The long-term objective of these studies is to understand the regulation of different Th subsets which can and will develop in response to antigen (Ag). The application proposes to determine expression of cell surface receptors and adhesion molecules, expression of regulatory proteins and cytokines and investigate other behavioral characteristics which distinguish the subsets. Most importantly, the application proposes to determine the factors which regulate the development of different subsets, concentrating on those which favor generation of particular developmental stages and of subsets committed to particular patterns of cytokine secretion within those stages. The basic approach will be to use TCR transgenic animals. Distinct subsets of Th will be generated from these naive cells both in vitro and in vivo using techniques developed during the previous project period. In vitro techniques will generated 4-day effectors that have Th0 (IL-2, IL-4, IFN-gamma), Th1 (IL-2, IFN-gamma) or Th2 (IL-4, IL-10) patterns of cytokine secretion. These effectors will be transferred to adoptive hosts to obtain memory cells with related properties and then the different memory populations will be stimulated in vitro to generate memory effectors. The activation requirements and response potentials of these various subsets will be compared, the cytokines will be investigated, antigen presenting cell (APC) populations and Ag doses/numbers which favor the development of the distinct subsets will also be studied. The lineage relationships will be studied among the different populations to determine which precursors are committed to a particular pattern of cytokine secretion and which are multipotential. Also memory cells will be generated in situ using adoptive hosts of transgenic T cells. Several approaches will be employed to study the regulation of CD4 memory. The application proposes to examine in vitro the role of TGF-beta in generating cells with a memory phenotype. The application proposes to evaluate the effect of TGF-beta on the survivability on populations of cells, on susceptibility of cells to apoptosis and on the potential of cells to expand. In vivo studies will examine the lifespan and turnover of memory CD4 cells, and the roles of AG and TGF-beta in memory generation and persistence. Overall these studies are likely to provide important indications of the host factors that are likely to influence the initial and long-term generation of distinct Th subsets. It is believed that the nature and amount of Th help which develops during a response to a particular Ag will plan the major role in determining both the magnitude of the ensuing immune response and the kind of the immune response that occurs. Understanding how the generation of Th subsets is itself regulated, should give us clues about potential therapeutic intervention to influence development of particular subsets when appropriate immune responses fail to develop or when ineffective or even counterproductive immune responses occur. Furthermore, as we gain a greater understanding of the in vivo persistence and function of Th subsets that have been generated in vitro and transferred to adoptive hosts, we may be able to devise well-focused protocols for stimulating autologous T cells in vitro and then reintroducing them to achieve the desired immunity.
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