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 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.

scholarly journals Secondary Rearrangements and Hypermutation Generate Sufficient B Cell Diversity to Mount Protective Antiviral Immunoglobulin Responses

1999 ◽  
Vol 189 (11) ◽  
pp. 1791-1798 ◽  
Author(s):  
Constantino López-Macías ◽  
Ulrich Kalinke ◽  
Marilia Cascalho ◽  
Matthias Wabl ◽  
Hans Hengartner ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Gene Replacement ◽  
Antibody Responses ◽  
Single Chain ◽  
Cell Repertoire ◽  
Region Gene ◽  
B Cell Repertoire ◽  
Single Chain Fv ◽  
Cell Diversity

Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of “quasimonoclonal” (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, ∼20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.

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.

scholarly journals B cell repertoire diversification precedes immunoglobulin receptor expression.

1983 ◽  
Vol 158 (5) ◽  
pp. 1733-1738 ◽  
Author(s):  
R L Riley ◽  
D E Wylie ◽  
N R Klinman
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Receptor Expression ◽  
Cell Repertoire ◽  
Immunoglobulin Receptor ◽  
Precursor Pool ◽  
Regulatory Processes ◽  
B Cell Repertoire

68 monoclonal antibodies specific for the hemagglutinin (HA) of the influenza virus, PR8, were obtained from sIg- bone marrow B cell precursors stimulated in splenic fragment cultures. Reactivity pattern (RP) analysis demonstrated that these anti-HA antibody responses included at least 29 distinguishable clonotypes. Comparison of the specificities of anti-HA antibodies obtained from sIg- bone marrow cells with those obtained from adult spleen cells indicates that the anti-HA repertoires of the two populations are comparable in diversity. Since the sIg- bone marrow B cell precursor pool presumably has not encountered V region-specific regulatory mechanisms in vivo, our data suggest that substantial diversification of the B cell repertoire precedes surface immunoglobulin (sIg) expression and subsequent interaction with environmental regulatory processes.

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

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.

scholarly journals Efficient Peripheral Clonal Elimination of B Lymphocytes in MRL/lpr Mice Bearing Autoantibody Transgenes

1998 ◽  
Vol 188 (5) ◽  
pp. 909-917 ◽  
Author(s):  
Jennifer A. Kench ◽  
David M. Russell ◽  
David Nemazee
Keyword(s):  
B Cells ◽  
B Cell ◽  
Genetic Background ◽  
Cell Tolerance ◽  
Cell Repertoire ◽  
B Cell Tolerance ◽  
B Cell Repertoire ◽  
Nuclear Antigens ◽  
Large Numbers ◽  
Liver And Kidney

Peripheral B cell tolerance was studied in mice of the autoimmune-prone, Fas-deficient MRL/ lpr.H-2d genetic background by introducing a transgene that directs expression of membrane-bound H-2Kb antigen to liver and kidney (MT-Kb) and a second transgene encoding antibody reactive with this antigen (3-83μδ, anti-Kk,b). Control immunoglobulin transgenic (Ig-Tg) MRL/lpr.H-2d mice lacking the Kb antigen had large numbers of splenic and lymph node B cells bearing the transgene-encoded specificity, whereas B cells of the double transgenic (Dbl-Tg) MRL/lpr.H-2d mice were deleted as efficiently as in Dbl-Tg mice of a nonautoimmune B10.D2 genetic background. In spite of the severely restricted peripheral B cell repertoire of the Ig-Tg MRL/lpr.H-2d mice, and notwithstanding deletion of the autospecific B cell population in the Dbl-Tg MRL/lpr.H-2d mice, both types of mice developed lymphoproliferation and exhibited elevated levels of IgG anti-chromatin autoantibodies. Interestingly, Dbl-Tg MRL/lpr.H-2d mice had a shorter lifespan than Ig-Tg MRL/lpr.H-2d mice, apparently as an indirect result of their relative B cell lymphopenia. These data suggest that in MRL/lpr mice peripheral B cell tolerance is not globally defective, but that certain B cells with receptors specific for nuclear antigens are regulated differently than are cells reactive to membrane autoantigens.

In vitro differentiation of reprogrammed murine somatic cells into hepatic precursor cells

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Tobias Cantz ◽  
Martina Bleidißel ◽  
Martin Stehling ◽  
Hans R. Schöler
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Es Cells ◽  
Marrow Cell ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Precursor Cells ◽  
Differentiation Potential ◽  
Teratoma Formation ◽  
Transplantation Experiments

Abstract Recently, a new approach to reprogram somatic cells into pluripotent stem cells was shown by fusion of somatic cells with embryonic stem (ES) cells, which results in a tetraploid karyotype. Normal hepatocytes are often polyploid, so we decided to investigate the differentiation potential of fusion hybrids into hepatic cells. We chose toxic milk mice (a model of Wilson's disease) and performed initial transplantation experiments using this potential cell therapy approach. Mononuclear bone marrow cells from Rosa26 mice were fused with OG2 (Oct4-GFP transgenic) ES cells. Unfused ES cells were eliminated by selection with G418 for OG2-Rosa26 hybrids and fusion-derived colonies could be subcloned. Using an endodermal differentiation protocol, hepatic precursor cells could be generated. After FACS depletion of contaminating Oct4-GFP-positive cells, the hepatic precursor cells were transplanted into immunosuppressed toxic milk mice by intrasplenic injection. However, five out of eight mice showed teratoma formation within 3–6 weeks after transplantation in the spleen and liver. In conclusion, a hepatic precursor cell type was achieved from mononuclear bone marrow cell-ES cell hybrids and preliminary transplantation experiments confirmed engraftment, but also showed teratoma formation, which needs to be excluded by using more stringent purification strategies.

scholarly journals Immunoglobulin heavy chain variable region gene usage in bone marrow transplant recipients: lack of somatic mutation indicates a maturational arrest [see comments]

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1873-1880 ◽  
Author(s):  
I Suzuki ◽  
EC Milner ◽  
AM Glas ◽  
WO Hufnagle ◽  
SP Rao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Bone Marrow Transplant ◽  
Healthy Subjects ◽  
Somatic Mutations ◽  
Variable Region ◽  
Marrow Transplant ◽  
B Cell Differentiation ◽  
Region Gene ◽  
Vh Genes

Abstract Many recipients of bone marrow transplant (BMT) make normal amounts of serum immunoglobulin but are deficient in generating specific antibody responses to exogenous stimuli. To determine if abnormal usage of VH genes contributes to this immunodeficiency, the usage of VH genes was determined in peripheral blood B cells of four BMT recipients, two of whom had developed chronic graft versus host disease. The pattern of usage of VH3 or VH4 genes assessed at either 90 days or approximately 1 year after transplant was similar to that observed in healthy subjects and was marked by the over utilization of two elements, one VH3 and one VH4. However, the repertoires of each of the four BMT recipients appeared to be less complex than the repertoires of healthy subjects. The differences were a consequence of the accumulation of somatic mutations among rearrangements in the controls but not in the BMT recipients. The failure to accumulate somatic mutations in rearranged VH genes is consistent with a defect in antigen driven B-cell responses. These results indicate the although the VH gene content of the repertoire has normalized by 90 days posttransplant, a maturational arrest in B-cell differentiation associated with antigen activation persists for at least 1 year after BMT.

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