Award details

Regulation of lymphocyte functions by the novel Cybr-cytohesin pathway

Reference	BB/C005090/1
Principal Investigator / Supervisor	Professor James Johnston
Co-Investigators / Co-Supervisors	Dr Adrien Kissenpfennig
Institution	Queen's University of Belfast
Department	Sch of Medicine, Dentistry & Biomed Sci
Funding type	Research
Value (£)	204,322
Status	Completed
Type	Research Grant
Start date	07/11/2005
End date	06/12/2009
Duration	49 months

Abstract

This proposal aims to establish the physiological role of the Cybr-Cytohesin pathway in the control of lymphocyte function. We hypothesise that because of its regulatory interaction with cytohesin-1; Cybr can directly affect the signalling pathway downstream of LFA-1 and therefore influence adhesion, co-stimulation, and proliferation of immune cells. A brief description of the techniques that will be used in the project is outlined below. Cloning and transfection - We will establish cell lines expressing full-length or mutants of Cybr using the Flp-In system (available through Invitrogen) which allows directed integration of expression vectors in pre-defined loci. The system is particularly suitable to compare different expression constructs. Upon establishment of the cell lines the expression of the construct will be tested by Western blot. The effects of the mutants will then be analysed by the methods outlined below. Ras activation and ERK phosphorylation- The activation of the Ras pathway will be tested in transfected cells and T cells from Cybr transgenic mice stimulated with anti-CD3 or PMA. Ras activation will be assayed using a GST-fusion protein of the Ras-binding domain of Raf. Pulled-down lysates will be analysed by Western blot using a Ras antibody. Increased amounts of active Ras will result in enhanced function of the MAP kinases and consequently of ERK 1 and 2 which will be analysed by western blot using phospho-specific antibodies. Electromobility shift assays (EMSA)- Cells from wild type or Cybr transgenic mice will be incubated with anti-CD3 and then with anti-mouse antibody at different time-points or PMA plus lonomycin as a control. Nuclear extracts will be incubated with end-labelled double stranded oligonucleotide probes for NFATc and fractionated on a polyacrylamide gel. DNA-bound oligonucleodite will be revealed by autoradiography. For these series of experiments requiring manipulation of cell lysates at low temperature we have requested thepurchase of a refrigerated microcentrifuge. Cell growth Assays- Thymocytes proliferation will be assessed by thymidine incorporation assay in cells treated with IL-1 and CD3. Cells will be stimulated for 24 hours and pulsed with 3H thymidine. Similarly, T cells proliferation will be assessed after purification from transgenic mice and stimulation with anti-CD3. Calcium Flux measurement- We will assess TCR induced calcium mobilisation by loading Cybr-transgenic T cells with the calcium-sensitive dye Indo-1 and then incubating for 30 min with anti-CD3. Calcium influx will be detected by flow cytometry. A baseline will be initially collected and fluorescence changes will be observed after cross-linking the TCR. Adhesion assays- We will perform adhesion assays on substrates including ICAM-1, ICAM-2 or Fibronectin. We will use purified stably transfected Jurkat cells and T cells from wild-type and Cybr transgenic mice. Cells will be stimulated with PMA, or adding Mg2+-EGTA to discriminate between in inside-out signalling versus outside-in signalling. We will also assess the adhesive response upon TCR cross linking with anti CD3 and evaluate the effect of the LFA-1 pathway by using an anti LFA-1 (KIM 127). Chemotaxis assays- Lymphocytes from Cybr transgenic mice will be placed in the top wells of a 96 well chemotaxis plate chamber fitted with a 5-micron pore-size filter. Bottom wells will be filled with T cell chemokines such as SDF-1, CCL4, CCL21 at different concentrations. The plate will be incubated for various time points and the number of cells migrating to the lower well be evaluated by MTT staining. Many of these techniques are being used in the laboratory or in adjacent labs and the constructs and cells needed are already available in the lab. The mouse colony in currently being established and the appropriate number of mice will be available by the time the project will start.

Summary

unavailable

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	Immunology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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