Proliferation and functional activation of most cells in the immune system are controlled by the responses of activated T cells. At least three classes of T cells are known: killer T cells, and two types of helper T cells that secrete distinct sets of polypeptide hormones. All three types of T cells have many features in common, including the antigen-recognition structures that trigger their activation and a shared site of differentiation from common precursors. Upon stimulation, however, each of these three classes of cells initiates the accumulation of a different set of transcripts involved in effector function, e.g. interleukin-2 (IL2) for type 1 helpers and interleukin-4 (IL4) for type 2 helpers. This proposal is to investigate critically the molecular basis for these different responses. It is prompted by our results of in situ hybridization studies of response gene inducibility in primary cell populations, and transfection experiments to identify cell type-specific activators. These studies suggest that T cells are capable of transcribing inappropriate as well as appropriate """"""""response"""""""" genes, depending on the conditions of stimulation. We plan to determine whether individual cells in fact have the potential for multiple types of functions, and if so, what mechanisms operate normally to restrict it. In situ hybridization, quantitative probe protection, and flow cytometric phenotypic analysis will be used to identify the cells able to express RNA for IL2, IL4, and the killer-cell serine esterase CCP1. Defined populations of cells will be activated with ligands to membrane receptors or with membrane-permeable pharmacological agents, in the presence or absence of defined exogenous lymphokines. The time scale will be brief enough to permit calculation of the frequency of cells with dual responses or cells with mutually exclusive responses. Different kinetics of mRNA accumulation will be analyzed by nuclear runoff to estimate transcription rates and by actinomycin D chase to estimate stability of the RNA. In parallel, we will look directly for cell-type-specific regulatory proteins in gene transfer experiments with the murine IL2 gene in tissue culture cells and transgenic mice.
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