scholarly journals IL-7R signalling activates widespread VH and DH gene usage to drive antibody diversity in bone marrow B cells

2020 ◽  
Author(s):  
Amanda Baizan-Edge ◽  
Bryony A. Stubbs ◽  
Michael J. T. Stubbington ◽  
Daniel J. Bolland ◽  
Kristina Tabbada ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Gene Selection ◽  
De Novo ◽  
Fetal Liver ◽  
Antisense Transcription ◽  
Cell Repertoire ◽  
Antibody Diversity ◽  
Interleukin 7

AbstractGeneration of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain (Igh) locus. It has been proposed that interleukin-7 receptor (IL-7R) signalling is necessary for Igh recombination, but this has been challenging to partition from the receptor’s role in B cell survival and proliferation. By generating the first detailed description of the Igh repertoire of murine IL-7Rα-/- bone marrow B cells, we demonstrate that IL-7R signalling profoundly influences VH gene selection during VH-to-DJH recombination. We find skewing towards usage of 3’ VH genes during de novo VH-to-DJH recombination that is more severe than the fetal liver (FL) B cell repertoire, and we now show a role for IL-7R signalling in DH-to-JH recombination. Transcriptome and accessibility analyses suggests reduced expression of B lineage-specific transcription factors (TFs) and their targets, and loss of DH and VH antisense transcription in IL-7Rα-/- B cells. These results refute models suggesting that IL-7R signalling is only required for survival and proliferation, and demonstrate a pivotal role in shaping the Igh repertoire by activating underpinning epigenetic mechanisms.

Interleukin-3 and interleukin-7 are alternative growth factors for the same B-cell precursors in the mouse

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2045-2051 ◽  
Author(s):  
TH Winkler ◽  
F Melchers ◽  
AG Rolink
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Stromal Cells ◽  
Fetal Liver ◽  
Surrogate Light Chain ◽  
Interleukin 7 ◽  
Precursor B Cells

Clones and lines of precursor (pre) B cells can be established by limiting dilutions of unseparated cell suspensions of fetal liver or bone marrow on stromal cells in the presence of interleukin (IL)-7. When IL-3 is used instead of IL-7, cultures are regularly overgrown by different precursor cells of the myeloid lineage, as well as by adherent cells that inhibit pre-B-cell expansion. However, in the presence of either IL-7 or IL-3, clones of pre-B cells can be established on stroma cells at frequencies near one in one when the cultures are initiated with cell sorter purified CD45RO (B220)+/c-kit+ fetal liver or bone marrow derived pre-B cells. Clones grown on stromal cells in the presence of IL-7 can be regrown in IL-3, and vice versa. Pre-B cells that proliferate on stromal cells in the presence of IL-7 or IL-3 have the same phenotype, ie, are B220+ c-kit+, CD43+, and surrogate light chain+. Removal of the growth factors (IL-7, respectively IL-3) from the cultures results in differentiation to surface immunoglobulin (slg) positive, c-kit-, CD43-, surrogate light chain-B cells, a fraction of which is lipopolysaccharide (LPS) responsive as shown by IgM secretion. These results show that IL-7 and IL-3 stimulate largely overlapping populations of precursor B cells from bone marrow to proliferate for long periods of time in the presence of stromal cells. Thus, IL-7 and IL-3 are alternative growth factors for the same pre-BI cell.

scholarly journals Expression of phosphorylcholine-specific B cells during murine development

1977 ◽  
Vol 146 (4) ◽  
pp. 933-948 ◽  
Author(s):  
NH Sigal ◽  
AR Pickard ◽  
ES Metcalf ◽  
PJ Gearhart ◽  
NR Klinman
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Minor Role ◽  
Cell Repertoire ◽  
Antibody Diversity ◽  
Maternal Influences ◽  
B Cell Repertoire ◽  
A Minor ◽  
Kinetics Of

The TEPC 15 (T15) clonotype, a putatively germline antibody specificity, does not appear in the neonatal B-cell repertoire until approximately 1 wk of age. This report extends this observation by the demonstration that (a) the T15 clonotype follows similar kinetics of appearance in germfree as well as conventionally-reared mice; (b) maternal influences and genetic background play a minor role in the development of the T15 clonotype since CBFI neonates raised by C57BL/6 or BALB/c mothers acquire the T15 clonotype at the same time in ontogeny as BALB/c neonates; (c) the lack of phosphorylcholine (PC)-specific B cells shortly after birth is reflected in a dearth of PC-binding cells in the neonate as well; and (d) no PC-specifc B cells are found in 19-day fetal liver or in bone marrow until 7 days of life, coincident with their appearance in the spleen. These findings, along with a previous report that PC-specific splenic B cells are tolerizable as late as day 10 after birth, confirm the invariant, late occurrence of the T15 clonotype and support a highly- ordered, rigorously predetermined mechanism for the acquisition of the B- cell repertoire. The results are discussed in light of other studies on the ontogeny of B-cell specificity, and in terms of the implications on the mechanism by which antibody diversity is generated.

scholarly journals Transfer of antigen-encoding bone marrow under immune-preserving conditions deletes mature antigen-specific B cells in recipients and inhibits antigen-specific antibody production

2019 ◽  
Author(s):  
Jeremy F. Brooks ◽  
Janet M. Davies ◽  
James W. Wells ◽  
Raymond J. Steptoe
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Antibody Production ◽  
Specific Antibody ◽  
De Novo ◽  
Antigen Expression ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Specific Antibody Production

SummaryPathological activation and collaboration of T and B cells underlies pathogenic autoantibody responses. Existing treatments for autoimmune disease cause non-specific immunosuppression and induction of antigen-specific tolerance remains an elusive goal. Many immunotherapies aim to manipulate the T-cell component of T-B interplay but few directly target B cells. One possible means to specifically target B cells is the transfer of gene-engineered BM that, once engrafted, gives rise to widespread specific and tolerogenic antigen expression within the hematopoietic system. Gene-engineered bone marrow encoding ubiquitous ovalbumin expression was transferred after low-dose (300cGy) immune-preserving irradiation. B-cell responsiveness was monitored by analyzing ovalbumin-specific antibody production after immunization with ovalbumin/complete Freund’s adjuvant. Ovalbumin-specific B cells and their response to immunization were analyzed using multi-tetramer staining. When antigen-encoding bone marrow was transferred under immune-preserving conditions, cognate antigen-specific B cells were purged from the recipient’s pre-existing B cell repertoire as well as the repertoire that arose after bone marrow transfer. OVA-specific B-cell deletion was apparent within the established host B-cell repertoire as well as that developing after gene-engineered bone marrow transfer. OVA-specific antibody production was substantially inhibited by transfer of OVA-encoding BM and activation of OVA-specific B cells, germinal centre formation and subsequent OVA-specific plasmablast differentiation were all inhibited. Low levels of gene-engineered bone marrow chimerism were sufficient to limit antigen-specific antibody production. These data show that antigen-specific B cells within an established B-cell repertoire are susceptible to de novo tolerance induction and this can be achieved by transfer of gene-engineered bone marrow. This adds further dimensions to the utility of antigen-encoding bone marrow transfer as an immunotherapeutic tool.

B Cell Development in the Absence of PU.1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 226-226 ◽  
Author(s):  
Min Ye ◽  
Olga Ermaermakova-Cirilli ◽  
Thomas Graf
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Target Genes ◽  
Fetal Liver ◽  
Cell Formation ◽  
Cell Development ◽  
B Cell Development ◽  
B Lineage

Abstract Mice deficient of the ETS-family transcription factor PU.1 lack B cells as well as macrophages. While most macrophage specific genes are known to be regulated by high levels of PU.1, the reason for the defect in B cell formation is not known. Here we analyzed a mouse strain in which a floxed version of the PU.1 gene, surrounding exon 4 and 5, which encode the DNA, binding and PEST domains (developed by C. Somoza and D. Tenen), was excised by Cre mediated recombination. As expected, this strain lacks both B cells and macrophages and die at birth. Surprisingly, however, we were able to establish lymphoid cell lines from fetal livers of these mice (day 14 to day 18), which proliferated on S17 stromal cells supplemented with IL-7 and stem cell factor. These cells expressed the B lineage cell surface markers CD19, CD43, BP-1 and CD24, but not B220. They also expressed B cell transcription factors, EBF, E47, Pax5, and their target genes, Rag1, IL7R, λ5 and v-preB, as detected by RT-PCR, exhibited DJ and VDJ immunoglobulin heavy chain rearrangements, and expressed IgM after IL-7 withdrawal. We then tested the effect of PU.1 deletion in B cells in adult animals by crossing the floxed PU.1 strain with a CD19 Cre mouse line. The spleen and peripheral blood (but not bone marrow) of these mice contained B cells that were CD19+ IgMlow, IgDhigh but B220 negative and instead expressed CD43. Thus PU.1 is not essential for immunoglobulin production and late B cell development. Although PU.1−/− fetal liver cells can give rise to cells, resembling Pre-B in vitro, the process of B cell formation was delayed by almost 12 days, compared with wt fetal liver, and the efficiency was reduced approximately 25-fold. In addition, PU.1 deficient B cells demonstrated an impaired ability to engraft into the bone marrow, when injected into irradiated SCID mice. We have found that PU.1 deficient B progenitors showed reduced or undetectable levels of the SDF1 receptor CXCR4, a receptor that has been implicated in B cell homing. Taken together, our observations suggest that PU.1 plays two different roles during B cell development: for early B cell formation and for proper migration and engraftment, which might be mediated through regulation of CXCR4 expression.

scholarly journals Hematopoietic supportive functions of mouse bone marrow and fetal liver microenvironment: dissection of granulocyte, B-lymphocyte, and hematopoietic progenitor support at the stroma cell clone level

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4596-4606 ◽  
Author(s):  
C Friedrich ◽  
E Zausch ◽  
SP Sugrue ◽  
JC Gutierrez-Ramos
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Adhesion Molecule ◽  
Stromal Cell ◽  
Cell Clone ◽  
Fetal Liver ◽  
Hematopoietic Progenitors ◽  
Cell Clones ◽  
Stimulating Factor

We dissected the functions of the microenvironment of bone marrow (BM) and fetal liver (FL) at the cellular level by cloning individual stromal calls and characterizing their phenotypical and functional features. Stromal cell clones derived from FL are large in size (mean forward light scatter intensity [mFSC] of 450), express the surface antigen Thy-1 but not Sca-1 and 6 out of 6 are able to differentiate into fat accumulating adipocytes. BM derived stromal cell clones are either small (mFSC of 250) or large (mFSC of 450), express Sca-1 but not Thy-1 and only 2 out of 7 differentiate towards adipocytes. Heterogeneity in terms of vascular adhesion molecule-1, intracellular adhesion molecule-1 and heat stable antigen expression was found among the different cell clones. Functional assays using long- and short-term cocultures of stromal and hematopoietic calls revealed: (1) the capacity of 8 out of 12 stromal cell clones to support the expansion of primitive hematopoietic progenitors (colony forming unit spleen day 12) more than 10 weeks. Fat accumulation but not expression of stem cell factor by stromal cells did correlate with this supportive function. (2) Better support of granulocyte maturation and proliferation by BM- compared to FL-derived stromal cell clones. However, stromal cell clones from both organs expressed macrophage-colony stimulating factor. (3) The ability of 4 out of 12 stromal cell clones (derived from both, FL and BM) to support the expansion of Interleukin-7 dependent pre-B cells from the BM. Pre-B cell growth stimulating factor was not restricted to supporters. (4) Mutual exclusiveness of myeloid and lymphoid support in that a given stromal cell clone supported either pre B-cell or granulocyte expansion. Experiments comparing the support of BM- and FL-derived hematopoietic progenitors showed identical responses of late (B220+/c-kit-) but strikingly different responses of early (B220+/c-kit+) pre-B cells, revealing different proliferation requirements for FL- versus BM- derived early pre-B cells in vitro.

scholarly journals Interactions Between c-kit and Stem Cell Factor Are Not Required for B-Cell Development In Vivo

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 518-525 ◽  
Author(s):  
Shunichi Takeda ◽  
Takeyuki Shimizu ◽  
Hans-Reimer Rodewald
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Critical Role ◽  
Cell Development ◽  
B Cell Development ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract The receptor-type tyrosine kinase, c-kit is expressed in hematopoietic stem cells (HSC), myeloid, and lymphoid precursors. In c-kit ligand-deficient mice, absolute numbers of HSC are mildly reduced suggesting that c-kit is not essential for HSC development. However, c-kit− HSC cannot form spleen colonies or reconstitute hematopoietic functions in lethally irradiated recipient mice. Based on in in vitro experiments, a critical role of c-kit in B-cell development was suggested. Here we have investigated the B-cell development of c-kitnull mutant (W/W ) mice in vivo. Furthermore, day 13 fetal liver cells from wild type or W/W mice were transferred into immunodeficient RAG-2−/− mice. Surprisingly, transferred c-kit− cells gave rise to all stages of immature B cells in the bone marrow and subsequently to mature conventional B2, as well as B1, type B cells in the recipients to the same extent as transferred wild type cells. Hence, in contrast to important roles of c-kit in the expansion of HSC and the generation of erythroid and myeloid lineages and T-cell precursors, c-kit− HSC can colonize the recipient bone marrow and differentiate into B cells in the absence of c-kit.

scholarly journals Autoantibodies inhibit interleukin-7-mediated proliferation and are associated with the age-dependent loss of pre-B cells in autoimmune New Zealand Black Mice

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3289-3296 ◽  
Author(s):  
MS Merchant ◽  
BA Garvy ◽  
RL Riley
Keyword(s):  
Bone Marrow ◽  
New Zealand ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Igm Antibodies ◽  
Interleukin 7 ◽  
Heat Stable Antigen ◽  
B Cell Precursors

Surface IgM+B220+ B cell precursors can be categorized as either leukosialin (CD43/S7) negative (late stage pre-B cells) or positive (pro-B/early pre-B cells). In autoimmune New Zealand Black (NZB) mice, bone marrow small pre-B cells (IgM-CD43-B220+) and pro-B/early pre-B cells (IgM-CD43+B220+) declined significantly with age. In particular, subpopulations of pro-B/early pre-B cells expressing the heat stable antigen (HSA) were found in lower proportions with age. Significant decreases in interleukin-7 (IL-7) colony forming units (CFU) were also seen in NZB mice by 6 to 8 months of age and accompanied alterations in the numbers of pro-B and pre-B cells in bone marrow. Concomitant with reduced numbers of B lineage precursor cells and IL-7 CFU in vivo, NZB mice produced serum IgM antibodies that strongly inhibited IL-7 CFU responses in vitro. Two monoclonal IgM antibodies (5G9, 2F5) derived from LPS stimulated 10-month-old NZB splenocytes recognized pre-B cell surface antigens on both pre-B cell lines and on IL-7 stimulated bone marrow pro-B/pre-B cells. However, these monoclonal antibodies (MoAb) failed to significantly stain ex vivo bone marrow cells. The 5G9 and 2F5 MoAbs also partially inhibited IL-7 CFU in vitro. These results suggest that NZB bone marrow becomes increasingly deficient in B cell precursors and especially in IL-7 responsive pre-B cells with age. IgM serum antibodies and monoclonal IgM antibodies derived from older NZB mice inhibit pre-B cell growth to IL-7. The production of such autoantibodies may interfere with B cell development in aging NZB mice by preventing IL-7-mediated proliferation.

scholarly journals Role of variable region gene expression and environmental selection in determining the antiphosphorylcholine B cell repertoire.

1983 ◽  
Vol 158 (6) ◽  
pp. 1948-1961 ◽  
Author(s):  
N R Klinman ◽  
M R Stone
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Variable Region ◽  
Precursor Cells ◽  
Cell Repertoire ◽  
Region Gene ◽  
B Cell Repertoire

To evaluate the role of environmental selective processes, as opposed to variable region gene expression, in the determination of B cell repertoire expression, we have assessed the phosphorylcholine (PC)-specific repertoire of precursor cells that remain in bone marrow cell populations after the removal of surface immunoglobulin (sIg)-bearing cells. Such cells are assumed to represent a stage in B cell maturation before the expression of sIg, and thus at a time when they have not as yet interfaced with environmental influences that operate through sIg receptors such as antigenic stimulation, tolerance, or antiidiotypic regulation. The repertoire as expressed in these cells, therefore, should reflect the readout of immunoglobulin variable region genes as they are expressed in progenitors to B cells. The results of these studies indicate that, as in mature primary B cell pools of BALB/c mice, the majority of PC-responsive sIg- bone marrow cells are of the T15 clonotype. Thus, environmental selective mechanisms would not appear to be required for the high frequency of B cells of the T15 idiotype in the primary B cell repertoire of BALB/c mice. Analysis of the sIg- bone marrow cells in (CBA/N X BALB/c)F1 male mice demonstrated that the deficit of PC-responsive mature B cells, which is a characteristic of this murine strain, must occur after receptor expression, since a normal frequency of PC-responsive and T15-expressing cells is present in their sIg- bone marrow population. Finally, these same mice were used to obtain bone marrow cell preparations from individual leg bones, so as to permit an analysis of the occurrence of T15+ and T15- clonotypes within individual bone marrow populations. The findings from these studies indicate that T15+ B cells occur as a high frequency event within bone marrow generative cell pools. Furthermore, bone marrow populations that are positive for PC-responsive precursor cells often display multiple copies of such precursor cells that are exclusively either T15+ or T15-. This finding indicates that clonal expansion of cells within the B cell lineage apparently occurs before immunoglobulin receptor acquisition.

scholarly journals Sheepish B cells: evidence for antigen-independent antibody diversification in humans and mice

2008 ◽  
Vol 205 (6) ◽  
pp. 1251-1254 ◽  
Author(s):  
David Tarlinton
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
Germinal Centers ◽  
B Cell Development ◽  
Cell Populations ◽  
Antibody Diversity ◽  
Antibody Diversification

Antibody diversity is first generated by rearrangement of immunoglobulin (Ig) genes during B cell development in the bone marrow, and later by antigen-driven diversification in germinal centers (GCs). New data in humans and mice now identify specific B cell populations that may have undergone antigen-independent hypermutation outside GCs.

scholarly journals BCL6 Is Critical for the Development of a Diverse Primary B Cell Repertoire.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 91-91
Author(s):  
Cihangir Duy ◽  
J. Jessica Yu ◽  
Srividya Swaminathan ◽  
Rahul R. Nahar ◽  
Soo-mi Kweon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
B Cells ◽  
B Cell ◽  
Transcriptional Repression ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Repertoire ◽  
Self Renewal ◽  
B Cell Repertoire

Abstract Abstract 91 Through DNA strand breaks resulting from somatic hypermutation and class-switch recombination, germinal center (GC) B cells are exposed to a high level of DNA damage stress. At the GC stage of development, B cells are protected against apoptosis by specific expression of BCL6, which functions as transcriptional repressor of genes in the DNA damage response pathway. In the absence of BCL6, GC formation is abrogated. During normal B cell development, BCL6 expression was only found in GCs, where the secondary B cell repertoire is shaped. Extensive DNA damage, however, also occurs during the development of the primary B cell repertoire in the bone marrow. B cell precursors in the bone marrow sustain DNA damage during V(D)J recombination at immunoglobulin heavy and light chain loci. It is currently unclear, through which mechanisms early B cell precursors are protected against extensive DNA damage stress caused by V(D)J recombination. Here we report that BCL6 plays a critical role during early B cell development by protecting pre-B cells from DNA damage-induced apoptosis during V(D)J recombination. At the transition from IL7-dependent to IL7-independent stages of B cell development, BCL6 is activated, reaches similar expression levels as in GC B cells. Compared to IL7-dependent pro-B cells and large cycling pre-B cells, BCL6 mRNA and protein levels in IL7-independent small resting pre-B cells were increased by 60- to 90-fold, respectively. We identified STAT5 as a critical negative regulator of BCL6 downstream of IL7 receptor signaling in pre-B cells. Expression of a constitutively active STAT5 mutant prevented BCL6 upregulation in differentiating pre-B cells at the transition from IL7-dependent to IL7-independent stages of B cell development. BCL6 function was then tested in bone marrow precursor cells from BCL6−/− and BCL6+/+ mice: Comparing the gene expression pattern of BCL6−/− and BCL6+/+ pre-B cells, we found that BCL6 is required for transcriptional repression of the ARF (Cdkn2a), p21 (Cdkn1a), Gadd45a and p53 genes, which all contribute to cellular senescence and cell cycle arrest. In agreement with gene expression analyses, ChIP-chip and single-locus q-ChIP studies identified ARF (Cdkn2a), p21 (Cdkn1a), Gadd45a and p53 as transcriptional targets of BCL6 in pre-B cells. BCL6-dependent transcriptional repression of these genes in pre-B cells is critical because BCL6+/+ but not BCL6−/− pre-B cells were capable to proliferate in vitro and to form pre-B cell colonies in semisolid agar. Of note, peptide-inhibition of BCL6 suppressed growth and colony formation in ARF+/+ but not ARF−/− pre-B cells, suggesting that ARF-deficiency rescues lack of BCL6 function. We conclude that BCL6-mediated transcriptional repression of ARF is critical for pre-B cell self-renewal. To determine whether BCL6 function is also required for normal early B cell differentiation in vivo, we performed a comprehensive analysis of B cell differentiation stages in bone marrow from BCL6−/− and BCL6+/+ mice. In agreement with previous studies, the overall number of B cell precursors in the bone marrow was only slightly reduced and pro-B cell and large pre-B cell populations were normal. However, the pools of small-resting pre-B cells and new emigrant B cells were reduced in BCL6−/− mice by 3- and 7-fold, respectively. While the overall numbers of mature B cells in BCL6−/− mice were normal, we found that their clonal repertoire was extremely restricted. Using spectratype analysis, we found a broad polyclonal primary B cell repertoire in BCL6+/+ mice, whereas the B cell repertoire in their BCL6−/− counterparts was strictly oligoclonal. We conclude that pre-B cell self-renewal and polyclonal B cell production critically depends on BCL6. While the self-renewal defect of BCL6-deficient pre-B cells can be numerically compensated by increased proliferation at later stages of development, the diversity of the B cell repertoire in BCL6−/− mice is permanently restricted. We conclude that BCL6 is required for pre-B cell self-renewal and the formation of a normal polyclonal B cell repertoire. Disclosures: No relevant conflicts of interest to declare.

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