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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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μH chain is detectable on the surface of λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− pre–B cells. (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 drinking water for 7 d. Cells were expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for an additional 18 h in the presence of IL-7 with (shaded) or without Tet (unshaded). Cells were harvested and stained with biotinylated Abs against μH chain (goat polyclonal), pre-BCR (SL156), or SLC components (LM34, VP245, and R5). Surface-bound biotin was amplified and revealed by EAS and streptavidin-PE. Propidium iodide negative cells were analyzed by flow cytometry. (B) CD19+ BM cells were isolated by MACS from SLC−/− or Rag1−/− (control) mice and expanded on stromal cells in medium containing IL-7 for 4 d. Cells were harvested and stained with FITC-labeled Abs against κ/λLC and biotinylated Abs against μHC (goat polyclonal) or λ5 (LM34). Surface-bound biotin was amplified and revealed aforementioned. Numbers represent the percentage of cells within the quadrants.
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fig5: μH chain is detectable on the surface of λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− pre–B cells. (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 drinking water for 7 d. Cells were expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for an additional 18 h in the presence of IL-7 with (shaded) or without Tet (unshaded). Cells were harvested and stained with biotinylated Abs against μH chain (goat polyclonal), pre-BCR (SL156), or SLC components (LM34, VP245, and R5). Surface-bound biotin was amplified and revealed by EAS and streptavidin-PE. Propidium iodide negative cells were analyzed by flow cytometry. (B) CD19+ BM cells were isolated by MACS from SLC−/− or Rag1−/− (control) mice and expanded on stromal cells in medium containing IL-7 for 4 d. Cells were harvested and stained with FITC-labeled Abs against κ/λLC and biotinylated Abs against μHC (goat polyclonal) or λ5 (LM34). Surface-bound biotin was amplified and revealed aforementioned. Numbers represent the percentage of cells within the quadrants.

Mentions: To allow detection of low levels of surface μHC complexes, we used biotinylated Abs in combination with EAS. We analyzed surface expression of transgenic μHC and SLC on CD19+ BM cells from tet-μHC, tet-μHC λ5−/−, and tet-μHC VpreB1−/−VpreB2−/− mice. Cells were isolated and expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for 18 h in the presence (Fig. 5 A, shaded) or absence (Fig. 5 A, unshaded) of Tet. Expression of the transgenic μHC in the absence of Tet was confirmed by parallel cytoplasmic staining for μHC (unpublished data).


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)

μH chain is detectable on the surface of λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− pre–B cells. (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 drinking water for 7 d. Cells were expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for an additional 18 h in the presence of IL-7 with (shaded) or without Tet (unshaded). Cells were harvested and stained with biotinylated Abs against μH chain (goat polyclonal), pre-BCR (SL156), or SLC components (LM34, VP245, and R5). Surface-bound biotin was amplified and revealed by EAS and streptavidin-PE. Propidium iodide negative cells were analyzed by flow cytometry. (B) CD19+ BM cells were isolated by MACS from SLC−/− or Rag1−/− (control) mice and expanded on stromal cells in medium containing IL-7 for 4 d. Cells were harvested and stained with FITC-labeled Abs against κ/λLC and biotinylated Abs against μHC (goat polyclonal) or λ5 (LM34). Surface-bound biotin was amplified and revealed aforementioned. Numbers represent the percentage of cells within the quadrants.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: μH chain is detectable on the surface of λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− pre–B cells. (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 drinking water for 7 d. Cells were expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for an additional 18 h in the presence of IL-7 with (shaded) or without Tet (unshaded). Cells were harvested and stained with biotinylated Abs against μH chain (goat polyclonal), pre-BCR (SL156), or SLC components (LM34, VP245, and R5). Surface-bound biotin was amplified and revealed by EAS and streptavidin-PE. Propidium iodide negative cells were analyzed by flow cytometry. (B) CD19+ BM cells were isolated by MACS from SLC−/− or Rag1−/− (control) mice and expanded on stromal cells in medium containing IL-7 for 4 d. Cells were harvested and stained with FITC-labeled Abs against κ/λLC and biotinylated Abs against μHC (goat polyclonal) or λ5 (LM34). Surface-bound biotin was amplified and revealed aforementioned. Numbers represent the percentage of cells within the quadrants.
Mentions: To allow detection of low levels of surface μHC complexes, we used biotinylated Abs in combination with EAS. We analyzed surface expression of transgenic μHC and SLC on CD19+ BM cells from tet-μHC, tet-μHC λ5−/−, and tet-μHC VpreB1−/−VpreB2−/− mice. Cells were isolated and expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for 18 h in the presence (Fig. 5 A, shaded) or absence (Fig. 5 A, unshaded) of Tet. Expression of the transgenic μHC in the absence of Tet was confirmed by parallel cytoplasmic staining for μHC (unpublished data).

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