Flow cytometric measurement of mutant T cells with altered expression of TCR: detecting somatic mutations in humans and mice.

Abstract

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-alpha or -beta 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 TCRalphabeta/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.