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Mol. Cells 2012; 34(5): 481-493

Published online November 30, 2012

https://doi.org/10.1007/s10059-012-0246-4

© The Korean Society for Molecular and Cellular Biology

Thymic Epithelial Requirement for γδ T Cell Development Revealed in the Cell Ablation Transgenic System with TSCOT Promoter

Gwanghee Lee1,3, Ki Yeon Kim, Cheong-Hee Chang2, and Moon Gyo Kim*

Department of Biological Sciences, Inha University, Incheon 402-701, Korea, 1Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA, 2Department of Microbiology and Immunology, The University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA, 3Present address: Department of Physiological Chemistry, Genentech, South San Francisco, CA 94080, USA

Correspondence to : *Correspondence: mgkim@inha.ac.kr

Received: September 14, 2012; Accepted: October 10, 2012

Abstract

In order to investigate the role of thymic epithelial cell (TEC) subsets during T-cell development, we established a new transgenic system, enabling inducible cell-specific ablation as well as marking the TEC subsets using bicistronic bacterial nitroreductase and EGFP genes. Two different lengths of the TSCOT promoter in transgenic mice, named 3.1T-NE and 9.1T-NE, drive EGFP expression into TECs. In adult life, EGFP expression was located in the medulla with a smaller 3.1 kb TSCOT promoter, while it was maintained in the cortex with a 9.1 kb promoter, suggesting putative TEC specific as well as compartment specific cis elements within two promoters. Nitroreductase induced cell death was specific without bystander killing upon the treatment of prodrugs such as nitrofurantoin and metronidazol. The degree of cell death was dependent on the dose of the prodrug in the cell and the fetal thymic organ cultures (FTOCs). Fetal thymic stromal populations were analyzed based on the expression levels of EpCAM, MHCII, CDR1 and/or UEA-1. EGFP expression patterns varied among subsets indicat-ing the differential TSCOT promoter activity in each TEC subset. Prodrug treatment in FTOCs reduced the numbers of total and subsets of thymocytes. A CD4+CD8+ double positive cell population was highly susceptible in both transgenic lines. Surprisingly, there was a distinct reduction in gamma delta T cell population only in the 9.1T-NE thymus, indicating that they require a NTR-EGFP expressing TEC population. Therefore, these results support a division of labor within TEC subsets for the alpha beta? and gamma delta lineage specification.

Keywords EGFP, fetal thymic organ culture, gamma delta T cell, thymic epithelial cell, transgenic mouse

Article

Research Article

Mol. Cells 2012; 34(5): 481-493

Published online November 30, 2012 https://doi.org/10.1007/s10059-012-0246-4

Copyright © The Korean Society for Molecular and Cellular Biology.

Thymic Epithelial Requirement for γδ T Cell Development Revealed in the Cell Ablation Transgenic System with TSCOT Promoter

Gwanghee Lee1,3, Ki Yeon Kim, Cheong-Hee Chang2, and Moon Gyo Kim*

Department of Biological Sciences, Inha University, Incheon 402-701, Korea, 1Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA, 2Department of Microbiology and Immunology, The University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA, 3Present address: Department of Physiological Chemistry, Genentech, South San Francisco, CA 94080, USA

Correspondence to:*Correspondence: mgkim@inha.ac.kr

Received: September 14, 2012; Accepted: October 10, 2012

Abstract

In order to investigate the role of thymic epithelial cell (TEC) subsets during T-cell development, we established a new transgenic system, enabling inducible cell-specific ablation as well as marking the TEC subsets using bicistronic bacterial nitroreductase and EGFP genes. Two different lengths of the TSCOT promoter in transgenic mice, named 3.1T-NE and 9.1T-NE, drive EGFP expression into TECs. In adult life, EGFP expression was located in the medulla with a smaller 3.1 kb TSCOT promoter, while it was maintained in the cortex with a 9.1 kb promoter, suggesting putative TEC specific as well as compartment specific cis elements within two promoters. Nitroreductase induced cell death was specific without bystander killing upon the treatment of prodrugs such as nitrofurantoin and metronidazol. The degree of cell death was dependent on the dose of the prodrug in the cell and the fetal thymic organ cultures (FTOCs). Fetal thymic stromal populations were analyzed based on the expression levels of EpCAM, MHCII, CDR1 and/or UEA-1. EGFP expression patterns varied among subsets indicat-ing the differential TSCOT promoter activity in each TEC subset. Prodrug treatment in FTOCs reduced the numbers of total and subsets of thymocytes. A CD4+CD8+ double positive cell population was highly susceptible in both transgenic lines. Surprisingly, there was a distinct reduction in gamma delta T cell population only in the 9.1T-NE thymus, indicating that they require a NTR-EGFP expressing TEC population. Therefore, these results support a division of labor within TEC subsets for the alpha beta? and gamma delta lineage specification.

Keywords: EGFP, fetal thymic organ culture, gamma delta T cell, thymic epithelial cell, transgenic mouse

Mol. Cells
Aug 31, 2022 Vol.45 No.8, pp. 513~602
COVER PICTURE
Cryo-EM structure of human porphyrin transporter ABCB6 (main figure) shows that binding of hemin (inset, magenta) in concert with two glutathione molecules (cyan) primes ABCB6 for high ATP turnover (Kim et al., pp. 575-587).

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