During aging, lymphocyte development and functions become compromised, resulting in age- dependent increases in susceptibility to infectious agents. The decline in T cell function is the most clear and striking of the immune system defects related to aging, resulting in lower and slower immune responses This study will use live cell imaging and Forster (or fluorescence) Resonance Energy Transfer (FRET) microscopy to investigate changes in formation of the immunological synapse and molecular interactions within the synapse during aging. The induction of the interaction between the TCR and co-receptor, CD4 or CD8, will be investigated during aging. The form of the immunological synapse and interactions between TCR and coreceptor within the synapse cells will be investigated in T cells from aged or young TCR transgenic animals. The cells will be transfected with genes to express fluorescent chimeric proteins and live cell imaging and FRET microscopy will be used to analyze the induction and kinetics of the TCR-coreceptor interaction. Reversible protein highlighting, with a photo-reactivatable fluorescent protein will be used to measure the movement kinetics of TCR or coreceptor on the cell surface or in the synapse. T cells require recognition of self-peptide-MHC (pMHC) complexes in order to aid in activation by limiting quantities of antigenic pMHC. Changes in this requirement during aging of the T cell population will be assayed. Changes in the sensitivity of the T cells to limited quantities of antigen will be measured. A fluorescent marker protein for lipid rafts will be used to compare raft dynamics in young or aged T cells. Movement of TCR and or co-receptor proteins will be analyzed at the same time, so that raft dynamics can be related to formation of the synapse. Reversible protein highlighting will be used to visualize movement of TCR or coreceptor proteins into and out of rafts. Possible age-related changes in the transfer of membrane components from the antigen presenting cell to the T cell (""""""""trogocytosis"""""""") will be analyzed as a measure of changes in T cell membrane properties. The immune response particularly that of T cells, declines with age, leaving the individual more susceptible to infection. This project aims to determine how changes in the T cell's recognition structures occur during aging, which is important in understanding how age-related defects could be prevented. ? ? ? ?
| Fu, Guo; Gascoigne, Nicholas R J (2009) Multiplexed labeling of samples with cell tracking dyes facilitates rapid and accurate internally controlled calcium flux measurement by flow cytometry. J Immunol Methods 350:194-9 |
