The role of JAK1 and JAK3 in CD8⁺ effector T cells

The aim of this project was to explore the role of the tyrosine kinases JAK1 and JAK3 in cytokine signalling, focusing on interleukin-2 signalling in CD8⁺ effector T lymphocytes. Initial experiments compared the effects of the pan JAK1/JAK3 inhibitor tofacitinib, the selective JAK1 inhibitor GSK186, and the selective JAK3 inhibitor GSK192 on IL-2 control of effector CD8+ cytotoxic T cells (CTL). On the basis of these preliminary data, a detailed analysis of the effect of tofacitinib on effector CD8⁺ T lymphocytes was performed. <br/><br/>Phosphorylation events regulated by tofacitinib were identified using mass spectrometry analysis of SILAC (stable isotope labelling with amino acids in cell culture) labelled CTL. Tofacitinib regulated a selective number of phosphorylation sites, with less than 1.2% of the CTL phosphoproteome significantly regulated by tofacitinib treatment following 4hrs tofacitinib treatment. Proteins with downregulated phosphorylation sites were enriched in functions related to the Jak-STAT signalling, regulation of gene expression, and MAPK signalling cascades. Proteins with upregulated phosphorylations were also enriched in functions related to regulation of gene transcription. <br/><br/>The proteome of tofacitinib treated CTL was defined by label free mass spectrometry. Approximately 4.5% of the CTL proteome was significantly regulated following 24 hours tofacitinib treatment, suggesting tofacitinib regulates the expression of a selective subset of proteins. Tofacitinib treatment resulted in the downregulation of proteins involved in ribosome biosynthesis, steroid biosynthesis, regulation of transcription and the cell cycle; and the upregulation of proteins with hydrolase activity, and with roles in the lysosome and extracellular exosomes. The phosphoproteomic and proteomic data demonstrates that JAK kinase dependent IL-2 signalling regulates essential processes in CTL by controlling a selective number of phosphorylation events and proteins. Validation of proteins identified as regulated following tofacitinib treatment identified new targets of IL-2 signalling in CTL, including the transcription factor NFIL3. NFIL3 was shown to be upregulated in CD8⁺ T lymphocytes following stimulation with IL-2 and regulated perforin and CD62L expression, suggesting a role in the regulation of CTL effector function.<br/>

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2016

Author dc:creator

• Rollings, Christina

Advisor dc:contributor.advisor

• Cantrell, Doreen

• IL-2 signalling
• CD8 T cells
• jak kinases
• tofacitinib
• NFIL3

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/03bc71f9-1291-4e6f-9478-11b9b6227f3f

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/03bc71f9-1291-4e6f-9478-11b9b6227f3f