TRANSCRIPTIONAL REGULATION OF INTERLEUKIN-2 GENE-EXPRESSION BY CD69-GENERATED SIGNALS

The 5' flanking region of the human interleukin (IL)-2 gene was invest igated for enhancer activity in response to CD69-generated signals, us ing a chloramphenicol acetyltransferase (CAT)-driven transient express ion system in Jurkat cells. The region extending from -317 to +47 rela tive to the initiation site of IL-2 gene transcription was shown to co ntain sequences able to respond to CD69 cross-linking, by enhancing by about 100% a phorbol 12-myristate 13-acetate (PMA)-plus-ionomycin sti mulation of CAT activity. A similar increase in CAT activity produced by PMA-plus-anti-CD3 mAb was induced by CD69 crosslinking, while a 200 % increase over that obtained by PMA-plus-anti-CD28 mAb stimulation wa s seen. Analysis of enhancer deletion mutants revealed that proximal A P-1, OCT-1/octamer-associated protein and nuclear factor of activated T cells (NFAT) binding regions were all necessary to allow CD69-mediat ed enhancement of CAT activity. By gel mobility shift analysis, cyclos porin A-sensitive NFAT-binding induction and enhancement of AP-1 bindi ng activity could be detected in nuclear extracts of both Jurkat and p eripheral blood T cells after simultaneous CD69 and protein kinase C s timulation. Finally, CD69-mediated signals could increase NFAT and AP- 1 binding activity following PMA and ionomycin stimulation in peripher al blood T cells. Collectively, these data suggest that CD69-generated signals participate in the control of the IL-2 gene expression at the transcriptional level, likely acting through NFAT and AP-1 transcript ion factor complexes.