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Updated: Aug 8, 2026
Isolation, Identification, and Purification of Murine Thymic Epithelial Cells
Published on: August 8, 2014
Medullary thymic epithelium expresses a ligand for CTLA4 in situ and in vitro
A J Nelson1, S Hosier, W Brady
1Department of Biological Structure, University of Washington, Seattle 98195.
Insights
The study identified B7 as the primary ligand for CTLA4 on murine thymus cells. This ligand is mainly found on medullary epithelial and dendritic cells, with expression developing during gestation.
Area of Science:
- Immunology
- Developmental Biology
- Cell Biology
Background:
- Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is a key regulator of T-cell activation.
- Understanding CTLA4 ligand distribution in the thymus is crucial for comprehending T-cell development and tolerance.
- The extracellular domain of CTLA4 fused to an Ig C gamma 1 chain (CTLA4-Ig) serves as a valuable tool for identifying CTLA4 ligands.
Purpose of the Study:
- To investigate the distribution and characteristics of CTLA4 ligands within the murine thymus.
- To determine the identity of the CTLA4 ligand expressed by thymic epithelial cells.
- To analyze the developmental regulation of CTLA4 ligand expression in the thymus.
Main Methods:
- Two-color immunofluorescence staining of murine thymus tissue using CTLA4-Ig fusion protein.
- Analysis of CTLA4-Ig binding sensitivity to tissue fixation methods.
- Developmental studies examining ligand expression from gestational day 14 onwards.
- Characterization of thymic epithelial cell lines using polymerase chain reaction (PCR) and immunoprecipitation.
Main Results:
- CTLA4-Ig binding was observed on medullary thymic epithelial cells and dendritic cells, co-expressing MHC class II and ICAM-1.
- Ligand binding was highly sensitive to the tertiary structure of the tissue, indicating a conformational epitope.
- Expression of CTLA4 ligands was developmentally regulated, increasing from gestational day 14 to term.
- PCR analysis confirmed the presence of B7 mRNA in medullary thymic epithelial cell lines.
- Immunoprecipitation identified a 65-70 kDa protein consistent with B7 as the CTLA4 ligand.
Conclusions:
- The primary ligand for CTLA4 in the murine thymus is B7, expressed by medullary epithelial cells and dendritic cells.
- CTLA4 ligand expression in the thymus is developmentally regulated and primarily localized to the medullary compartment.
- These findings provide insights into the role of CTLA4-mediated signaling in thymic T-cell selection and immune tolerance.
Abstract:
A fusion protein consisting of the extracellular domain of CTLA4 and an Ig C gamma 1 chain (CTLA4-Ig) was used to examine the distribution of the ligands for CTLA4 within the murine thymus and to characterize the nature of these ligands. Two-color immunofluorescence of thymus tissue revealed binding of the fusion protein to medullary thymic epithelial cells and dendritic cells within the corticomedullary and medullary areas of the thymus. Medullary cells binding the fusion protein also expressed MHC class II products and ICAM-1. Thymus tissue sections treated with cross-linking fixatives, such as glutaraldehyde, paraformaldehyde, or 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide no longer bound the CTLA4 fusion protein, indicating that binding was very sensitive to the tertiary structure of the tissue ligand. The ability of thymic tissue to bind the fusion protein was developmentally regulated. At day 14 of gestation, only scattered single cells were labeled. Clusters of labeled cells, which were detected by day 16 of gestation, increased in frequency with advancing gestational age. Consistent with the in situ labeling studies, CTLA4-Ig also labeled several thymic epithelial cell lines previously shown to have a medullary phenotype. Polymerase chain reaction analysis of mRNA extracted from these cells indicated they contained mRNA for B7, a known counter receptor for CTLA4 and CD28. Immunoprecipitation of 125I-labeled thymic epithelial cells with the CTLA4-Ig detected a M(r) 65,000 to 70,000 species under reducing conditions, consistent with previous studies of B7. These data suggest that the ligand for CTLA4 expressed by thymic epithelial cells in vitro is B7 and that the expression of this ligand in situ is largely restricted to the medullary compartment and is associated with epithelial cells and dendritic cells.
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