Abstract The Wilms tumor protein, WT-1 is expressed in over 60% of serous adenocarcinomas of the ovary. Its expression has been hypothesized to be critical for the growth or survival of tumorigenic stem cells. In this study, we have assessed the capacity of in vitro generated T-cells specific for a series of immunogenic WT1 peptide epitopes presented by different HLA class I alleles to prevent the outgrowth of two human ovarian adenocarcinoma cell lines expressing either low (SKOV3-A2) or high (OVCAR3) levels of WT1 by FACS in NOD/SCID mice. For this study, epitope-specific HLA restricted WT1-CTLs were generated from PBMC of 4 normal donors by in vitro sensitization with autologous EBV BLCL loaded with a pool of 141 15-mers overlapping by 11aa and spanning the entire sequence of the WT1 protein. WT1-CTL restricted by HLA alleles expressed on the OVCAR3 and SKOV-3 lines were pre-incubated in vitro for 8 hours at different E:T ratios (0:1, 5:1, 10:1, 50:1, 100:1) with 0.05×10⁁6 ovarian carcinoma cells transduced to express a luciferase reporter gene. The cell mixtures were injected i.p. into NOD/SCID mice. Tumor growth was monitored weekly by intensity of their bioluminescent signal. In all animals injected with the tumor cells alone the bioluminescent signal could be detected in the abdomen by day 10-15 and increased steadily through 60 days of observation (by which time, all mice died). The tumor engraftment was either markedly inhibited (SKOV3-A2WT1low) or completely abrogated (in OVCAR-3WT1high) by pre-incubation at 100:1 E:T ratio and was correlated with higher survival of the animals (80%) over a period of 120 days. In the animals injected with tumor cells pre-incubated with the WT1-CTLs at a 50:1 and 10:1 E:T ratios, tumor growth was suppressed as reflected by weaker bioluminescent signal in the abdomen which increased much later in the course of the study. Pre-incubation of the tumor cells with WT1-CTL at a 5:1 E:T ratio did not significantly affect tumor engraftment and growth or survival of the mice. WT1-CTL specific for the 398-406LKTHTTRTHT epitope presented by the A0201 allele induced significantly greater suppression of tumor growth than T-cells specific for the (−125)-(−117)RQRPHPGAL peptide that can be presented by either HLA-A0301 or B0702. T cells specific for different WT1 epitopes administered i.v. at a dose of 2.5×10⁁6 cells/animal inhibited the growth of the pre-established OVCAR3 tumor xenografts inoculated i.p. as compared to the growth of the same tumors in control animals. Mice treated with WT-1 specific T-cells also had a significantly extended survival. Our results provide evidence that tumorigenic ovarian carcinoma cells expressing WT1+ are susceptible to eradication in vivo by high doses of WT1-CTL. However, the effectiveness of these T cells may differ depending on the level of WT1 expressed by the tumor cells and peptide epitope of WT1 targeted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5515. doi:10.1158/1538-7445.AM2011-5515