Functional and Molecular Comparison of Anergic and Regulatory T Lymphocytes Birgit Knoechel, Jens Lohr, Shirley Zhu, Lisa Wong, Donglei Hu, Lara Ausubel and Abul K. Abbas This information is current as of September 29, 2021. J Immunol 2006; 176:6473-6483; ; doi: 10.4049/jimmunol.176.11.6473 http://www.jimmunol.org/content/176/11/6473 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2006/05/18/176.11.6473.DC1 Material References This article cites 54 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/176/11/6473.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 29, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Functional and Molecular Comparison of Anergic and Regulatory T Lymphocytes1 Birgit Knoechel,2* Jens Lohr,2* Shirley Zhu,† Lisa Wong,† Donglei Hu,† Lara Ausubel,3* and Abul K. Abbas4* Tolerance in vivo is maintained by multiple mechanisms that function to prevent autoimmunity. An encounter of CD4؉ T cells with a circulating self-Ag leads to partial thymic deletion, the development of CD25؉ regulatory T cells (Tregs), and functional anergy in the surviving CD25؊ population. We have compared anergic and regulatory T cells of the same Ag specificity generated in vivo by the systemic self-Ag. Anergic cells are unresponsive to the self-Ag that induces tolerance, but upon transfer into a new host and immunization, anergic cells can induce a pathologic autoimmune reaction against tissue expressing the same Ag. Tregs, in contrast, are incapable of mediating harmful reactions. To define the basis of this functional difference, we have compared gene expression profiles of anergic and regulatory T cells. These analyses show that Tregs express a distinct molecular signature, but Downloaded from anergic cells largely lack such a profile. Anergic cells express transcripts that are associated with effector differentiation, e.g., the effector cytokines IL-4 and IFN-␥. Anergic cells do not produce these cytokines in response to self-Ag, because the cells exhibit a proximal signaling block in response to TCR engagement. Thus, anergy reflects an aborted activation pathway that can readily be reversed, resulting in pathologic effector cell responses, whereas Treg development follows a distinct developmental pathway that extinguishes effector functions. The Journal of Immunology, 2006, 176: 6473–6483. http://www.jimmunol.org/ cell tolerance to self-Ags is maintained by three principal To address these questions, we have generated a Tg mouse that mechanisms: anergy (functional unresponsiveness), dele- expresses a secreted form of OVA (sOVA) in the circulation T tion (apoptotic death), and suppression by regulatory T (sOVA Tg mouse) and crossed these animals with TCR Tg mice lymphocytes (1–7). Many of the studies on which this conclusion expressing the DO11.10 (referred to hereafter as DO11) OVA- is based have relied on cloned cell lines or polyclonal stimuli (8– specific TCR. This is the first experimental system in which both 10). Much less has been done to address the mechanisms of T cell anergic and regulatory T cells are induced by a single self-Ag in tolerance induced in normal T cells by self-Ags, especially in vivo. the same Ag-specific population, and thus it is possible to formally 5 by guest on September 29, 2021 Transgenic (Tg) mouse models are valuable for defining the fates compare the two cell populations. In this work we show that an- of T cells that encounter their cognate Ag in different forms. A ergic and regulatory T cells can be distinguished on the basis of large number of studies have shown that deletion, anergy, and function and gene expression profiles. Microarray analysis re- ϩ suppression by CD25 regulatory T cells (Tregs) are all demon- vealed that anergic CD4ϩ T cells express very few specific genes. strable with Ag receptor Tg T cells (11–17). However, few studies However, they express abundant mRNA for cytokine genes such have formally compared the properties of anergic and regulatory T as Ifn-␥ and Il4 that are typically assigned to Th cells with effector cells induced in one T cell population by one Ag. In particular, function, suggesting that anergic cells in this setting may be given the considerable interest in inducing T cell anergy as a ther- “poised” to become true effector cells. We demonstrate that this apeutic strategy, it is important to ask whether this may be more or differentiation also occurs if tolerance is induced de novo when T less effective than generating Tregs. It is also unclear whether the cells newly encounter a tolerogenic Ag in an adoptive transfer biochemical and molecular characteristics of anergic and regula- model (18). Similar to T cells that have encountered self-Ag during tory T cells are distinct or show some overlap. development, the transferred T cells express IFN-␥ mRNA within four days after encounter with the self-Ag. Anergic T cells can *Department of Pathology and †Diabetes Center, University of California San Fran- even be triggered to secrete IFN-␥ upon stimulation in vivo. How- cisco School of Medicine, San Francisco, California 94143 ever, at the same time anergic cells develop a proximal signaling ϩ Received for publication August 9, 2005. Accepted for publication March 15, 2006. defect leading to decreased Ca2 mobilization upon TCR stimu- The costs of publication of this article were defrayed in part by the payment of page lation. Importantly, we show that the state of anergy can be broken charges. This article must therefore be hereby marked advertisement in accordance in that anergic T cells are unresponsive to the systemic self-Ag but with 18 U.S.C. Section 1734 solely to indicate this fact. retain their ability to trigger severe pathologic autoimmunity. In 1 This work was supported by National Institutes of Health Grant PO1 AI35297 and Deutsche Forschungsgemeinschaft Fellowships KN 533/1-1 and LO 808/1-1. contrast, regulatory T cells are incapable of causing harmful au- 2 These authors contributed equally to this work. toimmune reactions, and they express a large number of genes that 3 Current address: City of Hope Medical Center, Duarte, CA 91010. are specific to this lineage. Our studies suggest that anergy induc- tion is a double-edged sword in which a signaling block and 4 Address correspondence and reprint requests to Dr. Abul K. Abbas, Department of Pathology, University of California San Francisco, 505 Parnassus Avenue, M590, San aborted effector responses develop simultaneously and that this Francisco, CA 94143-0511. E-mail address: [email protected] balance may be easily tipped. Regulatory T cells, in contrast, are 5 Abbreviations used in this paper: Tg, transgenic; HPRT, hypoxanthine phosphori- incapable of effector responses, suggesting that they develop by a bosyltransferase; mOVA, membrane-bound OVA; sOVA, soluble form of OVA; RIP, rat insulin promoter; RMA, robust multiarray average; Treg, CD25ϩ regulatory T distinct pathway. These data have implications for a variety of cell. approaches attempting to induce tolerance in vivo. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 6474 ANERGIC AND REGULATORY T LYMPHOCYTES Materials and Methods from DO11 mice were used as responders and stimulated on 25,000 APCs with 1 ␮g/ml OVA. The input of responder cells remained constant. KJ1- Mice ϩ ϩ ϩ ϩ ϩ Ϫ 26 CD4 CD25 and KJ1-26 CD4 CD25 T cells from double-Tg mice All experimental mice were used at 6–12 wk of age. All mice were age and were used as suppressors and, starting from equal numbers, were titrated in sex matched Ϯ2 wk. BALB/c mice were purchased from Charles River 1/4 dilutions. [3H]Thymidine (1 ␮Ci/well) was added during the last 16 h Laboratory. Tg mice expressing the DO11 TCR specific for the chicken of culture, and incorporation was measured by scintillation counting after OVA peptide (OVA323–339) in the context of the MHC class II molecule 72 h of culture. I-Ad were obtained from K. Murphy (Washington University, St. Louis, MO). RNA purification and amplification sOVA Tg mice express a soluble form of OVA in the serum under KJ1-26ϩCD4ϩCD25Ϫ (anergic) and CD25ϩ (Treg) T cells from DO11 ϫ control of the metallothionein promoter I and were generated by cloning sOVA Tg and KJ1-26ϩCD4ϩCD25Ϫ cells from DO11 mice (naive) were the OVA cDNA into the metallothionein locus control region expression sorted from lymph nodes. Activated cells were recovered as cycled KJ1– vector 2999 (kindly provided by R. D. Palmiter (University of Washington, 26ϩCD4ϩ cells from BALB/c mice that had been transferred with 5 ϫ 106 Seattle, WA) (19) and injecting the construct into blastocysts from FVB CFSE-labeled DO11 cells and immunized with 200 ␮g of OVA in IFA 4 mice. Founders were screened for OVA expression by Southern blotting days before sorting. Cells from up to four mice were pooled for each and ELISA (anti-OVA; Research Diagnostics), and one founder expressing ϳ individual sample per group.