Reports of research work funded by grants prior to 2013
Victoria University of Wellington
Lymphocyte-platelet Conjugates in Stable Atherosclerotic Disease
L Johnston, A La Flamme, S Harding and P Larsen,
School of Biological Sciences
Background and Study Aims
Lymphocytes, particularly T-cells and platelets are key immune cells in the development and destabilisation of atherosclerotic plaques respectively. However, the interactions between lymphocytes and platelets in atherosclerosis and how these interactions modulate plaque development are not well characterised. Our initial work demonstrates that platelet-T cell aggregates in patients with peripheral vascular disease are different in character than in healthy subjects – with an enhanced CD4 effector T cell population in vascular disease but comparable CD8 population. Furthermore, platelet – CD4 T cell binding was increased in subjects with peripheral vascular disease. This raises the question of how platelets influence CD4 T cell development and activity in atherosclerosis and if blocking platelet-T cell interactions can abrogate these changes.
Aim 1. To assess the effect of platelet interactions on CD4 T cell proliferation, cytokine production and activation state.
Aim 2. To assess the effect of antiplatelet therapy on platelet – CD4 T cell interactions.
To investigate the effect of inhibiting platelets on lymphocyte phenotypes, 12 antiplatelet naïve vascular patients and 12 healthy patients with no known cardiovascular disease or on any cardiovascular medications will be recruited. Subjects will then be loaded with 60mg of Prasugrel and continued on a maintenance dose of 10mg/day for 14 days. On day 14 a further 50mL of blood will be collected. To investigate the effects of platelets on CD4 T cell phenotypes and proliferation CD4 T cells will be extracted using a Dynal® CD4 untouched negative isolation kit and cultured with or without autologous platelets. Anti-CD3 bound to 96 well plates and soluble CD28 will be used to stimulate T cells and PMA and ionomycin to induce cytokine production. To measure the effect of platelets on T cell proliferation, T cells will be incubated with CellTrace Violet Cell Proliferation stain at the start of culturing, and analysed using flow cytometry. A protein secretion inhibitor such as BD GolgiStop will be added to stop the release of cytokines in preparation for intracellular cytokine analysis. Supernatant from T cell cultures will be collected for measurement of IFN–γ levels by ELISA. To look at the specific T helper subtypes involved, the extracellular marker CD4 will be paired with cytokine markers for IFN-γ (Th1), IL17A (Th17) and transcription factor FoxP3 (Treg). By pairing extracellular activation markers with intracellular cytokine and proliferation markers, this protocol will allow us to determine which phenotype the platelets are driving a T cell towards and their effect on T cell proliferation and cytokine production.
The study is utilising intracellular cytokine staining, proliferation dye and flow cytometry to identify proliferating CD4 cells and the T helper cell phenotypes. This staining method and appropriate culturing conditions have now been fully optimised. With optimisation of the experimental methods complete, the screening and recruitment of participants is underway. Healthy volunteers have been recruited and experiments are underway in this cohort. In the vascular patient group, screening of the carotid surveillance list is being conducted and initial contact by letters is being made with potential patients.