DNA Methylation Is a Nonredundant Repressor of the Th2 Effector Program Karen W. Makar and Christopher B. Wilson This information is current as J Immunol 2004; 173:4402-4406; ; of September 29, 2021. doi: 10.4049/jimmunol.173.7.4402 http://www.jimmunol.org/content/173/7/4402 Downloaded from References This article cites 16 articles, 5 of which you can access for free at: http://www.jimmunol.org/content/173/7/4402.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology DNA Methylation Is a Nonredundant Repressor of the Th2 Effector Program1 Karen W. Makar2 and Christopher B. Wilson3 The extent to which DNA methylation contributes to proper regulation of murine T cell effector function is unclear. In this study, we show that in the absence of the maintenance DNA methyltransferase Dnmt1, silencing of IL-4, IL-5, IL-13, and IL-10 in CD8 T cells was abolished, and expression of these Th2 cytokines increased as much as 1000-fold compared with that of control CD8 ,T cells. Th2 cytokine expression also increased in Dnmt1؊/؊ CD4 T cells, but the increase (ϳ20–40-fold for IL-4 and IL-10 5-fold for IL-5 and IL-13) was less than for CD8 T cells. As a result, both Dnmt1؊/؊ CD4 and CD8 T cells expressed high and> comparable amounts of Th2 cytokines. Loss of Dnmt1 had more subtle effects on IL-2 (<5-fold increase) and IFN-␥ (ϳ5–10-fold increase) expression and did not affect the normal bias for greater IL-2 expression by CD4 T cells and greater IFN-␥ expression by CD8 T cells, nor the exclusive expression of perforin and granzyme B by the CD8 T cells. These results indicate that Dnmt1 Downloaded from and DNA methylation are necessary to prevent cell autonomous Th2 cytokine expression in CD8 T cells but are not essential for maintaining proper T cell subset-specific expression of Th1 or CTL effectors. We also found that the expression of Th2 cytokines by Dnmt1؊/؊ T cells was appropriately up-regulated in Th2 conditions and down-regulated in Th1 conditions, indicating that transcription factors and DNA methylation are complementary and nonredundant mechanisms by which the Th2 effector pro- gram is regulated. The Journal of Immunology, 2004, 173: 4402–4406. http://www.jimmunol.org/ eritable programs of tissue-specific gene expression are several specific loci, including the Ifng and Il4-Il13 loci (2, 3). We controlled in part through epigenetic mechanisms, and show that the expression of Th2 effector cytokines is broadly and H several studies suggest a role for DNA methylation in substantially derepressed in T cells lacking Dnmt1. Despite this, the epigenetic regulation of cytokine gene expression. Analysis of Th1 and Th2 cytokine expression is appropriately up- and down- DNA methylation in the Il4-Il13 locus has demonstrated that the regulated in Dnmt1Ϫ/Ϫ T cells in response to polarizing culture locus is highly methylated in naive CD4 and CD8 T cells and fully conditions. These results support the notion that the Th2 cytokine polarized Th1 cell lines (1, 2). As CD4 T cells differentiate into program is coordinately silenced in a DNA methylation-dependent Th2 effectors, DNA methylation is slowly lost, and fully polarized manner that is separable from cytokine signaling pathways, which Ϫ Ϫ Th2 cell lines have almost no DNA methylation at this locus (1, 2). remain intact and functional in Dnmt1 / T cells. by guest on September 29, 2021 IL-4 expression is up-regulated in CD4 and CD8 T cells with re- duced DNA methylation, either due to a lack of the maintenance Materials and Methods DNA methyltransferase Dnmt1 (2, 3) or following treatment with Mice the DNA methylation inhibitor 5-azacytidine (1, 4, 5). DNA meth- 2lox ylation has also been implicated in the regulation of other cyto- The generation and initial characterization of the CD4Cre Dnmt mice has been described previously (2, 3). Age- and sex-matched ␥ ϩ kines, such as IFN- (6), IL-2 (7), and IL-3 (8), and in the regu- CD4CreDnmt2lox/ littermates were used as controls. These studies were lation of other T cell lineage genes, such as perforin (9) and CD8 approved by the University of Washington Animal Care and Use (3, 10), suggesting that DNA methylation may help to establish Committee. and/or maintain the different effector responses exhibited by CD4 Ϫ/Ϫ and CD8 T cells. Analysis of gene expression in Dnmt1 and control T cells In this report, we address the contribution of DNA methylation Naive T cells were purified from spleen and lymph nodes as described 6 and DNA methyltransferase 1 (Dnmt1) in the regulation of T cell elsewhere (2) and cultured at 1 ϫ 10 cells/ml on plates coated with anti- ␮ ␮ effector functions using T cells from CD4CreDnmt12lox mice in CD3 (5 g/ml) and anti-CD28 (10 g/ml) in IMDM supplemented with 10% FBS, penicillin, streptomycin, 50 ␮M 2-ME, and 10 ng/ml IL-2 (Chi- which the Dnmt1 gene is efficiently deleted in double-positive thy- ron, Emeryville, CA). Medium for type 2-polarizing culture conditions mocytes. T cells from these mice do not express Dnmt1 and have contained 10 ng/ml IL-4 (R&D Systems, Minneapolis, MN), 10 ␮g/ml markedly reduced levels of DNA methylation, both globally and at anti-IL-12 (BioSource International, Camarillo, CA), and 10 ␮g/ml anti- IFN-␥ (BioSource International); for type 1-polarizing conditions, medium contained 10 ␮g/ml anti-IL4 and 5 ng/ml IL-12 (R&D Systems); for neu- tral conditions, medium contained 20 ␮g/ml anti-IL-4, 20 ␮g/ml anti-IL- Department of Immunology, University of Washington, Seattle, WA 98195 12, and 20 ␮g/ml anti-IFN-␥. Activated cells were restimulated with 50 ␮ Received for publication May 11, 2004. Accepted for publication July 21, 2004. ng/ml PMA and 0.7 M ionomycin (Sigma-Aldrich, St. Louis, MO) for 4–6 h before harvesting for gene expression analysis. RNA was isolated The costs of publication of this article were defrayed in part by the payment of page and gene expression was measured by real-time quantitative PCR as de- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. scribed previously (2). TaqMan probes were used for all genes except for Il10, Rog, and Eomes, which were quantified using Brilliant SYBR Green 1 This work was supported by awards from the National Institutes of Health QPCR reagents (Stratagene, La Jolla, CA). Primer and probe sequences for (GM20865, HD18184, HD39454) and the March of Dimes. Il4, Il5, IL13, Gata3, and Tbx21 (T-bet) were as described in Ref. 11; Actb 2 Current address: Dendreon Corporation, 3005 1st Avenue, Seattle, WA 98121. (␤-actin) and Ifng were as described in Ref. 2. Sequences for all other Ј 3 Address correspondence and reprint requests to Dr. Christopher B. Wilson, Depart- primers and probes were as follows: perforin forward, 5 -CAG GTC AGG ment of Immunology, Campus Box 357650, University of Washington, 1959 NE CCA GCA TAA GAG-3Ј; perforin reverse, 5Ј-TGG TTG GTG ACC TTT Pacific Street, Seattle, WA 98195. E-mail address: [email protected] GAA TCC-3Ј; perforin probe, 5Ј-6-FAM-AGC CAT GAT TCA TGC Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 4403 CAG TGT GAG TGC-TAMRA-3Ј; granzyme B forward, 5Ј-GCC TTC Dnmt1 might more broadly disrupt normal patterns of T cell sub- TTC CTC TCC TAG AGG TTA A-3Ј; granzyme B reverse, 5Ј-CGG AAG set-specific effector gene expression. However, this was not the Ј Ј GCC GCC TAG GT-3 ; granzyme B probe, 5 -6-FAM-CAG CGT CAA case. IFN-␥ expression increased ϳ5–10-fold in both CD4 and GAG TGT CCT TGC TCT CT-TAMRA-3Ј; Eomes forward, 5Ј-CAC Ϫ/Ϫ ϳ GGC TTC AGA AAA TGA CA-3Ј; Eomes reverse, 5Ј-CTC TGT TGG CD8 Dnmt1 T cells, and IL-2 expression increased 5-fold in GGT GAG AGG AG-3Ј; ROG forward, 5Ј-CTC TCT GGA GTC AGA CD8 T cells lacking Dnmt1, but the loss of Dnmt1 did not alter the ATC AGC TGG-3Ј; ROG reverse, 5Ј-AGC GCT GAG GAC AGA GGC normal T cell subset-specific patterns of expression. Control and TAC AGG-3Ј; IL-10 forward, 5Ј-GGT TGC CAA GCC TTA TCG GA-3Ј; Dnmt1Ϫ/Ϫ CD8 T cells consistently expressed higher levels of and IL-10 reverse, 5Ј-ACC TGC TCC ACT GCC TTG CT-3Ј. IFN-␥ than CD4 T cells from the same mice, while the converse was true for CD4 T cells (Fig. 1B). In addition, neither perforin nor Results Ϫ Ϫ granzyme B were up-regulated in Dnmt1 / CD4 T cells, dem- Loss of DNA methylation broadly and preferentially derepresses onstrating that Dnmt1Ϫ/Ϫ CD4 T cells have not acquired a CD8 the Th2 effector program in CD8 T cells CTL-like gene expression profile (Fig.