Defective Th1 Cytokine Gene Transcription in CD4  and CD8  T Cells from Wiskott-Aldrich Syndrome Patients 1 Sara Trifari,* Giovanni Sitia, † Alessandro Aiuti,* Samantha Scaramuzza,* Francesco Marangoni,* ‡ Luca G. Guidotti, †§ Silvana Martino, ¶ Paola Saracco, ¶ Luigi D. Notarangelo,  Maria-Grazia Roncarolo, 2 * ‡ and Loı ¨c Dupre ´* Wiskott-Aldrich syndrome (WAS) protein (WASP) plays a key role in TCR-mediated activation and immunological synapse formation. However, the effects of WASP deficiency on effector functions of human CD4  and CD8  T cells remain to be determined. In this study, we report that TCR/CD28-driven proliferation and secretion of IL-2, IFN-, and TNF- are strongly reduced in CD8  T cells from WAS patients, compared with healthy donor CD8  T cells. Furthermore, WAS CD4  T cells secrete low levels of IL-2 and fail to produce IFN- and TNF-, while the production of IL-4, IL-5, and IL-10 is only minimally affected. Defective IL-2 and IFN- production persists after culture of naive WAS CD4  T cells in Th1-polarizing conditions. The defect in Th1 cytokine production by WAS CD4  and CD8  T cells is also present at the transcriptional level, as shown by reduced IL-2 and IFN- mRNA transcripts after TCR/CD28 triggering. The reduced transcription of Th1 cytokine genes in WAS CD4  T cells is associated with a defective induction of T-bet mRNA and a reduction in the early nuclear recruitment of NFAT-1, while the defective activation of WAS CD8  T cells correlates with reduced nuclear recruitment of both NFAT-1 and NFAT-2. Together, our data indicate that WASP regulates the transcriptional activation of T cells and is required specifically for Th1 cytokine production. The Journal of Immunology, 2006, 177: 7451–7461. W iskott-Aldrich syndrome (WAS) 3 protein (WASP) is expressed in cells of hemopoietic origin and is in- volved in actin cytoskeleton remodeling, thereby playing a key role in chemotaxis, adhesion, phagocytosis, and traf- fic of various hemopoietic cells (1). In addition, WASP controls the assembly of synaptic structures among different cells of the immune system (2, 3). In T cells, upon triggering of TCR and costimulatory molecules, WASP is recruited to the immunological synapse and is required for its organization (4, 5). Regulation of WASP localization is under the control of WASP-interacting pro- tein and Nck adaptor protein/SH2-domain containing leukocyte protein of 76 kDa (6 – 8), while its activation status is modulated by binding to the small Rho GTPase Cdc42 and by phosphoryla- tion (9 –13). The active form of WASP induces actin nucleation through binding to actin-related protein 2/3 (14). The molecular mechanisms by which WASP integrates multiple signals to pro- mote actin polymerization and control immunological synapse as- sembly have been extensively studied. However, the role of WASP in controlling downstream signaling and, more in general, T cell functions is still largely unknown. Mutations in the WAS gene in humans may lead to different clinical phenotypes, ranging from WAS (15), characterized by mi- crothrombocytopenia, eczema, immunodeficiency, autoimmune disorders, and high incidence of lymphoid malignancies (1), to X-linked thrombocytopenia (XLT), a milder form of the disease with minimal or no immunodeficiency (16, 17). A strong correla- tion between expression of WASP in lymphocytes and clinical phenotype has been recently reported in patients carrying WAS gene mutations (18, 19). Although WASP deficiency affects the functions of both lymphoid and myeloid cells, results obtained from the follow-up of WAS patients who underwent HLA- matched hemopoietic stem cell transplant, and from the analysis of WAS patients with spontaneous genetic reversion, indicate that WASP expression confers a selective growth advantage to T cells only (20, 21). Consistently, it has been shown that WAS patients have a reduction in the numbers of circulating T lymphocytes, especially within the naive compartment (22). WAS patients suffer from recurrent infections caused by encapsulated bacteria, viruses, and fungi (19), suggesting an impairment in cellular-mediated im- munity. Moreover, they have a high susceptibility to develop lym- phomas (19) which could be due to poor immune surveillance. However, T cell functions are not completely abrogated in WAS patients, as illustrated by the high frequency of T cell-mediated autoimmune manifestations (23). T cells from WAS patients pro- liferate poorly, secrete low levels of IL-2 in response to anti-CD3 mAb and display abnormal cell morphology (4, 24, 25). Defective secretion of IL-2, IFN-, TNF-, and IL-4 in response to TCR or TCR/CD28-mediated stimulation has been shown in WASP knockout mice (26 –29). Although proximal TCR signaling is not significantly affected in T cells from WASP knockout mice (26), WASP seems to be required for downstream events, including ac- tivation of NFAT-1 (NFAT-p), NFAT-2 (NFAT-c), and expres- sion of Fos (13, 30, 31). Similar results have been obtained in NK *San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; † Immunopathogen- esis of Liver Infections Unit, San Raffaele Scientific Institute, Milan, Italy; ‡ Vita- Salute San Raffaele University, Milan, Italy; § Department of Molecular and Experi- mental Medicine, The Scripps Research Institute, La Jolla, CA 92037; ¶ Department of Pediatrics, University of Turin, Turin, Italy; and  Department of Pediatrics, Uni- versity of Brescia, Spedali Civili, Brescia, Italy Received for publication March 8, 2006. Accepted for publication August 16, 2006. 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 This work was supported by grants from the Italian Telethon Foundation and the Italian Ministry of University and Research (MIUR/FIRB No. RBNE03FMCJ-008). 2 Address correspondence and reprint requests to Dr. Maria-Grazia Roncarolo, San Raffaele Telethon Institute for Gene Therapy, Via Olgettina 58, 20132, Milan, Italy. E-mail address: m.roncarolo@hsr.it 3 Abbreviations used in this paper: WAS, Wiskott-Aldrich syndrome; WASP, WAS protein; h, human; XLT, X-linked thrombocytopenia; TPA, 12-O-tetradecanoylphor- bol-13-acetate; RPA, RNase protection assay; HD, healthy donor. The Journal of Immunology Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00