Immunology Letters 132 (2010) 61–68 Contents lists available at ScienceDirect Immunology Letters journal homepage: www.elsevier.com/locate/ IL-2-deprivation and TGF- are two non-redundant suppressor mechanisms of CD4 + CD25 + regulatory T cell which jointly restrain CD4 + CD25 - cell activation Guohua Wang a,b,1 , Mithun Khattar a,1 , Zhiyong Guo a,c,1 , Yoshihiro Miyahara a , Sean P. Linkes a , Zongquan Sun b , Xiaoshun He c , Stanislaw M. Stepkowski a,∗ , Wenhao Chen a,∗ a Department of Medical Microbiology and Immunology, University of Toledo College of Medicine, Toledo, OH 43614, United States b Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China c Organ Transplant Center, 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, PR China article info Article history: Received 18 November 2009 Received in revised form 17 May 2010 Accepted 1 June 2010 Available online 8 June 2010 Keywords: IL-2 TGF- Suppressor mechanism Regulatory T cell summary The benefits of immunotherapy by regulatory T (Treg) cells are unpredictable partially due to the uncer- tainty of their suppressive mechanism. In fact, various suppressive mechanisms have been proposed but each remains controversial. To better understand Treg-mediated suppression, we have investigated fac- tors which may influence the suppressive effects. In an in vitro suppression assay, over-expression of anti-apoptotic Bcl2 enhancing survival of conventional T responder cells (Tconvs) did not subvert Treg- mediated suppression. In contrast, enhancing activation of Tconvs by increasing the potency of calcium signals completely abrogated Treg-mediated suppression. While Tregs were incapable of suppressing already activated Tconvs, they prevented expression of activation markers on naïve Tconvs during acti- vation, thereby indicating that Tregs mediate suppression through controlling early activation stage. Interestingly, IL-2 deprivation or TGF-, two suppressive mechanisms, did not effectively inhibit Tconv activation and proliferation when applied alone. In contrast, IL-2 deprivation combined with TGF- sup- pressed Tconv activation as potently as Tregs. More importantly, in the transwell system, that separates Tregs from Tconvs, TGF- contributed to Treg suppression under IL-2 depriving condition. In conclusion, these two suppressive mechanisms acting in concert may be necessary to effectively restrain the early activation of Tconvs. © 2010 Elsevier B.V. All rights reserved. 1. Introduction While CD4 + T cells play a central role in establishing and max- imizing various immune responses, they also contain a unique subpopulation that suppresses these responses. Sakaguchi et al. identified CD25 (IL-2R) as a marker for this CD4 + suppressor cell population, named regulatory T cell (Treg). Their study showed that CD4 + CD25 + Tregs prevented CD4 + CD25 - cell-mediated autoim- mune diseases in athymic nude mice [1]. In 2003, the same and other groups demonstrated that the Foxp3 transcription fac- tor was predominantly expressed in CD4 + CD25 + cells. The stable expression of Foxp3 is critical for establishing and maintaining Treg lineage [2–5]. Mutations of the Foxp3 gene can result in lethal autoimmunity both in mouse and human [2–5], proving the requirement for Tregs in maintaining self-tolerance. ∗ Corresponding authors at: Department of Medical Microbiology and Immunol- ogy, University of Toledo-Health Science Campus, 3000 Arlington Avenue, HEB 210, Toledo, OH 43614, United States. Tel.: +1 419 383 6673; fax: +1 419 383 3002. E-mail addresses: Stanislaw.stepkowski@utoledo.edu (S.M. Stepkowski), Wenhao.chen@utoledo.edu (W. Chen). 1 These authors contributed equally to this work. Given its suppressive nature, it is of great interest to explore the therapeutic effects of Tregs in immune diseases. However, not fully defined suppressive mechanisms of Tregs shroud potential barriers in developing Treg-based immunotherapy. In fact, mul- tiple suppressor mechanisms have been proposed but their exact roles are subjects of intense debates. Tregs suppress conventional T cells (Tconvs) by secreting suppressive cytokines (TGF-, IL-10, and IL-35) [6–8], by competing with Tconv for IL-2 [9], by expressing suppressive molecules i.e. galectin-1 on their cell surface [10], or by killing effector T cells [11]. In addition, Tregs have been shown to suppress APCs, which in turn fail to activate Tconvs, as Tregs express cell surface molecules (e.g. CTLA-4, LAG-3, CD39, and Nrp- 1) that directly influence APC function [8,12]. Not surprisingly, these increasing number of mechanisms has been associated with numerous controversial issues related to the fact that abrogating only one mechanism may not reverse Treg suppression [13–15]. It is possible and rather likely that two or more timely interacting mech- anisms produce effective Treg suppression. Hence, we explored different conditions that may limit inhibitory effects and exam- ined how coordinated suppressive factors may produce optimal suppression. Difficulties had emerged from observations that Tregs in totally TGF-1-deficient mice were either protective or nonfunctional in 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2010.06.001