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Generation of a transplantable murine tumor model expressing the human breast cancer associated tumor antigen NY-BR-1 in HLA-DRB1*0401-transgenic mice

Das, Krishna

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Abstract

Prevalence of mutations and deregulated gene expression has resulted in a wide variety of tumor antigens which can be targeted for immunotherapy. NY-BR-1 is a breast cancer associated differentiation antigen which is overexpressed in more than 60% of breast cancers. In this study we established the first NY-BR-1 expressing transplantable tumor model using the murine mammary adenocarcinoma cell line EO771 as parental cells for the generation of NY-BR-1-expressing transfectants (EONY cells) which are compatible for transplantation into HLA-DRB1*0401tg mice. Since NY-BR-1 is not endogenously expressed in these mice, transplantation of NY-BR-1 expressing tumors led to the induction of CD4+ and CD8+ T cells specific for HLA-DR*0401- and H2-Db-restcried NY-BR-1 epitopes, respectively, previously identified in our lab. We also generated MHC I knockout cell lines with the aim of using them to study NK cell responses or as parental lines for transection of human MHC I molecules. Since several HLA-restricted NY-BR-1-specific T cell epitopes have been described, these cells could serve as an important tool to study the efficacy of using the epitopes as peptide vaccines and studying the synergistic effect of induced CD8+ and CD4+ T cells in HLA-transgenic mice co-expressing human MHC I and MHC II molecules. Additionally, we also confirmed that previously identified H2-Db-restrcited epitope was indeed naturally processed based on detection of CD8+ T cells which specifically recognized this epitope in mice following transplantation of NY-BR-1 expressing tumors or upon immunization with global NY-BR-1 antigen. We also observed that immunization with Ad.NY-BR-1 was more suitable for generating a NY-BR-1 specific CD8+ T cell line compared to immunization with nonameric peptide. We also obtained evidence suggesting that the H2-Db-restricted CTL epitope might actually be identical to the core sequence of one of the previously identified HLA-DRB1*0301-restricted CD4+ T cell epitopes. Thus binding of the CTL epitope to HLA-DRB1*0301 molecules on feeder cells might have resulted in predominant restimulation of HLA-DR3B1*0401-restricted CD4+ T cells in vitro. Cancer immunotherapy involving T cells have focused primarily on cytotoxic CD8+ T cells. However, a body of evidence has emerged in the recent past that argues for including CD4+ T cells because they not only eliminate tumor cells directly but also greatly enhance the outcome of cancer immunotherapy by various ways. One of the mechanisms by which CD4+ T cells can contribute to successful immunotherapy is by repolarizing tumor associated macrophages (TAMs) into a less immunosuppressive phenotype in an antigen dependent fashion. In this study we could demonstrate that NY-BR-1 specific CD4+ T cells could repolarize M2-like macrophages towards an M1-like phenotype upon antigen specific interaction. However, the majority of the CD4+ T cells infiltrating the EONY tumors appeared to have tumor promoting functions as depletion of CD4+ T cells resulted in delayed tumor growth and was accompanied by switch in the polarization status of TAMs towards an M-1like phenotype. Though Ad.NY-BR-1 immunization resulted in induction of NY-BR-1 specific CD4+ T cells and slight changes in polarization of intra-tumoral macrophages; it did not seem to drastically change the proportion of tumor-promoting CD4+ T cells in the tumor since the protective effect and switch in TAM phenotype was observed in both control virus and Ad.NY-BR-1 immunized mice. Therefore, antigen-specific instruction of TAMs by NY-BR-1-specific CD4+ T cells could not be demonstrated in vivo, possibly due to the prevalence of tumor infiltrating CD4+ T cells with a tumor promoting phenotype occurring in the EO771/NY-BR-1 model established within this thesis.

Item Type: Dissertation
Supervisor: Eichmüller, Prof. Dr. Stefan
Date of thesis defense: 28 July 2017
Date Deposited: 02 Aug 2017 09:29
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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