Document Type

Theses, Ph.D

Rights

Available under a Creative Commons Attribution Non-Commercial Share Alike 4.0 International Licence

Disciplines

1.6 BIOLOGICAL SCIENCES

Publication Details

Successfully submitted for the award of Doctor of Philosophy (Ph.D) to the Technological University Dublin, 2013.

Abstract

The activation of naive T cells is essential for the initiation of the adaptive immunity. One promising approach to overcome immunological tolerance involves augmenting endogenous T cell-mediated immunity by interrupting the T cell down-regulatory pathways. In this project a gene therapy delivery system to overexpress small interfering RNA was used to silence the inhibitory signals induced by CTLA4. A small hairpin RNA duplex designed to silence expression of CTLA4 was successfully cloned into a plasmid. A stably transfected T cell line that is expressing constitutively the CTLA4 siRNA has been developed as a model to understand the role of CTLA4 in functional assays. The expression of the mature siRNA in this new cell line was confirmed upon optimization of a real-time quantification by a stem loop RT-PCR method and the downregulation of CTLA4 was detected at the protein and RNA level. The generated cell lines were used to study the role of CTLA4 in T cell activation pathways. It was determined that CTLA4 crosslinking downregulated IL-2 production in the control cell lines. However, silencing of CTLA4 abrogated this effect in the cell line expressing the CTLA4 siRNA. These results suggested that it is possible to modulate T cell activation using RNA interference for CTLA4. CTLA4 crosslinking in combination with CD3 and CD28 ligation did not seem to have an effect on the phosphorylation of ERK or other proteins, which take part in the proximal T cell signaling, like ZAP-70, Lck, SLP-76 and LAT. However, it was found that the c-Jun N-terminal kinase (JNK) activation is up-regulated in the control cell lines. In the stable cell line with silenced CTLA4 expression such effect was not ii observed, thus suggesting a role of CTLA4 as a negative regulator in T cell activation via the up-regulation of JNK.

DOI

https://doi.org/10.21427/D7R595

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