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

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.

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

Blood ◽

10.1182/blood.v79.7.1695.bloodjournal7971695 ◽

1992 ◽

Vol 79 (7) ◽

pp. 1695-1703 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v79.7.1695.1695 ◽

1992 ◽

Vol 79 (7) ◽

pp. 1695-1703 ◽

Cited By ~ 28

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

Abstract 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.

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Analysis of immunoglobulin and T-cell receptor gene rearrangements in human fetal bone marrow B lineage cells

Blood ◽

10.1182/blood.v76.6.1196.1196 ◽

1990 ◽

Vol 76 (6) ◽

pp. 1196-1200 ◽

Cited By ~ 6

Author(s):

TW LeBien ◽

RL Elstrom ◽

M Moseley ◽

JH Kersey ◽

F Griesinger

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

T Cell Receptor ◽

High Frequency ◽

Cell Receptor ◽

Gene Rearrangements ◽

Fetal Bone ◽

B Lineage ◽

B Lineage Cells

Abstract Fetal bone marrow B lineage cells representing multiple stages of B cell development were isolated by two-color cell sorting and analyzed for immunoglobulin H and T-cell receptor (TCR) gamma and delta gene rearrangements. Analysis of CD10+/surface mu- cells using a JH probe revealed a high frequency of rearrangements; some of these rearrangements used the 3′ D region gene DQ52. Analysis of CD10+/surface mu- cells revealed no detectable TCR-gamma or -delta rearrangements, nor were TCR-delta rearrangements detected in CD10+/surface mu+ cells, despite the limited repertoire of these genes. These observations are surprising given the high frequency of TCR delta/gamma rearrangements in B cell precursor acute lymphoblastic leukemia, and identify a potential difference in patterns of gene rearrangement that distinguish normal and leukemic B cell precursors.

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Suppression of B lymphopoiesis during normal pregnancy.

Journal of Experimental Medicine ◽

10.1084/jem.178.5.1507 ◽

1993 ◽

Vol 178 (5) ◽

pp. 1507-1515 ◽

Cited By ~ 153

Author(s):

K L Medina ◽

G Smithson ◽

P W Kincade

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

B Lymphocyte ◽

B Lymphopoiesis ◽

Color Flow ◽

Heat Stable ◽

Brdu Labeling ◽

Pregnant Mice ◽

B Lineage

We describe a dramatic reduction in numbers and activity of committed B lymphocyte precursors in the bone marrow of normal pregnant mice. Changes in cells responsive to IL-7 were evident as early as 6.5 d of pregnancy and values were < 10% of normal at parturition. B lineage precursors, identified by display of CD45R and absence of surface IgM, were also substantially depressed, and subpopulations representing different stages in the B lineage were assessed by three-color flow cytometry. Early pro-B cells are medium to large in size and have been previously characterized by low expression of the heat-stable antigen (HSA). This category of cells was not reduced, and in fact may have been slightly elevated, during pregnancy. In contrast, all subsequent populations of B lineage precursors, defined by patterns of expression of heat-stable and CD43 antigens, were substantially depressed. The immediate precursors of B cells (small pre-B cells) were identified by small size, expression of CD45R, absence of CD43, and lack of surface IgM. These were the most reduced of any phenotypically defined population in bone marrow. Numbers of newly formed B cells, characterized by the presence of sIgM, but not sIgD, were also diminished. However, B cells with a mature phenotype (sIgM+, sIgD+) were present in normal to somewhat elevated numbers. Mitogen-responsive B cells clonable in a semisolid agar assay were not significantly affected. A bromodeoxyuridine (BrdU) labeling technique was used to evaluate mitotic activity, which revealed an increased proportion of long-lived lymphocytes in the bone marrow of pregnant mice. These observations indicate that B lymphopoiesis is markedly downregulated during pregnancy and that all precursor populations beyond the early pro-B cell stage are affected. The pregnancy-related changes in bone marrow were selective for B lineage precursors, as cells expressing myeloid and erythroid markers were not reduced. In spleen, evidence was obtained for partial depletion of one subset of B cells. These cells, which have been reported to be recent immigrants from marrow, are characterized as having high levels of sIgM and HSA. Changes in other major B lymphocyte subsets in the spleen were less remarkable. When considered with results from the BrdU labeling procedure, the findings indicate that both production and export of lymphocytes from marrow may be substantially decreased. Numbers of B cell precursors were higher in postpartum animals whose litters were removed at birth, suggesting that lactation may prolong regeneration of lymphocyte production.(ABSTRACT TRUNCATED AT 400 WORDS)

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Analysis of immunoglobulin and T-cell receptor gene rearrangements in human fetal bone marrow B lineage cells

Blood ◽

10.1182/blood.v76.6.1196.bloodjournal7661196 ◽

1990 ◽

Vol 76 (6) ◽

pp. 1196-1200 ◽

Cited By ~ 1

Author(s):

TW LeBien ◽

RL Elstrom ◽

M Moseley ◽

JH Kersey ◽

F Griesinger

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

T Cell Receptor ◽

High Frequency ◽

Cell Receptor ◽

Gene Rearrangements ◽

Fetal Bone ◽

B Lineage ◽

B Lineage Cells

Fetal bone marrow B lineage cells representing multiple stages of B cell development were isolated by two-color cell sorting and analyzed for immunoglobulin H and T-cell receptor (TCR) gamma and delta gene rearrangements. Analysis of CD10+/surface mu- cells using a JH probe revealed a high frequency of rearrangements; some of these rearrangements used the 3′ D region gene DQ52. Analysis of CD10+/surface mu- cells revealed no detectable TCR-gamma or -delta rearrangements, nor were TCR-delta rearrangements detected in CD10+/surface mu+ cells, despite the limited repertoire of these genes. These observations are surprising given the high frequency of TCR delta/gamma rearrangements in B cell precursor acute lymphoblastic leukemia, and identify a potential difference in patterns of gene rearrangement that distinguish normal and leukemic B cell precursors.

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Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow.

Journal of Experimental Medicine ◽

10.1084/jem.173.5.1213 ◽

1991 ◽

Vol 173 (5) ◽

pp. 1213-1225 ◽

Cited By ~ 1077

Author(s):

R R Hardy ◽

C E Carmack ◽

S A Shinton ◽

J D Kemp ◽

K Hayakawa

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Polymerase Chain Reaction Amplification ◽

Cell Surface Expression ◽

Cell Contact ◽

Mouse Bone Marrow ◽

Heat Stable Antigen ◽

B Lineage ◽

B Lineage Cells

We have resolved B220+ IgM- B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin, CD43), BP-1, and 30F1 (heat stable antigen). Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction, lacking S7, consists of pre-B cells whereas the others, expressing S7, include B lineage cells before pre-B. These S7+ fractions, provisionally termed Fr. A, Fr. B, and Fr. C, can differentiate in a stromal layer culture system. Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7- pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1, DFL16.1, and Jk1, we find that the Ig genes of Fr. A are in germline configuration, whereas Fr. B and C are pro-B cell stages with increasing D-J rearrangement, but no V-D-J. Finally, functional analysis demonstrates that the proliferative response to IL-7, an early B lineage growth factor, is restricted to S7+ stages and, furthermore, that an additional, cell contact-mediated signal is essential for survival of Fr. A.

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Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3562-3568.1990 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3562-3568

Author(s):

M Principato ◽

J L Cleveland ◽

U R Rapp ◽

K L Holmes ◽

J H Pierce ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Bone Marrow Cells ◽

Hematopoietic Differentiation ◽

Developmental Relationships ◽

Murine Bone Marrow ◽

B Lineage ◽

Marrow Cells

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.

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TP53INP1 deficiency maintains murine B lymphopoiesis in aged bone marrow through redox-controlled IL-7R/STAT5 signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809980116 ◽

2018 ◽

Vol 116 (1) ◽

pp. 211-216 ◽

Cited By ~ 2

Author(s):

Bochra Zidi ◽

Christelle Vincent-Fabert ◽

Laurent Pouyet ◽

Marion Seillier ◽

Amelle Vandevelde ◽

...

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

B Cells ◽

B Cell ◽

Immune Cell ◽

B Lymphopoiesis ◽

Hematopoietic Stem ◽

Chronic Oxidative Stress ◽

The Impact ◽

Deficient Mice

Bone marrow (BM) produces all blood and immune cells deriving from hematopoietic stem cells (HSCs). The decrease of immune cell production during aging is one of the features of immunosenescence. The impact of redox dysregulation in BM aging is still poorly understood. Here we use TP53INP1-deficient (KO) mice endowed with chronic oxidative stress to assess the influence of aging-associated redox alterations in BM homeostasis. We show that TP53INP1 deletion has no impact on aging-related accumulation of HSCs. In contrast, the aging-related contraction of the lymphoid compartment is mitigated in TP53INP1 KO mice. B cells that accumulate in old KO BM are differentiating cells that can mature into functional B cells. Importantly, this phenotype results from B cell-intrinsic events associated with defective redox control. Finally, we show that oxidative stress in aged TP53INP1-deficient mice maintains STAT5 expression and activation in early B cells, driving high Pax5 expression, which provides a molecular mechanism for maintenance of B cell development upon aging.

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B-cell depletion reactivates B lymphopoiesis in the BM and rejuvenates the B lineage in aging

Blood ◽

10.1182/blood-2010-09-307983 ◽

2011 ◽

Vol 117 (11) ◽

pp. 3104-3112 ◽

Cited By ~ 51

Author(s):

Zohar Keren ◽

Shulamit Naor ◽

Shahar Nussbaum ◽

Karin Golan ◽

Tomer Itkin ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Antibody Responses ◽

Peripheral Compartment ◽

B Cell Depletion ◽

B Lymphopoiesis ◽

Age Related ◽

B Lineage ◽

Old Mice ◽

Age Related Changes

Abstract Aging is associated with a decline in B-lymphopoiesis in the bone marrow and accumulation of long-lived B cells in the periphery. These changes decrease the body's ability to mount protective antibody responses. We show here that age-related changes in the B lineage are mediated by the accumulating long-lived B cells. Thus, depletion of B cells in old mice was followed by expansion of multipotent primitive progenitors and common lymphoid progenitors, a revival of B-lymphopoiesis in the bone marrow, and generation of a rejuvenated peripheral compartment that enhanced the animal's immune responsiveness to antigenic stimulation. Collectively, our results suggest that immunosenescence in the B-lineage is not irreversible and that depletion of the long-lived B cells in old mice rejuvenates the B-lineage and enhances immune competence.

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

Blood ◽

10.1182/blood.v110.11.1334.1334 ◽

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.

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