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Impaired T-cell function has been noted in tumor-infiltrating lymphocytes (TIL). Recently, loss of function was found to be associated with modifications in T-cell receptor complex (TCR)-mediated signaling. A common feature is loss or reduced expression levels of the signaling chain, TCRzeta. We evaluated whether loss of function in TIL and tumor-associated lymphocytes (TAL) from patients with ovarian cancer is associated with changes in TCRzeta expression, and which factors can cause these defects.TIL and TAL were isolated from multiple patients and evaluated for their proliferative capacity by stimulation with a polyclonal stimulus. In addition, expression of TCRzeta and CD3epsilon was evaluated in fresh TIL and TAL by the Western blot technique. Finally, various conditions within a tumor environment were tested for their effect on TCRzeta and CD3epsilon.TIL, but not TAL, were significantly impaired in their proliferative response, even when both populations were derived from the same patient (P.05). Reduced proliferation levels were associated with loss of expression of TCRzeta but not of CD3epsilon. Exposure of normal T cells to relative ischemia or heat shock, or culture in medium without IL-2, did not significantly reduce expression of TCRzeta compared with CD3epsilon. However, coculture of T cells with tumor-derived macrophages or tumor-derived factors led to a selective loss of TCRzeta compared with CD3epsilon (P.05). Further analysis suggested that oxides such as hydrogen peroxide secreted by macrophages may be responsible for loss of TCRzeta and high molecular weight factors secreted by certain tumors.TIL but not TAL show impaired T-cell function, which is associated with loss of TCRzeta. In addition to macrophages secreting oxides, loss of TCRzeta may be caused by tumor-derived soluble factors.

The protooncogene HER2/neu encodes a 185-kDa transmembrane protein with extensive homology to the epidermal growth factor receptor. It is overexpressed in several human cancers of epithelial origin, such as pancreatic cancer. Previously, we demonstrated that cytotoxic T lymphocytes (CTL) derived from breast, ovarian, and non-small cell lung cancer recognized a peptide derived from HER2/neu. To evaluate whether this HLA-A2-binding peptide is a tumor-associated antigen (TAA) in pancreatic cancer, the ability of HER2/neu-reactive CTL to lyse human pancreatic carcinoma cells was tested. CTL were generated from tumor-associated T lymphocytes from HLA-A2+ HER2/neu+ breast and ovarian cancer patients. All CTL recognized autologous and allogeneic HER2/ neu+ tumor cells in an HLA-A2-restricted fashion. Furthermore, all CTL recognized p654-662 (GP2) derived from HER2/neu. These CTL also recognized HER2/neu+ pancreatic cancer cells in an HLA-A2-restricted fashion. HER2/neu+ HLA-A2- pancreatic cancer were not or only poorly lysed. Repeated stimulation of HLA-A2+ PBL from pancreatic cancer patients using the HER2/neu-derived peptide resulted in specific recognition of this peptide and, more importantly, HER2/neu+ pancreatic tumors in an HLA-A2-restricted fashion. Autologous HLA-A2+ fibroblasts or HLA-A2+ malignant melanoma cells were not recognized. HLA-A2- peptide-stimulated T lymphocytes showed no significant cytotoxicity. These results demonstrate that this HER2/neu-derived peptide is a shared TAA among several adenocarcinomas including pancreatic carcinoma, suggesting a common mechanism of recognition of these human tumors by T lymphocytes. The identification of the HER2/neu-derived peptide GP2 as a TAA in pancreatic cancer provides an opportunity for the design of novel immunotherapy and vaccine strategies.

Cytotoxic T-cell (CTL) cultures were generated from five ovarian cancer patients (OvCTL) and from three breast cancer patients (BrCTL). All CTL lines were T-cell receptor (TcR) alphabeta+ and predominantly CD8+ (73 +/- 13%). These CTL lines preferentially recognized autologous tumor cells in an HLA class I-restricted, and in part HLA-A2-restricted, manner. In addition, the CTL lines recognized allogeneic HLA-A2+ ovarian and breast tumor cells. Specific recognition was determined by T-cell-mediated cytotoxicity as well as cytokine release. Coculture of irradiated autologous tumor cells with OvCTL induced secretion of IFN-gamma, GM-CSF and TNF-alpha, but not IL-4, indicating a T helper-1-type response. Similar results were obtained when OvCTL and BrCTL were stimulated with histologically matched HLA-A2+ tumor cells. Also, BrCTL stimulated with HLA-A2+ but not HLA-A2- ovarian tumor cells produced significant levels of GM-CSF and TNF-alpha. Finally, the Her2/neu peptide p654-662, earlier identified as a tumor antigen in both ovarian and breast cancer, induced cytotoxicity as well as the specific release of IFN-gamma and TNF-alpha but not IL-4 by OvCTL and BrCTL. Thus, tumor-specific recognition by CTL was verified by cytotoxicity and cytokine release. The secretion of Th1-like cytokines as opposed to Th2-like cytokines suggest that therapeutically OvCTL and BrCTL could potentially enhance the endogenous immune response to tumor.