The HER2/neu oncoprotein and dendritic cells in immunity against tumors

Sammanfattning: It is now well established that small peptide epitopes bound to the major histocompatibility complex (MHC), derived from processed proteins synthesized by the tumor cells, can be recognized as antigens by cytotoxic T lymphocytes (CTLs). A number of tumor antigens recognized by CTLs on human melanomas have been defined, but less is known about antigens from other tumor types. Carcinoma specific tumor antigens, including the HER2/neu oncoprotein which is over-expressed in a high proportion of carcinomas, were recently described as potential targets for T cellbased therapy. Dendritic cells (DCs) have been identified as the most effective antigen-presenting cells. DCs possess an exceptional capability to capture antigens (Ags), process and present antigenic peptide fragments, and induce immune responses in T cells. I have here established a method to produce peptide specific CTLs by the use of peptide-pulsed autologous DCs from the ascites of patients with ovarian carcinomas, and used this method to define new HLA A2.1 restricted CTL epitopes from the HER2/neu protein. Through the use of a computer algorithm we searched the HER2/neu protein for potential HLA A2.1 binding CTL epitopes, and those with high "scores" were synthesized and tested for binding to HLA A2.1. Four novel epitopes were identified which were able to elicit CTLs that specifically killed peptidesensitized target cells and, most importantly, a HER2/neu transfected cell line, autologous tumor cells and HLA-A2+, HER2/neu+ allogeneic renal and colon carcinoma cell lines. I also confirm that HER2/neu is over-expressed in several melanoma lines, and as a new finding, demonstrate that some of these lines are sensitive to HER2/neu specific CTLs. Two of the identified HER2/neu epitopes were also found to be recognized by HLA-A2. 1 -restricted, gastric cancer-specific CTL lines produced by repetitive stimulation of tumor-associated lymphocytes (TALs) with autologous tumor cells in vitro. Our findings implicate and broaden the potential application of HER2/neu based immunotherapy and vaccine strategies against different kinds of tumors. DCs-cocultured with autologous ovarian tumor cells were effective stimulators of tumor-specific CTLs, and I analyzed the mechanism by which these DCs acquired tumor antigens. DCs demonstrated significant cytotoxicity towards autologous or allogeneic ovarian tumor cells, which was mediated by a mechanism which was independent of Ca2+ and which at least partly involved the Fas/FasL pathway. The cytotoxicity of DCs against ovarian tumor cells resulted in apoptosis of these tumor cells and eventually in engulfment of the apoptotic tumor cells by the DCs. These results demonstrate that DCs can kill autologous ovarian tumor cells via the Fas/FasL pathway and that this may have important consequences for their ability to stimulate tumor specific CTLs. I also showed that the cytokine IFN-[gamma] could negatively influence the differentiation and function of monocyte-derived DCs by affecting their expression of the surface molecules CD80, CD1a and CD4, which are involved in antigen-presentation. This indicates that a negative feed-back regulation exists between T cells and DCs, where T cells stimulated by DCs will produce IFN-[gamma], which will in turn shut of DC function.