Malignant T cells are CD4/CD8 double-positive and aberrantly express early markers of T-cell development. (A) Flow cytometric analysis of single-cell suspensions of BM, spleen, and thymus of a representative diseased mouse demonstrates a CD4/CD8 double-positive population in all 3 tissues, with some cells becoming single-positive for CD8. The percentages of cells are indicated in each quadrant. (B) Flow cytometric analysis of single-cell suspensions of the thymus from 2 diseased mice demonstrates variable phenotypes. Plots were gated on live cells and stained for CD4 and CD8 (top panel). The gated tumor cell population was analyzed for the expression of CD25 and CD44 (bottom panel). The percentages of cells of interest are indicated. (C) T-ALL cells express RAG1, RAG2, and pre–T-α. Tumor and control cDNA was amplified using primer for RAG1, RAG2, pre–T-α, and GAPDH, electrophoresed through a 2% agarose gel and stained with ethidium bromide. M indicates molecular weight marker; 1, KM donor 1; and W, water.