Novel approaches to identify T cell-recognized tumor antigens and to redirect T cells for adoptive immunotherapy
AdvisorHarris, David T.
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PublisherThe University of Arizona.
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AbstractTumor antigens (Ags) and adoptive immunotherapy are two important topics in tumor immunology. Traditional methods for identifying T cell-recognized tumor antigens and adoptive therapy using antigen-specific T cells are laborious and difficult. Rapid developments in molecular biology and immunology have allowed us to design novel strategies to achieve these two goals more efficiently. A novel strategy, SING (S̲I̲gnal transduction molecule-mediated, N̲FAT-controlled, G̲FP expression), for cloning T-cell recognized tumor Ags, was designed using Ag-specific T cells. The SING system is an artificial Ag presentation system, in which a mouse T cell line BW5147 has been manipulated to respond to stimulation by Ag-specific TCR (the resultant BW5147 cells are named BS cells). Either Ag peptide-pulsed or Ag-expressing BS cells could become transiently fluorescent (GFP⁺) and puromycin resistant after TCR engagement. In combination with retrovirally mediated functional genomics, the SING strategy should allow us to isolate antigen-expressing (GFP⁺) cells directly and retrieve sequences coding for tumor antigens by PCR amplification of genomic DNA from GFP⁺ BS cells. To investigate whether three-domain single chain T cell receptors (3D-scTCR) are able to redirect T cells to recognize tumor cells, multiple scTCR constructs were constructed and retrovirally transduced into T cells. The effects of CD8, CD28 and the complete CD3 complex on scTCR-induced T cell activation were also determined. Compared with full-length TCR (flTCR)-modified T cells and native CTLs, seTCR-modified T cells had high thresholds for response to Ag stimulation. After adoptive transfer of TCR (either scTCR or flTCR)-modified T cells into tumor-bearing mice, the in vivo tumor growth was controlled to some extent, although most TCR-modified T cell recipient mice didn't show significant signs of anti-tumor effects. This result suggests the possible application of scTCR- as well as fITCR-modified T cells for adoptive immunotherapy. Finally, to accomplish the above goals, we systematically investigated the optimal conditions for transduction of murine primary T cells as well as T cell lines. Our results showed that successful infection of murine primary T cells required a combination of high titer (>10⁷ CFU/ml) of ecotropic retroviral vectors and proper timing of infection (within 24 hours after mitogen stimulation).
Degree ProgramGraduate College
Microbiology and Immunology