Elucidating the molecular mechanisms of p110δ activation in T cell antigen receptor signalling

Phosphoinositide 3-kinases (PI3Ks) are activated in immune cells downstream of the antigen recognition receptors, the signals from which are crucial for the development, differentiation and activation of lymphocytes. Engagement of the T cell antigen receptor (TCR) initiates a signalling cascade leading to the rapid induction of PI3K activity. The class IA catalytic isoform p110δ is known to be the dominant PI3K involved in signal transduction downstream of the TCR. However, the mechanism by which the TCR recruits and activates p110δ remains unresolved. This study has used a combination of proteomic, biochemical and gene editing approaches to identify proteins that are involved in the regulation of p110δ activity during TCR signalling. This work has optimised the AviTag affinity purification system for the isolation of endogenous p110 isoforms from primary murine lymphocytes. A quantitative proteomics approach was subsequently used to identify endogenous proteins that interact with p110δ in primary activated CD4+ T cells upon TCR stimulation. This analysis has identified multiple adaptor proteins and co-stimulatory receptors that may regulate p110δ activity at the TCR signalosome. This study has also developed a CRISPR/Cas9-mediated gene editing approach to disrupt targets of interest in primary murine activated T cells via ribonucleoprotein (RNP) nucleofection. This system was used to investigate the roles of the p110δ-associated proteins in TCR-induced PI3K signalling and cytokine production in activated T cells. Collectively, this work has demonstrated the potential of proteomic analysis coupled with CRISPR/Cas9 gene editing for the identification and interrogation of signalling pathway components in primary T lymphocytes. Using this approach, this study has uncovered molecular details of p110δ recruitment and activation downstream of the TCR.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2019

Author dc:creator

• Luff, Daisy Helen

Advisor dc:contributor.advisor

• Okkenhaug, Klaus

• PI3K
• TCR Signalling
• Proteomics
• CRISPR/Cas9
• T Cells
• p110δ
• TCR
• Interactomics
• Lymphocyte Signalling
• AviTag

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b0b3121c-3bd8-4eb5-9673-d24ccf72b9aa/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.38233

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/291052