Defense Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Gordon Ginder

Abstract

The human Class Ib gene, HLA-E inhibits both Natural Killer Cells and a subset of CD8+ cytotoxic T lymphocytes by engaging the CD94/NKG2A inhibitory receptor. IFN-γ induces the expression of HLA-E as well as Class Ia molecules, which are required for the killing of target cells. Since HLA-E has negative effects on immune killing of target cells, we have sought to identify locus specific mechanisms of IFN-γ induction in order to identify molecular targets for selective activation of Class Ia genes, but not HLA-E. We have previously identified a unique upstream IFN-γ response region in the HLA-E promoter and showed that GATA-1 is required for its function in the K562 leukemic cell line. We have now examined the effect of GATA family members on IFN-γ induction of HLA-E in other cell types. HLA-E CAT reporter gene assays demonstrate that tumor cells that express GATA factors as determined by western blot and quantitative PCR, mediate a 2.4 to 4.0 fold enhanced response to IFN-γ stimulation. Functional constructs containing mutations of the core nucleotides in the GATA binding site had a 4.8 fold decreased response to IFN-γ in A2780 cells and a 8.5 to 14.0 fold decreased response to IFN-γ in SKOV3 cells. Knockdown of GATA-6 using siRNA resulted in a 40% decrease in HLA-E induction in Seg1 cells and a 30% decrease in HLA-E induction in HCT116 cells. Tetracycline regulated shRNA knockdown of GATA-6 expression in the SKOV3 cell line revealed a 3 fold decrease in the IFN-γ response of HLA-E reporter driven constructs. Additionally we observed a decreased IFN-γ response in SKOV3 cells transfected with siRNA specific for CBP and IRF-9. We conclude that GATA factors play a tissue specific role in regulation of IFN-γ mediated HLA-E expression and that IRF-9 may be a target for the differential manipulation of classical MHC and HLA-E.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2010

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