T-ALL in Sur-TCR-Tg mice is composed of transformed thymocytes that express and signal via the transgenic TCR. (A) Flow cytometry of splenocytes harvested from representative WT, Sur-TCR-Tg preleukemic mice (∼6 weeks of age), and Sur-TCR-Tg T-ALL mice. T-ALL cells consistently express CD3, CD24, and TRBV8, usually express CD25 and typically do not express CD4 or CD8. Similar results were observed in at least 3 separate experiments involving >6 animals of each type. (B) Sur-TCR-Tg T-ALL signal in response to the Sur_20-28 peptide. T-ALL cell lines derived from a representative Sur-TCR-Tg leukemic mouse were transduced with NFAT-eGFP, then exposed to the irrelevant control E7 peptide, Sur_20-28, or PMA/Ca ionophore. Nuclear translocation of NFAT-eGFP as identified via microscopy is shown in a representative field (immunofluoresence images) and data are summarized from 10 fields for each condition. Four independent experiments using 2 separate T-ALL cell lines were tested with similar results. (C) TRAV gene expression in representative WT and Sur-TCR-Tg preleukemic mice reveal a diverse repertoire. T-ALL harvested from spleen of a representative T-ALL mouse expresses an oligoclonal repertoire of TRAV genes, whereas a cell line derived from leukemic splenocytes is clonal, as evidenced by a single native TRAV gene (TRAV12 arrow). Preleukemic spleen, leukemic spleen, and the T-ALL cell lines (but not WT) express the transgenic TRAV8, designated with a star. This experiment was repeated at least 3 times using 3 different leukemic and WT animals as well as 3 separate leukemic lines with similar results.