Isolation, expansion, cloning and characterization of specific anti-tumor cytolytic T-cell lymphocytes (CTL) derived from a patient with ewing's sarcoma (ES)

The optimal treatment for Ewings sarcoma remains suboptimal, especially for patients with advanced disease. Novel immune mediated therapies, such as the use of anti-tumor CTL, may provide useful therapeutic approaches to be explored. For CTL to be effective, several conditions are thought to be necessary: 1 ) a tumor associated or specific antigen must be presented in the context of HLA molecules expressed on the tumor, 2) cellular adhesion molecules (ICAM 1) must be present on the tumor cell surface, and 3) costimulatory signals must be delivered to T-Cells. To evaluate whether anti-ES tumor specific lymphocyte clones could be generated from patients with ES, a patient with ES of the chest wall had tumor cells placed in culture. The resultant tumor cell line expressed MIC2 and a 177bp EWS-FLI transcript detected by RT-PCR, both characteristic of ES tumors. This cell line also expressed ICAM 1 and HLA Class 1. To evaluate the contribution of CD80 co-stimulatory molecules, 3 subclones of the parent line were created using retrovirally mediated transfection with constructs containing cDNA for CD80 (sense or anti-sense) or vector only. Four months after termination of chemotherapy, autologous peripheral blood mononuclear cells (PBMC) were stimulated in culture with one of the following irradiated, autologous tumor cell clones: native, CD80 +, anti-sense transfectants, or vector only transfectants. PBMC were added at a 10:1 ratio and cultured in media containing 20 U/ml IL-2. After 3 weeks, cultures were tested for specific CTL activity using standard Cr51 release assay. Targets consisted of the same clones which served as stimulators. At a 50:1 E:T ratio, 40-50% specific lysis was found for all targets, except for CD80 expressing targets which demonstrated specific lysis of 15%. An autologous lymphoblastoid cell line (LCL) was not killed by any of the PBMC cultured. The NKtarget, K562, and an allogeneic LCL were not killed. PBMC clones were tested for CTL activity. One of 3 CTL clones, 1C4, had specific anti-tumor cytolytic activity and was shown by flow cytometric analysis to be CD3+, CD4+, CDS-, CD56-. Pre-incubating native tumor with anti-Class I HLA inhibitory antibody resulted in a 52% decrease in CTL activity. These data demonstrate the possibility of generating anti-ES CTLs. Further exploitation of immune-mediated therapy for patients with ES based on such experiments can be envisioned.