REGULATORY ELEMENTS OF INTERLEUKIN-2 GENE EXPRESSION

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Our long-term interest is a better understanding of molecular mechanisms involved T lumphocyte activation and proliferation. In this proposal we focus specifically on defining sequences in the 5'-flanking region of the human interleukin-2 (IL2) gene that participate in IL2 gene expression in normal and leukemic T lymphoid lines and whether trans-acting factors function in these responses. In particular we will examine 1) IL2 gene activation and subsequent down regulation by mitogenes and phorbol esters (PHA/PMA), antibodies to T cell surface antigens T3, T11 and 9.3, and ionophores; 2) inhibition of IL2 expreession by cyclosporina A; 3) cell specific restriction of IL2 expression to certain T lymphoid cells; 4) cycloheximide-mediated superinduction of Il2 mRNA; 5) whether there is species specific IL2 expression in murine and gibbon lymphoid lines, and 6) the potential role of trans-active factors in the apparent lack of normal IL2 expression in the gibbon leukemia line, MLA144. The basic approach will be to test the expression of the bacterial "reporter" enzyme gene chloramphenicol acetyl transferase (CAT) fused to 5'-flanking regions of IL2 (pIL2CAT). We have demonstrated regulatory behavior in these constructs which parallels the endogenous IL2 gene in the mitogen-inducible human T cell line, J32. Mutations constructed in the Il2 5'-flanking sequences of pIL2CAT would permit a comparison of transfected normal and mutant plasmids to define important regulatory sequences. 5' deletions, linker-scanner and site-directed mutants will allow precise delineation of responsible residues. CAT expression data will be supplemented by S1 nuclease protection of CAT mRNAs to confirm that alterations in CAT activity reflect transcriptional regulation of properly initiated "IL2 transcripts." Moreover, competition experiments using cotransfection of normal or altered pIL2CAT with other genes linked to normal or altered IL2 5'-flanking sequences should permit us to determine if trans-acting factors play a role in any Il2 gene responses, a first step toward the isolation and further study of what may be crucial intracellular molecules in a host of regulated gene events. The central role of Il2 in T lymphocyte activation, expansion and other lyumphocyte responses, along with the marked effect on allograft retention of the Il2 inhibitor, cyclosporin A, make the study of Il2 gene expression extremely important for understanding and potentially manipulating immune responses.