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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 μHC-expressing BM pre–B cells in the absence of SLC components and conventional LC. CD19+ BM cells isolated from C57BL/6, VpreB1/2−/−, or VpreB1/2−/− λ5−/− (SLC−/−) mice were stained for surface c-kit or CD25, followed by fixation and permeabilization to detect intracellular κ/λLC and μHC in combination with TdT or βGal (isotype control). Cells positive for conventional LC were excluded from the analysis. (A) Cells positive for c-kit (i.e., pro–B cells) and negative (G1) or positive (G2) for cytoplasmic μHC were analyzed separately for TdT expression (unshaded). Staining for βGal was used as isotype control (shaded). (B) Cells positive for surface CD25 and cytoplasmic μHC (i.e., pre–B cells) were analyzed as in A. Numbers in the dot plots indicate the percentage of cells within the gates. Numbers in the histograms represent mean fluorescence intensities for TdT or βGal (shaded). The purity of CD19+ enriched cells was 96 (C57BL/6), 91 (VpreB1/2−/−), and 89% (SLC−/−) in this experiment. Representative results of two FACS® analyses are shown.
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fig2: TdT is down-regulated in μHC-expressing BM pre–B cells in the absence of SLC components and conventional LC. CD19+ BM cells isolated from C57BL/6, VpreB1/2−/−, or VpreB1/2−/− λ5−/− (SLC−/−) mice were stained for surface c-kit or CD25, followed by fixation and permeabilization to detect intracellular κ/λLC and μHC in combination with TdT or βGal (isotype control). Cells positive for conventional LC were excluded from the analysis. (A) Cells positive for c-kit (i.e., pro–B cells) and negative (G1) or positive (G2) for cytoplasmic μHC were analyzed separately for TdT expression (unshaded). Staining for βGal was used as isotype control (shaded). (B) Cells positive for surface CD25 and cytoplasmic μHC (i.e., pre–B cells) were analyzed as in A. Numbers in the dot plots indicate the percentage of cells within the gates. Numbers in the histograms represent mean fluorescence intensities for TdT or βGal (shaded). The purity of CD19+ enriched cells was 96 (C57BL/6), 91 (VpreB1/2−/−), and 89% (SLC−/−) in this experiment. Representative results of two FACS® analyses are shown.

Mentions: In wild-type mice, as expected, most c-kit+μHC− pro–B cells showed high levels of TdT expression, whereas most c-kit+μHC+ cells had down-regulated TdT (Fig. 2 A, top), presumably because they already express the pre-BCR (23). As expected, in VpreB1−/− VpreB2−/− and SLC−/− mice, most c-kit+μHC− cells were positive for TdT (Fig. 2 A, middle and bottom). However, in contrast with wild-type cells, TdT levels stay elevated in most c-kit+μHC+ cells from both VpreB1−/−VpreB2−/− and SLC−/− mice.


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 μHC-expressing BM pre–B cells in the absence of SLC components and conventional LC. CD19+ BM cells isolated from C57BL/6, VpreB1/2−/−, or VpreB1/2−/− λ5−/− (SLC−/−) mice were stained for surface c-kit or CD25, followed by fixation and permeabilization to detect intracellular κ/λLC and μHC in combination with TdT or βGal (isotype control). Cells positive for conventional LC were excluded from the analysis. (A) Cells positive for c-kit (i.e., pro–B cells) and negative (G1) or positive (G2) for cytoplasmic μHC were analyzed separately for TdT expression (unshaded). Staining for βGal was used as isotype control (shaded). (B) Cells positive for surface CD25 and cytoplasmic μHC (i.e., pre–B cells) were analyzed as in A. Numbers in the dot plots indicate the percentage of cells within the gates. Numbers in the histograms represent mean fluorescence intensities for TdT or βGal (shaded). The purity of CD19+ enriched cells was 96 (C57BL/6), 91 (VpreB1/2−/−), and 89% (SLC−/−) in this experiment. Representative results of two FACS® analyses are shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: TdT is down-regulated in μHC-expressing BM pre–B cells in the absence of SLC components and conventional LC. CD19+ BM cells isolated from C57BL/6, VpreB1/2−/−, or VpreB1/2−/− λ5−/− (SLC−/−) mice were stained for surface c-kit or CD25, followed by fixation and permeabilization to detect intracellular κ/λLC and μHC in combination with TdT or βGal (isotype control). Cells positive for conventional LC were excluded from the analysis. (A) Cells positive for c-kit (i.e., pro–B cells) and negative (G1) or positive (G2) for cytoplasmic μHC were analyzed separately for TdT expression (unshaded). Staining for βGal was used as isotype control (shaded). (B) Cells positive for surface CD25 and cytoplasmic μHC (i.e., pre–B cells) were analyzed as in A. Numbers in the dot plots indicate the percentage of cells within the gates. Numbers in the histograms represent mean fluorescence intensities for TdT or βGal (shaded). The purity of CD19+ enriched cells was 96 (C57BL/6), 91 (VpreB1/2−/−), and 89% (SLC−/−) in this experiment. Representative results of two FACS® analyses are shown.
Mentions: In wild-type mice, as expected, most c-kit+μHC− pro–B cells showed high levels of TdT expression, whereas most c-kit+μHC+ cells had down-regulated TdT (Fig. 2 A, top), presumably because they already express the pre-BCR (23). As expected, in VpreB1−/− VpreB2−/− and SLC−/− mice, most c-kit+μHC− cells were positive for TdT (Fig. 2 A, middle and bottom). However, in contrast with wild-type cells, TdT levels stay elevated in most c-kit+μHC+ cells from both VpreB1−/−VpreB2−/− and SLC−/− mice.

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