Abstract A149: Assay development to validate RNAi candidates targeting FoxP3 transcription factor in adaptive regulatory T cell.
Authors: Bajgelman Marcio C. , Brenneman Randall J. , Seales Dawn , Berezhnoy Alexey , Carvalho Ana Carolina P. , Gilboa Eli 
Published: August 2014

Abstract
Regulatory T cells (Treg) play an important role controlling the balance between tolerance and surveillance. Regarding its potential to inhibit T cell proliferation, Treg can antagonize antitumor immunity. Therefore, inhibition of Treg can potentiate antitumor immunity.
There are several strategies to inhibit Treg function or traffic to tumor site; however these approaches aren't specific for Treg and can impair antitumor response. In this way, the exploration of molecular targets could assure more selectivity for Treg inhibition. The FoxP3 transcription factor is considered an important Treg marker and a master key which is associated with the determination of immunosuppressive phenotype. Silencing FoxP3 expression can lead inactivation of immunosuppressive phenotype of Tregs.
Here in we developed an assay to validate RNAi candidates targeting FoxP3. The assay is based in two steps. In the first step we generate shRNA candidates that are cloned in expression vectors. These vectors are co-transfected with a luciferase reporter vector encoding synthetic targets, in cell culture. A decrease in luciferase activity indicates efficiency of the shRNA sequence. Using this screening tool we could find shRNA candidates that exhibited more than 95% of knockdown efficiency. In the second step we evaluated the activity of the best shRNA candidates to knockdown endogenous FoxP3 in primary cells. We generated lentiviral vectors encoding both the shRNA and an expression cassette of CD90.1 reporter gene to transduce primary CD4 T cells. These transduced cells were incubated with a TGF beta cocktail which induces FoxP3 expression and adaptive Treg generation.
FoxP3 expression was checked by flow cytometry, gating CD90.1 positive cells. We found up to 75% of inhibition, for the RNAi candidate #183. On the other hand, we also observed that a highly efficient shRNA sequence indicated by the luciferase reporter assay could be inefficient to silence the endogenous FoxP3 in primary cells.
We conclude this assay is a reliable tool which can be used to generate RNAi to knockdown FoxP3 expression in primary cells, overcoming issues associated with transfection or transduction efficiency. Transduced cells can be labeled on cell surface with an ati-CD90.1 marker and stained for membrane, intracellular markers or even other kind of analysis as cell cycle. The validated RNAi candidates are active against the endogenous target and can be used to explore strategies to inhibit the immunosuppressive phenotype of regulatory T cells and to potentiate antitumor immunity.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A149.
Citation Format: Marcio C. Bajgelman, Randall J. Brenneman, Dawn Seales, Alexey Berezhnoy, Ana Carolina P. Carvalho, Eli Gilboa. Assay development to validate RNAi candidates targeting FoxP3 transcription factor in adaptive regulatory T cell. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A149.