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Regulation of B cell development by antigen receptors
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47.

During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown.

After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A.

Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A. 

SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM.

Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

Place, publisher, year, edition, pages
Umeå: Print & Media , 2011. , 60 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1409
Keyword [en]
calcium, calmodulin, e-proteins, E2A, B cell development, antigen receptor signaling, surrogate light chains, RAG, allelic exclusion, V(D)J recombination, AID, negative feedback regulation, plasma cell differentiation
Identifiers
URN: urn:nbn:se:umu:diva-40819ISBN: 978-91-7459-167-5OAI: oai:DiVA.org:umu-40819DiVA: diva2:403018
Public defence
2011-04-08, Major Groove, Inst Molekylärbiologi, By 6L, NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2011-03-18 Created: 2011-03-10 Last updated: 2011-03-18Bibliographically approved
List of papers
1. Calmodulin inhibition of E2A stops expression of surrogate light chains of the pre-B-cell receptor and CD19
Open this publication in new window or tab >>Calmodulin inhibition of E2A stops expression of surrogate light chains of the pre-B-cell receptor and CD19
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2010 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 47, no 5, 1031-1038 p.Article in journal (Refereed) Published
Abstract [en]

To create antibody diversity, B lymphocyte development is characterized by the ordered rearrangement of first immunoglobulin (Ig) heavy chain gene segments and then Ig light-chain gene segments. Early in B-cell development, expression of a pre-B-cell receptor (pre-BCR) composed of membrane-bound Ig heavy chain protein associated with surrogate light-chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces clonal expansion, but it also turns off transcription of the genes for the SLC proteins lambda5 and VpreB, which limits this proliferation. Here we show that signaling from the pre-BCR rapidly down-regulates lambda5 and VpreB and also the co-receptor CD19 in primary pre-B-cells. We show that calcium (Ca(2+)) signaling is essential for this silencing of the SLC and CD19 genes. The SLC genes are activated by the E2A transcription factor, and we show that E2A is required for pre-BCR-mediated regulation of the genes. E2A mutated in its binding site for the Ca(2+) sensor protein calmodulin, and thus with calmodulin-resistant DNA binding, makes lambda5, VpreB and CD19 expression resistant to the inhibition following pre-BCR activation. Thus, Ca(2+) down-regulates SLC and CD19 gene expression upon pre-BCR activation through inhibition of E2A by Ca(2+)/calmodulin.

Keyword
Pre-BCR; Calcium signaling; Cell proliferation; Gene regulation; E2A; Calmodulin; Surrogate light chains; Signal transduction
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-32844 (URN)10.1016/j.molimm.2009.11.015 (DOI)000274945100010 ()20022378 (PubMedID)
Available from: 2010-03-29 Created: 2010-03-29 Last updated: 2011-03-18Bibliographically approved
2. Regulation of allelic exclusion of IgH by calmodulin inhibition of E2A
Open this publication in new window or tab >>Regulation of allelic exclusion of IgH by calmodulin inhibition of E2A
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:umu:diva-40817 (URN)
Available from: 2011-03-10 Created: 2011-03-10 Last updated: 2011-03-18Bibliographically approved
3. B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins
Open this publication in new window or tab >>B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins
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2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 4, 1267-1272 p.Article in journal (Refereed) Published
Abstract [en]

Upon encountering antigens, B-lymphocytes can adapt to produce a highly specific and potent antibody response. Somatic hypermutation, which introduces point mutations in the variable regions of antibody genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both somatic hypermutation and CSR. The mutagenic AID enzyme has to be tightly controlled. Here, we show that engagement of the membrane-bound antibodies of the B-cell receptor (BCR), which signals that good antibody affinity has been reached, inhibits AID gene expression and that calcium (Ca(2+)) signaling is essential for this inhibition. Moreover, we show that overexpression of the Ca(2+) sensor protein calmodulin inhibits AID gene expression, and that the transcription factor E2A is required for regulation of the AID gene by the BCR. E2A mutated in the binding site for calmodulin, and thus showing calmodulin-resistant DNA binding, makes AID expression resistant to the inhibition through BCR activation. Thus, BCR activation inhibits AID gene expression through Ca(2+)/calmodulin inhibition of E2A.

Keyword
activation-induced cytidine deaminase, BCR, calcium, E2A
Identifiers
urn:nbn:se:umu:diva-20779 (URN)10.1073/pnas.0708220105 (DOI)18203819 (PubMedID)
Available from: 2009-03-25 Created: 2009-03-25 Last updated: 2011-03-18Bibliographically approved
4. Negative feedback regulation of antigen receptors through calmodulin inhibition of E2A
Open this publication in new window or tab >>Negative feedback regulation of antigen receptors through calmodulin inhibition of E2A
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:umu:diva-40818 (URN)
Available from: 2011-03-10 Created: 2011-03-10 Last updated: 2011-03-18Bibliographically approved
5. Initiation of antigen receptor-dependent differentiation into plasma cells by calmodulin inhibition of E2A
Open this publication in new window or tab >>Initiation of antigen receptor-dependent differentiation into plasma cells by calmodulin inhibition of E2A
2009 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 183, no 2, 1179-1187 p.Article in journal (Refereed) Published
Abstract [en]

Differentiation of B lymphocytes into Ab-secreting plasmablasts and plasma cells is Ag driven. The interaction of Ag with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. However, not much is known about the coupling between BCR activation and the shift in transcription factor network from that of a B cell to that of ASC differentiation. Our genome-wide analysis shows that Ab-secreting cell differentiation of mouse B cells is induced by BCR activation through very fast regulatory events from the BCR. We identify activation of IFN regulatory factor-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B gene expression as immediate early events. Furthermore, the transcription factor E2A is required for the rapid key down-regulations after BCR activation, and the Ca(2+) sensor protein calmodulin has the corresponding regulatory effect as BCR activation. Moreover, mutants in the calmodulin binding site of E2A show that Ca(2+) signaling through calmodulin inhibition of E2A is essential for the rapid down-regulation of immediate early genes after BCR activation in initiation of plasma cell differentiation.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-32845 (URN)10.4049/jimmunol.0900455 (DOI)19553523 (PubMedID)
Available from: 2010-03-29 Created: 2010-03-29 Last updated: 2011-03-18Bibliographically approved

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Hauser, Jannek
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