Signaling via the survivin-reactive TCR contributes to leukemogenesis. (A) Cell images taken from 4 separate cells designated as “high similarity scores” vs “low similarity scores” are shown. Cells with high similarity scores were from Sur-TCR-Tg mice and cell with low similarity scores were from WT controls. (B) Thymocytes from 6-week Sur-TCR-Tg (black) and WT (gray) mice were harvested and stained immediately using cell-surface antibodies, then fixed/permeabilized and stained with intracellular NFAT antibody. Lin⁻ cells were gated and the frequency of DN thymocytes from Sur-TCR-Tg⁺ (black) and WT (gray) exhibiting nuclear translocation of NFAT is shown using the NFAT/DAPI similarity score histogram overlay. Representative result from 1 mouse is shown for each condition. In this analysis, a larger score indicates a greater degree of signal correlation between NFAT channel and nuclear channel and thus, translocation. Translocated cells are defined as those with a similarity score above 1. Similar results were seen in >2 separate experiments containing at least 3 animals per group. (C) Thymocytes with NFAT nuclear translocation (similarity score > 1.0) by ImageStream^X analysis in thymocyte subsets of Sur-TCR-Tg mice, compared with WT mice (top). Among the DN subsets, Sur-TCR-Tg showed significantly more NFAT nuclear translocated cells in DN2 and DN4 subset compared with WT mice (bottom). Similar results were obtained in 2 separate experiments with at least 3 animals per group. (D) Sur-TCR-Tg mice were bred with β2M^−/− mice to generate Sur-TCR-Tg⁺ β2M^−/− mice, then animals were monitored for the development of T-ALL for 20 months. Kaplan-Meier analysis reveals significantly diminished death due to T-ALL in Sur-TCR-Tg⁺ β2M^−/− mice compared with those Sur-TCR-Tg⁺β2M^+/− mice (P = .006).