In vitro priming and expansion of donor-derived T cells result in ETCs reactive against C1498 only when they were primed with recipient-derived DCs_pulsed. Donor-derived ETCs primed with R- or D-DCs_pulsed were further characterized in vitro. (A) Expansion rates of ETCs either primed on R- DCs_pulsed (◆) or D-DCs_pulsed () were determined using the trypan blue exclusion method for viability assessment. Expansion rates of up to 10-fold were obtained within 4 days. Values are shown plus or minus SE (n = 3). Similar expansion rates were achieved after priming with R-or D-DCs_pulsed. (B) The phenotype of ETCs and naive donor splenocytes were characterized by flow cytometric analysis. In the uppermost panels, events were gated on live cells by forward-side scatter exclusion and at least 15 000 live events were acquired. In the bottom panels, data from events gated on live CD8⁺ cells stained with the indicated antibodies are depicted. (C) The ETCs obtained after priming with R-DCs_pulsed (black continuous line) or D-DCs_pulsed (gray continuous line) and donor splenocytes (black dotted line) were stained to study intracellular Granzyme B and surface CD95L expression. Data from events gated on CD8⁺ live cells of the indicated populations are depicted. (D) Left panel: ETCs primed on R-DCs_pulsed (◆) demonstrated robust anti-C1498 responses in vitro, whereas D-DCs_pulsed () failed to mediate significant cytotoxicity (P < .05). Right panel: Cytotoxicity assay performed after blocking target MHC-I (H2D^b, H2K^b) led to a complete loss of the response indicating a cytotoxic activity mediated by CD8⁺ T cells. Values are shown plus or minus SE.