Distinct Molecular Program Imposed on CD4 + T Cell Targets by CD4+CD25+ Regulatory T Cells

This information is current as Teresa L. Sukiennicki and Deborah J. Fowell of September 28, 2021. J Immunol 2006; 177:6952-6961; ; doi: 10.4049/jimmunol.177.10.6952 http://www.jimmunol.org/content/177/10/6952 Downloaded from

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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

Distinct Molecular Program Imposed on CD4؉ T Cell Targets by CD4؉CD25؉ Regulatory T Cells1

Teresa L. Sukiennicki and Deborah J. Fowell2

CD4؉CD25؉ regulatory T cells (Tregs) are key modulators of immunity, but their mechanism of action is unclear. To elucidate the molecular consequences of Treg encounter, we analyzed changes in gene expression in CD4؉ T cell targets activated in the presence or absence of CD4؉CD25؉ Tregs. Tregs did not alter the early activation program of CD4؉ T cells, but had reversed many of the activation-induced changes by 36 h. It is not known whether Tregs simply induce a set of transcriptional changes common to other nonproliferative states or whether instead Tregs mediate a distinct biological activity. Therefore, we compared the gene profile of T cells following Treg encounter with that of T cells made anergic, TGF-␤-treated, or IL-2-deprived; all possible modes of Treg action. Strikingly, all genes down-regulated in suppressed cells were indeed common to these nonproliferative

states. In contrast, Treg encounter led to elevated expression of a unique set of genes in the target T cells. Although different from Downloaded from the nonproliferative states tested, the Treg-imposed gene program is exemplified by expression of many genes associated with growth arrest or inhibition of proliferation. We suggest that Tregs function by the induction of a distinct set of negative regulatory factors that initiate or maintain target T cells in a nonproliferative state. The Journal of Immunology, 2006, 177: 6952–6961.

aturally occurring regulatory T cells (Tregs)3 are impor- via Treg-produced granzyme B and/or perforin (21, 22) have been

tant modulators of immune responses. Initially demon- proposed as additional modes of Treg action. Thus, Tregs may http://www.jimmunol.org/ N strated to prevent autoimmunity, they have since been as- have multiple ways in which to disable a developing immune re- sociated with the regulation of immune responses to pathogens, sponse, and the mode of action may depend on the context of both tumors, and transplantation Ags (1–3). Their influence on CD4ϩ T the target and Treg activation signals. cells has been studied best, although direct or indirect effects on Tregs cause a block in proliferation, differentiation, and effector CD8ϩ T cells, B cells, and innate cell types including dendritic cells T cell function, but beyond this the molecular changes in target T also have been reported. Tregs suppress many activities of target cells cell biology following Treg encounter are ill-defined. IL-2 tran- including proliferation, IL-2 production (4, 5), T cell differentiation, scription is down-regulated in the target T cells (4, 5, 12) but it is and effector T cell migration and cytokine secretion (6–8). not known whether the IL-2 gene modulation is the primary target,

Little is known about how Tregs mediate their suppressive ef- or a downstream consequence, of Treg suppression. The strength by guest on September 28, 2021 fects. They require activation through their TCR to acquire full of the target T cell activation signal appears to play a role in their functional competence and, in vitro, must be in close proximity to susceptibility to suppression, with increased costimulation (5, 23) their targets (or APC) to mediate suppression (4, 5). The expres- and/or Ag concentration (24) rendering the target T cells refractory sion of CD25 at high levels on Tregs suggests that they may work to Treg suppression. It is unclear how these additional signals pro- through competitive consumption of IL-2 (9), although the ability tect against Treg action. Recent data suggest that the NFAT and of Tregs to suppress IL-2R-deficient T cells (10) and in the pres- Cbl-b pathways in the target population are important for Treg ence of excess exogenous IL-2 (11, 12) suggests that IL-2 com- action, since Tregs were unable to block the proliferation of CD4ϩ petition is not essential for suppression. IL-10 and TGF-␤ are key T cell targets that lacked expression of these signal molecules (25, immunomodulators in in vivo models of Treg suppression pro- 26). Therefore, Tregs may directly target these signaling pathways duced by the Tregs themselves or induced in other cell types by to facilitate suppression. Tregs (13–16). Modulation of APC function by Tregs (17–19), Postulating that molecular changes in the suppressed cell may anergy induction (20), and direct cytotoxicity of immune targets yield clues to the functional state induced in targets by Tregs and/or the mechanism of Treg activity, we used microarray tech- nology to determine changes in target T cell gene expression fol- Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, University of Roch- lowing Treg-mediated suppression. Differences in gene expression ester, Rochester, NY 14642 in the suppressed cells evolved over time, with only 12 genes ϩ Ϫ Received for publication April 21, 2006. Accepted for publication August 25, 2006. differentially expressed between CD4 CD25 target cells cul- The costs of publication of this article were defrayed in part by the payment of page tured in the presence (suppressed) or absence (nonsuppressed) of charges. This article must therefore be hereby marked advertisement in accordance Tregs at 12 h and 242 genes at 36 h. The kinetics of expression of with 18 U.S.C. Section 1734 solely to indicate this fact. many of these genes suggested a pattern of aborted activation in 1 This work was supported by the Juvenile Diabetes Research Foundation, Research the presence of Tregs. That is, similar transcriptional changes oc- Grant 1-2000-609 (to D.J.F.) and National Institutes of Health Training Grant AI- 07169 (to T.L.S.). curred in the presence or absence of Tregs early after activation, 2 Address correspondence and reprint requests to Dr. Deborah J. Fowell, David H. but the presence of Tregs reversed many of these activation-in- Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical duced changes by 36 h. To determine whether Tregs induced dis- Sciences, Department of Microbiology and Immunology, University of Rochester, tinct changes in target T cells, we examined cells that had been 601 Elmwood Avenue, Box 609, Rochester, NY 14642. E-mail address: ␤ [email protected] anergized, deprived of IL-2, or treated with TGF- for expression 3 Abbreviations used in this paper: Treg, regulatory T cell; 7-AAD, 7-aminoactino- of a panel of genes found to be differentially expressed following mycin D; KLF, Kruppel family; GRP1, general receptor of phosphoinositide 1. a Treg encounter. All of the down-regulated genes in suppressed

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 6953 cells were similarly affected under one or more of the nonprolif- Quantitative real-time PCR erative conditions tested, suggesting that Tregs do not function by Unamplified cDNA was generated from mRNA using random hexamers inhibiting a discrete panel of target T cell genes. In contrast, a and Superscript II (Invitrogen Life Technologies). cDNA was loaded in unique set of genes was more highly expressed in target T cells duplicate onto custom-designed TaqMan Low-Density Arrays (Applied following Treg encounter but not in T cells inactivated in other Biosystems). Data were analyzed using Sequence Detection System soft- ways. This global analysis of gene expression in suppressed cells ware (Applied Biosystems) with 18S ribosomal RNA as the endogenous control. supports the idea that Treg-induced suppression is a distinct mo- lecular process. Results Kinetic changes in target T cell activation in the presence Materials and Methods of Tregs Mice The timing and downstream consequences of Treg encounter for BALB/c Thy1.2ϩ mice were purchased from The Jackson Laboratory or ϩ the target T cell are poorly understood. We have recently defined the National Cancer Institute. BALB/c Thy1.1 mice were bred in house. a narrow kinetic window in which Tregs act on CD4ϩ T cell tar- Mice were housed under pathogen-free conditions and all animal protocols were approved by the University Committee for Animal Resources at the gets, occurring within the first 6–12 h of target T cell activation in University of Rochester Medical School. vitro (12). To obtain a better understanding of how the target T cell changes over time, we analyzed the expression of early activation Media, Abs, and reagents markers on CD4ϩCD25Ϫ target T cells in the presence or absence ϩ ϩ

Cells were cultured in RPMI 1640 with 10% heat-inactivated FCS of allelically marked CD4 CD25 Tregs. In the first 6 h, target T Downloaded from (HyClone), 50 ␮M 2-ME (Bio-Rad), 2 mM L-glutamine (Invitrogen Life cells up-regulated the expression of CD69 and CD25 similarly in Technologies), and 100 U/ml penicillin and streptomycin (Invitrogen Life the presence or absence of Tregs (Fig. 1a), demonstrating that Technologies). All Abs for flow cytometry were purchased from Tregs do not block initial activation signals. By 12 h, however, the eBioscience, biotin anti-mouse CD25 (clone 7D4) and 7-aminoactinomy- cin D (7-AAD) from BD Pharmingen, and recombinant human TGF-␤1 percentage of cells expressing high levels of these markers began and rIL-12 from PeproTech. Human IL-2 was obtained from the National to decline in the presence of Tregs, with the differential expression Institutes of Health Research and Reference Reagent Program. being most evident by 36 h. Given recent reports of the ability of http://www.jimmunol.org/ Tregs to express cytotoxic machinery and to kill target T cells (21, Cell purification and culture 22), loss of activation marker expression at the population level CD4ϩCD25Ϫ and CD4ϩCD25ϩ cells were isolated by magnetic separation could have been due to the preferential death of those cells that had ϩ Ϫ (Miltenyi Biotec) as described previously (12). CD4 CD25 cells (1 ϫ initiated activation in the presence of Tregs. However, analysis of 105) were stimulated with 1 ␮g/ml soluble anti-CD3 and 1 ϫ 105 T- depleted splenocytes with and without 0.5–1 ϫ 105 CD4ϩCD25ϩ cells. viability revealed little cell death in cultures containing Tregs Where indicated, the CD25Ϫ cells were prelabeled with CFSE (Molecular (Fig. 1b). The vast majority of cells remained viable (87% 7-AAD Probes). In some cases, 10 ng/ml recombinant human TGF-␤1 was added negative with Tregs and 90% without Tregs at 36 h) with similar at the start of culture. IL-2 was blocked 6 h into the culture (5 ␮g/ml target cell yields at 12 and 36 h in the presence or absence of anti-IL-2 mAb) based on our previous observations on the kinetics of IL-2 ϩ Tregs. Thus, Tregs inhibit the proliferation of CD4 T cells in by guest on September 28, 2021 down-regulation by Tregs (12). Parallel cultures were set up for confirma- tion that the Tregs were functional and that the anti-IL-2, TGF-␤, and vitro without blocking initial activation signals or inducing con- anergy-inducing conditions indeed inhibited proliferation. Proliferation siderable cell death. was measured during the last6hofa72-h culture by incorporation of [3H]thymidine (1␮Ci).

Anergy induction Anergy was induced as described previously (27) in Th1 cells generated from CD4ϩCD25Ϫ cells isolated from DO11.10 TCR-C␣Ϫ/Ϫ BALB/c mice. Briefly, CD4ϩCD25Ϫ cells were stimulated with 1 ␮M pOVA

(OVA323–339 peptide) and APC in the presence of 10 U/ml recombinant human IL-2, 10 ng/ml rIL-12, and 40 mg/ml anti-IL-4 mAb. Th1-primed cells were anergized by stimulation at 1 ϫ 106 cells/ml with 1 ␮M iono- mycin in complete RPMI 1640 for 16 h at 37oC.

Microarray sample preparation and analysis CD4ϩCD25ϪThy1.1ϩ cells were stimulated in the presence or absence of CD4ϩCD25ϩThy1.2ϩ cells as described above. At 0, 12, and 36 h, CD4ϩCD25Ϫ cells were isolated by flow cytometric sorting of 7-AADϪCD4ϩThy1.1ϩ cells (98.5–99.5% CD4ϩThy1.1ϩ). Total RNA was isolated using TRIzol (Invitrogen Life Technologies). RNA was am- plified and biotin-labeled by the MessageAmp kit procedure (Ambion) us- ing a T7-oligo(dT) primer. Fragmented, biotinylated cRNA was hybridized at 45°C for 16 h with GeneChip Mouse Genome 430A 2.0 arrays (Affymetrix). The following standard Affymetrix biotinylated cRNAs were spiked into the hybridization solution: bioB, bioC, bioD, and cre. Two separate sets of biological samples were isolated at 12 and 36 h and were run on separate arrays. FIGURE 1. CD4ϩ T cells are activated and remain viable in the pres- Data were analyzed using Microarray Suite 5.0 (Affymetrix) and ence of CD4ϩCD25ϩ T cells. a, Surface expression of CD69 and CD25 Gene-Traffic software (RMA and GCRMA algorithms; Iobian/Stratagene). ϩ Ϫ ϩ was determined for CD4 CD25 Thy1.1 target T cells stimulated with Genes were selected from the first set of arrays that were at least 2-fold and without CD4ϩCD25ϩThy1.2ϩ T cells. All plots are gated on differentially expressed between cells cultured in the presence or absence ϩ ϩ of Tregs at each time point. Genes with poor replicates (less than a 1.5-fold CD4 Thy1.1 target T cells, unstimulated cells (dotted lines). b, Viability difference in expression in the second experiment) were discarded. A list of of CFSE-labeled target T cells stimulated as in a, by exclusion of 7-AAD, ϩ differentially expressed genes was generated by the intersection of genes gated on Thy1.1 target T cells. Numbers indicate the percentage of cells with good replicates determined using the three algorithms. in each quadrant. 6954 DISTINCT MOLECULAR SIGNATURE IMPOSED BY Tregs

Changes in target T cell gene expression following regulatory indicates their respective kinetic pattern of gene expression T cell encounter (groups 1–6). To gain a molecular understanding of the altered state of the target Interestingly, the aborted-activation group 1A (Fig. 3a) included T cells following activation in the presence of Tregs, we positively quiescence-associated genes of the Kruppel family (KLF-2 and selected viable Thy1.1ϩ target T cells by FACS from cultures with KLF-3), which regulate cell proliferation and differentiation. (suppressed) and without (nonsuppressed) Thy1.2ϩ Tregs at 0, 12, KLF-2 is expressed in resting, unstimulated lymphocytes and its and 36 h, purified mRNA and, performed microarray analysis of rapid down-regulation appears to be a necessary step for lympho- gene expression using Affymetrix oligonucleotide arrays. Two in- cyte activation (28). Both suppressed and nonsuppressed cells dependent isolations of target T cells from cultures with and with- down-regulated KLF-2 and KLF-3 at 12 h, but although down- out Tregs were analyzed on individual microarrays. Suppressive regulated expression of KLF-2 and KLF-3 was maintained in non- activity was verified in parallel cultures by [3H]thymidine incor- suppressed cells, the suppressed cells re-expressed these genes by poration (data not shown). Consistent with our functional data, 36 h. This may be indicative of a loss of activation stimulus re- there were strikingly few differences between CD4ϩ T cell targets quired to maintain reduced expression of KLFs or the positive in the presence or absence of Tregs at 12 h, whereas the effect of regulation of KLFs’ re-expression by Tregs (29). In the other Treg encounter was readily apparent at 36 h (Fig. 2). To identify aborted-activation group, group 1B (Fig. 3b), expression of many strong candidate genes for suppression, microarray data were an- molecules characteristic of T cell activation and differentiation, alyzed using three different algorithms (MAS 5.0, RMA, and such as CD25, was initiated in both populations at 12 h but down- GCRMA) and genes were selected for further analysis from the regulated in suppressed cells at 36 h. The aborted activation pattern intersection of these individual algorithm lists. After 12 h, only 12 was also seen by real-time RT-PCR analysis of expression of a Downloaded from genes were reproducibly differentially expressed in suppressed number of immune activation and differentiation genes (Fig. 3f ). cells and mostly represented quantitative differences between sup- Thus, our gene profiling approach confirms the initial functional pressed and nonsuppressed targets (data not shown). A larger num- data, suggesting that early T cell activation proceeds largely un- ber of genes were differentially expressed at 36 h (242 genes, 91 perturbed in the presence of Tregs, but that Tregs disrupt the main- genes at higher levels and 151 genes at lower levels in suppressed tenance/progression of the activation program. target cells, Table I, all genes for which a functional class has been Many of the genes expressed at lower levels in suppressed cells http://www.jimmunol.org/ determined, 184 of 242 total genes). at 36 h reflected a general down-regulation of metabolic pathways: DNA/RNA binding, transcription, translation, and protein modifi- Kinetic patterns of gene expression cations (Table I). A large group (n ϭ 36; Table I) of these genes expressed at lower levels in suppressed cells have been designated Many more genes were differentially expressed in suppressed tar- as Myc target genes (ͳwww.myccancergene.org/site/mycTargetDB. get T cells at 36 h than at 12 h (Fig. 2). The kinetics of expression aspʹ), Myc also being down-regulated in suppressed cells. In ad- was suggestive of a profile of abortive activation on Treg encoun- dition, all differentially expressed mRNAs for secreted proteins ter, in that activation-induced changes in gene expression at 12 h were down-regulated including immune effector molecules such as

were similar in target cells in the presence or absence of Tregs, but by guest on September 28, 2021 IL-4, Ccl5 (RANTES), and granzymes (Table I). Down-regulated this activation profile was subsequently reversed only in the pres- cell surface proteins included a number of cytokine receptor chains ence of Tregs (36 h). This profile was seen for almost half of the (IL-2R␣, IL-12R␤2, and IL-7r) and molecules associated with T genes differentially expressed at 36 h (120 of 242, 49.6%; groups cell activation (Gp49a, Ly6c, CD24a, and CD62P) consistent with 1A and 1B). Fig. 3 shows examples of this pattern of expression the aborted activation phenotype. In contrast, a number of genes for genes initially down-regulated (Fig. 3a, group 1A) or up-reg- encoding cell surface molecules associated with down-regulation ulated (Fig. 3b, group 1B) compared with unstimulated cells. Other of proliferation were more highly expressed in suppressed cells groupings included genes specifically induced at 36 h in sup- (Tspan32, TGF-␤r2, and Timp2). Interestingly, we found little ev- pressed cells (Fig. 3c, group 2; 22 of 242, 9%) or specifically idence for Treg targeting of transcription of genes involved in down-regulated at 36 h in suppressed cells (Fig. 3d, group 3; 37 of regulation of the cell cycle or apoptotic pathways at 36 h. 242, 15%). In addition, a number of genes failed to be induced at 12 or 36 h in suppressed cultures but were specifically up-regu- Confirmation by quantitative RT-PCR lated in nonsuppressed cells at 36 h (Fig. 3e, group 4; 37 of 242, We designed custom low-density arrays (Applied Biosystems) for 15%). Table I lists all of the differentially expressed genes for real-time PCR analysis of expression of 71 of the most differen- which a functional class has been assigned (184 of 242 genes) and tially expressed genes identified by microarray at 36 h. This se- lected panel of genes was used to first verify the molecular profile of T cells activated in the presence of Tregs (Fig. 4) and, second, to serve as a molecular signature of Treg encounter for comparison to other nonproliferating states (Fig. 5). Of the selected panel of genes identified by microarray, 23 (68%) of 34 genes expressed more highly and 34 (92%) of 37 genes expressed at lower levels in suppressed cells vs nonsup- pressed cells at 36 h and remained differentially expressed in sup- pressed target T cells at 36 h by real-time RT-PCR (see Fig. 4 for the genes confirmed). Our 12-h microarray data were very variable between samples, suggesting that this time point may be close to FIGURE 2. Treg-induced changes in gene expression in target T cells over time. mRNA was isolated from CD4ϩCD25Ϫ Thy1.1ϩ T cells stim- the induction point of suppression. The observed variability prob- ulated for 12 or 36 h in the presence (suppressed) or absence (nonsup- ably reflected the asynchrony of the activation and/or suppression pressed) of CD4ϩCD25ϩ Thy1.2ϩ T cells and analyzed on Affymetrix events. To identify likely gene candidates that are causative, rather mouse genome 430A 2.0 arrays. Signal intensities (GCRMA algorithm) for than a consequence, of suppression, we analyzed our 36-h gene each probe set were plotted against each other on a log scale. program for early expression at 12 h by real-time RT-PCR (Fig. 4). The Journal of Immunology 6955

Table I. Functional groupings and kinetic pattern of expression designations of genes differentially expressed in T cells cultured in the presence of CD4ϩCD25ϩ T cells at 36 ha

Gene Function Higher Lower Gene Function Higher Lower

Secreted I121 (Ϫ29.73) 1B General metabolism Ephx1 (8.44) 1A Mlstd2 (Ϫ21.23) 3 Gzmb (Ϫ20.36) 1B Smpd13a (7.69) 6 Dld (Ϫ7.31) 3 Cc15 (RANTES) (Ϫ15.04) 1B Qprt (7.3) 2 Asns (Ϫ7.15)b 1B Gzma (Ϫ9.92) 1B Sesn1 (4.95) 1A Ppat (Ϫ3.85)b 3 I14 (Ϫ8.59) 1B Cmah (4.54) 6 Hmger (Ϫ3.47) 3 Lif (Ϫ7.96) 1B Mgst2 (4.23) 1A Cyp51 (Ϫ2.71)b 4 Pbef1 (Ϫ6.04) 1B Hsd11b1 (4.08) 1A Lss (Ϫ2.61) 1B Tgn (Ϫ2.70) 6 St6gal1 (3.61) 1A Sc4mol (Ϫ2.45) 4 Mical1 (3.45) 6 Shmt2 (Ϫ2.20) 1B Cell surface or Cd7 (11.89) 2 Gp49a (Ϫ23.19) 4 Eno3 (3.45) 2 Txnrd1 (Ϫ2.19) 6 Pglyrp1 (7.49) 2 Ly6c (Ϫ14.10) 4 b extracellular Dhrs8 (3.34) 6 Pyp (Ϫ2.17) 6 1A Ϫ 3 Timp2 (5.88) Cd24a (HAS) ( 10.10) Acas21 (3.12) 6 1A Ϫ 1B Tspan32 (Tssc6, Phemx) (5.84) Ly6a (Sca1) ( 8.74) Cnp1 (2.98) 6 Emp3 (5.78) 1A Bst2 (Ϫ6.39) 1B Galgt1 (2.91) 5 Fas1 (4.77) 6 F2r (Ϫ5.05)b 1B Cox7a21 (2.33) 1A Cd96 (4.23) 1A I17r (Ϫ5.04) 6 Cd97 (3.93) 1A Itgb1 (Ϫ4.73) 3 b Translation Eif5b (Ϫ12.21) 3 Ms4a4c (3.92) 2 II12rb2 (Ϫ4.24) 4 Prkr (Ϫ5.54) 4 Itgb7 (3.46) 1A Selp (CD62P) (Ϫ3.85) 4 Etf1 (Ϫ5.43)b 3 Tnfrsf7 (CD27) (3.42) 5 II2ra (CD25) (Ϫ3.64) 1B Iars (Ϫ5.42)b 3 Ms4a4b (3.38) 5 Eprs (Ϫ4.86)b 3 Ifnar2 (3.32) 1A Downloaded from Ϫ 1B Slamf6 (Ly108) (2.73) 6 Rsl1d1 ( 3.42) Ϫ 3 Tgfbr2 (2.60) 6 Dars ( 3.38) Nars (Ϫ2.57) 1B Eefle1 (Ϫ2.50) 1B Signal transduction Sbk1 (11.65) 2 Map3k8 (Ϫ27.43) 3 b Pdlim4 (10.92) 1A Ifi202b (Ϫ16.02) 4 Protein binding/ Sgne1 (54.24) 1A Tgm2 (Ϫ24.13) 1B Rasa3 (5.83) 1A Ier3 (Ϫ11.02) 4 b modification/ Ramp1 (9.68) 1A Plod2 (Ϫ19.80) 4 Bin1 (4.85) 1A Pmaip1 (Noxa) (Ϫ7.89) 1B degradation Ctsd (3.18) 2 Egln3 (Ϫ18.57) 4 Inad1 (4.67) 1A Gbp1 (Ϫ4.72) 1B Usp3 (3.03) 1A Usp18 (Ϫ17.85) 4 Pscd3 (4.64) 1A Jub (Ϫ3.70) 4 http://www.jimmunol.org/ 6 Ϫ 4 Pycard (Asc) (3.43) 2 Plk4 (Ϫ2.93) 6 Pdlim2 (2.86) Glp2 ( 17.41) Ϫ b 1B Arhgap9 (3.09) 1A Socs2 (Ϫ2.78) 6 Tpp2 ( 15.73) Ϫ 3 Gpsm3 (2.88) 1A Strap (Ϫ2.50) 1B Cul2 ( 6.63) Cacybp (Ϫ6.59)b 3 Ikbke (2.88) 6 Hspca (Ϫ6.14)b 1B Ppmlm (2.65) 1A Srp54 (Ϫ5.08)b 3 Ero11 (Ϫ3.60) 1B Transcription Klf3 (20.46) 1A Prdm1 (Blimp1) (Ϫ12.41) 1B b Hspa5 (Ϫ3.58) 3 Pou2af1 (Bob1, Obf-1) (13.30) 2 Atf3 (Ϫ10.88) 1B Usp1 (Ϫ3.54) 1B Mxd4 (12.74) 1A Smarca5 (Ϫ10.86) 3 b Tmed7 (Ϫ3.48) 3 Id3 (8.06) 2 Rbbp8 (Ϫ10.16)b 4 Hsp105 (Ϫ3.23) 6 Elk3 (6.73) 1A Crem (Ϫ9.63) 1B b Casp6 (Ϫ3.12) 1B Klf2 (5.61) 1A Ssb (Ϫ7.93)b 3 Hspa9a (Ϫ2.97) 3 Dbp (4.70) 2 Trim30 (Rpt1) (Ϫ5.86) 4 by guest on September 28, 2021 Tra1 (Ϫ2.93) 4 Nr4a1 (4.12) 5 Nfil3 (Ϫ4.66)b 6 b Usp14 (Ϫ2.59) 6 Mk11 (3.66) 1A Dnajc2 (MIDA1) (Ϫ3.95) 3 Tnpo3 (Ϫ2.47) 3 Churc1 (2.98) 2 Stat2 (Ϫ3.95) 4 Tcerg1 (Ϫ3.78) 3 2 Ϫ 4 Rbpsuh (CBF1) (Ϫ3.68) 1B DNA/RNA binding Msh5 (4.87) Kif3a ( 22.71) Top1 (Ϫ10.28)b 3 Crabp2 (Ϫ3.50) 1B Ϫ 3 Cdk8 (Ϫ3.41) 3 Ddx46 ( 9.49) Ϫ 1B Irf4 (Ϫ3.15) 1B No15 ( 9.36) Ϫ 3 Nolc1 (Ϫ2.98) 1B Mphosph10 ( 8.73) b Ϫ 4 Etv6 (Ϫ2.93) 1B Hells ( 7.60) Ϫ 6 Sap30 (Ϫ2.92) 1B Oas3 ( 6.53) b Ϫ 3 Tardbp (Ϫ2.86) 3 Xpot ( 6.26) Nc1 (Ϫ6.25)b 3 Gtf2h1 (TFIIH) (Ϫ2.79) 3 Ϫ 6 Suzl2 (Ϫ2.73) 4 D11Lgp2e ( 5.21) Ϫ 3 Myc (Ϫ2.36) 6 Rev11 ( 4.66) b Isg20 (Ϫ4.63)b 6 Csda (Ϫ2.22) 1B b Rp130 (Ϫ4.38)b 3 Syncrip (Ϫ4.13) 1B Small molecule Syt11 (8.42) 2 Gja1 (Ϫ114.83) 4 Rnu22 (Ϫ4.12) 3 Sfxn3 (6.77) 1A Slc16a1 (Ϫ4.28)b 3 binding/transport Recc1 (Ϫ3.66) 1B 1A Ϫ 1B 0610039P13Rik (5.83) Clic4 ( 3.76) Orc11 (Ϫ3.36) 1B S100a6 (5.55) 1A Kcnk5 (Ϫ2.74) 4 Hnrpu (Ϫ2.74) 3 Kcnmb4 (4.28) 2 Slc7a5 (Ϫ2.59)b 1B Ddx18 (Ϫ2.43) 1B Tpcn1 (3.59) 1A b S100a10 (3.52) 2 Cytoskeleton Pacsin1 (10.19) 1A Tmc6 (3.35) 1A Synpo (7.55) 2 S100a13 (3.27) 2 associated Add1 (2.61) 1A

a Genes were placed into columns based on whether they were expressed at a higher or lower level in suppressed cells compared to nonsuppressed cells at 36 h. Numbers in parentheses indicate the changes in expression in the presence of Tregs (fold change based on analysis of microarray data using the GCRMA algorithm). Numbers in bold at right of each column are the pattern designations for kinetic gene expression: group 1A, aborted activation, gene expression down on initial activation (12 h) in both suppressed and nonsuppressed cells compared to unstimulated cells but back up at 36 h in suppressed only; group 1B, aborted activation gene expression was upregulated on initial activation (12 h) in both suppressed and nonsuppressed cells compared to unstimulated cells but was down-modulated at 36 h in suppressed only; group 2, uniquely induced in suppressed cells at 36 h; group 3, uniquely down-regulated in suppressed cells at 36 h; group 4, uniquely induced in nonsuppressed cells at 36 h; group 5, uniquely down-regulated in nonsuppressed cells at 36 h; and group 6, quantitative changes, no difference in pattern of expression over time in suppressed and nonsuppressed cells. Genes of unknown function (n ϭ 22 higher, 36 lower) are not shown. b Myc target genes.

The higher resolution afforded by real-time RT-PCR revealed that expression differences were not identified by our microarray anal- many of the genes confirmed as differentially expressed at 36 h ysis given the use of a strict 2-fold cutoff and the selection of genes were already differentially expressed by 12 h. It is likely that these from the intersection of three separate algorithms. The genes 6956 DISTINCT MOLECULAR SIGNATURE IMPOSED BY Tregs

FIGURE 3. Kinetic gene expression patterns in target cells cultured in the presence CD4ϩCD25ϩ T cells. a and c, Representative genes differentially expressed at a higher level in suppressed targets at 36 h. b, d, and e, Representative genes differentially expressed at a lower level in suppressed cells at 36 h. Group 1, aborted activation; group 1A, gene expression down-regulated on initial activation (12 h) in both suppressed and nonsuppressed cells compared with unstimulated cells but genes were re-expressed in suppressed cells by 36 h; group 1B, genes were activated at 12 h in both suppressed and nonsuppressed cells but down-regulated only in nonsuppressed cells at 36 h; group 2, uniquely induced in suppressed cells at 36 h; group 3, uniquely Downloaded from down-regulated in suppressed cells at 36 h; and group 4, uniquely induced in nonsuppressed cells at 36 h. Genes in groups 2–4 were unchanged at 12 h in both nonsuppressed or suppressed cells relative to unstimulated cells (2-fold cutoff). f, mRNA expression for immune-response genes was analyzed by real-time RT-PCR in suppressed and nonsuppressed CD4ϩCD25Ϫ T cells at 12 and 36 h relative to unstimulated cells. *, GATA3 and T-bet were not identified as differentially expressed in the original microarray screen using a strict 2-fold cutoff.

expressed more highly in suppressed compared with nonsup- up-regulation or maintenance of gene expression represented po- http://www.jimmunol.org/ pressed T cells at 36 h (Fig. 4, a–d) could be subdivided into genes tential initiators of suppression and contained a number of tran- up-regulated or sustained only in suppressed cells early at 12 h scriptional regulators, such as Pou2af1 (also known as BOB.1, relative to nonsuppressed cells (Fig. 4, a and b) or genes up- OCA-B) and Id3, and signal transduction modifiers, such as regulated in suppressed cells late at 36 h (Fig. 4, c and d). The S100a10 and Sbk. In contrast to the microarray data (Fig. 3a), genes differentially expressed early in suppressed cells through Tspan 32 (also known as Tssc6 or Phemx) was up-regulated at by guest on September 28, 2021

FIGURE 4. Quantitative RT-PCR confirmation of a molecular signature for Treg encounter. The 57 genes listed were confirmed as differentially expressed in suppressed target cells at 36 h by quantitative RT-PCR. Genes were divided into groups according to whether they were expressed at a higher level (n ϭ 23) or a lower level (n ϭ 34) in the suppressed cells compared with the nonsuppressed cells by microarray at 36 h. Real-time PCR data showing gene expression for suppressed and nonsuppressed cells at 0, 12, and 36 h are shown for single genes representative of each kinetic profile. All genes that fit that pattern are listed in the boxes to the right of each graph. Expression levels of mRNA are relative to nonsuppressed cells at 12 h. Results represent one of two similar experiments. The Journal of Immunology 6957 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Tregs induce a distinct gene program in target T cells. CD4ϩCD25Ϫ T cells were stimulated with and without CD4ϩCD25ϩ T cells, or under other inhibitory conditions: IL-2 blockade, TGF-␤ exposure, or ionomycin-induced anergy. Expression of genes differentially expressed in target T cells following CD4ϩCD25ϩ encounter (suppressed signature, Fig. 4) was measured by real-time RT-PCR. a, Genes were grouped according to whether they were expressed at lower or higher levels in suppressed cells or in cells exposed to each of the inhibitory conditions vs nonsuppressed cells at 36 h (2-fold cutoff). Pie charts show the percentage of genes that were similarly affected under each condition. The one gene expressed more highly in suppressed, anergized, and TGF-␤-treated cells is CD7. b, Genes uniquely expressed at a higher level in Treg-suppressed cells. Genes associated with growth arrest or inhibition of proliferation in lymphocytes (bold) or other cells types (bold italics). c, Genes expressed at a higher level only in Treg-suppressed and anergized cells. d, Genes expressed at a lower or higher level in suppressed cells grouped according to the kinetic patterns of gene expression described in Table I: group 1A, aborted activation, gene expression down on initial activation (12 h) in both suppressed and nonsuppressed cells compared with unstimulated cells but back up at 36 h in suppressed only; group 1B, aborted activation gene expression was up-regulated on initial activation (12 h) in both suppressed and nonsuppressed cells compared with unstimulated cells but was down-modulated at 36 h in suppressed only; group 2, uniquely induced in suppressed cells at 36 h; group 3, uniquely down-regulated in suppressed cells at 36 h; group 4, uniquely induced in nonsuppressed cells at 36 h; group 5, uniquely down-regulated in nonsuppressed cells at 36 h; and group 6, quantitative changes, no difference in pattern of expression over time in suppressed and nonsuppressed cells.

12 h by quantitative RT-PCR and has been implicated in the due to enhanced expression in the nonsuppressed cells, as shown negative regulation of T cell proliferation. In contrast, genes for CD25 (Fig. 4e). Very few genes were specifically down-reg- up-regulated late (Fig. 4, c and d) may be required to maintain the ulated at 12 h in suppressed cells (Fig. 4f), perhaps suggestive of suppressed state, such as the quiescence-associated genes Klf2 and this time point being at the early stages of Treg suppression. In- Ramp1, a component of the immunosuppressive calcitonin terestingly, nearly half of the genes down-regulated at 36 h in receptor. suppressed cells were, in contrast, more highly expressed (through Kinetic analysis of the genes expressed at lower levels in sup- induction or maintenance) in suppressed cells compared with non- pressed cells at 36 h (Fig. 4, e–h) revealed some interesting pat- suppressed cells at 12 h (Fig. 4, g and h). These genes may also be terns of expression. Half of the genes appeared to be of the aborted important initial regulators of the suppressed state and include the activation group, where genes were similarly regulated in both transcriptional regulator Cdk8 and the suppressor of cytokine- suppressed and nonsuppressed cells at 12 h but diverged at 36 h signaling Socs2 (Fig. 4g). 6958 DISTINCT MOLECULAR SIGNATURE IMPOSED BY Tregs

Little overlap with the gene expression profiles known for other IL-2, GATA3, and T-bet in suppressed cells (Fig. 3f) as also re- nonproliferative states ported for TGF-␤-exposed T cells (35). In contrast, there was little Although little is known about the mechanisms by which Tregs correlation between genes associated with suppression and those ␤ inhibit an immune response, it has been hypothesized that they genes reported to be induced by TGF- treatment, such as Smad7 may deprive cells of IL-2 (9), induce anergy (20), or act through and Foxp3 (37, 38), neither of which was differentially up-regu- the action of cell surface-associated TGF-␤ (30). Indeed, these lated in T cell targets following Treg encounter (microarray data). manipulations all lead to a common cellular outcome, that of a nonproliferative state. Comparison of the genes reported to be as- A distinct molecular signature in target cells following sociated with anergy, IL-2-deprivation, and TGF-␤ treatment with Treg encounter our own for Treg encounter at 36 h highlighted a striking disparity between gene expression in suppressed cells and published genes It is not clear whether Treg encounter simply terminates target T associated with IL-2 deprivation, anergy, or TGF-␤ exposure cell activation or whether Tregs induce a distinct change in the (Table II) (31–35). The expression of many anergy-associated molecular program of the target T cells. Comparison to genes asso- genes remained unchanged in target T cells cultured with Tregs, ciated with nonproliferating cells from the current literature (Table II) relative to target T cells in the absence of Tregs, including the suggested that Tregs did indeed impose a unique gene program in ubiquitin ligases, Itch, Cbl-b, and Rnf128 (also known as GRAIL) target cells. However, such cross-laboratory comparison suffers (27, 36). We found little correlation between suppression and from variability between studies with respect to cell type and ex- changes in IL-2-deprivation-associated genes including those that perimental protocol. Therefore, to directly compare gene profiles control cell cycle and cell death, such as Cdkn1b (also known as of our suppressed cells with other nonproliferative states we iso- Downloaded from p27kip), Bcl2, and Bim. Overlap was seen however with those lated mRNA from CD4ϩCD25Ϫ T cell cultures where T cell pro- genes down-regulated on TGF-␤ exposure including the immune- liferation had been blocked by Treg encounter or via treatment response molecules granzyme B, IL-4, and the IL-12R␤2 chain. In with TGF-␤, deprivation of IL-2, or induction of anergy (by iono- addition, subsequent real-time PCR analysis revealed decreases in mycin treatment as described by Rao (27)). These treated cells http://www.jimmunol.org/

Table II. Little Treg-mediated modulation of genes (36 h) reported in the literature to be associated with anergy, IL-2-deprivation, and TGF-␤ exposurea

Anergy Associated IL-2 Deprivation Associated

Positive correlation No correlation Positive correlation No correlation

(CD7 (1) Arf6 (؊) Atf5 (Ϫ Myc (2) ApoE (Ϫ) Bbc3 (Ϫ) by guest on September 28, 2021 S100a10 (1) Bax (Ϫ) Bcl2 (Ϫ) (Fasl (1) Casp3 (؊) Bid3 (Ϫ (Cb1-b (؊) Bim (؊ (Cdkn1b (p27kip1) (Ϫ) Capp5 (؊ (Ctla4 (؊) Cdkn1b (p27kip1) (Ϫ (Dgka (؊) Dapk2 (؊ (Egr2 (؊) Gilz (؊ (Fyn (؊) Itm2b (؊ (Gbp3 (؊) Nrgn (؊ IL-2 (Ϫ) Pim1 (Ϫ) (IL-10 (؊ (Itch (؊ (Itgb2 (؊ (Iarid2 (؊ Lck (؊) TGF-␤-associated genes (Ldh1 (؊ Pdcd1 (؊) Positive correlation No correlation Pgam1 (Ϫ) Ϫ (Pim1 ( ) Gzmb (2) Atp1b1 (؊ (Rab10 (؊) IL-12Rb2 (2) Cc15 (2 (Slc3a2 (؊) IL-4 (2) Cdkn1b (p27kip1) (؊ (RNF128 (GRAIL) (؊) Myc (2) Cdkn2b (p15INK4b) (Ϫ (Sath1 (؊) Nfil3 (2) C2ta (Ϫ ؊ Socs2 (2) Foxp3 ( ) Ϫ Tle4 (Ϫ) Gata3 ( ) IL-2 (Ϫ) (Tnfrsf1b (؊ Satb1 (Ϫ) ؊ (Tnfsf9 (4-1BBL) ( ) Smad7 (؊ (Tob1 (؊) Tbx21 (T-bet) (Ϫ (Traf5 (؊) Tnfsf9 (4-IBBL) (؊ (Zfpn1a1 (Ikaros) (؊) Vim (؊ a Genes are grouped according to whether they were reported in the literature to be associated with anergy, IL-2 deprivation, or TGF-␤. The effect of Tregs on expression of these genes in CD4ϩCD25Ϫ target cells was determined by microarray analysis (GCRMA). If Tregs had the same effect on expression as reported for the indicated inhibitory condition, the gene was placed in the positive correlation column. Symbols in the parentheses indicate the effect that Tregs had on gene expression relative to nonsuppressed cells: increased (1), decreased (2), or unchanged (Ϫ). Bold, genes reported in the literature to be up-regulated. The Journal of Immunology 6959 were analyzed by real-time RT-PCR for appropriate gene expres- aborted activation whereby Tregs did not markedly prevent the sion patterns. Our anergic cells had up-regulated expression of Fas normal activation program of CD4ϩ T cells during the first 12 h ligand, Itch, GRAIL, PD-1, and IL-10 and down-regulated Myc, but thereafter reversed many of the changes induced by activation. whereas our TGF-␤-treated cells had up-regulated expression of Our previous kinetic analysis of Treg action at the functional level Smad7 and Foxp3 and down-regulated Myc, IL-2, Statb1, (IL-2 production) suggested a similar pattern of initial activation GATA3, and T-bet. We performed real-time RT-PCR to compare followed by Treg-mediated shutdown of target T cell activation the effect of these treatments to that of Treg encounter using our (12). The kinetics of Treg action could be driven by the time molecular signature of Treg suppression: the panel of the 57 genes needed for Tregs to acquire activity or by the time required for confirmed to be differentially expressed in target T cells on Treg target T cells to reach a suppressible state. We favor the latter encounter at 36 h (Fig. 4). scenario, since preactivation of the CD4ϩCD25ϩ population did Surprisingly, all genes expressed at lower levels in suppressed T not change the timing of suppression, at least at the level of IL-2 cells (n ϭ 34) were shared with one or all of the nonproliferative modulation (12). The time point at which Tregs appear to act is an states (Fig. 5a; see Fig. 4 for genes analyzed). Within this group, interesting one, in agreement with recent studies looking at the the majority of genes (71%) were down-regulated following Treg length of TCR-signaling time that is required for T cell prolifer- encounter, IL-2 deprivation, and TGF-␤ treatment, whereas 26% ation and function in vitro and in vivo (39, 40). Indeed, premature of genes were similarly regulated in all nonproliferative states. The termination of signaling in the first 12 h in vitro (39) led to the majority of these genes were of the aborted activation group 1B blockade of proliferation and cytokine production, a functional (Fig. 5d). This notable commonality suggests that Tregs do not state that may resemble that of Treg encounter. Recent in vivo regulate target T cell activity by the suppression or down-modu- observations that Tregs may limit the number or duration of stable Downloaded from lation of distinct genes. target T cell-APC interactions (18, 19) correspond well with our In striking contrast, a distinct set of genes was expressed at gene profile of aborted activation. higher levels in T cells following Treg encounter (Fig. 5) with 61% There was a remarkable concordance between genes expressed of genes uniquely modulated by Treg encounter (14 of 23) (Fig. at a reduced level in our Treg-suppressed cells and genes down- 5b). Moreover, in many cases, the elevated gene expression in regulated under each of the other nonproliferative states analyzed

suppressed cells was countered by a concomitant decrease in gene (Fig. 5a). Indeed, real-time PCR analysis of 34 genes expressed at http://www.jimmunol.org/ expression in target cells deprived of IL-2 or exposed to TGF-␤. lower levels in suppressed T cells failed to find a single gene that Thus, Treg encounter appears to lead to the elevated expression of was uniquely decreased following Treg encounter. Given that one a distinct set of genes that distinguishes the Treg-induced nonpro- of the functional outcomes of Treg suppression is an IL-2 deficit, liferative state from that of IL-2 deprivation, TGF-␤ exposure, or it was not surprising to find that all genes examined that were anergy. Those genes at higher levels in suppressed cells only were down-regulated on Treg encounter were also down-regulated on predominantly of the aborted activation group, group 1A (Fig. 5d). IL-2 deprivation in our hands. Therefore, the molecular profile Noticeably, many of the more highly expressed genes restricted to identified here for genes expressed at lower levels in suppressed Treg encounter have been reported to negatively regulate cellular cells appears to represent a common end stage for cells driven into proliferation or differentiation (9 of 14 genes) (Fig 5b, bold, and a nonproliferative state under the conditions tested. by guest on September 28, 2021 see Discussion). Therefore, we suggest that Tregs disable their However, our data support the idea that Treg encounter does not target T cells in part by the induction or maintenance of expression simply terminate target T cell activation via common pathways of of a unique set of genes that inhibit progression toward the pro- proliferative inhibition, such as IL-2 deprivation or TGF-␤ expo- liferative state. sure. In stark contrast to the genes inhibited after Treg encounter, Of the remaining genes that were more highly expressed in sup- 61% (14 of 23) of genes expressed at a higher level in suppressed pressed cells, 35% (8 of 23) were affected in a similar fashion only cells were unique to Treg encounter. Notably, this group was en- in anergized cells (Fig. 5c), perhaps reflecting similarities in the riched (9 of 14) for gene products that have been associated with pathways for ionomycin-induced anergy and Treg suppression. In- growth arrest and/or inhibition of proliferation in lymphocytes or terestingly, the genes expressed more highly in suppressed and other cell types (Fig. 5b, genes in bold). A smaller fraction of anergized cells contained a higher number of genes uniquely up- genes (3 of 8) was associated with inhibition of proliferative cells regulated under these conditions (group 2, Fig. 5d), again suggest- in the suppressed and anergized group (Fig. 5c, genes in bold). The ing shared components in the pathways to these two nonprofilera- most differentially expressed gene was the neuroendocrine protein tive states. Sgne1 (also known as 7B2) whose expression has been associated with the inhibition of pituitary cell proliferation following TGF-␤ Discussion treatment, but its role in lymphocytes is not known (41). Although It is well established that the CD4ϩCD25ϩFoxp3ϩ population of we did not see TGF-␤ induction of Sgne1 expression in CD4 T regulatory T cells plays an important role in modulating immune cells, Sgne1 was highly expressed in both nonproliferating, un- responses to both self and foreign Ag. However, their mechanism stimulated and Treg-suppressed CD4ϩ T cells. Id3 was induced by of action remains elusive. Our analysis of transcriptional changes 12 h in suppressed cells and is an inhibitor of basic-helix-loop- in target CD4ϩCD25Ϫ T cells following activation in the presence helix protein transcription factors important for cellular prolifera- of CD4ϩCD25ϩ T cells provides the first insight into molecular tion and differentiation (42). With relevance to immune regulation, events in the target T cells following Treg encounter. The outcome Id3 deficiency in T cells led to the development of a Sjo¨gren-like of Treg action appears to manifest at the transcriptional level at a syndrome (43). relatively late stage in T cell activation. Kinetic analysis revealed Several of the genes unique to Treg encounter have functions in substantial changes in gene expression in target T cells after stim- lymphocyte growth arrest or immunosuppression. Both Tspan32 ulation for 12 h, and these new activation-induced patterns were (also known as Tssc6 or Phemx) and Ramp1 have been directly similar in the presence or absence of CD4ϩCD25ϩ T cells. How- linked with the inhibition of IL-2 production by T cells (29, 44) ever, by 36 h, a large group of genes was identified as being dif- and are found more highly expressed in suppressed cells but not ferentially expressed following Treg encounter. Examination of other nonproliferative states here. Interestingly, Tspan32 is in- changes in individual genes over time revealed a general pattern of duced early in the suppression process whereas Ramp1 comes up 6960 DISTINCT MOLECULAR SIGNATURE IMPOSED BY Tregs later, suggesting possible inductive and maintenance roles in sup- help with microarray analysis. Special thanks to Tim Mosmann, Jim Miller, pression, respectively (Fig. 4, a and c). Two members of the Ms4a Nick Crispe, and Ben Segal for insightful comments on this manuscript. family of receptors (Ms4a4b and Ms4a4c) are more highly ex- pressed in suppressed cells. Interestingly, the best characterized Disclosures Ms4a member is CD20, cross-linking of which can also lead to The authors have no financial conflict of interest. growth arrest (45). Ramp1, which is uniquely increased in Treg- References suppressed cells at 36 h compared with the other nonproliferative 1. Sakaguchi, S. 2005. Naturally arising Foxp3-expressing CD25ϩCD4ϩ regulatory states tested, is a component of the calcitonin receptor that re- T cells in immunological tolerance to self and non-self. Nat. Immunol. 6: sponds to the neuropeptide calcitonin gene-related peptide that has 345–352. 2. von Boehmer, H. 2005. Mechanisms of suppression by suppressor T cells. Nat. immunosuppressive activity (46). 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