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Examination of Thymic Positive and Negative Selection by Flow Cytometry
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Examination of Thymic Positive and Negative Selection by Flow Cytometry

Published on: October 8, 2012

Claudin-4 induction by E-protein activity in later stages of CD4/8 double-positive thymocytes to increase positive

Yohei Kawai1, Yoko Hamazaki, Harumi Fujita

  • 1Department of Immunology and Cell Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.

Proceedings of the National Academy of Sciences of the United States of America
|February 18, 2011
PubMed

Insights

Claudin-4 (Cld4) unexpectedly functions in T-cell development within the thymus, independent of its usual barrier role. This protein enhances T-cell receptor signaling and aids in the selection of developing T cells.

Area of Science:

  • Immunology
  • Cell Biology
  • Molecular Biology

Background:

  • Claudins (Clds) are integral membrane proteins forming tight junctions in epithelial cells, crucial for barrier function.
  • Claudin-4 (Cldn4) expression is typically associated with epithelial tissues and tight junctions.

Purpose of the Study:

  • To investigate the unexpected expression and function of Claudin-4 (Cld4) in thymic lymphocytes.
  • To elucidate the role of Cld4 in T-cell development and T-cell receptor (TCR) signaling.

Main Methods:

  • Analysis of Cld4 expression in thymocytes from various genetically modified mouse models (MHC-I(-/-) II(-/-), Rorγ(-/-), E47-deficient).
  • Chromatin immunoprecipitation (ChIP) to identify transcription factor binding to the Cldn4 promoter.
  • Functional assays including T-cell receptor costimulation, cell surface staining, and fetal thymic organ culture with Cldn4 knockdown.

Main Results:

  • Cld4 is unexpectedly expressed in a subset of CD4/CD8 double-positive (DP) thymocytes, independent of tight junctions.
  • Cld4 expression is regulated by E-proteins (E2A, HEB) and influenced by specific genetic backgrounds.
  • Cld4 exhibits potent TCR costimulatory activity, is recruited to the immunological synapse, and its knockdown impairs the generation of single-positive T cells.

Conclusions:

  • Claudin-4 plays a novel role in T-cell development, specifically enhancing T-cell receptor signaling and positive selection in DP thymocytes.
  • E-protein activity induces Cld4 expression in later DP stages, contributing to efficient T-cell maturation.
  • This study uncovers a previously unrecognized function for a Claudin family protein in the immune system.

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