The goal of this project is to describe, quantitatively, the human lymphocyte response to mitogens and specific antigens and to define the amplification kinetics of the responding subpopulations in vitro. This study differs from all others in that these responses will be measured by determining both culture cellularity and 3HtdR incorporation. Furthermore, culture conditions will be used in which lymphocytes have been shown, by direct cell counting, to proliferate exponentially with a doubling time of 20 hrs for several days when stimulated with PHA and specific antigens. Response kinetics will be defined by counting cells in culture and identification of cycling cells through single and double label radioautography and liquid scintillation counting. Means of improving culture conditions to support lymphocyte growth will be further investigated. Using improved techniques designed in our laboratory, we will measure lymphocyte cell cycle parameters, recruitment into cycle, their proliferative capacity and the differentiation kinetics after stimulation by mitogens and specific antigens. The differentiated effector cells will also be investigated in detail. These investigations will provide important information about the proliferative capabilities of lymphocytes as well as to identify artifacts introduced by in vitro methodology. The combined results will provide important insights into the regulation of these responses at the cellular level. This will be accomplished by measuring the antigen reactive population size using both intact lymphocyte suspensions as well as separated lymphocyte subpopulations. Using the latter isolated subpopulations, recombined in a controlled manner, the conditions and cell populations involved in the response can be evaluated. Once these conditions for recruitment of the responsive cells into cycle have been defined, measurement of their proliferative capacity to specific soluble antigens and to alloantigens will provide important information on both their amplification potential as well as the practical use of this technology to expand the antigen reactive cell pool to obtain relatively pure populations of these cells. Finally, the role of the progeny which leave cell cycle and accumulate in culture in both the regulation of the proliferative response as well as in the mediation of immune function can be evaluated.