scholarly journals B Lineage Cells in ANCA-Associated Vasculitis

2021 ◽  
Vol 23 (1) ◽  
pp. 387
Author(s):  
Ana Merino-Vico ◽  
Jan Piet van Hamburg ◽  
Sander W. Tas
Keyword(s):  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Systemic Autoimmune Disease ◽  
Therapeutic Effects ◽  
Signalling Molecules ◽  
Anca Associated Vasculitis ◽  
B Lineage ◽  
Anti Cd20 ◽  
B Lineage Cells

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease that affects small sized blood vessels and can lead to serious complications in the lungs and kidneys. The prominent presence of ANCA autoantibodies in this disease implicates B cells in its pathogenesis, as these are the precursors of the ANCA-producing plasma cells (PCs). Further evidence supporting the potential role of B lineage cells in vasculitis are the increased B cell cytokine levels and the dysregulated B cell populations in patients. Confirmation of the contribution of B cells to pathology arose from the beneficial effect of anti-CD20 therapy (i.e., rituximab) in AAV patients. These anti-CD20 antibodies deplete circulating B cells, which results in amelioration of disease. However, not all patients respond completely, and this treatment does not target PCs, which can maintain ANCA production. Hence, it is important to develop more specific therapies for AAV patients. Intracellular signalling pathways may be potential therapeutic targets as they can show (disease-specific) alterations in certain B lineage cells, including pathogenic B cells, and contribute to differentiation and survival of PCs. Preliminary data on the inhibition of certain signalling molecules downstream of receptors specific for B lineage cells show promising therapeutic effects. In this narrative review, B cell specific receptors and their downstream signalling molecules that may contribute to pathology in AAV are discussed, including the potential to therapeutically target these pathways.

scholarly journals Multiple Myeloma Includes Phenotypically Defined Subsets of Clonotypic CD20+ B Cells that Persist during Treatment with Rituximab

2008 ◽  
Vol 2 ◽  
pp. CMO.S615 ◽  
Author(s):  
Linda M. Pilarski ◽  
Eva Baigorri ◽  
Michael J. Mant ◽  
Patrick M. Pilarski ◽  
Penelope Adamson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Subset ◽  
Light Scatter ◽  
Cell Compartments ◽  
B Lineage ◽  
B Cell Subsets ◽  
Anti Cd20

Potential progenitor B cell compartments in multiple myeloma (MM) are clinically important. MM B cells and some circulating MM plasma cells express CD20, predicting their clearance by treatment with anti-CD20. Here we describe two types of clonotypic CD20+ B cell in peripheral blood of myeloma patients, identified by their expression of CD19 and CD20 epitopes, their expression of CD45RA and their light scatter properties. Thus, the circulating component of the MM clone includes at least two distinct CD19+ CD20+ B cell compartments, as well as CD138+CD20+ plasma cells. To determine whether either or both B cell subsets and the CD20+ plasma cell subset were depleted by anti-CD20 therapy, they were evaluated before, during and after treatment of patients with rituximab (anti-CD20), followed by quantifying B cell subsets over a 5 month period during and after treatment. Overall, all three types of circulating B lineage cells persist despite treatment with rituximab. The inability of rituximab to prolong survival in MM may result from this failure to deplete CD20+ B and plasma cells in MM.

Targeting Long-Lived Plasma Cells Is Essential For Treating Hemophilia A Inhibitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 927-927
Author(s):  
Chao Lien Liu ◽  
Meghan Lyle ◽  
Simon Shin ◽  
Carol H. Miao
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Immune Responses ◽  
B Cell ◽  
Plasma Cells ◽  
Hemophilia A ◽  
Combined Treatment ◽  
Therapeutic Effects ◽  
Anti Cd20

Abstract The most critical and challenging problem for therapeutic treatment in hemophilia A patients is due to the formation of Inhibitory antibodies. It is hypothesized that long-lived plasma cells (LLPCs) play an important role in the persistent production of anti-FVIII antibodies in hemophilia A (HemA) inhibitor patients. The migration of plasma cells to the BM, where they become the LLPCs, is largely controlled by an interaction between the C-X-C type chemokine ligand 12 (CXCL12) produced by bone marrow (BM) stromal cells and its receptor CXC receptor4 (CXCR4; CD184) on plasma cells surface. Our previous data showed that administration of anti-murine CD20 (IgG2a) alone can deplete B cells significantly and reduce anti-FVIII inhibitor titers transiently in FVIII plasmid-treated HemA mice with pre-existing inhibitors, however, complete tolerance to FVIII was not achieved probably due to the persistence of LLPCs. We sought novel therapeutic strategies that target CXCL12/CXCR4 pathway to reduce/eliminate LLPCs and achieve the goal for long-term tolerance to FVIII in the HemA inhibitor mice. AMD3100, the CXCR4 antagonist, plus G-CSF inhibit the interaction of CXCL12 and CXCR4, thus facilitating the mobilization of stem cells and blocking the homing and retention of LLPCs. The combined treatment strategy used anti-CD20, G-CSF and AMD3100 to target B cells plus with the specific IL-2/IL-2mAb (JES6-1) complexes to target T cells for preventing both T and B cell-dependent anti-FVIII immune responses. Two groups of FVIII-primed inhibitor mice were treated with different combined immunomodulation regimens: (1) IL-2 complexes+AMD3100+G-CSF+anti-CD20, (2) AMD3100+G-CSF+anti-CD20. Control mouse groups were treated with each of the single regimens and FVIII only, or untreated as the naive control. All the treatments were administered one cycle per two weeks for 6 weeks and the therapeutic effects (FVIII activities) as well as immune responses (anti-FVIII inhibitors) were evaluated at different time points after treatment. Significant expansion of Treg cells reaching a 5∼7-fold increase on the peak days (day 3-7 after treatment) was observed in the IL-2/IL-2mAb complexes treated groups, whereas ∼95% of B cell populations were depleted in the anti-CD20 treated groups. In addition, administration of AMD3100 plus G-CSF significantly reduced circulating and bone marrow CXCR4+ plasma cells by flow cytometry analysis as well as decreased FVIII-specific plasma cells in bone marrow by ELISPOT assay. Except for the control groups, the two mouse groups treated with combined immunosuppressive regimens showed a significant reduction of inhibitory titers following the treatment. Long-term responses are being followed and second challenge with FVIII plasmid will be used to evaluate the induction of long term tolerance to FVIII. These combination regimens are highly promising in modulating/eliminating pre-existing anti-FVIII antibodies and inducing long-term tolerance in FVIII primed subjects. Disclosures: No relevant conflicts of interest to declare.

Cellular side of acquired immunity (B cells)

2013 ◽  
pp. 371-378
Author(s):  
Thomas Dörner ◽  
Peter E. Lipsky
Keyword(s):  
B Cells ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Plasma Cells ◽  
Adaptive Immune System ◽  
Adaptive Immune ◽  
B Cell Homeostasis ◽  
Anti Cd20 ◽  
Antigen Presenting

B cells have gained interest in rheumatoid arthritis (RA) beyond being the precursors of antibody-producing plasma cells since they are also a broader component of the adaptive immune system. They are capable of functioning as antigen-presenting cells for T-cell activation and can produce an array of cytokines. Disturbances of peripheral B-cell homeostasis together with the formation of ectopic lymphoid neogenesis within the inflamed synovium appears to be a characteristic of patients with RA. Enhanced generation of memory B cells and autoreactive plasma cells producing IgM-RF and ACPA-IgG antibodies together with formation of immune complexes contribute to the maintenance of RA, whereas treatment with B-cell-directed anti-CD20 and CLTA4-Ig therapy provides clinical benefit.

scholarly journals Regulated Expression of the Eph-Related Receptor Tyrosine Kinase Hek11 in Early Human B Lymphopoiesis

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3613-3622 ◽  
Author(s):  
Hans-Christian Aasheim ◽  
Leon W.M.M. Terstappen ◽  
Ton Logtenberg
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Expression Patterns ◽  
B Lymphopoiesis ◽  
Fetal Bone ◽  
Degenerate Oligonucleotide ◽  
Regulated Expression ◽  
B Lineage ◽  
B Lineage Cells

Abstract Members of the large Eph family of receptor tyrosine kinases (RTKs) display temporally and spatially restricted expression patterns during embryogenesis, suggesting a role in various developmental processes. We have begun to investigate the expression of members of this receptor family during human hematopoiesis, in particular B lymphopoiesis. Expression of Eph RTKs in cells of the B-lymphoid lineage was assessed by using degenerate oligonucleotide primers based on stretches of conserved nucleic acid sequences in members of the Eph family. First, the content of Eph-family RTKs was assessed in freshly sorted fetal bone marrow pro–B cells. This population was found to harbor transcripts of the Hek8 and Hek11 members of this gene family. Subsequent analysis of expression of these genes in B cells representing various differentiation and ontogenic stages showed that the Hek8 transcript is constitutively present in all fetal and adult B-lineage cells, with high levels of expression in peripheral blood B cells. In contrast, the Hek11 transcript was exclusively found in fetal bone marrow pro–B cells and pre–B cells, but not in more mature fetal B-lineage cells. All adult B-lineage cells, from early pro–B cells to end-stage plasma cells, lacked Hek11 transcripts. The developmentally regulated expression of Hek11 during fetal B lymphopoiesis suggests a role for this gene in pre/pro–B cell expansion and/or differentiation and defines a difference in progenitor B cell populations isolated from fetal versus adult human bone marrow.

B Cell Lineage-Specific Deletion of Icsbp/IRF8 Reveals Roles for IRF8 in the Regulation of Marginal Zone and Follicular B Cell Development.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1334-1334
Author(s):  
Hongsheng Wang ◽  
Jianxun Feng ◽  
Chang Hoon Lee ◽  
Herbert Morse
Keyword(s):  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Development ◽  
B Cell Development ◽  
Conditional Knockout ◽  
Myelogenous Leukemia ◽  
Exon 2 ◽  
B Lineage ◽  
B Lineage Cells

Abstract Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a transcription factor that expresses in T cells, B cells and macrophages and plays a role in myeloid development. Targeted deletion of IRF8 in mice (IRF8−/−) induced progressive increase in the numbers of granulocytes in various lymphoid organs and development of a syndrome similar to human chronic myelogenous leukemia. In addition to defective development of macrophages and dendritic cells, B cell development was also impaired in IRF8−/− mice. This includes decreased numbers of early B cells, expanded marginal zone (MZ) B cells and diminished follicular (OF) B2 cells. Because abnormal myeloid cells could alter microenvironment required for normal B cell development, we have generated IRF8 conditional knockout mice to specifically investigate the function of IRF8 in B lineage cells. Mice were engineered to have exon 2, encoding the DNA binding domain of IRF8, flanked by loxP sites (designated IRF8f/+). These mice were then crossed with the CD19Cre strain in which the expression of Cre-recombinase is controlled by the endogenous CD19 locus. Homozygous mice (designated (IRF8f/f x Cre)F1) underwent germline excision of IRF8 in CD19+ B lineage cells. As a result, there was no detectable mRNA and protein of IRF8 in their splenic B cells. Flow cytometry analysis revealed expanded MZ B cells and reduced OF B2 cells in the spleen of (IRF8f/f x Cre)F1 mice. Interestingly, the expression level of CD23 on OF B cells was significantly decreased in (IRF8f/f x Cre)F1 mice, indicating that IRF8 is required for maintaining a normal OF phenotype. In the peritoneum of (IRF8f/f x Cre)F1 mice, while the numbers of B1a and B2 cells were slightly decreased, the number of B1b cells was slightly increased. Furthermore, BXH2 mice carrying a mutation (C915T) in the Icsbp1 gene exhibited similar expansion of MZ B cells and low expression of CD23 in OF B cells. Taken together, these analyses indicate that IRF8 is required for development of normal MZ and B2 cells.

miRNA Analysis Identifies a Unique Expression in Waldenström Macroglobulinemia B Cells and Plasma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 620-620
Author(s):  
Sherine F. Elsawa ◽  
Anne J Novak ◽  
Deanna Grote ◽  
Thomas E Witzig ◽  
Stephen M. Ansell
Keyword(s):  
B Cells ◽  
B Cell ◽  
Expression Pattern ◽  
Mirna Expression ◽  
Target Genes ◽  
Plasma Cells ◽  
Mirna Signature ◽  
Variance Stabilization ◽  
Healthy Donors ◽  
B Lineage

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that are approximately 20–22 nucleotides with critical functions in cell growth, survival, and differentiation. These conserved sequences can regulate expression of multiple genes and are often tissue specific and dysregulated in malignancies. Thus, miRNA profiling has been used to create signatures for many solid tumors. These profiles have been used to classify tumors and to help predict survival and outcome. In the present study, we utilized the DiscovArray miRNA profiling service (Asuragen Services, Austin, TX) which utilizes a custom-manufactured Affymetrix GeneChip® from Ambion that covers miRNAs derived from the Sanger miRBase (http://microrna.sanger.ac.uk/sequences/index.shtml) and over 11,000 predicted miRNAs derived from published reports. The signal processing implemented was a multi-step process involving probe-specific signal detection calls, background estimation and correction, constant variance stabilization and global normalization. For each probe, an estimated background value was subtracted derived from the median signal of a set of G-C-matched anti-genomic controls. Arrays within a specific experiment were normalized together according to variance stabilization method. Detection calls were based on a Wilcoxon rank-sum test of the miRNA probe signal compared to the distribution of signals from GC-content matched anti-genomic probes. For statistical hypothesis testing, a two-sample t-test, with assumption of equal variance, was applied. One-way ANOVA was used for multiple group comparison. Probes were considered to be differentially expressed based on two criteria: a p-value of < 0.001 and glog2 difference > 1. miRNA expression was analyzed in all malignant B lineage cells (CD19+ CD138+) (n=8), malignant B cells alone (CD19+) (n=6) and plasma cells alone (CD138+) (n=3) from Waldenström macroglobulinemia (WM) patients. The expression was compared to malignant CD19+ B cells from chronic lymphocytic leukemia (CLL) patients (n=5), malignant plasma cells (CD138+) from multiple myeloma (MM) patients (n=5) and to B lineage cells (CD19+ CD138+) (n=4), CD19+ B-lymphocytes (n=3) and CD138+ plasma cells (n=6) from healthy donors. Data analysis based on a total of approximately 11,000 miRNAs analyzed shows that CD19+ CD138+ cells (double sorting) from WM patients did not cluster as a unique group. Some samples had a pattern similar to CLL, some similar to MM and others similar to CD19+ CD138+ cells from healthy controls. This lack of clear signature was observed by others in gene expression profiling and CGH arrays. We therefore hypothesized this lack of clustering was due to the lymphoplasmacytic nature of WM cells and therefore we analyzed B cells (CD19+) and plasma cells (CD138+) separately. miRNA expression in B cells (CD19+) identifies a signature in normal B cells that is absent in both WM B cells (CD19+) and CLL cells. There is also a set of miRNAs that are absent in normal B cells that are expressed in WM B cells and CLL. In addition, WM B cells had a unique miRNA signature that is unique compared to CLL and normal B cells. An additional set of miRNAs were expressed and clustered only in CLL patients. Similar to B cells, plasma cell (CD138+) analysis in WM, MM and healthy donors shows a clustering pattern that identifies normal plasma cells from MM plasma cells. WM plasma cells had a miRNA signature that is unique only to WM patients, however, a subset of miRNAs shared an expression pattern with MM plasma cells. While miRNAs can target multiple genes, some of the genes that are targets of the miRNAs identified in this analysis include XBP-1, Blimp-1, IRF-4, Bcl-6 and TACI. These target genes are known to be important in B cell and plasma cell development. In summary, we have analyzed miRNA expression in malignant B cells (CD19+) and malignant plasma cells (CD138+) from WM patients and compared their expression pattern to their normal counterpart as well as malignant counterpart in CLL B cells and MM plasma cells. Our analysis shows that WM B cells have a miRNA signature unique to WM only and one that is shared by CLL cells. Similarly, WM plasma cells have a unique miRNA signature but also has some miRNAs that are shared by malignant plasma cells in MM. These miRNAs target genes involved in B cell differentiation. Analysis of the functional roles of these miRNAs will and their regulation will further our understanding of the regulation of B cells development in normal and malignant conditions.

scholarly journals Dynamics and localization of early B-lymphocyte precursor cells (pro-B cells) in the bone marrow of scid mice

Blood ◽  
1992 ◽  
Vol 79 (7) ◽  
pp. 1695-1703 ◽  
Author(s):  
DG Osmond ◽  
N Kim ◽  
R Manoukian ◽  
RA Phillips ◽  
SA Rico-Vargas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Scid Mice ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Normal Numbers ◽  
Cytoplasmic Inclusions ◽  
B Lineage ◽  
Immunofluorescence Labeling ◽  
B Lineage Cells

Mice homozygous for the scid (severe combined immunodeficiency) mutation are generally unable to produce B lymphocytes, a condition attributed to defective rearrangement of immunoglobulin genes in precursor B cells. Some early B-lineage cells are present in the bone marrow (BM), however. In scid mice, we defined three subsets of early progenitor B cells lacking mu heavy chains (pro-B cells) based on the expression of terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein: (a) early pro-B cells (TdT+B220-), (b) intermediate pro-B cells (TdT+B220+), and (c) late pro-B cells (TdT-B220+). Double immunofluorescence labeling of BM cell suspensions has shown normal numbers of early and intermediate pro-B cells, substantially reduced numbers of late pro-B cells, and an absence of pre-B cells and B cells. Early and intermediate pro-B cells accumulated in metaphase in near- normal numbers after intraperitoneal (IP) vincristine administration. B220+ pro-B cells have been localized in BM sections by the binding of intravenously (IV) administered 125I monoclonal antibody (MoAb) 14.8, detected by light and electron microscope radioautography. Many B220+ cells were located peripherally in the bone-lining cell layers associated with stromal reticular cells. More centrally located B220+ cells were frequently associated with macrophages containing prominent cytoplasmic inclusions. Occasional B220+ cells were present in venous sinusoids. These results demonstrate that many pro-B cells in scid mice occupy microenvironments in the BM near the surrounding bone. The pro-B cells maintain normal rates of production during stages of presumptive mu heavy-chain gene rearrangement, apparently unaffected by the absence of a mature B cell pool. Nearly all defective cells then abort at the late pro-B cell stage and are deleted, apparently by macrophages. The findings contribute to models of in vivo differentiation, regulation, localization, and selection of early B-lineage cells in the BM.

CD127 Expression Confers Enhanced Survival on Human B-Lineage Cells; Implications for the Role of IL-7 Signaling in Human B Lymphopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1329-1329
Author(s):  
Sonja E. Johnson ◽  
J. Donald Capra ◽  
Tucker W. LeBien
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cord Blood ◽  
B Cell ◽  
Immunofluorescent Staining ◽  
Segment Length ◽  
B Lineage ◽  
B Cell Precursors ◽  
Cd127 Expression ◽  
B Lineage Cells

Abstract The IL-7Rα chain (CD127) is expressed on some, but not all, CD19+/surface μ-B-lineage cells isolated from fetal and adult marrow, and umbilical cord blood, but the functional consequences of CD127 expression in early human B cell development during all stages of human life are unresolved. We have described a xenogeneic culture model for generating CD19+ B-lineage cells from CD34+ cord blood stem cells following a 4 wk co-culture on murine MS-5 stromal cells (Johnson SE et al., 2005 J Immunol.). The majority of these cells express a pro-B cell phenotype i.e., CD19+/CD10+/CD20lo/CD21−/CD22+/CD24+/pre-BCR-. Moreover, 1–4% express surface μ heavy chains (HC), and some express κ or λ light chains. We have also reported that 20–40% of CD19+ cells emerging in the xenogeneic culture express CD127, CD127+ cells are more blastic than CD127- cells, and development of CD19+ cells is substantially blocked in xenogeneic cultures treated with anti-murine IL-7 (Johnson SE, ibid). The goal of the present study was to further characterize the developmental potential and patterns of gene expression in CD19+/CD127+ and CD19+/CD127− cells that emerge in xenogeneic cultures. We hypothesized that CD127 expression defines a subset of CD19+ B cell precursors with heightened potential to become mature B-lymphocytes. CD19+/CD127+ and CD19+CD127− populations that emerged in 4 wk xenogeneic cultures were sorted to > 95% purity by FACS. Immunofluorescent staining showed that surface and intracellular μ HC positive cells were present in both sorted populations at frequencies of 3–4%. Using an RT-PCR-based sequencing approach, we analyzed approximately 100 IgM V region sequences from both CD19+/CD127+ and CD19+/CD127− FACS-purified populations. The results indicated that the sequences from both populations were: non-mutated and used VH4, D and J gene segments that were similar to what would be expected in a cord blood B cell repertoire, and exhibited no statistically significant difference in N segment length, CDR3 length, or CDR3 charges. These results suggest that the amplified sequences were likely derived from the 3–4% cytoplasmic μ HC+ pre-B cells present in both sorted populations. The immunofluorescent staining and VDJH rearrangement sequence results suggest that large CD19+/CD127+/cytoplasmic μ HC+ pre-B cells may differentiate into small CD19+/CD127−/cytoplasmic μ HC+ cells in the xenogeneic culture. This was further supported by showing that intra-tibial injection of FACS-purified CD19+/CD127+ and CD19+/CD127− cells into sub-lethally irradiated NOD-SCID mice led to the appearance of surface μ HC+ cells from both sorted populations. Western blotting of sorted CD19+/CD127+ and CD19+/CD127− cells showed the presence of Bcl-2, p-GSK3β (specifically p-Ser9, the residue phosphorylated by AKT) and pERK1/2 in CD19+/CD127+ cells, while these proteins/phosphoproteins were undetectable in CD19+/CD127− cells. By contrast, expression of the cell cycle inhibitor p27KIP1 was elevated in CD19+/CD127− cells compared to CD19+/CD127+ cells. Furthermore, FACS-purified CD19+/CD127+ cells survived longer than CD19+/CD127− cells when cultured in the absence of MS-5, and the survival of the former was enhanced by stimulation with IL-7. The collective results indicate that CD127 expression identifies a population of normal human CD19+ B cell precursors with a pattern of gene expression and survival/proliferation attributes consistent with a crucial role in the development of the B-lymphocyte arm of the human immune system.

Charting the Early Stages of Human B Lymphopoiesis Using the IL-7 Receptor as a Navigational Marker.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 95-95
Author(s):  
Sonja E. Nodland ◽  
Anna A Bajer ◽  
Tucker W. LeBien
Keyword(s):  
B Cells ◽  
Cord Blood ◽  
B Cell ◽  
Low Frequency ◽  
Cell Development ◽  
Hematopoietic Stem ◽  
Developmental Potential ◽  
Cord Blood Cd34 ◽  
B Lineage ◽  
B Lineage Cells

Abstract Abstract 95 The identity and developmental potential of human lymphoid progenitors (LP) restricted to differentiate into T, B and/or NK cells at the exclusion of myeloid/erythroid lineages is not completely understood. Although LP from cord blood and marrow been described, there is no consensus as to the phenotype of LP committed to the B-lineage from either tissue source, or the identity of the cytokine signals that regulate lineage commitment of these progenitors. Resolution of this question has implications for identifying potential leukemic stem cells in acute lymphoblastic leukemia, as well as for lymphocyte reconstitution in the setting of cord blood and marrow transplantation. One of the challenges in conducting a comprehensive cellular and molecular characterization of LP is their low frequency in normal marrow and cord blood. The xenogeneic human hematopoietic stem cell/murine stromal cell (MS-5) model of lymphohematopoiesis has been shown to support the development of all human lymphoid lineages from human CD34+ hematopoietic progenitor cells (HPC). Therefore, the goal of the current study was to use the MS-5 model as a cellular resource to identify and characterize candidate LP subsets, and to evaluate the expression and function of the IL-7 receptor in B-lineage cell development. We initially determined whether expression of CD127 (the IL-7 receptor alpha chain) and CD34 were sufficient to define CD19- LP with enhanced B lymphopoietic potential. Xenogeneic cultures were initiated by plating cord blood CD34+ HPC onto MS-5, and CD19- lymphohematopoietic cells were characterized by polychromatic flow cytometry. Gating on CD19-/CD14-/CD15- cells revealed four populations: CD19-/CD34+/CD127-, CD19-/CD34+/CD127+, CD19-/CD34-/CD127+, and CD19-/CD34-/CD127-. CD19-/CD34+/CD127- cells were the predominant population during the first week of culture but sharply declined thereafter. A low frequency CD19-/CD34+/CD127+ population was first detected at 5-7 days and largely disappeared by 2 wks. The CD19-/CD34-/CD127+ population was initially detected at 1 wk, and underwent a bi-phasic increase and decrease over the following 3 wks. Evaluation of sorted fractions by quantitative PCR showed that the onset of expression of RAG1, RAG2, TDT, CD79A, VPREB, IGλ LIKE, and EBF-1 were coincident with onset of CD127 expression. Differentiation of CD34+/CD127+ to CD34-/CD127+ LP was accompanied by an ∼ 50-fold increase in expression of PAX5 and CD19, suggesting that the latter population was the immediate precursor of CD19+ B-lineage cells. When the three populations were FACS-purified on day 14 and re-plated on MS-5, a progression from CD34+/CD127- > CD34+/CD127+ > CD34-/CD127+ was suggested by the kinetics of CD19+ cell development. We have previously shown that neutralization of MS-5 produced murine IL-7 reduces the development of CD19+ cells from cord blood CD34+ HPC by ∼ 80%. When fetal liver, pediatric marrow or adult marrow CD34+ cells were used to initiate xenogeneic cultures, we observed a similar dependency on IL-7 for CD19+ cell development. However, neutralization of murine IL-7 had no effect on the development of CD34-/CD127+/CD19- LP. Surprisingly, independent of the source of CD34+ HPC used to initiate xenogeneic cultures, B-lineage cells expressing cell surface IgM developed in the absence of the canonical CD34+/CD19+ pro-B cell population present in human marrow. The absence of CD19+/CD34+ pro-B cells was not due to MS-5 xenogeneic culture conditions being non-permissive, since CD19+/CD34+ pro-B cells FACS-purified from fresh pediatric marrow and plated on MS-5 survived and differentiated over 3-4 wks of culture into surface IgM+/IgD+ naive B cells. Additionally, CD34 was stably expressed on CD19- cells in xenogeneic cultures for ∼ 2 wks. We conclude that expression of CD127 on CD19- LP and CD19+ B-lineage cells may define an early continuum in human B cell development, which at least partially encompasses a pathway that either bypasses or is distinct from development of canonical CD34+/CD19+ pro-B cells. Disclosures: No relevant conflicts of interest to declare.

B-lineage colonies from normal, human bone marrow are initiated by B cells and their progenitors

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 961-970 ◽  
Author(s):  
K McGinnes ◽  
M Letarte ◽  
CJ Paige
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Plasma Cells ◽  
Present Report ◽  
Colony Assay ◽  
Normal Human ◽  
B Lineage ◽  
Cell Colony

Abstract We have recently described a reproducible method whereby colonies containing cells that secrete immunoglobulin (Ig) can be grown from normal, human, adult bone marrow samples. The present report characterizes the cells that initiate these colonies. It is shown that all clonogenic cells express the CD19 surface antigen, as removal of these cells before plating in the B-cell colony assay abolished the subsequent growth of plaque-forming, B-lineage colonies. Cells from both the CD10+ and CD20+ B-lineage subpopulations initiated the growth of B-cell colonies, as removal of either subset resulted in a 50% reduction in the number of resulting B-cell colonies. The removal of activated B cells (CD23+), plasma cells (PCA-1+), or myeloid cells (CD13+) did not lead to a significant depletion in B-cell colony formation. Pre-B cells that were not yet committed to Ig light chain expression were also able to differentiate and proliferate into Ig- secreting colonies under the culture conditions used. Colonies initiated by these light chain uncommitted cells were distinguished using a replicate protein immunoblotting technique, which detects the simultaneous secretion of Ig kappa and Ig lambda from single colonies. These experiments provide evidence that the CD10 antigen is expressed on B-lineage cells before Ig light chain commitment, whereas CD20 is not. In conclusion, this B-cell colony assay provides a system for studying the differentiation of bone marrow-derived B cells and their precursors into Ig-secreting cells.

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