CUTTING EDGE IMMUNOLOGY THE OF JOURNAL Cutting Edge: A Regulatory T Cell-Dependent Novel Function of CD25 (IL-2R) Controlling Memory CD8  T Cell Homeostasis 1 Rahul Sharma, Lingjie Zheng, Umesh S. Deshmukh, Wael N. Jarjour, Sun-sang J. Sung, Shu Man Fu, 2 and Shyr-Te Ju 2,3 A massive systemic expansion of CD8  memory T (T M ) cells and a remarkable increase in circulating IL-2 were observed only in IL-2R (CD25) knockout (KO) mice but not in IL-2 KO and scurfy mice, although all three mutants lack regulatory T (Treg) cells. However, both phenotypes were suppressed by the transfer of Treg cells. The data presented indicate that Treg cell deficiency drives naive T cells to T M cells. The lack of high-affinity IL-2R in IL-2R KO mice increases circulating IL-2 that is then preferentially used by CD8  T M cells through its abundant low-affinity IL-2R, resulting in systemic CD8  T M cell dominance. Our study demonstrates the critical control of CD8  T M cell homeostasis by a Treg cell-de- pendent novel function of CD25 and resolves its mecha- nism of action. The Journal of Immunology, 2007, 178: 1251–1255. U nder normal conditions, thymic selection generates two sets of T cells: a CD4 + CD25 + regulatory T (Treg) 4 cell set that suppresses Ag-specific responses and a CD4 + (and CD8 + ) CD25 - single-positive T cell set that responds to Ags in the host. The conditions required for the generation, maintenance, and functions of Treg cells have been defined (1, 2). Treg cell selection involves high-affinity TCR/ peptide plus MHC interaction and is skewed toward self-Ags (3). In addition, it requires the presence of the Foxp3 transcrip- tion factor (4). In the periphery, the maintenance of Treg cells requires high-affinity IL-2/IL-2R interaction (5) and TGF- (6). Thus, mice with targeted mutation of IL-2, IL-2R, IL- 2R (CD122), and TGF- are defective in Treg cell expres- sion. The requirement for high-affinity IL-2R for Treg cell maintenance puts CD25 as the major marker for Treg cells. The primary function of Treg cells is to keep immune re- sponses in check by inhibiting the activation of Ag-specific T cells, including autoimmune response (1, 2). However, recent studies based on mAb treatment have suggested that Treg cells also regulate CD8 + memory T (T M ) cell expression, but the precise mechanism remains unclear (7–9). Indeed, the loss of CD8 + T M cell homeostasis control in Treg cell-deficient strains has not been established. In this study, we describe our analysis of CD8 + T M cell homeostasis in three Treg cell-deficient strains: IL-2 knockout (KO), IL-2R KO, and scurfy. Interest- ingly, systemic CD8 + T M cell dominance was observed only in IL-2R KO mice, and only IL-2R KO mice displayed very high levels of serum IL-2, a factor critical to CD8 + T M cell ho- meostatic expansion. Significantly, both phenotypes were sup- pressed by CD4 + CD25 + Treg cells. Our study demonstrates the critical role of CD25 and the novel molecular mechanism in the CD25-mediated regulation of CD8 + T M cell homeostasis by controlling Treg cells and IL-2 expression. Materials and Methods Mice C57BL/6 (B6) mice, B6.Il2 +/- mice B6.Il2R +/- mice, B6.Cg-Foxp3 sf/x /J, and B6.SJL-Ptprc a Pepc b /BoyJ (B6.CD45.1) mice were obtained from The Jack- son Laboratory. B6.Il2 +/- mice and B6.Il2R +/- mice were bred to obtain B6.Il2 -/- (IL-2 KO) and B6.Il2R -/- (IL-2R KO) offspring, respectively. Scurfy mice were obtained by breeding female B6.Foxp3 sf/x mice with male B6 mice. Flow cytometric analysis Single-cell suspensions were prepared from blood and various lymphoid and nonlymphoid organs as described (10). Cells were stained with fluorescently labeled Abs and analyzed by flow cytometry. FITC-, PE-, PE-Cy5-, or biotin- conjugated mAb against CD4 (clone GK1.5), CD8 (clone 53-6.7), Thy-1.2 (clone 30H12), CD45.1 (clone A20), CD44 (clone IM7), CD62L (clone MEL-14), CD69 (clone H1.2F3), IL-2R (clone PC61), and Ly-6C were ob- tained from BD Biosciences. Alexa 488-conjugated streptavidin was obtained from Invitrogen Life Technologies. Intracellular Foxp3 was detected using the Foxp3 staining kit (eBioscience). The total cell number for CD4 + and CD8 + T cells in each sample was determined as described previously (10). Division of Rheumatology and Immunology, Department of Internal Medicine, Univer- sity of Virginia, Charlottesville, VA 22908 Received for publication September 27, 2006. Accepted for publication November 29, 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 National Institutes of Health Grants AI-036938, DE-017579 and AR-051203 (to S.-T.J.), AR-045222, AR-047988 and AR-049449 (to S.M.F.), HL-70065 (to S.-S.J.S.), AR-051391 (to U.S.D.), and DK-059850 (to W.N.J.). 2 S.M.F. and S.-T.J. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Shyr-Te Ju, Division of Rheu- matology and Immunology, Department of Internal Medicine, P.O. Box 800412, Old Medical School Building, Room 5777, University of Virginia, Charlottesville, VA 22908-0412. E-mail address: sj8r@virginia.edu 4 Abbreviations used in this paper: Treg, regulatory T; KO, knockout; T M , memory T; T N , naive T. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org