Gene engineering of allogeneic T cells with antileukemia reactive TCR genes augments the GVL effect in vitro and vivo. TCR-transduced allogeneic or syngeneic T cells were further characterized in vitro. (A) Donor-derived allogeneic (B10.A) or syngeneic (B6) T cells were transduced with the OT-I TCR, primed on tumor lysate-pulsed recipient-type DCs (B6) for 2 days, stimulated with αCD3/αCD28-coated microspheres for one day, and further expanded for 2 days with cytokines (IL-2/IL-7). GFP⁺CD8⁺ T cells of allogeneic (■) and syngeneic (□) origin are represented in the graph. Results from one representative experiment are shown. Expansion rates of approximately 50-fold were obtained after day 7 of culture. Flow cytometric plots represent increasing rates of GFP⁺CD8⁺ expression during the generation process: after transduction (day 2), after priming on DCs (day 4), and of the final T-cell product (day 7). (B) Representative plots of allogeneic or syngeneic TCR-transduced T cells from the final T-cell product (day 7). The upper 2 plots were gated on live cells by forward side scatter exclusion and stained for CD8; the bottom plots were gated on GFP⁺CD8⁺ T cells and stained for the introduced OT-I TCR chains Vβ5/Vα2. (C) Further phenotypic features known to impact in vivo function are shown on syngeneic (top row) or allogeneic GFP⁺CD8⁺ (bottom row) T cells. (D) Gene-modified T cells were assessed for cytotoxicity at the stated effector/target ratios in a JAM assay. TCR-transduced allogeneic (■) and syngeneic (□) T cells displayed a nearly identical cytotoxicity profile against C1498-OVA (solid line). Antigen nonexpressing C1498 target cells were used as controls (dashed line) (---■---/---□---). Values are mean ± SE. One representative experiment of 4 is shown. (E) Lethally irradiated B6 recipients were reconstituted with 15 × 10⁶ TCD B10.A BM and received 1.2 × 10⁶ C1498-OVA cells intravenously on day 56 after HCT. On day 57, recipients were treated with increasing doses of either TCR-transduced B10.A T cells (left) or nontransduced controls (right). Differences in survival after treatment with TCR-transduced T cells or phosphate-buffered saline (PBS): P < .001 between 40 × 10⁶ T cells vs PBS. P < .05 between 20 × 10⁶ vs PBS. P = not significant (n.s.) between 10 × 10⁶ and 5 × 10⁶ T cells vs PBS. P values for nontransduced T cells vs PBS: P < .05 between 40 × 10⁶ vs PBS. P = not significant (n.s.) for all other groups vs PBS. n = 9 or 10 per group.