(A-B) Flow cytometric measurement of mispairing of (A) human F5 TCR chains in human peripheral blood T cells and (B) murine OTI TCR chains in murine splenic T cells. Cells were transduced with only TCRα (left panel) or only TCRβ (right panel) such that expression of the introduced chain on the cell surface required mispairing with endogenous TCR chains. Representative histograms are displayed above bar graphs quantifying means ± SD for 3 technical replicates. (C-D) Mouse model of TCR gene transfer-induced graft-vs-host disease (TI-GvHD). Mice were irradiated on day -1, administered TCR-transduced T cells on day 0, and injected twice daily with IL-2 from day 10–12. Weight was monitored daily and mice were euthanized at 85% initial body weight. Results are aggregated from 3 independent experiments. Respective group sizes, n = 9, 15, 15, and 25. *p<0.05, ****p<0.0001, ns, not significant. (C) Percent initial body weight of surviving mice at 16 days after T cell administration. (D) Kaplan-Meier survival curve. (E) Tumor size in mice administered TCR-transduced T cells and then injected with ovalbumin-expressing E.G7 thymoma tumor cells. Respective group sizes, n = 9, 6, 10, and 10. Mean ± SD for each group is overlaid on traces for individual animals. (F) Tumor size in mice (n = 10) administered OTI dsTCR-transduced T cells and then injected on opposing flanks with EL4 (control) or E.G7 (target) thymoma tumor cells.