The importance of HLA class-I-restricted, CD8+CTL in the immune response to virus infections is unquestioned. Despite years of investigation, the intricacies of the initial immune response to viral pthogens, and the subsequent development of memory are incompletely understood. While much of our knowledge derives from work in animal models, recent advances have allowed the intimate dissection of human cellular immune responses to viruses. We can now visualize epitope-specific CTL directly from clinical samples by several techniques. These advances will allow us to differentiate memory from effector CTL, and begin to dissect the mechanisms that regulate the inter-conversion of these cells. We will also be able to precisely quantify individual clones of memory and effector CTL at the signle cell level and assess the functional capability and clonal heterogeneity of these cells. To complete these goals we propose to use multiparameter flow cytometry linked to functional assays to determine the magnitude of the CTL response, and develop markers that will allow for the differentiation of individual effector and memory virus-specific CTL. Potential targets of this aim will be: a) TCR surface expression and internalization in response to TCR engagement b) Granzyme and perforin expression and release in response to TCR engagement c) Expression of mediators of apoptosis and susceptibility to antigen induced programmed cell death d) CD28 expression and responsiveness to co-stimulatory signals e) Surface expression of activation markers and adhesion molecules In addition we propose to use these markers to quantitatively determine the TCR-defined clonal complexity and functional heterogeneity of virus-specific CD8+CTL at the single cell level. Within this aim we intend to: a) Determine the number of heterogeneity of TCR-defined clonotypes of memory and effector CTL during acute, chronic, and convalescent viral infections b) Determine the role of altered peptide ligands in changing the response of individual T cells to TCR signaling during chronic viral infection c) Define other functional differences between memory and effector CTL, and determine if there is hierarchy of functional capacity of these cells.
