Novel approaches to identify T cell-recognized tumor antigens and to redirect T cells for adoptive immunotherapy

Persistent Link:
http://hdl.handle.net/10150/280461
Title:
Novel approaches to identify T cell-recognized tumor antigens and to redirect T cells for adoptive immunotherapy
Author:
Zhang, Tong
Issue Date:
2003
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Tumor 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).
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Molecular.; Health Sciences, Immunology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Microbiology and Immunology
Degree Grantor:
University of Arizona
Advisor:
Harris, David T.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleNovel approaches to identify T cell-recognized tumor antigens and to redirect T cells for adoptive immunotherapyen_US
dc.creatorZhang, Tongen_US
dc.contributor.authorZhang, Tongen_US
dc.date.issued2003en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.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).en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Molecular.en_US
dc.subjectHealth Sciences, Immunology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorHarris, David T.en_US
dc.identifier.proquest3108972en_US
dc.identifier.bibrecord.b44846678en_US
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