scholarly journals Ku70 Is Required for Late B Cell Development and Immunoglobulin Heavy Chain Class Switching

1998 ◽  
Vol 187 (12) ◽  
pp. 2081-2089 ◽  
Author(s):  
John P. Manis ◽  
Yansong Gu ◽  
Rusty Lansford ◽  
Eiichiro Sonoda ◽  
Roger Ferrini ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Strand Break ◽  
Cell Development ◽  
Class Switching ◽  
Class Switch ◽  
Igg Isotypes ◽  
Rag 1

Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement. Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination. Ku70−/− (K70T) mice, like recombination activating gene (RAG)-1– or RAG-2–deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J. Seidl, G.A. Rathbun, C. Zhu, J.P. Manis, N. van der Stoep, L. Davidson, H.L. Cheng, J.M. Sekiguchi, K. Frank, et al. 1997. Immunity. 7:653–665; Ouyang, H., A. Nussenzweig, A. Kurimasa, V.C. Soares, X. Li, C. Cordon-Cardo, W. Li, N. Cheong, M. Nussenzweig, G. Iliakis, et al. 1997. J. Exp. Med. 186:921–929). Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a functionally rearranged VH(D)JH and Ig λ light chain transgene (referred to as K70T/HL mice). Previously, we have shown that B cells from R1T or R2T mice carrying these rearranged Ig genes (R1T/HL or R2T/HL mice) can undergo CSR to IgG isotypes (Lansford, R., J. Manis, E. Sonoda, K. Rajewsky, and F. Alt. 1998. Int. Immunol. 10:325–332). K70T/HL mice had significant numbers of peripheral surface IgM+ B cells, which generated serum IgM levels similar to those of R2T/HL mice. However, in contrast to R2T/HL mice, K70T/HL mice had no detectable serum IgG isotypes. In vitro culture of K70T/HL B cells with agents that induce CSR in normal or R2T/HL B cells did lead to the induction of germline CH transcripts, indicating that initial signaling pathways for CSR were intact in K70T/HL cells. However, treatment with such agents did not lead to detectable CSR by K70T/HL B cells, and instead, led to cell death within 72 h. We conclude that Ku70 is required for the generation of B cells that have undergone Ig HC class switching. Potential roles for Ku70 in the CSR process are discussed.

Mucosal Epithelial Cells Initiate Frontline Immunoglobulin Class Switching through an SLPI-Regulated Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3898-3898
Author(s):  
Andrea Cerutti ◽  
Bing He ◽  
April Chiu ◽  
Meimei Shan ◽  
Paul Santini ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
B Cells ◽  
Epithelial Cells ◽  
B Cell ◽  
Antibody Production ◽  
Cytidine Deaminase ◽  
Dna Recombination ◽  
Class Switching ◽  
Class Switch

Abstract Introduction. Class switching from IgM to IgG and IgA is central to immunity against microbes and usually occurs in draining lymph nodes and requires activation of B cells by CD4+ T cells expressing CD40 ligand. Growing evidence indicates that B cells can mount frontline IgG and IgA responses at mucosal sites of entry through an alternative CD40-independent pathway involving B cell-activating factor of the TNF family (BAFF, also known as BLyS) and a proliferation-inducing ligand (APRIL). These innate factors are usually produced by dendritic cells and stimulate B cells through at least three distinct receptors. Together with dendritic cells, epithelial cells have a key position at the host-environment interface. Therefore, we asked whether epithelial cells play a role in frontline antibody production. Methods. Tonsillar tissue sections from healthy donors were analyzed for expression of activation-induced cytidine deaminase (AID) by immunohistochemistry and in situ hybridization. A simplified in vitro model reproducing the geometry of mucosal surfaces was used to evaluate the role of epithelial cells in class switching. Briefly, primary epithelial cells and B cells were cultured in the upper and lower chambers, respectively, of a trans-well system. Monocyte-derived dendritic cells were positioned on a filter separating the two chambers. Various microbial product analogues were used to mimic infection. RNA interference was performed to knockdown BAFF in epithelial cells. AID expression, CSR, antibody production and signaling were evaluated in B cells as reported (Litinsky et al., Nat. Immunol.2002, 3:822–829; Qiao et al., Nat. Immunol.2006, 7:302–310). Results. We found that the upper respiratory mucosa of healthy subjects comprised intraepithelial pockets filled with B cells expressing AID, a DNA-editing enzyme associated with ongoing class switch DNA recombination (CSR). Epithelial cells released innate class switch-inducing factors, including BAFF, after sensing microbial products through TLRs, thereby inducing AID expression, CSR, and ultimately IgG and IgA production in neighboring B cells. Epithelial cell-induced antibodies comprised polyreactive IgG and IgA capable of recognizing multiple microbial determinants. Intraepithelial class switching was enhanced by thymic stromal lymphopoietin (TSLP), an epithelial IL-7-like cytokine that augments the innate B cell-licensing functions of dendritic cells, and restrained by secretory leukocyte protease inhibitor (SLPI), an epithelial alarm antiprotease that suppresses AID expression in activated B cells. Conclusions. The present findings indicate that epithelial cells function as non-immune sentinels capable to autonomously orchestrate compartmentalized IgG and IgA responses at the interface between host and environment. This implies that mucosal vaccines should activate both epithelial and immune cells to elicit optimal antibody production.

scholarly journals Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

2002 ◽  
Vol 9 (2) ◽  
pp. 86-95 ◽  
Author(s):  
Denise A. Kaminski ◽  
John J. Letterio ◽  
Peter D. Burrows
Keyword(s):  
B Cells ◽  
Growth Factor ◽  
B Cell ◽  
Transforming Growth Factor ◽  
Plasma Cells ◽  
Population Analysis ◽  
Cell Development ◽  
B Cell Development

Transforming growth factor β (TGFβ) can inhibit thein vitroproliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/-mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1-pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell developmentin vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.

scholarly journals Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development

1997 ◽  
Vol 185 (4) ◽  
pp. 609-620 ◽  
Author(s):  
Andrei Constantinescu ◽  
Mark S. Schlissel
Keyword(s):  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Light Chain ◽  
Immunoglobulin Heavy Chain ◽  
Cell Development ◽  
B Cell Development ◽  
Allelic Exclusion ◽  
Chain Locus ◽  
Heavy Chain Locus

The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an Eμ-myc transgene.

scholarly journals Identification of an Essential Cytoplasmic Region of Interleukin-7 Receptor α Subunit in B-Cell Development

2018 ◽  
Vol 19 (9) ◽  
pp. 2522 ◽  
Author(s):  
Hirotake Kasai ◽  
Taku Kuwabara ◽  
Yukihide Matsui ◽  
Koichi Nakajima ◽  
Motonari Kondo
Keyword(s):  
B Cells ◽  
B Cell ◽  
Myeloid Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Α Subunit ◽  
Interleukin 7 ◽  
In Cells

Interleukin-7 (IL-7) is essential for lymphocyte development. To identify the functional subdomains in the cytoplasmic tail of the IL-7 receptor (IL-7R) α chain, here, we constructed a series of IL-7Rα deletion mutants. We found that IL-7Rα-deficient hematopoietic progenitor cells (HPCs) gave rise to B cells both in vitro and in vivo when a wild-type (WT) IL-7Rα chain was introduced; however, no B cells were observed under the same conditions from IL-7Rα-deficient HPCs with introduction of the exogenous IL-7Rα subunit, which lacked the amino acid region at positions 414–441 (d414–441 mutant). Signal transducer and activator of transcription 5 (STAT5) was phosphorylated in cells with the d414–441 mutant, similar to that in WT cells, in response to IL-7 stimulation. In contrast, more truncated STAT5 (tSTAT5) was generated in cells with the d414–441 mutant than in WT cells. Additionally, the introduction of exogenous tSTAT5 blocked B lymphopoiesis but not myeloid cell development from WT HPCs in vivo. These results suggested that amino acids 414–441 in the IL-7Rα chain formed a critical subdomain necessary for the supportive roles of IL-7 in B-cell development.

scholarly journals A Dual Role for Src Homology 2 Domain–Containing Inositol-5-Phosphatase (Ship) in Immunity

2000 ◽  
Vol 191 (5) ◽  
pp. 781-794 ◽  
Author(s):  
Cheryl D. Helgason ◽  
Christian P. Kalberer ◽  
Jacqueline E. Damen ◽  
Suzanne M. Chappel ◽  
Nicolas Pineault ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Lymphoid Development ◽  
Src Homology 2 ◽  
Src Homology ◽  
Src Homology 2 Domain ◽  
B Lymphoid

In this report, we demonstrate that the Src homology 2 domain–containing inositol-5-phosphatase (SHIP) plays a critical role in regulating both B cell development and responsiveness to antigen stimulation. SHIP−/− mice exhibit a transplantable alteration in B lymphoid development that results in reduced numbers of precursor B (fraction C) and immature B cells in the bone marrow. In vitro, purified SHIP−/− B cells exhibit enhanced proliferation in response to B cell receptor stimulation in both the presence and absence of Fcγ receptor IIB coligation. This enhancement is associated with increased phosphorylation of both mitogen-activated protein kinase and Akt, as well as with increased survival and cell cycling. SHIP−/− mice manifest elevated serum immunoglobulin (Ig) levels and an exaggerated IgG response to the T cell–independent type 2 antigen trinitrophenyl Ficoll. However, only altered B cell development was apparent upon transplantation into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice. The in vitro hyperresponsiveness, together with the in vivo findings, suggests that SHIP regulates B lymphoid development and antigen responsiveness by both intrinsic and extrinsic mechanisms.

Mutator Genes UDG and AID Appear To Be Normal in Class-Switch Deficient and Clonally Homogeneous Waldenstrom’s Macroglobulinemias Having Either Germline or Hypermutated Clonotypic IgH VDJ.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1359-1359
Author(s):  
Jitra Kriangkum ◽  
Brian J. Taylor ◽  
Erin R. Strachan ◽  
Steven P. Treon ◽  
Michael J. Mant ◽  
...  
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Splice Variants ◽  
Cytidine Deaminase ◽  
Essential Elements ◽  
Class Switching ◽  
Conserved Sequence ◽  
Class Switch ◽  
Mutator Genes

Abstract Clonotypic B cells of Waldenstrom’s macroglobulinemia (WM) are characterized as CD20+IgM+IgD+ cells that are usually somatically mutated in IgH VDJ but for some patients, the clonotypic IgH VDJ is germline (unmutated).For both mutated and unmutated clones, WM lack ongoing somatic hypermutation (SHM) and class switch recombination (CSR). This may be due to abnormalities in switching and/or mutator genes. To understand the nature of unswitched tumor B cells, uracil DNA glycosylase (UDG) and activation-induced cytidine deaminase (AID), the two essential elements for CSR, were analysed in WM. Analysis of 12 WM clones characterized by somatic hypermutation showed that the mutation profile of VH genes had normal transition/transversion ratios at C or G, and thus did not suggest UDG abnormalities. Expression of AID was determined by single stage RT-PCR. Out of 14 patients studied (2 unmutated and 12 mutated VH clones), two of them (WM1-01 and WM1-08,with mutation rates of 0% and 6.2% respectively) gave positive bands. In WM1-01, despite having a germline IgH VDJ, AID is consistently expressed in two bone marrow samples collected three years apart and from which the identical unmutated clonotypic VDJ sequence was isolated. Full-length (FL) AID transcripts of WM have a conserved sequence, thus ruling out the possibility of functional defects due to point mutation. In addition, detection of AID in an unmutated VH clone suggested that lack of SHM does not result from an inability to produce AID. In addition to FL transcripts, three other splice variants were identified in both patients. Single cell analysis indicated that only a small compartment (10% or less), not all, of clonotypic B cells expressed AID, and multiple isoforms may be detectable in individual cells. Whether these splice variants that contain truncated C-terminal ends play a role in the regulation of CSR in WM remains to be investigated. Splice variants, nevertheless, may not characterize tumor B cells since up to 10% of AID-expressing normal activated B cells (n=3) also carried them. In vitro activation of clonotypic WM B cells by CD40L and IL4, using conditions that induced CSR in normal B cells, did not yield detectable class switching in WM B cells. In cultures of B cells from WM, the number of non-clonal B cells increased but the clonotypic B cells did not appear to expand, as indicated by the reduction of clonotypic IgM transcript at 5-days of culture. Thus, as well as failing to undergo somatic mutation or class switching, WM tumor B cells appear unresponsive to CD40L+IL4. They may be fundamentally unresponsive to signals for class switching and their clonal expansion may depend upon alternate signaling pathways.

scholarly journals The Earliest Step in B Lineage Differentiation from Common Lymphoid Progenitors Is Critically Dependent upon Interleukin 7

2002 ◽  
Vol 196 (5) ◽  
pp. 705-711 ◽  
Author(s):  
Juli P. Miller ◽  
David Izon ◽  
William DeMuth ◽  
Rachel Gerstein ◽  
Avinash Bhandoola ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Gamma Chain ◽  
Lineage Differentiation ◽  
Lymphoid Progenitors ◽  
B Lineage

Little is known about the signals that promote early B lineage differentiation from common lymphoid progenitors (CLPs). Using a stromal-free culture system, we show that interleukin (IL)-7 is sufficient to promote the in vitro differentiation of CLPs into B220+ CD19+ B lineage progenitors. Consistent with current models of early B cell development, surface expression of B220 was initiated before CD19 and was accompanied by the loss of T lineage potential. To address whether IL-7 receptor (R) activity is essential for early B lineage development in vivo, we examined the frequencies of CLPs and downstream pre–pro- and pro-B cells in adult mice lacking either the α chain or the common gamma chain (γc) of the IL-7R. The data indicate that although γc−/− mice have normal frequencies of CLPs, both γc−/− and IL-7Rα−/− mice lack detectable numbers of all downstream early B lineage precursors, including pre–pro-B cells. These findings challenge previous notions regarding the point in B cell development affected by the loss of IL-7R signaling and suggest that IL-7 plays a key and requisite role during the earliest phases of B cell development.

Differential expression of CD21 identifies developmentally and functionally distinct subsets of human transitional B cells

Blood ◽  
2010 ◽  
Vol 115 (3) ◽  
pp. 519-529 ◽  
Author(s):  
Santi Suryani ◽  
David A. Fulcher ◽  
Brigitte Santner-Nanan ◽  
Ralph Nanan ◽  
Melanie Wong ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Differential Expression ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Repertoire ◽  
Molecular Features ◽  
Transitional B Cells ◽  
Transitional Cells

Abstract The transitional stage of B-cell development represents an important step where autoreactive cells are deleted, allowing the generation of a mature functional B-cell repertoire. In mice, 3 subsets of transitional B cells have been identified. In contrast, most studies of human transitional B cells have focused on a single subset defined as CD24hiCD38hi B cells. Here, we have identified 2 subsets of human transitional B cells based on the differential expression of CD21. CD21hi transitional cells displayed higher expression of CD23, CD44, and IgD, and exhibited greater proliferation and Ig secretion in vitro than CD21lo transitional B cells. In contrast, the CD21lo subset expressed elevated levels of LEF1, a transcription factor highly expressed by immature lymphocytes, and produced higher amounts of autoreactive Ab. These phenotypic, functional, and molecular features suggest that CD21lo transitional B cells are less mature than the CD21hi subset. This was confirmed by analyzing X-linked agammaglobulinemia patients and the kinetics of B-cell reconstitution after stem cell transplantation, which revealed that the development of CD21lo transitional B cells preceded that of CD21hi transitional cells. These findings provide important insights into the process of human B-cell development and have implications for understanding the processes underlying perturbed B-cell maturation in autoimmune and immunodeficient conditions.

scholarly journals Iκb Kinase α Is Essential for Mature B Cell Development and Function

2001 ◽  
Vol 193 (4) ◽  
pp. 417-426 ◽  
Author(s):  
Tsuneyasu Kaisho ◽  
Kiyoshi Takeda ◽  
Tohru Tsujimura ◽  
Taro Kawai ◽  
Fumiko Nomura ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Cell Development ◽  
B Cell Development ◽  
Cytokine Signaling ◽  
Iκb Kinase ◽  
And Function

IκB kinase (IKK) α and β phosphorylate IκB proteins and activate the transcription factor, nuclear factor (NF)-κB. Although both are highly homologous kinases, gene targeting experiments revealed their differential roles in vivo. IKKα is involved in skin and limb morphogenesis, whereas IKKβ is essential for cytokine signaling. To elucidate in vivo roles of IKKα in hematopoietic cells, we have generated bone marrow chimeras by transferring control and IKKα-deficient fetal liver cells. The mature B cell population was decreased in IKKα−/− chimeras. IKKα−/− chimeras also exhibited a decrease of serum immunoglobulin basal level and impaired antigen-specific immune responses. Histologically, they also manifested marked disruption of germinal center formation and splenic microarchitectures that depend on mature B cells. IKKα−/− B cells not only showed impairment of survival and mitogenic responses in vitro, accompanied by decreased, although inducible, NF-κB activity, but also increased turnover rate in vivo. In addition, transgene expression of bcl-2 could only partially rescue impaired B cell development in IKKα−/− chimeras. Taken together, these results demonstrate that IKKα is critically involved in the prevention of cell death and functional development of mature B cells.

scholarly journals Arhgap25 deficiency leads to decreased numbers of peripheral blood B cells and defective germinal center reactions

2020 ◽  
Author(s):  
Silke E. Lindner ◽  
Colt A. Egelston ◽  
Stephanie M. Huard ◽  
Peter P. Lee ◽  
Leo D. Wang
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Peripheral Blood ◽  
Cell Development ◽  
B Cell Development ◽  
Dependent Manner ◽  
B Cell Differentiation ◽  
Nucleotide Exchange

ABSTRACTRho family GTPases are critical for normal B cell development and function and their activity is regulated by a large and complex network of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, the role of GAPs in B cell development is poorly understood. Here we show that the novel Rac-GAP ARHGAP25 is important for B cell development in mice in a CXCR4-dependent manner. We show that Arhgap25 deficiency leads to a significant decrease in peripheral blood B cell numbers, as well as defects in mature B cell differentiation. Arhgap25-/- B cells respond to antigen stimulation in vitro and in vivo but have impaired germinal center formation and decreased IgG1 class switching. Additionally, Arhgap25-/- B cells exhibit increased chemotaxis to CXCL12. Taken together, these studies demonstrate an important role for Arhgap25 in peripheral B cell development and antigen response.

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