Enhanced T Cell Proliferation in Mice Lacking the p85 Subunit of Phosphoinositide 3-Kinase 1 Jonathan A. Deane,* Matthew J. Trifilo,* Claudine M. Yballe, † Sangdun Choi, ‡ Thomas E. Lane,* and David A. Fruman 2 * Phosphoinositide 3-kinase activation is important for lymphocyte proliferation and survival. Disrupting the gene that encodes the major phosphoinositide 3-kinase regulatory isoform p85 impairs B cell development and proliferation. However, T cell functions are intact in the absence of p85. In this study, we test the hypothesis that the related isoform p85 is an essential regulatory subunit for T cell signaling. Unexpectedly, T cells lacking p85 showed a marked increase in proliferation and decreased death when stimulated with anti-CD3 plus IL-2. Both CD4  and CD8  T cells completed more cell divisions. Transcriptional profiling revealed reduced levels of caspase-6 mRNA in p85-deficient T cells, which was paralleled by reduced caspase-6 enzyme activity. Increased T cell accumulation was also observed in vivo following infection of p85-deficient mice with mouse hepatitis virus. Together, these results suggest a unique role for p85 in limiting T cell expansion. The Journal of Immunology, 2004, 172: 6615– 6625. P hosphoinositide 3-kinases (PI3Ks) 3 phosphorylate inositol phospholipids, thereby promoting membrane association of certain cytoplasmic proteins that specifically bind to PI3K lipid products (1–3). PI3K signaling promotes proliferation and survival in many cell types, both normal and transformed (3, 4). Cellular levels of PI3K products increase following engage- ment of the B cell receptor (BCR) and the TCR (5, 6). Elegant microscopy studies from two groups recently demonstrated that the PI3K lipid product phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) accumulates at the T cell plasma membrane and is concen- trated at the APC contact zone in an Ag-dependent manner (7, 8). Inhibitors of PI3K enzyme activity (wortmannin, LY294002) block Ag receptor-mediated proliferation of both T and B cells, illustrating the importance of PI3K signaling (9 –12). Inhibitor studies also have demonstrated a role for PI3K in cytokine-driven proliferation and survival of lymphocytes (2, 11, 13). PI3K appears to regulate both shared and distinct pathways in T and B cells. For example, activation of Akt is PI3K dependent in both cell types (14), whereas calcium mobilization is more dependent on PI3K in B cells than in T cells (8, 15). There are four subgroups of PI3K with distinct functions in cells (1, 3, 16). Class I A PI3Ks, which function downstream of activated tyrosine kinases, are heterodimers composed of a catalytic subunit and a tightly associated regulatory subunit. The regulatory subunit influences both the activity and localization of the catalytic sub- unit. There are three known genes for each component of the het- erodimer. The Pik3ca, Pik3cb, and Pik3cd genes encode the cat- alytic subunits p110, p110, and p110. The Pik3r1, Pik3r2, and Pik3r3 genes encode regulatory subunits p85, p85, and p55. Two additional products of the Pik3r1 gene encode the proteins p55 and p50. The different regulatory subunits can associate with the catalytic subunits interchangeably, but have unique tissue distributions. Each regulatory subunit possesses two highly con- served Src homology 2 (SH2) domains and a p110-binding domain in the C-terminal portion. The smaller isoforms possess short N- terminal segments before the first SH2 domain. The N-terminal portions of p85 and p85 have a similar set of domains (SH3, RhoGAP homology, proline rich), but less primary sequence iden- tity, suggesting that this region may mediate distinct functions. Lymphocytes express each of the catalytic isoforms as well as the regulatory isoforms p85 and p85 (11, 14). T cells also ex- press p50. Genetic studies in mice have begun to elucidate the specific function of class I A PI3K isoforms in lymphocytes. Mice lacking only p85 or lacking all variants of Pik3r1 (p85, p55, and p50) show comparable immune phenotypes (11, 17). Spe- cifically, B cell development and proliferation are impaired, whereas T cell development and function are apparently normal. In contrast, a functional p110 catalytic subunit is required for opti- mal proliferation of both T and B cells (14, 18, 19). The demon- stration that T cells require class I A PI3K signaling, but not p85 or its variants, suggested that p85 may be the essential class I A regulatory subunit for T cell proliferation and development. In this study, we test this hypothesis by analyzing lymphocyte develop- ment and proliferation in mice lacking p85. We show that B cell development and function in p85 knockout mice are indistin- guishable from wild type. In contrast, we observe a surprising enhancement of proliferation in p85-deficient T cells in vitro and a sustained expansion of T cells in vivo following viral infection. Materials and Methods Mice The generation of mice lacking p85 (Pik3r2 null) was described in detail elsewhere (20). Mice were maintained in a mixed background (C57BL/6  129SvEv) for the experiments shown in this work and were studied at 2– 4 *Department of Molecular Biology and Biochemistry, University of California, Ir- vine, CA 92697; † Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215; and ‡ Division of Biology, California Institute of Tech- nology, Pasadena, CA 91125 Received for publication June 30, 2003. Accepted for publication March 29, 2004. 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 National Institutes of Health Grants to D.A.F. (AI50831) and T.E.L. (NS41429), and a National Multiple Sclerosis Society Grant (R63278-A-3) to T.E.L. 2 Address correspondence and reprint requests to Dr. David A. Fruman, University of California, Department of Molecular Biology and Biochemistry, 3242 McGaugh Hall, Irvine, CA 92697-3900. E-mail address: dfruman@uci.edu 3 Abbreviations used in this paper: PI3K, phosphoinositide 3-kinase; AICD, activa- tion-induced cell death; BCR, B cell receptor; MHV, mouse hepatitis virus; NP, nitrophenol; PIP 3 , phosphatidylinositol-3,4,5-trisphosphate; PLC, phospholipase C; SH, Src homology. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00