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Surface mu heavy chain signals down-regulation of the V(D)J-recombinase machinery in the absence of surrogate light chain components.

Galler GR, Mundt C, Parker M, Pelanda R, Mårtensson IL, Winkler TH - J. Exp. Med. (2004)

Bottom Line: Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion.It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele.Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells.

View Article: PubMed Central - PubMed

Affiliation: Hematopoiesis Unit, Nikolaus-Fiebiger-Center, Friedrich-Alexander University, Glueckstrasse 6, 91054 Erlangen, Germany.

ABSTRACT
Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible muHC transgene in Rag2-/- pro-B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-alpha. Furthermore, in wild-type mice and in mice lacking either lambda5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in muHC+LC- pre-B cells. Surprisingly, muHC without LC is expressed on the surface of pro-/pre-B cells from lambda5-/-, VpreB1-/-VpreB2-/-, and SLC-/- mice. Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.

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TdT is down-regulated in pre–B cells after de novo synthesis of transgenic μHC in the absence of λ5 or VpreB1/2. (A) CD19+ BM cells were isolated by MACS from tet-μHC, tet-μHC λ5−/−, and tet-μHC VpreB1−/−VpreB2−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the presence (top) or absence (bottom) of Tet. After 24 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT, Ku70, or βGal (isotype control). Fluorescence was determined by flow cytometry. Numbers within the gates represent mean fluorescence intensities for βGal, TdT, and Ku70, respectively. (B) CD19+ BM cells were isolated by MACS from tet-μHC λ5−/− Ig-α−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the absence of Tet. After 48 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT or βGal (isotype control). Numbers within the gates represent mean fluorescence intensities for βGal and TdT, respectively.
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fig1: TdT is down-regulated in pre–B cells after de novo synthesis of transgenic μHC in the absence of λ5 or VpreB1/2. (A) CD19+ BM cells were isolated by MACS from tet-μHC, tet-μHC λ5−/−, and tet-μHC VpreB1−/−VpreB2−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the presence (top) or absence (bottom) of Tet. After 24 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT, Ku70, or βGal (isotype control). Fluorescence was determined by flow cytometry. Numbers within the gates represent mean fluorescence intensities for βGal, TdT, and Ku70, respectively. (B) CD19+ BM cells were isolated by MACS from tet-μHC λ5−/− Ig-α−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the absence of Tet. After 48 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT or βGal (isotype control). Numbers within the gates represent mean fluorescence intensities for βGal and TdT, respectively.

Mentions: To confirm a causal connection, we took advantage of a tetracycline-controlled μHC transgene that can be expressed in the absence of endogenous V(D)J rearrangements, called tet-μHC (4). In these mice, μHC expression is completely suppressed, and B cell development is blocked at the pro–B cell stage after treatment with tetracycline in the drinking water for 7 d (4). CD19+ BM pro–B cells from these mice were cultured on stromal cells in the presence of IL-7. Expression of the transgenic μHC was induced by omitting tetracycline. After 24 h, the cells were harvested, stained for intracellular μHC and TDT expression, and analyzed by FACS®. Fig. 1 A demonstrates that μHC positive cells (−Tet) showed a marked decrease in TdT protein level compared with μHC negative cells (−Tet). Surprisingly, after μHC induction (−Tet), a similar, though less pronounced down-regulation of TdT was observed in cells that lack the SLC components λ5 or VpreB1/2, and, hence, cannot express a complete pre-BCR complex. Parallel staining for Ku70, an ubiquitously expressed protein involved in V(D)J recombination, as well as general DNA repair (22), revealed no decrease in μHC positive cells, confirming the specificity of μHC-mediated TdT down-regulation in the absence of λ5 or VpreB1/2. Comparable results were obtained with cells cultured in the absence of IL-7 (unpublished data). Thus, de novo expression of μHC is causally involved in the down-regulation of TdT and, unexpectedly, de novo expression of a μHC can signal the down-regulation of TdT in the absence of the pre-BCR.


Surface mu heavy chain signals down-regulation of the V(D)J-recombinase machinery in the absence of surrogate light chain components.

Galler GR, Mundt C, Parker M, Pelanda R, Mårtensson IL, Winkler TH - J. Exp. Med. (2004)

TdT is down-regulated in pre–B cells after de novo synthesis of transgenic μHC in the absence of λ5 or VpreB1/2. (A) CD19+ BM cells were isolated by MACS from tet-μHC, tet-μHC λ5−/−, and tet-μHC VpreB1−/−VpreB2−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the presence (top) or absence (bottom) of Tet. After 24 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT, Ku70, or βGal (isotype control). Fluorescence was determined by flow cytometry. Numbers within the gates represent mean fluorescence intensities for βGal, TdT, and Ku70, respectively. (B) CD19+ BM cells were isolated by MACS from tet-μHC λ5−/− Ig-α−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the absence of Tet. After 48 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT or βGal (isotype control). Numbers within the gates represent mean fluorescence intensities for βGal and TdT, respectively.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211789&req=5

fig1: TdT is down-regulated in pre–B cells after de novo synthesis of transgenic μHC in the absence of λ5 or VpreB1/2. (A) CD19+ BM cells were isolated by MACS from tet-μHC, tet-μHC λ5−/−, and tet-μHC VpreB1−/−VpreB2−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the presence (top) or absence (bottom) of Tet. After 24 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT, Ku70, or βGal (isotype control). Fluorescence was determined by flow cytometry. Numbers within the gates represent mean fluorescence intensities for βGal, TdT, and Ku70, respectively. (B) CD19+ BM cells were isolated by MACS from tet-μHC λ5−/− Ig-α−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the absence of Tet. After 48 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT or βGal (isotype control). Numbers within the gates represent mean fluorescence intensities for βGal and TdT, respectively.
Mentions: To confirm a causal connection, we took advantage of a tetracycline-controlled μHC transgene that can be expressed in the absence of endogenous V(D)J rearrangements, called tet-μHC (4). In these mice, μHC expression is completely suppressed, and B cell development is blocked at the pro–B cell stage after treatment with tetracycline in the drinking water for 7 d (4). CD19+ BM pro–B cells from these mice were cultured on stromal cells in the presence of IL-7. Expression of the transgenic μHC was induced by omitting tetracycline. After 24 h, the cells were harvested, stained for intracellular μHC and TDT expression, and analyzed by FACS®. Fig. 1 A demonstrates that μHC positive cells (−Tet) showed a marked decrease in TdT protein level compared with μHC negative cells (−Tet). Surprisingly, after μHC induction (−Tet), a similar, though less pronounced down-regulation of TdT was observed in cells that lack the SLC components λ5 or VpreB1/2, and, hence, cannot express a complete pre-BCR complex. Parallel staining for Ku70, an ubiquitously expressed protein involved in V(D)J recombination, as well as general DNA repair (22), revealed no decrease in μHC positive cells, confirming the specificity of μHC-mediated TdT down-regulation in the absence of λ5 or VpreB1/2. Comparable results were obtained with cells cultured in the absence of IL-7 (unpublished data). Thus, de novo expression of μHC is causally involved in the down-regulation of TdT and, unexpectedly, de novo expression of a μHC can signal the down-regulation of TdT in the absence of the pre-BCR.

Bottom Line: Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion.It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele.Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells.

View Article: PubMed Central - PubMed

Affiliation: Hematopoiesis Unit, Nikolaus-Fiebiger-Center, Friedrich-Alexander University, Glueckstrasse 6, 91054 Erlangen, Germany.

ABSTRACT
Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible muHC transgene in Rag2-/- pro-B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-alpha. Furthermore, in wild-type mice and in mice lacking either lambda5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in muHC+LC- pre-B cells. Surprisingly, muHC without LC is expressed on the surface of pro-/pre-B cells from lambda5-/-, VpreB1-/-VpreB2-/-, and SLC-/- mice. Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.

Show MeSH
Related in: MedlinePlus