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The immune system plays an important role in protection and recovery against cancer. With the discovery of tumor-associated antigens more than a decade ago, the application of immunotherapy against tumors, which is focused intensively on anti-tumor vaccination, has been challenging. Although vaccines have revolutionized public health by preventing the development of many important infectious diseases, to date, it has been much more difficult to develop effective vaccines to prevent cancer, or in the treatment of patients with existing disease. In spite of this, significant interest has been generated by recent advances in the areas of immunology and cancer biology, which have led to more sophisticated and promising vaccine strategies than those previously available. Cancer vaccines typically consist of a source of cancer-associated material (antigen), along with other components, to further stimulate the immune response against the antigen. The challenge has been to find better antigens, as well as to package the antigen in such a way as to enhance the patient's immune system to fight tumor cells expressing the appropriate antigen. Therefore, efficient screening and useful monitoring systems for analyzing antigen-specific T cells is important toward discovering novel disease-associated antigens, understanding cellular immune responses against these tumor-specific molecules, and establishing effective anti-tumor vaccination.

In this chapter, we introduce a newly established system for the analysis of antigen-specific T cells, which is based on the technology using artificial antigen presenting cell expressing human leukocyte antigen (HLA)-A*201 coupled to the enhanced green fluorescent protein (GFP) (HmyA2GFP cells) (1). This system is related to the cellular immune response between antigen presenting cells (APC) and CD8+ T cells, and thought to be an advanced method to monitor T cell directed vaccine trials in cancer patients as well as to analyze cellular immune responses against tumor-associated antigens. During the process of antigen recognition by T cells, antigen-specific T cells recognize peptides of defined length that are bound to major histocompatibility complex (MHC) molecules on APC, and after antigen recognition, the peptide-MHC clusters are acquired by CD8+ T cells and internalized through T cell receptor (TCR) (2, 3). Because this internalization is based on the specificity of peptide-MHC-TCR recognition, we have postulated that the acquisition of peptide/MHC complexes by T cells can be used for the quantitative detection of antigen-specific T cell populations, identification of antigen-specific T cell epitopes, and for analysis of T cell responses after antigen-stimulation. In the following sections, we demonstrate the methods for the generation of artificial APC and the acquisition of peptide/HLA complexes by T cell clones. We utilize this system to quantitate antigen-specific T cell populations from bulk peripheral blood mononuclear cells (PBMC) of patients. Moreover, we extend this system to define novel virus-specific CD8+ T cell immunodominant epitopes.

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