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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA039605-05
Application #
3178777
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1985-01-01
Project End
1993-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
078731668
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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Garrity, P A; Chen, D; Rothenberg, E V et al. (1994) Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors. Mol Cell Biol 14:2159-69
Chen, D; Rothenberg, E V (1993) Molecular basis for developmental changes in interleukin-2 gene inducibility. Mol Cell Biol 13:228-37
Rothenberg, E V (1992) The development of functionally responsive T cells. Adv Immunol 51:85-214
Novak, T J; Yoshimura, F K; Rothenberg, E V (1992) In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters. Mol Cell Biol 12:1515-27
Rothenberg, E V (1990) Death and transfiguration of cortical thymocytes: a reconsideration. Immunol Today 11:116-9
Novak, T J; Chen, D; Rothenberg, E V (1990) Interleukin-1 synergy with phosphoinositide pathway agonists for induction of interleukin-2 gene expression: molecular basis of costimulation. Mol Cell Biol 10:6325-34
Novak, T J; Rothenberg, E V (1990) cAMP inhibits induction of interleukin 2 but not of interleukin 4 in T cells. Proc Natl Acad Sci U S A 87:9353-7
Novak, T J; White, P M; Rothenberg, E V (1990) Regulatory anatomy of the murine interleukin-2 gene. Nucleic Acids Res 18:4523-33
Rothenberg, E V; Diamond, R A; Pepper, K A et al. (1990) IL-2 gene inducibility in T cells before T cell receptor expression. Changes in signaling pathways and gene expression requirements during intrathymic maturation. J Immunol 144:1614-24

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