Out of all malignancies, breast cancer is the second most common cancer worldwide and the leading cause of cancer related death in females. In recent years it has been shown that the anti-tumor vaccination might be a feasible approach for the treatment of certain cancer types, including breast cancer. It is of great interest to develop immunotherapies which not only prevent further dissemination by the tumor cells, but also eliminate tumor tissue and impair the function of immune-suppressive cells, such as regulatory T cells (Tregs), in the tumor microenvironment. Not only antigen-specific cytotoxic CD8+ T cells (CTLs) but also CD4+ T cells show a great capacity to facilitate a specific anti-tumor immune response. Furthermore, successful immunological eradication of tumors depends on the presence of activated tumor antigen-specific CD4+ effector T cells as documented by numerous reports. The differentiation antigen NY-BR-1 has been described to be expressed in 60% of all invasive mammary carcinomas. Since NY-BR-1 protein levels are highly elevated in malignant breast tissues compared to healthy breast tissues, NY-BR-1 might represent a suitable target antigen for T cell based immunotherapy approaches against breast cancer. The aim of this project was to identify novel MHC-I- and MHC-II-restricted T cell epitopes derived from the breast cancer associated antigen NY-BR-1. A NY-BR-1-specific peptide library was utilized to screen for the presence of MHC-I and MHC-II- restricted T cells in HLA-DRB1*0301- transgenic mice (DR3tg mice) and HLA-DRB1*0401-transgenic mice (DR4tg mice), after global NY-BR-1-DNA vaccination. Splenocytes of immunized mice were screened ex vivo for a NY-BR- 1-specific T cell response against a synthetic peptide library covering the entire NY-BR-1 protein. So far, novel NY-BR-1-specific, HLA-A2-restricted CD8+ T cell epitopes could not be identified. However, the first NY-BR-1-derived, HLA-DRB1*0301-restricted peptides (BR1-1347, BR1-88, BR1-1238) and the first NY-BR-1 derived, HLA-DRB1*0401-restricted peptides (BR1-537, BR1- 1242, BR1-656/-775) were identified in DR3tg mice and DR4tg mice, respectively. Stable murine CD4+ T cell lines specific for five new epitopes could be established from peptide-immunized DR3tg mice / DR4tg mice, and HLA-DR-restriction of the cell lines was confirmed on peptide loaded T2/DR3 and T2/DR4 target cells in vitro. Furhtermore, endogenous processing of HLA-DRB1*0301-restricted NY-BR-1-derived epitopes BR1-88, BR1-1347 and of the HLA-DRB1*0401-restricted NY-BR-1-derived epitopes BR1-537, BR1-1242 could be confirmed by specific recognition of human dendritic cells loaded with cell lysates of melanoma cell line Ma-Mel73a infected with Ad5-NY-BR-1. CD4+ T cells specific for the NY-BR-1 derived, HLADRB1* 0301-restricted peptides BR1-88, BR1-1347, BR1-1238 and for the HLA-DRB1*0401- restricted peptides BR1-537, BR1-1242, BR1-656/-775 were detected among PBMCs of breast cancer patients stimulated with the respective peptide in vitro for 24 days. Furthermore, CD4+ T cells with the same specificities were also detected among PBMCs of HLA-matched healthy donors, however, frequencies of antigen-specific CD4+ T cells were higher in the peripheral blood of breast cancer patients compared to healthy donors. The findings of this thesis, such as the identified NY-BR-1-specific, HLA-DRB1*0301-/*0401- restricted CD4+ T cell epitopes, might be used to generate NY-BR-1-specific tetramers which could be applied to monitor immune-responses in breast cancer patients with a tumor expressing the NY-BR-1 antigen. Moreover, the new NY-BR-1-specific, CD4+ T cell epitopes could be applied to expand NY-BR-1-specific autologous CD4+ T cells for an adoptive T cell transfer. Cloning of high affinity TCRs, specific for the newly identified epitopes, to generate TCR-transduced CD4+ T cells for adoptive T cell transfer might be another application for the findings obtained in this work. NY-BR-1-specific therapeutic vaccines could be designed by combination of CTL and CD4+ T cell epitopes to induce NY-BR-1-specific CD8+ T cells as well as NY-BR-1-specific CD4+ T cells. Infect, CD4+ T cells are not only important to sustain a functional CD8+ T cell response, but might also target MHC-II expressing tumor associated macrophages (TAMs) presenting NYBR- 1-specific epitopes on MHC-II, thereby contributing to anti-tumor immunity.
|Supervisor:||Eichmüller, Prof. Dr. Stefan|
|Date of thesis defense:||18 February 2014|
|Date Deposited:||28 Mar 2014 12:53|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||500 Natural sciences and mathematics|
|Controlled Keywords:||Immunotherapy, T cell epitope, HLA-transgenic mice, breast cancer, tumor antigen|