Enumeration and characterization of memory cells in the TH compartment.

There is a great deal of interest in understanding how helper T cells differentiate in vivo and exert their regulatory role on a developing, immune response. Essential to development of protective immunity is the development of memory T cells. To study memory T cells in vivo we first need the means to identify and characterize these cells as they develop in their complex microenvironments. We have developed a method which allows us to directly purify both primary and memory helper T cells from the draining lymph nodes of mice as they respond to pigeon cytochrome c in vivo. Junctional sequences from these populations and from individual T cells show a strong selection for CDR3 length and residues characteristic of antigen binding. Overall these studies support a model of progressive clonal maturation with the memory T cell repertoire being more homogeneous than that of the primary response. There is some suggestion that affinity maturation may take place after repeated immunization, but on a more modest scale than that seen for antibodies. Finally we present the use of two new technologies that promise to greatly expand the analysis of immune responses in vivo. The use of flow cytometry with simultaneous detection of five and six fluorescence parameters helps to reliably resolve rare subsets of antigen-specific cells in order to understand the progression of their differentiation in vivo. Lastly, we have developed peptide/MHC tetramers as a new class of staining reagent that has wide applicability in the study of T cell responses in vivo.