Dissecting early calcium signalling-events in controlling effector function of T lymphocytes

Supervisors: Dr Marco Fritzsche and Prof Tao Dong

Background

Calcium (Ca2+) signalling is a pivotal event promoting adaptive immune responses initiated by binding of antigens to antigen receptors, resulting ultimately in the formation of the immunological synapse (IS). The IS is a specialised interface at the cell-cell contact between lymphocytes and antigen presenting cells (APCs), which has been unleashed to enhance T-cell function in cancer immunotherapy or restrained to suppress inappropriate immune responses in autoimmune diseases. 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 effects of early calcium signalling on the IS and its relationship to long-term effector function of T lymphocytes. Additionally, despite our knowledge of molecular calcium release during the activation of T cells, the exact mechanisms controlling IS formation remain elusive. These shortcomings precluded understanding of the mechanisms by which calcium-signalling-events may modulate the quality of the IS and T lymphocyte long-term effector signalling. A remedy to this dilemma are recently developed more advanced calcium microscopy techniques, such as dual-colour calcium quantifications. Specifically, we recently developed a new calcium assay and a second generation analysis software tool CalQuo (1,2), that allows the systematic parallel large population-level quantification of global intracellular calcium responses in thousands of T lymphocytes. This research project aims to employ the dual-colour calcium assay to the study of calcium-signalling-events in T lymphocyte clones interacting with target APCs loaded with a multitude of different affinity antigens. We will initially investigate the dynamics of the above detailed players in primary T cell clones interacting with protein-functionalised glass surfaces and then extend our studies to primary T lymphocytes interacting with target APCs. Introducing a third-colour allows the quantification of killing events in response to non-self antigens loaded APCs.

Project

We aim to use a recently developed dual-colour calcium microscope technology coupled with a T-cell clone library to highlight novel details of the relationship of early calcium signalling and long-term effector signalling. We intend to use the a set of well-characterised T-cell clones recognising a range of cancer and viral specific antigens and with different functionality (i.e.: potentials to kill the target cells) (3), which offers the opportunity to investigate the relationship between the strength of TCR engagement, its effect on formation of the IS, and long-term effects on effector function.

Training Opportunities

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 optical microscopy and dual-colour calcium measurement techniques, and MATLAB-based programming. 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 responses in health and disease.

Theme

Immunology, T-cell clones, calcium signalling, long-term signalling, actin cytoskeleton, optical microscopy.

Publications

  1. M Fritzsche, RA Fernandes, H Colin-York, AM Santos, SF Lee, BC Lagerholm, SJ Davis, and C Eggeling, CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses, Sci Reports, 2015.
  2. AM Lee, H Colin-York, M Fritzsche, CalQuo2: Automated Fourier-space, population-level quantification of global intracellular calcium responses, Sci Reports, 2017.
  3. LYH Lee, DLA Ha, C Simmons, MD de Jong, NVV Chau, R Schumacher, YC Peng, AJ McMichael, JJ Farrar, GL Smith, ARM Townsend, BA Askonas, S Rowland-Jones, and T Dong, Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals, J Clin Invest, 2012.

For further information please contact:  Dr Marco Fritzsche