This project will study several aspects of cannabinoid receptors in T cell responses to antigen, because of the immunosuppressive effects of cannabinoids on T cell mediated immunity, which has relevance to the ability of drug abusers to fight infections. The approach makes use of chimeric genes that produce fluorescent versions of cannabinoid receptor 2 (CB2) and T cell surface proteins, using variants of the green fluorescent protein (GFP). This allows direct monitoring of protein movement in living cells by fluorescence deconvolution microscopy. T cell receptor (TCR) and CD4 co-localize in the """"""""immunological synapse,"""""""" a structure that forms at the interface of a T cell interacting with an antigen-presenting cell. This occurs faster and more effectively with TCR agonist rather than antagonist ligands. Fluorescence resonance energy transfer (FRET) showed a direct interaction between TCR and CD4 with the agonist, but no interaction with the TCR antagonist. These experiments establish an experimental system to study how signaling through cannabinoid receptors affects T cell activation through formation of the immune synapse. Deconvolution fluorescent microscopy of live cells will be used to obtain high resolution, three dimensional, real-time information on the effect of cannabinoids on movement of CD3& and CD4 during T cell stimulation, and in particular, on the formation of the immunological synapse in response to an antigenic ligand. Fluorescent CB2 will be expressed in transfected cells. Time lapse experiments will be performed to monitor distribution of fluorescent CB2 following exposure to cannabinoids and to distinguish whether CB2 becomes internalized, clusters in the cell membrane, or is unaffected after cannabinoid recognition. We will determine if CB2 is included or excluded from the lipid rafts involved in signal transduction, and whether the distribution changes with ligand binding. CB2 expression will be related to early signaling events by using Ca2+ sensitive dyes. Expression of fluorescent CB2 will be compared in resting T cells and APC during T cell activation, with and without drug treatment. Expression of fluorescent CB2 in T cells will be in the presence of labeled TCR and/or co-receptor to determine whether CB2 is involved in formation of the immunological synapse, or if it remains outside of this structure. If the data show that CB2 co-localizes with TCR or CD4, FRET analysis will be performed to determine if there is a direct interaction. We will determine if CB2 and raft distribution is altered during T cell activation, and will compare this in the presence of CB2 ligand. We will express fluorescent CB2 in APC, to see how expression is affected by drug exposure and by interaction with T cells.
| Zal, Tomasz; Zal, M Anna; Gascoigne, Nicholas R J (2002) Inhibition of T cell receptor-coreceptor interactions by antagonist ligands visualized by live FRET imaging of the T-hybridoma immunological synapse. Immunity 16:521-34 |
