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Thymocyte maturation is regulated by the activity of the helix-loop-helix protein, E47.

Bain G, Quong MW, Soloff RS, Hedrick SM, Murre C - J. Exp. Med. (1999)

Bottom Line: Additionally, development of CD8 lineage T cells in an MHC class I-restricted TCR transgenic background is sensitive to the dosage of E47.Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells.Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I- and class II-restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I-restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection.

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Inhibition of E47 activity in a DP T cell line promotes differentiation. The 16610D9 DP T cell line was retrovirally infected with Id3 or control (S-003) virus. 48 h after infection, the cells were harvested and WCE was prepared as described from half of the cells while the remainder of the cells was stained with the indicated antibodies and analyzed by flow cytometry. (A) E2A binding activity was analyzed by electrophoretic mobility shift assay using a labeled μE5 oligo probe. Lanes 1–4, S-003–infected cells; lanes 5–8, Id3-infected cells. Extracts were preincubated with mAbs (Ab) against E47 (lanes 2 and 6), E12/HEB (lanes 3 and 7), or E12 (lanes 4 and 8) before the addition of the probe. The arrow at left indicates the E47-containing complex. (B) EGFP expression on the S-003 (top) and Id3 (bottom) infected cells. The gates used to define the GFP+ and GFP− populations are indicated in the histograms. (C) Infected cells were stained with the indicated antibodies and analyzed by flow cytometry. The left panels show staining patterns on the GFP+ cells, and the right panels show staining patterns on the GFP− cells within the same infection. Thin line indicates staining on the S-003–infected cells, and thick line indicates staining on the Id3-infected cells. (D) CD4/CD8 staining on GFP+ (top panels) and GFP− (bottom panels) from control (S-003) and Id3-infected cells. The percentage of CD4/CD8 DP cells (boxed area) is indicated in each plot.
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Figure 8: Inhibition of E47 activity in a DP T cell line promotes differentiation. The 16610D9 DP T cell line was retrovirally infected with Id3 or control (S-003) virus. 48 h after infection, the cells were harvested and WCE was prepared as described from half of the cells while the remainder of the cells was stained with the indicated antibodies and analyzed by flow cytometry. (A) E2A binding activity was analyzed by electrophoretic mobility shift assay using a labeled μE5 oligo probe. Lanes 1–4, S-003–infected cells; lanes 5–8, Id3-infected cells. Extracts were preincubated with mAbs (Ab) against E47 (lanes 2 and 6), E12/HEB (lanes 3 and 7), or E12 (lanes 4 and 8) before the addition of the probe. The arrow at left indicates the E47-containing complex. (B) EGFP expression on the S-003 (top) and Id3 (bottom) infected cells. The gates used to define the GFP+ and GFP− populations are indicated in the histograms. (C) Infected cells were stained with the indicated antibodies and analyzed by flow cytometry. The left panels show staining patterns on the GFP+ cells, and the right panels show staining patterns on the GFP− cells within the same infection. Thin line indicates staining on the S-003–infected cells, and thick line indicates staining on the Id3-infected cells. (D) CD4/CD8 staining on GFP+ (top panels) and GFP− (bottom panels) from control (S-003) and Id3-infected cells. The percentage of CD4/CD8 DP cells (boxed area) is indicated in each plot.

Mentions: As shown in Table , E2A-deficient DP thymocytes show reduced survival in vitro. However, the data described above suggest that the absence of E47 also promotes thymocyte positive selection. To test directly whether lowering the activity of E47 promotes maturation, we used a retroviral transduction system to study the effects of inhibiting E2A activity in an immature DP T cell line. The mouse cDNA for Id3, a negative regulator of E2A binding activity, was cloned into the retroviral vector S-003 14. This vector allows translation of both Id3 and enhanced green fluorescent protein (EGFP) from one retroviral transcript. Thus, retroviral transductants can be identified by the expression of EGFP. The S-003/Id3 and S-003 empty vector constructs were transfected into the φNX-eco retroviral packaging line, and supernatants from these cells were used to transduce virus into the T cells. The 16610D9 T cell line used was derived from a thymoma that developed spontaneously in a p53-deficient mouse and was adapted to culture. This T cell line expresses characteristics typical of DP thymocytes, including high levels of HSA, intermediate levels of TCR, and low levels of CD5 and CD44 (Fig. 8 C). In addition, the 16610D9 cells express significant levels of E2A binding activity (Fig. 8 A).


Thymocyte maturation is regulated by the activity of the helix-loop-helix protein, E47.

Bain G, Quong MW, Soloff RS, Hedrick SM, Murre C - J. Exp. Med. (1999)

Inhibition of E47 activity in a DP T cell line promotes differentiation. The 16610D9 DP T cell line was retrovirally infected with Id3 or control (S-003) virus. 48 h after infection, the cells were harvested and WCE was prepared as described from half of the cells while the remainder of the cells was stained with the indicated antibodies and analyzed by flow cytometry. (A) E2A binding activity was analyzed by electrophoretic mobility shift assay using a labeled μE5 oligo probe. Lanes 1–4, S-003–infected cells; lanes 5–8, Id3-infected cells. Extracts were preincubated with mAbs (Ab) against E47 (lanes 2 and 6), E12/HEB (lanes 3 and 7), or E12 (lanes 4 and 8) before the addition of the probe. The arrow at left indicates the E47-containing complex. (B) EGFP expression on the S-003 (top) and Id3 (bottom) infected cells. The gates used to define the GFP+ and GFP− populations are indicated in the histograms. (C) Infected cells were stained with the indicated antibodies and analyzed by flow cytometry. The left panels show staining patterns on the GFP+ cells, and the right panels show staining patterns on the GFP− cells within the same infection. Thin line indicates staining on the S-003–infected cells, and thick line indicates staining on the Id3-infected cells. (D) CD4/CD8 staining on GFP+ (top panels) and GFP− (bottom panels) from control (S-003) and Id3-infected cells. The percentage of CD4/CD8 DP cells (boxed area) is indicated in each plot.
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Figure 8: Inhibition of E47 activity in a DP T cell line promotes differentiation. The 16610D9 DP T cell line was retrovirally infected with Id3 or control (S-003) virus. 48 h after infection, the cells were harvested and WCE was prepared as described from half of the cells while the remainder of the cells was stained with the indicated antibodies and analyzed by flow cytometry. (A) E2A binding activity was analyzed by electrophoretic mobility shift assay using a labeled μE5 oligo probe. Lanes 1–4, S-003–infected cells; lanes 5–8, Id3-infected cells. Extracts were preincubated with mAbs (Ab) against E47 (lanes 2 and 6), E12/HEB (lanes 3 and 7), or E12 (lanes 4 and 8) before the addition of the probe. The arrow at left indicates the E47-containing complex. (B) EGFP expression on the S-003 (top) and Id3 (bottom) infected cells. The gates used to define the GFP+ and GFP− populations are indicated in the histograms. (C) Infected cells were stained with the indicated antibodies and analyzed by flow cytometry. The left panels show staining patterns on the GFP+ cells, and the right panels show staining patterns on the GFP− cells within the same infection. Thin line indicates staining on the S-003–infected cells, and thick line indicates staining on the Id3-infected cells. (D) CD4/CD8 staining on GFP+ (top panels) and GFP− (bottom panels) from control (S-003) and Id3-infected cells. The percentage of CD4/CD8 DP cells (boxed area) is indicated in each plot.
Mentions: As shown in Table , E2A-deficient DP thymocytes show reduced survival in vitro. However, the data described above suggest that the absence of E47 also promotes thymocyte positive selection. To test directly whether lowering the activity of E47 promotes maturation, we used a retroviral transduction system to study the effects of inhibiting E2A activity in an immature DP T cell line. The mouse cDNA for Id3, a negative regulator of E2A binding activity, was cloned into the retroviral vector S-003 14. This vector allows translation of both Id3 and enhanced green fluorescent protein (EGFP) from one retroviral transcript. Thus, retroviral transductants can be identified by the expression of EGFP. The S-003/Id3 and S-003 empty vector constructs were transfected into the φNX-eco retroviral packaging line, and supernatants from these cells were used to transduce virus into the T cells. The 16610D9 T cell line used was derived from a thymoma that developed spontaneously in a p53-deficient mouse and was adapted to culture. This T cell line expresses characteristics typical of DP thymocytes, including high levels of HSA, intermediate levels of TCR, and low levels of CD5 and CD44 (Fig. 8 C). In addition, the 16610D9 cells express significant levels of E2A binding activity (Fig. 8 A).

Bottom Line: Additionally, development of CD8 lineage T cells in an MHC class I-restricted TCR transgenic background is sensitive to the dosage of E47.Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells.Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I- and class II-restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I-restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection.

Show MeSH
Related in: MedlinePlus