Immunity to a number of viruses is known to be mediated by T cells, implying that many vaccines should be engineered to elicit strong T cell memory. While the phenomenon of functional T cell memory may be accounted for by increased frequencies of antigen specific cells with possibly heightened sensitivity, many of the mechanistic details of memory T cell development and maintenance remain unresolved. Precise analysis of memory T cells has been hampered by the absence of methods for detecting and purifying low frequency populations of antigen-specific T cells without resort to in vitro culture techniques. Recently described soluble tetramers of peptide-bound MHC complexes that label T cells according to the specificity of their antigen receptor provide a general solution to the problem. This technique will be applied to answer two specific questions on the nature of T cell memory, using well characterized CD8+ T cell responses to LCMV. 1) What is the lifespan of memory T cells? The MHC tetramers will provide definitive answers to this question through analysis of extremely pure populations of memory T cells (possessing several distinct antigen-specificities) generated during the course of physiologically relevant responses. As a corollary to the lifespan studies, the stability of the expression patterns of a large number of cell adhesion molecules, signaling molecules, and homing receptors will be analyzed on multiple antigen-specific populations within the same mouse. 2) Is the development of memory T cells dependent upon a distinct clonal selection step? If so, there will be detectable differences in the distribution of functional clonotypic T cell receptor (TCR) genes expressed by T cells involved in the primary response and those which form the memory compartment. These populations will be sorted using appropriate MHC tetramers. Pools of sorted cells specific for a single antigen will be analyzed by TCR spectratyping; sequences of functional TCR genes from single sorted cells will be analyzed using highly optimized RT-PCR techniques.
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