SAP Regulation of Follicular Helper CD4 T Cell Development and Humoral Immunity Is Independent of SLAM and Fyn Kinase 1 Megan M. McCausland,* Isharat Yusuf,* Hung Tran,* Nobuyuki Ono, † Yusuke Yanagi, ‡ and Shane Crotty 2 * Mutations in SH2D1A resulting in lack of SLAM-associated protein (SAP) expression cause the human genetic immunodeficiency X-linked lymphoproliferative disease. A severe block in germinal center development and lack of long-term humoral immunity is one of the most prominent phenotypes of SAP  mice. We show, in this study, that the germinal center block is due to an essential requirement for SAP expression in Ag-specific CD4 T cells to develop appropriate follicular helper T cell functions. It is unknown what signaling molecules are involved in regulation of SAP-dependent CD4 T cell help functions. SAP binds to the cytoplasmic tail of SLAM, and we show that SLAM is expressed on resting and activated CD4 T cells, as well as germinal center B cells. In addition, SAP can recruit Fyn kinase to SLAM. We have now examined the role(s) of the SLAM-SAP-Fyn signaling axis in in vivo CD4 T cell function and germinal center development. We observed normal germinal center development, long-lived plasma cell development, and Ab responses in SLAM / mice after a viral infection (lymphocytic choriomeningitis virus). In a separate series of experiments, we show that SAP is absolutely required in CD4 T cells to drive germinal center development, and that requirement does not depend on SAP-Fyn interactions, because CD4 T cells expressing SAP R78A are capable of supporting normal germinal center development. Therefore, a distinct SAP signaling pathway regulates follicular helper CD4 T cell differentiation, separate from the SLAM-SAP-Fyn signaling pathway regulating Th1/Th2 differentiation. The Journal of Immunology, 2007, 178: 817– 828. T he gene SH2D1A (SAP/DSHP) encodes SLAM-associ- ated protein (SAP). 3 Mutations in SH2D1A resulting in lack of SAP expression or destabilized SAP protein are the cause of the human genetic immunodeficiency X-linked lym- phoproliferative disease (XLP) (1–3). XLP patients exhibit a va- riety of immunological defects. The most notable and problematic symptoms of SAP-deficient humans are the inability to control EBV infection (as well as difficulties with other infections, includ- ing measles virus, Neisseria meningitidis, and vaccinia virus), the development of progressive hypogammaglobulinemia, and a heightened occurrence of B cell lymphomas (4 –7). Before recent improvements in diagnosis and treatment, most XLP patients died before reaching adulthood. SAP is a small SH2-domain protein expressed in CD4 T cells, CD8 T cells, NK cells, NKT cells, and some B cells (3, 8, 9). SAP is now known to play a role in an impressive array of lymphocyte functions. Examination of SAP-deficient mice revealed roles for SAP in CD8 and CD4 T cell proliferation (10 –14), T cell IFN- production (10, 11, 15), CD4 T cell Th1/Th2 differentiation (10, 11, 16, 17), and germinal center formation, and the generation of memory B cells (12, 18). Recent work has also shown that SAP is essential for the development of NKT cells, both in mice and hu- mans (19 –21). Work in XLP humans has confirmed the memory B cell defect and germinal center defect observed in SAP - mice (22– 24). Separately, a collection of studies in humans have shown that SAP plays multifaceted roles in the regulation of NK cell killing (25–27). How does SAP regulate or influence such a wide range of lymphocyte functions? SAP binds to the cytoplasmic tail of SLAM (CD150) via an ITAM/ITIM-related motif termed an immunoty- rosine switch motif (ITSM) (3, 28, 29). SLAM plays roles in cell adhesion (via homotypic interactions), costimulation, and cytokine synthesis (28, 30 –33). SLAM is also the primary receptor for mea- sles virus, a highly lymphotropic virus (34 –36). A cluster of seven receptors related to SLAM are closely grouped together on chromosome 1 of both mice and humans. These genes are termed the SLAM family of receptors: CD150 (SLAM), CD244 (2B4), CD229 (Ly9), CS1 (CRACC), CD48, CD84, and LY108 (NTB-A) (4, 5, 28). All of these genes encode surface receptors with at least two ITSM motifs in their cytoplasmic tail, except CD48, which has no cytoplasmic tail (28). Intriguingly, a major systemic lupus erythematosus genetic susceptibility locus maps to the SLAM family receptors in both human and mice (37, 38). SAP is known to bind to SLAM, CD244, CD229, CD84, and Ly108/NTB-A (3, 26, 39, 40). SAP binding to these receptors is not identical, because SAP can bind to SLAM in the presence or *Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; † Department of Medical Biophysics and Immunology, Princess Margaret Hospital, Toronto, Canada; and ‡ Department of Virology, Faculty of Med- icine, Kyushu University, Fukuoka, Japan Received for publication October 19, 2005. Accepted for publication October 27, 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 in part by a Cancer Research Institute Investigator Award (to S.C.) and a Pew Scholar Award (to S.C.). 2 Address correspondence and reprint requests to Dr. Shane Crotty, Division of Vac- cine Discovery, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92121. E-mail address: shane@liai.org 3 Abbreviations used in this paper: SAP, SLAM-associated protein; XLP, X-linked lymphoproliferative disease; ITSM, immunotyrosine switch motif; MHCII, MHC class II; LCMV, lymphocytic choriomeningitis virus; PNA, peanut lectin agglutinin; QPCR, quantitative real-time PCR; WT, wild type. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 The Journal of Immunology www.jimmunol.org