This proposal is aimed at understanding how alcohol activates T cell signaling pathways and how non-specific T cell activation could in turn lead to immunosuppression. We have used Jurkat cells stimulated by superantigen coated Raji cells as a ifiodel system for T cell activation and synapse formation. Our preliminary data indicates that 25-100 mM ethanol can cause Jurkat cells to adhere to Raji cells in the absence of superantigen. This adhesion depends on LFA-1 and is likely a consequence of triggering.T cell signaling pathways. Furthermore, ethanol induces the formation of a synapse that is essentially identical to that which forms when superantigen is present, a further indication that signaling pathways have been turned on. As a means of dissecting these signaling events, we propose to use the synapse and its components as molecular markers for T cell activation by ethanol. Our approach will be to use biochemical markers, mutant cells lines, and the three dimensional arrangement of key signaling proteins.at the synapse to monitor how ethanol intersects the normal T cell signaling system. Lipid rafts play an important role in T cell activation and it may be here that ethanol exerts it primary effects. To study this we will use fluorescence anisotropy measurements to determine the impact of ethanol on clustering of proteins lipid rafts. We will also analyze some of the consequences of activation such as T cell receptor downregulation to determine how ethanol modifies T cell responses after exposure. Having worked out the signaling pathway in Jurkat cells, we will look for similar activation events in murine T cells using a cloned allospecific cytotoxic T cell line (BM3.3). We will examine whether ethanol can cause adhesion and killing of target cells in the absence of antigen and we will determine if ethanol alters normal antigen-mediated target cell lysis. Jn an effort to abrogate the effects of ethanol, we will detemine whether inhibiting LFA-mediate adhesion or steps in the T cell activation pathway will block these effects.
| Combs, Jeffrey; Kim, Soo Jin; Tan, Sarah et al. (2006) Recruitment of dynein to the Jurkat immunological synapse. Proc Natl Acad Sci U S A 103:14883-8 |
