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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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Altered maturation of thymocytes in class I–restricted TCR transgenic mice expressing decreased levels of E47. Flow cytometric analysis of 4–6-wk-old E47-deficient female mice and littermate controls expressing the class I–restricted H-Y TCR transgene. (A) Thymocytes from E47 knockout and heterozygous littermate females were stained with anti-CD4–PE and anti-CD8α–FITC or with the anti-Vα3 antibody, T3.70, which recognizes the transgenic α chain. The numbers in each quadrant indicate the percentage of cells in that population, and the percentage of cells staining brightly for the transgenic α chain is indicated in the histograms. (B) Splenocytes (top panels) and lymph node cells (bottom panels) from female H-Y transgenics of the indicated E47 genotype were analyzed by staining with anti-CD4–PE and anti-CD8α–FITC. The numbers in each quadrant indicate the percentage of cells in that population. (C) Thymocytes from an E47 heterozygous and knockout littermate expressing the H-Y TCR transgene were analyzed by three-color flow cytometric analysis for expression of CD4, CD8, and either TCR-α/β or CD69. The boxed regions in the CD4 versus CD8 plots indicate the analysis gates used to define the DP population. In the histogram plots, the thin lines indicate staining on the E47 heterozygous H-Y DP cells, and the thick lines indicate staining on the E47-deficient H-Y DP cells. The third color antibody used in each case is indicated. (D) Thymocytes (top panels), splenocytes (middle panels), and lymph node cells (bottom panels) from female H-Y transgenics that are wild-type or heterozygous for E47 were analyzed by staining with antibodies to CD4 and CD8. The numbers in each quadrant indicate the percentage of cells in that population.
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Figure 4: Altered maturation of thymocytes in class I–restricted TCR transgenic mice expressing decreased levels of E47. Flow cytometric analysis of 4–6-wk-old E47-deficient female mice and littermate controls expressing the class I–restricted H-Y TCR transgene. (A) Thymocytes from E47 knockout and heterozygous littermate females were stained with anti-CD4–PE and anti-CD8α–FITC or with the anti-Vα3 antibody, T3.70, which recognizes the transgenic α chain. The numbers in each quadrant indicate the percentage of cells in that population, and the percentage of cells staining brightly for the transgenic α chain is indicated in the histograms. (B) Splenocytes (top panels) and lymph node cells (bottom panels) from female H-Y transgenics of the indicated E47 genotype were analyzed by staining with anti-CD4–PE and anti-CD8α–FITC. The numbers in each quadrant indicate the percentage of cells in that population. (C) Thymocytes from an E47 heterozygous and knockout littermate expressing the H-Y TCR transgene were analyzed by three-color flow cytometric analysis for expression of CD4, CD8, and either TCR-α/β or CD69. The boxed regions in the CD4 versus CD8 plots indicate the analysis gates used to define the DP population. In the histogram plots, the thin lines indicate staining on the E47 heterozygous H-Y DP cells, and the thick lines indicate staining on the E47-deficient H-Y DP cells. The third color antibody used in each case is indicated. (D) Thymocytes (top panels), splenocytes (middle panels), and lymph node cells (bottom panels) from female H-Y transgenics that are wild-type or heterozygous for E47 were analyzed by staining with antibodies to CD4 and CD8. The numbers in each quadrant indicate the percentage of cells in that population.

Mentions: To determine whether the absence of E47 also promotes increased selection of class I–restricted TCRs, we analyzed E47-deficient mice expressing the H-Y TCR transgene, which specifies reactivity with the male H-Y antigen presented by the H-2Db class I molecule 2728. Expression of the H-Y TCR transgene in females of the H-2b background leads to positive selection of CD8 SP thymocytes 2728. In contrast, thymocytes in male mice expressing the H-Y TCR are deleted at the DP stage. Negative selection mediated by the H-Y TCR transgene was unaffected by the absence of E47 (data not shown). However, E47−/−; H-Y female mice displayed an increased percentage of mature CD8 SP thymocytes compared with the E47 heterozygous littermates (56.6 vs. 33.6%; Fig. 4 A). This phenotype was accompanied by a significant decrease in the proportion of immature DP thymocytes (29.6 vs. 46%; Fig. 4 A). Because of continued rearrangement of endogenous α chain genes in TCR transgenic mice, a fraction of the thymocytes will express a TCR composed of the transgenic β chain and an endogenous α chain 252729. To analyze the proportion of cells expressing the transgenic TCR-α/β, we stained total thymocytes with the T3.70 antibody, which reacts specifically with the transgenic α chain molecule 30. Thymocytes isolated from E47−/−;H-Y mice contained almost twice the percentage of cells expressing high levels of both the transgenic α and β chains, consistent with a higher proportion of mature CD8 SP T cells (Fig. 4 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)

Altered maturation of thymocytes in class I–restricted TCR transgenic mice expressing decreased levels of E47. Flow cytometric analysis of 4–6-wk-old E47-deficient female mice and littermate controls expressing the class I–restricted H-Y TCR transgene. (A) Thymocytes from E47 knockout and heterozygous littermate females were stained with anti-CD4–PE and anti-CD8α–FITC or with the anti-Vα3 antibody, T3.70, which recognizes the transgenic α chain. The numbers in each quadrant indicate the percentage of cells in that population, and the percentage of cells staining brightly for the transgenic α chain is indicated in the histograms. (B) Splenocytes (top panels) and lymph node cells (bottom panels) from female H-Y transgenics of the indicated E47 genotype were analyzed by staining with anti-CD4–PE and anti-CD8α–FITC. The numbers in each quadrant indicate the percentage of cells in that population. (C) Thymocytes from an E47 heterozygous and knockout littermate expressing the H-Y TCR transgene were analyzed by three-color flow cytometric analysis for expression of CD4, CD8, and either TCR-α/β or CD69. The boxed regions in the CD4 versus CD8 plots indicate the analysis gates used to define the DP population. In the histogram plots, the thin lines indicate staining on the E47 heterozygous H-Y DP cells, and the thick lines indicate staining on the E47-deficient H-Y DP cells. The third color antibody used in each case is indicated. (D) Thymocytes (top panels), splenocytes (middle panels), and lymph node cells (bottom panels) from female H-Y transgenics that are wild-type or heterozygous for E47 were analyzed by staining with antibodies to CD4 and CD8. The numbers in each quadrant indicate the percentage of cells in that population.
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Related In: Results  -  Collection

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Figure 4: Altered maturation of thymocytes in class I–restricted TCR transgenic mice expressing decreased levels of E47. Flow cytometric analysis of 4–6-wk-old E47-deficient female mice and littermate controls expressing the class I–restricted H-Y TCR transgene. (A) Thymocytes from E47 knockout and heterozygous littermate females were stained with anti-CD4–PE and anti-CD8α–FITC or with the anti-Vα3 antibody, T3.70, which recognizes the transgenic α chain. The numbers in each quadrant indicate the percentage of cells in that population, and the percentage of cells staining brightly for the transgenic α chain is indicated in the histograms. (B) Splenocytes (top panels) and lymph node cells (bottom panels) from female H-Y transgenics of the indicated E47 genotype were analyzed by staining with anti-CD4–PE and anti-CD8α–FITC. The numbers in each quadrant indicate the percentage of cells in that population. (C) Thymocytes from an E47 heterozygous and knockout littermate expressing the H-Y TCR transgene were analyzed by three-color flow cytometric analysis for expression of CD4, CD8, and either TCR-α/β or CD69. The boxed regions in the CD4 versus CD8 plots indicate the analysis gates used to define the DP population. In the histogram plots, the thin lines indicate staining on the E47 heterozygous H-Y DP cells, and the thick lines indicate staining on the E47-deficient H-Y DP cells. The third color antibody used in each case is indicated. (D) Thymocytes (top panels), splenocytes (middle panels), and lymph node cells (bottom panels) from female H-Y transgenics that are wild-type or heterozygous for E47 were analyzed by staining with antibodies to CD4 and CD8. The numbers in each quadrant indicate the percentage of cells in that population.
Mentions: To determine whether the absence of E47 also promotes increased selection of class I–restricted TCRs, we analyzed E47-deficient mice expressing the H-Y TCR transgene, which specifies reactivity with the male H-Y antigen presented by the H-2Db class I molecule 2728. Expression of the H-Y TCR transgene in females of the H-2b background leads to positive selection of CD8 SP thymocytes 2728. In contrast, thymocytes in male mice expressing the H-Y TCR are deleted at the DP stage. Negative selection mediated by the H-Y TCR transgene was unaffected by the absence of E47 (data not shown). However, E47−/−; H-Y female mice displayed an increased percentage of mature CD8 SP thymocytes compared with the E47 heterozygous littermates (56.6 vs. 33.6%; Fig. 4 A). This phenotype was accompanied by a significant decrease in the proportion of immature DP thymocytes (29.6 vs. 46%; Fig. 4 A). Because of continued rearrangement of endogenous α chain genes in TCR transgenic mice, a fraction of the thymocytes will express a TCR composed of the transgenic β chain and an endogenous α chain 252729. To analyze the proportion of cells expressing the transgenic TCR-α/β, we stained total thymocytes with the T3.70 antibody, which reacts specifically with the transgenic α chain molecule 30. Thymocytes isolated from E47−/−;H-Y mice contained almost twice the percentage of cells expressing high levels of both the transgenic α and β chains, consistent with a higher proportion of mature CD8 SP T cells (Fig. 4 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