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Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter¹

Heather M. Gibson^*, Carrie J. Hedgcock^*, Barbara M. Aufiero^*, Adam J. Wilson^*, Mikehl S. Hafner^*, George C. Tsokos and Henry K. Wong^2,*

^* Department of Dermatology, Henry Ford Hospital, Detroit, MI 48202; and Rheumatology Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard University School of Medicine, Boston, MA 02115

CTLA-4 is a member of the costimulatory family, has homology to CD28, and binds the B7 family of ligands. Unlike CD28, CTLA-4 ligation transmits a negative signal in T cells. CTLA-4 expression, while inducible in most T cells, is expressed constitutively on T cells with a regulatory phenotype. The mechanism controlling CTLA-4 expression in human T cells is poorly characterized, thus we sought to better understand the mechanism of activation of the CTLA-4 gene. By cloning the 5' upstream promoter and creating promoter-deletion reporter constructs, we show that the proximal promoter is critical for activating the CTLA-4 gene. Within this region, we identify a NFAT consensus sequence that binds NFAT with high affinity that differs from other NFAT sequences and does not recruit AP-1. Analysis of the chromatin proteins in the native CTLA-4 gene shows that this promoter region becomes associated with acetylated histones by chromatin immunoprecipitation assays. In addition, NFAT1 binds to the promoter of the CTLA-4 gene after stimulation by chromatin immunoprecipitation. The functional requirement of the NFAT site for CTLA-4 transcription was demonstrated by mutations in the NFAT site that abolished the activity of the promoter. Furthermore, inhibitors of NFAT suppressed CTLA-4 gene expression, indicating that NFAT plays a critical role in regulating the induction of the CTLA-4 gene in lymphocytes. The identification of NFAT as a critical regulator of the CTLA-4 gene suggests that targeting NFAT function may lead to novel approaches to modulate the CTLA-4 gene to control the immune response.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ H.K.W. received a Clinical Career Development Award from the Dermatology Foundation and grant support from the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases (KO-8 AR47818). G.C.T. received funding support from the National Institutes of Health (National Institute of Allergy and Infectious Diseases) R01 AI 42269.

² Address correspondence and reprint requests to Dr. Henry K. Wong, Henry Ford Health System, One Ford Place 4-D, Detroit, MI 48202. E-mail address: hwong1{at}hfhs.org

³ Abbreviations used in this paper: Treg, regulatory T cell; CsA, cyclosporin A; SRE, serum response element; ChIP, chromatin immunoprecipitation; ds, double stranded.