+ + Dendritic Cells Control CD4 CD25 Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2 Katarina Kulhankova2, Todd Rouse1, Mohamed E. Nasr1, Elizabeth H. Field1,2* 1 Department of Veterans Affairs Medical Center, Iowa City, Iowa, United States of America, 2 Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa, United States of America Abstract CD4+CD25+Foxp3+ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4+ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-22/2 DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-22/2 DCs. Exogenous IL-2, added in excess of 500 U/ml to co- cultures with IL-22/2 DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function. Citation: Kulhankova K, Rouse T, Nasr ME, Field EH (2012) Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2. PLoS ONE 7(9): e43609. doi:10.1371/journal.pone.0043609 Editor: Derya Unutmaz, New York University, United States of America Received June 12, 2012; Accepted July 26, 2012; Published September 12, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This material is based upon work supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development with VA Merit Review Grant (EHF) and VA MREP Grant (MEN), and also NIH R21 DE19609- 01(EHF), NIH T32 AI007260-22 (KK). 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: elizabeth-field@uiowa.edu Introduction Natural CD4+CD25+Foxp3+ T regulatory cells (Tregs) comprise only about 1–10% of the pool of CD4+ cells, but because they develop and maintain peripheral tolerance to autoantigens, neo- antigens, and foreign antigens [1], [2] they are the primary cells responsible for limiting inflammatory adaptive immune responses. Moreover, their power can extend even to curtailing immunity to pathogens [3] and tumor antigens [4], [5]. Although therapies aimed at enhancing or preventing Treg function are being explored across medical disciplines, an inability to identify unique requirements for Treg activation has remained a barrier to their use in the management of immunologic disease. To date, agents known to expand and activate Tregs have risked enhancing conventional T cell contamination ex vivo [6] and conventional T cell activation in vivo [7]. Types of Treg-based therapies underway have included efforts to intensify immune tolerance to the autoantigen in type-1 diabetes patients [8], [9], [10], the donor- HLA antigen in solid organ transplant recipients [11], [12], and the host antigen in hematopoietic cell transplant recipients with graft-versus-host-disease [13]. The approach of such therapies has been to increase Treg activity by infusing ex vivo expanded Tregs or by introducing biologics or small-molecule chemical compounds that promote Treg development in vivo [14], [15]. In addition to the aforementioned indications, efforts are underway to modify cancer immunotherapy to decrease Treg activity [16], [17], since reports that conventional cancer immunotherapy may expand Tregs and accelerate tumor growth [18], [19], [20]. Collectively, the successful application of all Treg-based therapies will require a thorough knowledge of the mechanisms that control Treg function. Treg expansion and Treg fitness both contribute to Treg effectiveness in suppressing immune responses. In particular, the cytokine IL-2 is recognized as essential to Treg expansion and survival [21]. However, its role in Treg suppressor function has been less clear [22], in part because it is difficult to uncouple Treg function from Treg survival in vivo. The biological effect of IL-2 is mediated through its unique IL-2 receptor. The IL-2 receptor consists of three subunits: the CD25 alpha (a) chain, the CD122 beta (b) chain, and the CD132 common cytokine gamma (c) chain [23]. IL-2 binding to CD25 initiates the assembly of the high affinity IL-2 receptor signaling complex [24], [25]. Studies of knock-out (KO) mice show that in vivo Treg development and peripheral expansion require (i) IL-2 from a PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e43609