Influence of Membrane CD25 Stability on T Lymphocyte Activity: Implications for Immunoregulation Todd M. Brusko 1,2 , Clive H. Wasserfall 1 , Maigan A. Hulme 1 , Roniel Cabrera 4 , Desmond Schatz 3 , Mark A. Atkinson 1 * 1 Department of Pathology, University of Florida, Gainesville, Florida, United States of America, 2 Diabetes Center at the University of California San Francisco, San Francisco, California, United States of America, 3 Department of Pediatrics, University of Florida, Gainesville, Florida, United States of America, 4 Department of Medicine, University of Florida, Gainesville, Florida, United States of America Abstract Background: CD25, a component of the IL-2 receptor, is important in T cell proliferation, activation induced cell death, as well as the actions of both regulatory (Treg) and effector (Teff) T cells. Recent genome wide association studies have implicated the CD25 locus as an important region for genetic susceptibility to a number of autoimmune disorders, with serum levels of soluble CD25 receptor (sCD25) serving as a potential phenotypic marker for this association. However, the functional impact of CD25 cleavage, as well as the influence of sCD25 on immunoregulatory activities, remain largely unknown and form the basis of this effort. Methodology/Principal Findings: The generation of sCD25 by Treg (CD4 + CD25 + ) and Teff (CD4 + CD25 2 ) cells was examined during in vitro suppression assays, efforts that demonstrated constitutive and stable surface CD25 expression on Treg throughout the period of in vitro assessment. In contrast, Teff cells increased CD25 expression during the process of in vitro suppression, with supernatant sCD25 levels correlating to the amount of cellular proliferation. Interestingly, under serum- free conditions, Tregs partially lost their characteristic anergic and suppressive properties. sCD25 supplementation at physiological concentrations to serum free in vitro suppression assays reduced Teff proliferation without specifically influencing suppression. Indeed, sCD25 production within these cultures correlated with cell death. Conclusions/Significance: These results support the notion that sCD25 functions as both a surrogate marker of T cell activation as well as an indicator of subsequent cellular death. In addition, the role of CD25 in immunomodulation is likely dependent on the local inflammatory milieu, with molecules capable of modulating surface CD25 expression playing a key role in defining immune responsiveness. Citation: Brusko TM, Wasserfall CH, Hulme MA, Cabrera R, Schatz D, et al. (2009) Influence of Membrane CD25 Stability on T Lymphocyte Activity: Implications for Immunoregulation. PLoS ONE 4(11): e7980. doi:10.1371/journal.pone.0007980 Editor: Derya Unutmaz, New York University School of Medicine, United States of America Received July 7, 2009; Accepted October 3, 2009; Published November 24, 2009 Copyright: ß 2009 Brusko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These studies were supported, in part, by grants from The National Institutes of Health (AI42288 and AI39250), the American Diabetes Association, the Juvenile Diabetes Research Foundation (JDRF) (Post Doctoral Fellowship to TMB), and the Brehm Coalition for Type 1 Diabetes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: atkinson@ufl.edu Introduction Interleukin-2 (IL-2) has long been recognized for its role in promoting T cell proliferation in vitro, yet only recently has appreciation grown for its paradoxical role in maintaining peripheral tolerance in vivo. One key means by which IL-2 appears to promote tolerance involves its influence over CD4 + CD25 + T cells, often termed regulatory T cells (Treg). In 1995, Sakaguchi and colleagues identified the a-chain of the IL-2 receptor (CD25) as a putative marker of functional Treg [1]. The high-affinity, trimeric IL-2 receptor complex consists of three major sub-units; the a-chain (designated CD25), the b-chain (CD122), and the common cytokine receptor c-chain (CD132) [2]. These subunits, as well as other fragments of the molecule, appear to play important roles not only in immune physiology, but as markers of immune activity. The concept that the expression level and affinity of cell surface receptors dictates cellular fate was introduced as the quantal theory by Kendall A Smith [3]. In activated T cells, signaling through the IL-2 receptor results in increased high-affinity receptor expression and eventual activation induced cell death (AICD) through up-regulation of Fas and FasL [4,5]. It has been known since 1985 that production of the soluble form of CD25 (sCD25) associates with T cell activation in vitro [6]. In healthy individuals, sCD25 is present in serum at approximately 2 ng/ml, whereas increased serum sCD25 levels have been associated with lymphocyte activation in infection as well as in hematologic malignancies [7,8]. More recently, polymorphisms in the CD25 gene have been associated with autoimmune diseases such as type 1 diabetes, multiple sclerosis, and Graves’ disease; with measurable differences in serum levels of the soluble form of the corresponding molecule (i.e., sCD25) [9–12]. These data point to, but do not specifically identify, a functional role for sCD25 separate from its value as a biomarker of activation in serum. Indeed, several key questions regarding sCD25 remain unanswered. These include those asking what biological actions result in the formation of PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e7980