Dissecting the ability of PD-1 signalling-events in altering actin cytoskeleton dynamics in tumour infiltrating T lymphocytes
Supervisor: Dr Marco Fritzsche and Prof Vincenzo Cerundolo
Cancer cells often hamper tumour specific responses by dysregulating activation events in tumour infiltrating T cells. A pivotal event promoting normal T-cell activation is binding of MHC/peptide complexes to T-cell receptors (TCR) and subsequent actin cytoskeleton reorganisations resulting in receptor cluster formation, intracellular signalling, and ultimately immunological synapse (IS) formation. The IS is a specialised interface at the cell-cell contact between lymphocytes and antigen presenting cells (APCs). TCR mediated activation of T cells by APCs depends on actin assembly, while inhibition of actin dynamics arrests the IS and results in impaired T cell activation. Unexpectedly, only circular IS interfaces (with symmetric geometry) are stable and promote full activation of T cells, while non-symmetric interfaces impair organised IS formation (1,2). Historically, advances in understanding the mechanisms underlying key events in the IS have been driven by innovations in microscopy that provided greater spatial and temporal resolution coupled to appropriate immuno-chemical, genetic, and biochemical tools. However, until recently, state-of-the-art microscopy has not been sufficiently informative to dissect the underlying real-time spatiotemporal dynamics of the actin cytoskeleton. Additionally, despite our knowledge of molecular actin assembly, the exact mechanisms controlling actin dynamics during IS formation remain elusive. These shortcomings precluded understanding of the mechanisms by which inhibitory-signalling-events may modulate the quality of the IS. A remedy to this are recently developed more advanced microscopy techniques, such as super-resolution optical nanoscopy such as STED (stimulated emission depletion) and the unique TIRF-SIM (total internal reflection fluorescence- structured illumination microscope) (3,4), as well as actin-specific turnover measurements (5,6).
We aim to use advanced microscope technology coupled with original biochemical tools to highlight novel details of the spatiotemporal organisation and dynamics of the actin cytoskeleton in PD-1 dependent tumour persistence. We intend to use the well-characterised 1G4-TCR model system, which offers the opportunity to investigate the relationship between the strength of TCR engagement, its effect on PD-1 up-regulation, and downstream effect on actin remodelling at the IS.
This project will be based in the MRC Human Immunology Unit at the Weatherall Institute of Molecular Medicine, with access to state-of-the-art facilities. The project provides an opportunity for training in a broad range of different techniques, such as cell culture, molecular biology, and advanced microscopy, specifically including super-resolution optical microscopy and force measurement techniques such as STED and the unique TIRF-SIM. The disclosure of novel details of T-cell activation is an important line of basic immunological research that may translate into new approaches of modulating the immune response during infection.
Immunology, actin cytoskeleton, super-resolution STED microscopy, atomic force microscopy.
Sims TN et al, Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse, Cell 2007, 4, 773-85
Fife BT et al, Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR-induced stop signal, Nat Imm 2009, 10, 1185-92.
C. Eggeling, Lens-based fluorescence nanoscopy. Q Rev BiophyJ (2015).
D. Li, Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics, Science. 2015 Aug 28; 349(6251): aab3500.
Fritzsche M et al, Actin kinetics shapes cortical network structure and mechanics, Science Adv 2016, 2, e1501337.
M. Fritzsche, A. Lewalle, T. Duke, K. Kruse, G. Charras, Analysis of turnover dynamics of the submembranous actin cortex. Mol Biol Cell (2013).
For further information, please contact: Dr Marco Fritzsche