Flow Cytometric Measurement of Mutant T Cells

Spontaneously generated mutant T cells defective in T-cell receptor (TCR ) gene expression are detectable at the frequency of 10⁻⁴ in vivo, and the mutant fractions are dose-dependently increased by exposure to genotoxic substances such as ionizing radiation. Mutant cells with altered expression of TCR-α or -β among CD4⁺ T cells can be detected as CD3⁻ /CD4⁺ cells by two-color flow cytometry using anti-CD3 and anti-CD4 monoclonal antibodies labeled with different fluorescent dyes, because an incomplete TCRαβ/CD3 complex cannot be transported to the cellular membrane. This flow cytometric mutation assay can be applied to CD4⁺ T cells from human peripheral blood and mouse spleen. Methods for both preparation of target cells and detection of the mutant cells are described.