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Drug Selection of γδ T cells using miRNA target sequences

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Abstract

Gamma delta T cells constitute a small subset of circulating T lymphocytes in peripheral blood, and recognize antigens in an MHC-independent manner through their γδ T cell receptor. We sought to selectively expand ablatable γδ T cells using an inducible suicide switch, drug and miRNA selection strategies. Drug resistant plasmid encoding an inducible caspase 9 (iC9) suicide switch and a double mutant human dihydrofolate reductase (DHFR) gene was generated by ligating FLAG-tagged double mutant DHFR, T2A, and Myc-tagged iC9 (dDiC9) sequences. In order to selectively expand γδ T cells, 4 copies of miRNA10a target sequences were inserted in the 3’ UTR of the dDiC9 construct and the plasmids dDiC9-miRT10a3’ and dDiC9-miRT10a5’ were generated to hybridize with the miRNA10a in the 3’ and 5’ direction, respectively. PBMC cells were electroporated with dDiC9 constructs using the Sleeping Beauty transposase system. Electroporated cells were expanded on K562 Clone 4 artificial antigen presenting cells with IL-2, and under the selection of methotrexate (MTX) for 14 days. Analysis by flow cytometry determined that selective expansion of γδ T cells of >3-fold was achieved with the dDiC9-miRT10a5’ construct. However, drug selection conditions allowed for competition between γδ and  αβ T cells, instead of the expected competition between NK cells and γδ T cells. Nonetheless, we were able to selectively expand both γδ and αβ T cells in the presence of MTX. Analysis to test effectiveness of the caspase 9 suicide switch revealed that cells electroporated with dDiC9 constructs were effectively ablated after induction of apoptosis with a chemical inducer. To conclude, we have been successful in selectively expanding ablatable T cells using a suicide switch, drug and miRNA selection strategies.

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This  presentation is supported by the National Cancer Institute through the U54 CA096297/CA096300: UPR/MDACC Partnership for Excellence in Cancer Research Training Program.

Cite this work

Researchers should cite this work as follows:

  • Rosa Maria Santana Carrero (2015), "Drug Selection of γδ T cells using miRNA target sequences," https://ncihub.cancer.gov/resources/1119.

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