) maintenance and function. Breg associated markers such as CD39 and CD73 were also expressed by both populations. We also show that human blood and tonsillar precursor-like MZ B-cells were the main B-cell population to express elevated levels of CD83 and NR4A1-3 proteins ex vivo and without stimulation. Sorted tonsillar precursor-like MZ B-cells exerted regulatory activity on autologous activated CD4+ T-cells, and this was affected by a CD83 blocking reagent. We believe these observations shed light on the Breg potential of MZ populations, and identify NR4A1-3 as potential Breg markers, which as for Tregs, may be involved in stabilization of a regulatory status. Since expression and activity of these molecules can be modulated therapeutically, our findings may be useful in strategies aiming at modulation of Breg responses.

scholarly journals bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice.

1996 ◽  
Vol 183 (2) ◽  
pp. 381-391 ◽  
Author(s):  
D A Grillot ◽  
R Merino ◽  
J C Pena ◽  
W C Fanslow ◽  
F D Finkelman ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Cell Lineage ◽  
Cell Development ◽  
B Cell Development ◽  
Immunoglobulin M ◽  
Protective Mechanism ◽  
And Function ◽  
Peripheral B Cells

We have assessed during B cell development, the regulation and function of bcl-x, a member of the bcl-2 family of apoptosis regulatory genes. Here we show that Bcl-xL, a product of bcl-x, is expressed in pre-B cells but downregulated at the immature and mature stages of B cell development. Bcl-xL but not Bcl-2 is rapidly induced in peripheral B cells upon surface immunoglobulin M (IgM) cross-linking, CD40 signaling, or LPS stimulation. Transgenic mice that overexpressed Bcl-xL within the B cell lineage exhibited marked accumulation of peripheral B cells in lymphoid organs and enhanced survival of developing and mature B cells. B cell survival was further increased by simultaneous expression of bcl-xL and bcl-2 transgenes. These studies demonstrate that Bcl-2 and Bcl-xL are regulated differentially during B cell development and activation of mature B cells. Induction of Bcl-xL after signaling through surface IgM and CD40 appears to provide mature B cells with an additional protective mechanism against apoptotic signals associated with antigen-induced activation and proliferation.

IRF-4/MUM-1 Expression Is a Critical Switch in the Generation of Plasma Cells Versus Memory B-Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 337-337 ◽  
Author(s):  
Ulf Klein ◽  
Stefano Casola ◽  
Giorgio Cattoretti ◽  
Qiong Shen ◽  
Marie Lia ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Plasma Cells ◽  
Large Fraction ◽  
Variable Region ◽  
Memory B Cells ◽  
Critical Event ◽  
Tumor Subtypes

Abstract Most types of human B-cell lymphomas harbor somatically mutated Ig variable region genes, reflecting their origin from cells that have undergone the germinal center (GC) reaction of T-dependent immune responses. The lymphomas exhibit diverse phenotypes and clinical behaviors, likely as a consequence of differences both in the mechanisms of transformation and in the specific target cell. We have recently identified two distinct subsets of GC B-cells that may represent late stages of the GC-reaction and may be the precursors of plasma cells and memory B-cells. The corresponding subsets are characterized by downregulation of the GC-marker BCL6 and the alternative expression of IRF-4/MUM-1 or nuclear c-Rel. These two subsets seem to reflect distinct cellular programs which are altered during B-lymphomagenesis in various tumor subtypes which co-express BCL6, IRF-4 and nuclear c-Rel, an event never observed in normal B-cells. In order to gain insights into the physiologic role of IRF-4/MUM-1 and nuclear c-Rel in GC-development, we are ablating their expression specifically in mouse GC B-cells. Transgenic mice were generated that carry an IRF-4/MUM-1 null allele and a conditional IRF-4/MUM-1 allele which, following Cre-mediated deletion of the loxP-flanked promotor region and exons 1 and 2, expresses eGFP, thus allowing to track the development of the IRF-4/MUM-1 deficient cells at the single cell level. IRF-4/MUM-1fl/- mice were crossed with transgenic mice that express the Cre-recombinase specifically in B-cells undergoing class-switch to IgG1, an event occurring in a large fraction of GC B-cells. Upon immunization with sheep red blood cells or nitrophenyl-(NP)-KLH, mice unable to express IRF-4/MUM-1 in late GC B-cells (IRF-4/MUM-1fl/-/Cγ 1Cre/+), in contrast to control mice (IRF-4/MUM-1fl/+/Cγ 1Cre/+), did not develop plasma cells (IgG1+CD138+) in the peripheral lymphoid organs, blood, and bone marrow. On the other hand, the generation of memory B-cells appears normal since antigen-specific B-cells were present in the spleen (eGFP+B220+PNA-IgG1+) and blood (eGFP+B220+CD38+IgG1+). These results suggest that the IRF-4/MUM-1 gene product is required for the development of antigen-selected GC B-cells into plasma cells. We suggest that the expression of IRF-4/MUM-1 in a GC centrocyte is the critical event in the commitment of B-cells to differentiate into a plasma cell versus a memory B-cell, and are currently testing the role of nuclear c-Rel in the same process.

scholarly journals Activated PIK3CD drives innate B cell expansion yet limits B cell–intrinsic immune responses

2018 ◽  
Vol 215 (10) ◽  
pp. 2485-2496 ◽  
Author(s):  
Michelle N. Wray-Dutra ◽  
Fahd Al Qureshah ◽  
Genita Metzler ◽  
Mohamed Oukka ◽  
Richard G. James ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Immune Responses ◽  
B Cell ◽  
Plasma Cells ◽  
Recurrent Infection ◽  
Vaccine Responses ◽  
And Function ◽  
Natural Igm ◽  
The Impact

Activated PI3K-delta syndrome (APDS) is an immunodeficiency caused by gain-of-function mutations in PIK3CD. This disease exhibits complex immune phenotypes including increased IgM, recurrent infection, and impaired vaccine responses. To better understand the impact of B cells in this disease, we generated an inducible model of the common APDS mutation (hPIK3CD-E1021K; referred to as aPIK3CD) and intercrossed these mice with B cell–specific Cre models. Mb1-aPIK3CD mice exhibited bone marrow B lymphopenia and, conversely, expansion of the peripheral innate B1a and MZ B cell compartments. aPIK3CD B cells manifest increased pS6 and increased survival at several stages, without alterations in cycling, and baseline increases in plasma cells, natural IgM, and IgG3. Finally, Mb1-aPIK3CD mice exhibited blunted T cell–independent immune responses, and both AID- and CD21-aPIK3CD mice displayed reduced class-switched antibodies following T cell–dependent immunization. Thus, aPIK3CD alters B cell development and function and is counter-productive during immune responses, providing insight into B cell–intrinsic contributions to the APDS phenotype.

scholarly journals 3BP2 Deficiency Impairs the Response of B Cells, but Not T Cells, to Antigen Receptor Ligation

2006 ◽  
Vol 26 (14) ◽  
pp. 5214-5225 ◽  
Author(s):  
Miguel A. de la Fuente ◽  
Lalit Kumar ◽  
Bao Lu ◽  
Raif S. Geha
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Cycle Progression ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Normal Numbers ◽  
Tcr Signaling ◽  
And Function

ABSTRACT The adapter protein 3BP2 is expressed in lymphocytes; binds to Syk/ZAP-70, Vav, and phospholipase C-γ (PLC-γ); and is thought to be important for interleukin-2 gene transcription in T cells. To define the role of 3BP2 in lymphocyte development and function, we generated 3BP2-deficient mice. T-cell development, proliferation, cytokine secretion, and signaling in response to T-cell receptor (TCR) ligation were all normal in 3BP2−/− mice. 3BP2−/− mice had increased accumulation of pre-B cells in the bone marrow and a block in the progression of transitional B cells in the spleen from the T1 to the T2 stage, but normal numbers of mature B cells. B-cell proliferation, cell cycle progression, PLC-γ2 phosphorylation, calcium mobilization, NF-ATp dephosphorylation, and Erk and Jnk activation in response to B-cell receptor (BCR) ligation were all impaired. These results suggest that 3BP2 is important for BCR, but not for TCR signaling.

Frizzled 9 knock-out mice have abnormal B-cell development

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2487-2494 ◽  
Author(s):  
Erik A. Ranheim ◽  
Helen C. K. Kwan ◽  
Tannishtha Reya ◽  
Yu-Ker Wang ◽  
Irving L. Weissman ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Neurodevelopmental Disorder ◽  
Cell Development ◽  
B Cell Development ◽  
Normal Numbers ◽  
Self Renewal ◽  
Knock Out ◽  
Lymphoid Development

Abstract The binding of frizzled (Fzd) receptors by their Wnt ligands results in the inhibition of β-catenin degradation and subsequent transcription of β-catenin/LEF–inducible genes. The β-catenin pathway is known to be involved in development, tumorigenesis, and stem cell self-renewal. In humans, the FZD9 gene lies in the region of chromosome 7q11.23 deleted in the neurodevelopmental disorder, Williams-Beuren syndrome (WBS). Fzd9-/- mice show no obvious features of WBS, but reveal a role for Fzd9 in lymphoid development and maturation. Fzd9-/- mice show pronounced splenomegaly, thymic atrophy, and lymphadenopathy with age, with accumulation of plasma cells in lymph nodes. There is a depletion of developing B cells in the bone marrow (BM), particularly in the pre-B stage where immunoglobulin heavy chains are expressed and the cells are undergoing clonal expansion prior to light chain rearrangement. The pre-B defect is partially intrinsic to the hematopoietic system; as in competitive BM reconstitution studies, Fzd9-/--derived BM exhibits defective B-cell development when implanted into a wild-type host. Mature B cells are present in normal numbers in lymph node and spleen. These findings suggest a role for Fzd9 signaling in lymphoid development, particularly at points where B cells undergo self-renewal prior to further differentiation.

A NUP98-HOXD13 Fusion Gene Impairs B Lymphocyte Development and Function In a Mouse Model for Myelodysplastic Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 279-279
Author(s):  
Abdul Gafoor A. Puthiyaveetil ◽  
Bettina Heid ◽  
David L. Caudell
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
B Lymphocytes ◽  
Marginal Zone ◽  
Fusion Gene ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Cell Fractions ◽  
And Function

Abstract Abstract 279 Transgenic mice which express the fusion gene NUP98-HOXD13 (NHD13) have been shown to develop characteristic features of Myelodysplastic syndrome (MDS) including impaired hematopoietic differentiation and peripheral blood cytopenias in the presence of normocellular or hypercellular bone marrow (BM). It is evident that B-cells play a role in the progression of MDS by immune modulation or as direct targets of mutations resulting in ALL, or as cells that influence the BM microenvironment in which a neoplastic myeloid clone evolves. Choi and colleagues suggested a block in differentiation during the early development of B lymphocytes in the BM of NHD13 mice leading to lymphopenia consistent with the observation in some MDS patients. In this study, we sought to further delineate the role of NHD13 on B lymphocytes which escaped the initial differentiation block in the BM. We hypothesized that NHD13 impairs maturation and function of IgM+ B lymphocytes contributing to immunodeficiency. To study this, we performed blood smear examination, Complete Blood Counts (CBC), quantitative ELISA for antibody concentrations, and flow cytometric analysis of B cell fractions from the BM and spleen in 8–12 week-old transgenic and wild type (WT) mice. CBCs revealed significant lymphopenia and ELISA showed higher IgM concentrations (n=10, p<0.001), reduced levels of IgG1 (n=10, p<0.05) and IgE (n=10, p<0.01). The IgG2a, IgG2b, and IgG3 antibody levels were comparable to WT counterparts. Flow cytometric analysis of BM and splenic B cell fractions revealed reduced numbers of B cells in Hardy fractions D and F (n=10, p<0.01) indicative of impaired differentiation prior to these stages; splenic fractions in NHD13 mice were comparable to WT controls. Next, to assess the peripheral maturation and functional efficiency of B lymphocytes in the context of a comprehensive immune stimulation, a cohort of five WT and five preclinical transgenic mice were injected with 100 μ g dinitrophenol (DNP) followed by a booster dose on day 21. Mice were euthanized on day 28 and whole blood, spleen, lymphnodes and BM were harvested. CBC evaluation revealed significant lymphopenia in NHD13 mice (n=5, p<0.001). Quantitative ELISA for DNP specific antibodies showed comparable levels of serum IgM and significantly reduced levels of serum IgG1 (n=5, p<0.001), IgG2a (n=5, p<0.001), IgG2b (n=5, p<0.01), IgG3 (n=5, p<0.001) and IgE (n=5, p<0.01). Flow cytometric analysis of peripheral blood showed reduced numbers of B220+ IgM+ B cells (n=5, p<0.01), but comparable percentages of CD4+ and CD8+ T-cells. Detailed flow cytometric analysis of B-cell fractions in the BM and spleen of DNP-stimulated mice revealed a reduction in subpopulations of B lymphocytes. The earliest B cell lineage population, Pre-Pro B, was comparable to the WT controls. Hardy Pro B fraction B (n=5, p<0.001) and Pre B fractions E (n=5, p<0.01) and F (n=5, p<0.01) from BM of stimulated mice were significantly reduced in contrast to fractions C and C', which were higher (n=5, p<0.05 and p<0.001 respectively), indicative of cell growth arrest at these stages. Flow cytometry of splenic B-cell fractions from the DNP-stimulated mice showed significantly lower Transitional 1 (n=5, p<0.01), Follicular (n=5, p<0.05) and Marginal Zone (n=5, p<0.001) populations upon antigenic stimulation suggestive of defective clonal expansion of IgM+ cells even after escaping the block in the BM. Histopathology of the spleen revealed smaller lymphoid follicles with poorly developed mantle and marginal zone regions in the transgenic mice when compared to WT controls, consistent with the flow cytometric data. This study indicates that when NHD13 mice are immunologically challenged, B lymphocytes undergo impaired differentiation in the BM and maturation in the spleen, as well as reduced antibody class switching and subsequently lower antibody production. Analysis of B cell subsets during development and specific IgG/IgE antibody production, suggest that the NHD13 transgene might impair VDJ gene recombination and class switch recombination that are critical during these phases of B cell development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Selective expression of CD45 isoforms defines CALLA+ monoclonal B- lineage cells in peripheral blood from myeloma patients as late stage B cells

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 711-719 ◽  
Author(s):  
GS Jensen ◽  
MJ Mant ◽  
AJ Belch ◽  
JR Berenson ◽  
BA Ruether ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Plasma Cell ◽  
Late Stage ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Lower Proportion ◽  
Large Subset ◽  
B Lineage

Abstract The peripheral blood lymphocytes from 42 patients with multiple myeloma (MM) and 13 patients with monoclonal gammopathy of undetermined significance (MGUS) were studied by three-color immunofluorescence (IF) using antibodies directed to a broad range of B-cell markers (CD19, CD20, CD21, CD24), CALLA (CD10), PCA-1 (a plasma cell marker), and to the high and low molecular weight isoforms of the leukocyte common antigen, CD45RA (p205/220) and CD45RO (p 180). CD45RA is expressed on pre-B and B cells, and a transition from CD45RA to CD45RO defines differentiation towards plasma cells. Peripheral blood mononuclear cells (PBMC) from patients with myeloma included a large subset of B- lineage cells (mean of 39% to 45%) that were CALLA+ and PCA-1+ in all patients studied, including newly diagnosed patients and patients undergoing chemotherapy. Southern blot analysis indicated the presence of monoclonal Ig rearrangements in PBMC and a substantial reduction in the germ-line bands consistent with the presence of a large monoclonal B-cell subset. Avoidance of purification methods involving depletion of adherent cells was essential for detection of the abnormal B cells. Phenotypically, this abnormal B-cell population corresponded to late B or early pre-plasma cells (20% to 80% of PBMC), as defined by the concomitant expression of low densities of CD19 and CD20, moderate densities of CALLA and PCA-1, and strong expression of CD45RO on all B cells, with weakly coexpressed CD45RA on a small proportion. Heterogeneity in the expression of CD45RA and CD45RO within the abnormal B-cell population from any given patient suggested multiple differentiation stages. Abnormal B cells similar to those in MM were also detected in MGUS, although as a lower proportion of PBMC (26%). Abnormal B cells from patients with MGUS expressed predominantly the CD45RO isoform, but had a lower proportion of CALLA+ and PCA-1+ cells than were found on B cells from MM. This work indicates that the large subset of circulating monoclonal B lymphocytes from myeloma patients are at a late stage in B-cell differentiation, continuously progressing towards the plasma cell stage.

Expression of the IRTA1 receptor identifies intraepithelial and subepithelial marginal zone B cells of the mucosa-associated lymphoid tissue (MALT)

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3684-3692 ◽  
Author(s):  
Brunangelo Falini ◽  
Enrico Tiacci ◽  
Alessandra Pucciarini ◽  
Barbara Bigerna ◽  
Julia Kurth ◽  
...  
Keyword(s):  
B Cells ◽  
Lymph Nodes ◽  
B Cell ◽  
Cell Population ◽  
Lymphoid Tissue ◽  
Marginal Zone ◽  
Plasma Cells ◽  
Immunoglobulin Superfamily ◽  
Lymphoid Tissues ◽  
Marginal Zone B Cells

AbstractIRTA1 (immunoglobulin superfamily receptor translocation-associated 1) is a novel surface B-cell receptor related to Fc receptors, inhibitory receptor superfamily (IRS), and cell adhesion molecule (CAM) family members and we mapped for the first time its distribution in human lymphoid tissues, using newly generated specific antibodies. IRTA1 was selectively and consistently expressed by a B-cell population located underneath and within the tonsil epithelium and dome epithelium of Peyer patches (regarded as the anatomic equivalents of marginal zone). Similarly, in mucosa-associated lymphoid tissue (MALT) lymphomas IRTA1 was mainly expressed by tumor cells involved in lympho-epithelial lesions. In contrast, no or a low number of IRTA1+ cells was usually observed in the marginal zone of mesenteric lymph nodes and spleen. Interestingly, monocytoid B cells in reactive lymph nodes were strongly IRTA1+. Tonsil IRTA1+ cells expressed the memory B-cell marker CD27 but not mantle cell-, germinal center-, and plasma cell-associated molecules. Polymerase chain reaction (PCR) analysis of single tonsil IRTA1+ cells showed they represent a mixed B-cell population carrying mostly mutated, but also unmutated, IgV genes. The immunohistochemical finding in the tonsil epithelial areas of aggregates of IRTA1+ B cells closely adjacent to plasma cells surrounding small vessels suggests antigen-triggered in situ proliferation/differentiation of memory IRTA1+ cells into plasma cells. Collectively, these results suggest a role of IRTA1 in the immune function of B cells within epithelia. (Blood. 2003;102: 3684-3692)

scholarly journals Selective expression of CD45 isoforms defines CALLA+ monoclonal B- lineage cells in peripheral blood from myeloma patients as late stage B cells

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 711-719 ◽  
Author(s):  
GS Jensen ◽  
MJ Mant ◽  
AJ Belch ◽  
JR Berenson ◽  
BA Ruether ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Plasma Cell ◽  
Late Stage ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Lower Proportion ◽  
Large Subset ◽  
B Lineage

The peripheral blood lymphocytes from 42 patients with multiple myeloma (MM) and 13 patients with monoclonal gammopathy of undetermined significance (MGUS) were studied by three-color immunofluorescence (IF) using antibodies directed to a broad range of B-cell markers (CD19, CD20, CD21, CD24), CALLA (CD10), PCA-1 (a plasma cell marker), and to the high and low molecular weight isoforms of the leukocyte common antigen, CD45RA (p205/220) and CD45RO (p 180). CD45RA is expressed on pre-B and B cells, and a transition from CD45RA to CD45RO defines differentiation towards plasma cells. Peripheral blood mononuclear cells (PBMC) from patients with myeloma included a large subset of B- lineage cells (mean of 39% to 45%) that were CALLA+ and PCA-1+ in all patients studied, including newly diagnosed patients and patients undergoing chemotherapy. Southern blot analysis indicated the presence of monoclonal Ig rearrangements in PBMC and a substantial reduction in the germ-line bands consistent with the presence of a large monoclonal B-cell subset. Avoidance of purification methods involving depletion of adherent cells was essential for detection of the abnormal B cells. Phenotypically, this abnormal B-cell population corresponded to late B or early pre-plasma cells (20% to 80% of PBMC), as defined by the concomitant expression of low densities of CD19 and CD20, moderate densities of CALLA and PCA-1, and strong expression of CD45RO on all B cells, with weakly coexpressed CD45RA on a small proportion. Heterogeneity in the expression of CD45RA and CD45RO within the abnormal B-cell population from any given patient suggested multiple differentiation stages. Abnormal B cells similar to those in MM were also detected in MGUS, although as a lower proportion of PBMC (26%). Abnormal B cells from patients with MGUS expressed predominantly the CD45RO isoform, but had a lower proportion of CALLA+ and PCA-1+ cells than were found on B cells from MM. This work indicates that the large subset of circulating monoclonal B lymphocytes from myeloma patients are at a late stage in B-cell differentiation, continuously progressing towards the plasma cell stage.

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