Adoptive therapy with P1A-specific transgenic T cells selects for T cell–resistant tumor cells. (a) Treatment of large P1A-expressing tumors with transgenic T cells results in rapid shrinkage of tumors, followed by stagnation and resurgence of tumor growth. J558-Neo tumor cells (5 × 10⁶) were injected subcutaneously into RAG-2^–/– BALB/c mice. Two to three weeks later, when the tumors reached 1.2–1.4 cm in diameter, purified transgenic T cells (5 × 10⁶/mouse) were injected intravenously. The treatment was repeated on day 31 when the tumors had returned to their pretreatment size, as indicated by arrows. Mice were euthanized on day 39 (n = 4). (b) Tumor cells isolated after two treatments were resistant to cytolysis by activated P1A-specific T cells. Tumor cells were isolated from two different mice and were compared with their parental tumor J558-Neo cells for their susceptibility to cytolysis in a 6-hour cytotoxicity assay. Data shown are representative two experiments. A, AvaII; F, Fnu4HI; U, uncut. (c) Expression of P1A was not lost in the P1CTL-resistant tumor cells. First-strand DNA was generated from total RNA using reverse transcriptase and amplified with primers specific for either GAPDH (700-bp product) or P1A (500-bp product). (d) Normal expression of H-2L^d on the surface of CTL-resistant tumor cells. Histograms depict binding of second-step reagent (dotted lines) or H-2L^d–specific mAb followed by the second-step reagent (solid lines). E/T, effector to target ratio.