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The Journal of Immunology, 2006, 177: 6952-6961.
Copyright © 2006 by The American Association of Immunologists, Inc.

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

Teresa L. Sukiennicki and Deborah J. Fowell2

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, University of Rochester, Rochester, NY 14642


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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-β-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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Naturally occurring regulatory T cells (Tregs)3 are important modulators of immune responses. Initially demonstrated to prevent autoimmunity, they have since been associated with the regulation of immune responses to pathogens, tumors, and transplantation Ags (1, 2, 3). Their influence on CD4+ T cells has been studied best, although direct or indirect effects on CD8+ T cells, B cells, and innate cell types including dendritic cells also have been reported. Tregs suppress many activities of target cells including proliferation, IL-2 production (4, 5), T cell differentiation, and effector T cell migration and cytokine secretion (6, 7, 8).

Little is known about how Tregs mediate their suppressive effects. They require activation through their TCR to acquire full functional competence and, in vitro, must be in close proximity to their targets (or APC) to mediate suppression (4, 5). The expression of CD25 at high levels on Tregs suggests that they may work through competitive consumption of IL-2 (9), although the ability of Tregs to suppress IL-2R-deficient T cells (10) and in the presence of excess exogenous IL-2 (11, 12) suggests that IL-2 competition is not essential for suppression. IL-10 and TGF-β are key immunomodulators in in vivo models of Treg suppression produced by the Tregs themselves or induced in other cell types by Tregs (13, 14, 15, 16). Modulation of APC function by Tregs (17, 18, 19), anergy induction (20), and direct cytotoxicity of immune targets via Treg-produced granzyme B and/or perforin (21, 22) have been proposed as additional modes of Treg action. Thus, Tregs may have multiple ways in which to disable a developing immune response, and the mode of action may depend on the context of both the target and Treg activation signals.

Tregs cause a block in proliferation, differentiation, and effector T cell function, but beyond this the molecular changes in target T cell biology following Treg encounter are ill-defined. IL-2 transcription is down-regulated in the target T cells (4, 5, 12) but it is not known whether the IL-2 gene modulation is the primary target, or a downstream consequence, of Treg suppression. The strength of the target T cell activation signal appears to play a role in their susceptibility to suppression, with increased costimulation (5, 23) and/or Ag concentration (24) rendering the target T cells refractory to Treg suppression. It is unclear how these additional signals protect against Treg action. Recent data suggest that the NFAT and Cbl-b pathways in the target population are important for Treg action, since Tregs were unable to block the proliferation of CD4+ T cell targets that lacked expression of these signal molecules (25, 26). Therefore, Tregs may directly target these signaling pathways to facilitate suppression.

Postulating that molecular changes in the suppressed cell may yield clues to the functional state induced in targets by Tregs and/or the mechanism of Treg activity, we used microarray technology to determine changes in target T cell gene expression following Treg-mediated suppression. Differences in gene expression in the suppressed cells evolved over time, with only 12 genes differentially expressed between CD4+CD25– target cells cultured in the presence (suppressed) or absence (nonsuppressed) of Tregs at 12 h and 242 genes at 36 h. The kinetics of expression of many of these genes suggested a pattern of aborted activation in the presence of Tregs. That is, similar transcriptional changes occurred in the presence or absence of Tregs early after activation, but the presence of Tregs reversed many of these activation-induced changes by 36 h. To determine whether Tregs induced distinct changes in target T cells, we examined cells that had been anergized, deprived of IL-2, or treated with TGF-β for expression of a panel of genes found to be differentially expressed following a Treg encounter. All of the down-regulated genes in suppressed cells were similarly affected under one or more of the nonproliferative conditions tested, suggesting that Tregs do not function by inhibiting a discrete panel of target T cell genes. In contrast, a unique set of genes was more highly expressed in target T cells following Treg encounter but not in T cells inactivated in other ways. This global analysis of gene expression in suppressed cells supports the idea that Treg-induced suppression is a distinct molecular process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c Thy1.2+ mice were purchased from The Jackson Laboratory or the National Cancer Institute. BALB/c Thy1.1+ mice were bred in house. Mice were housed under pathogen-free conditions and all animal protocols were approved by the University Committee for Animal Resources at the University of Rochester Medical School.

Media, Abs, and reagents

Cells were cultured in RPMI 1640 with 10% heat-inactivated FCS (HyClone), 50 µM 2-ME (Bio-Rad), 2 mM L-glutamine (Invitrogen Life Technologies), and 100 U/ml penicillin and streptomycin (Invitrogen Life Technologies). All Abs for flow cytometry were purchased from eBioscience, biotin anti-mouse CD25 (clone 7D4) and 7-aminoactinomycin D (7-AAD) from BD Pharmingen, and recombinant human TGF-β1 and rIL-12 from PeproTech. Human IL-2 was obtained from the National Institutes of Health Research and Reference Reagent Program.

Cell purification and culture

CD4+CD25– and CD4+CD25+ cells were isolated by magnetic separation (Miltenyi Biotec) as described previously (12). CD4+CD25– cells (1 x 105) were stimulated with 1 µg/ml soluble anti-CD3 and 1 x 105 T- depleted splenocytes with and without 0.5–1 x 105 CD4+CD25+ cells. Where indicated, the CD25– cells were prelabeled with CFSE (Molecular Probes). In some cases, 10 ng/ml recombinant human TGF-β1 was added at the start of culture. IL-2 was blocked 6 h into the culture (5 µg/ml anti-IL-2 mAb) based on our previous observations on the kinetics of IL-2 down-regulation by Tregs (12). Parallel cultures were set up for confirmation that the Tregs were functional and that the anti-IL-2, TGF-β, and anergy-inducing conditions indeed inhibited proliferation. Proliferation was measured during the last 6 h of a 72-h culture by incorporation of [3H]thymidine (1µCi).

Anergy induction

Anergy was induced as described previously (27) in Th1 cells generated from CD4+CD25– cells isolated from DO11.10 TCR-C{alpha}–/– BALB/c mice. Briefly, CD4+CD25– cells were stimulated with 1 µM 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 x 106 cells/ml with 1 µM ionomycin in complete RPMI 1640 for 16 h at 37°C.

Microarray sample preparation and analysis

CD4+CD25–Thy1.1+ cells were stimulated in the presence or absence of CD4+CD25+Thy1.2+ cells as described above. At 0, 12, and 36 h, CD4+CD25– cells were isolated by flow cytometric sorting of 7-AAD–CD4+Thy1.1+ cells (98.5–99.5% CD4+Thy1.1+). Total RNA was isolated using TRIzol (Invitrogen Life Technologies). RNA was amplified and biotin-labeled by the MessageAmp kit procedure (Ambion) using 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.

Data were analyzed using Microarray Suite 5.0 (Affymetrix) and Gene-Traffic software (RMA and GCRMA algorithms; Iobian/Stratagene). Genes were selected from the first set of arrays that were at least 2-fold differentially expressed between cells cultured in the presence or absence of Tregs at each time point. Genes with poor replicates (less than a 1.5-fold difference in expression in the second experiment) were discarded. A list of differentially expressed genes was generated by the intersection of genes with good replicates determined using the three algorithms.

Quantitative real-time PCR

Unamplified cDNA was generated from mRNA using random hexamers and Superscript II (Invitrogen Life Technologies). cDNA was loaded in duplicate onto custom-designed TaqMan Low-Density Arrays (Applied Biosystems). Data were analyzed using Sequence Detection System software (Applied Biosystems) with 18S ribosomal RNA as the endogenous control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Kinetic changes in target T cell activation in the presence of Tregs

The timing and downstream consequences of Treg encounter for the target T cell are poorly understood. We have recently defined a narrow kinetic window in which Tregs act on CD4+ T cell targets, occurring within the first 6–12 h of target T cell activation in vitro (12). To obtain a better understanding of how the target T cell changes over time, we analyzed the expression of early activation markers on CD4+CD25– target T cells in the presence or absence of allelically marked CD4+CD25+ Tregs. In the first 6 h, target T cells up-regulated the expression of CD69 and CD25 similarly in the presence or absence of Tregs (Fig. 1a), demonstrating that Tregs do not block initial activation signals. By 12 h, however, the percentage of cells expressing high levels of these markers began to decline in the presence of Tregs, with the differential expression being most evident by 36 h. Given recent reports of the ability of Tregs to express cytotoxic machinery and to kill target T cells (21, 22), loss of activation marker expression at the population level could have been due to the preferential death of those cells that had initiated activation in the presence of Tregs. However, analysis of viability revealed little cell death in cultures containing Tregs (Fig. 1b). The vast majority of cells remained viable (87% 7-AAD negative with Tregs and 90% without Tregs at 36 h) with similar target cell yields at 12 and 36 h in the presence or absence of Tregs. Thus, Tregs inhibit the proliferation of CD4+ T cells in vitro without blocking initial activation signals or inducing considerable cell death.


Figure 1
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  		FIGURE 1. CD4+ T cells are activated and remain viable in the presence of CD4+CD25+ T cells. a, Surface expression of CD69 and CD25 was determined for CD4+CD25–Thy1.1+ target T cells stimulated with and without CD4+CD25+Thy1.2+ T cells. All plots are gated on CD4+Thy1.1+ target T cells, unstimulated cells (dotted lines). b, Viability of CFSE-labeled target T cells stimulated as in a, by exclusion of 7-AAD, gated on Thy1.1+ target T cells. Numbers indicate the percentage of cells in each quadrant.

 
Changes in target T cell gene expression following regulatory T cell encounter

To gain a molecular understanding of the altered state of the target T cells following activation in the presence of Tregs, we positively selected viable Thy1.1+ target T cells by FACS from cultures with (suppressed) and without (nonsuppressed) Thy1.2+ Tregs at 0, 12, and 36 h, purified mRNA and, performed microarray analysis of gene expression using Affymetrix oligonucleotide arrays. Two independent isolations of target T cells from cultures with and without Tregs were analyzed on individual microarrays. Suppressive activity was verified in parallel cultures by [3H]thymidine incorporation (data not shown). Consistent with our functional data, there were strikingly few differences between CD4+ T cell targets in the presence or absence of Tregs at 12 h, whereas the effect of Treg encounter was readily apparent at 36 h (Fig. 2). To identify strong candidate genes for suppression, microarray data were analyzed using three different algorithms (MAS 5.0, RMA, and GCRMA) and genes were selected for further analysis from the intersection of these individual algorithm lists. After 12 h, only 12 genes were reproducibly differentially expressed in suppressed cells and mostly represented quantitative differences between suppressed and nonsuppressed targets (data not shown). A larger number of genes were differentially expressed at 36 h (242 genes, 91 genes at higher levels and 151 genes at lower levels in suppressed target cells, Table I, all genes for which a functional class has been determined, 184 of 242 total genes).


Figure 2
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  		FIGURE 2. Treg-induced changes in gene expression in target T cells over time. mRNA was isolated from CD4+CD25– Thy1.1+ T cells stimulated for 12 or 36 h in the presence (suppressed) or absence (nonsuppressed) of CD4+CD25+ Thy1.2+ T cells and analyzed on Affymetrix mouse genome 430A 2.0 arrays. Signal intensities (GCRMA algorithm) for each probe set were plotted against each other on a log scale.

 

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  		Table I. Functional groupings and kinetic pattern of expression designations of genes differentially expressed in T cells cultured in the presence of CD4+CD25+ T cells at 36 ha

 
Kinetic patterns of gene expression

Many more genes were differentially expressed in suppressed target T cells at 36 h than at 12 h (Fig. 2). The kinetics of expression was suggestive of a profile of abortive activation on Treg encounter, in that activation-induced changes in gene expression at 12 h were similar in target cells in the presence or absence of Tregs, but this activation profile was subsequently reversed only in the presence of Tregs (36 h). This profile was seen for almost half of the genes differentially expressed at 36 h (120 of 242, 49.6%; groups 1A and 1B). Fig. 3 shows examples of this pattern of expression for genes initially down-regulated (Fig. 3a, group 1A) or up-regulated (Fig. 3b, group 1B) compared with unstimulated cells. Other groupings included genes specifically induced at 36 h in suppressed cells (Fig. 3c, group 2; 22 of 242, 9%) or specifically down-regulated at 36 h in suppressed cells (Fig. 3d, group 3; 37 of 242, 15%). In addition, a number of genes failed to be induced at 12 or 36 h in suppressed cultures but were specifically up-regulated in nonsuppressed cells at 36 h (Fig. 3e, group 4; 37 of 242, 15%). Table I lists all of the differentially expressed genes for which a functional class has been assigned (184 of 242 genes) and indicates their respective kinetic pattern of gene expression (groups 1–6).


Figure 3
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  		FIGURE 3. Kinetic gene expression patterns in target cells cultured in the presence CD4+CD25+ 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 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 CD4+CD25– 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.

 
Interestingly, the aborted-activation group 1A (Fig. 3a) included quiescence-associated genes of the Kruppel family (KLF-2 and KLF-3), which regulate cell proliferation and differentiation. KLF-2 is expressed in resting, unstimulated lymphocytes and its rapid down-regulation appears to be a necessary step for lymphocyte activation (28). Both suppressed and nonsuppressed cells down-regulated KLF-2 and KLF-3 at 12 h, but although down-regulated expression of KLF-2 and KLF-3 was maintained in nonsuppressed cells, the suppressed cells re-expressed these genes by 36 h. This may be indicative of a loss of activation stimulus required to maintain reduced expression of KLFs or the positive regulation of KLFs’ re-expression by Tregs (29). In the other aborted-activation group, group 1B (Fig. 3b), expression of many molecules characteristic of T cell activation and differentiation, such as CD25, was initiated in both populations at 12 h but down-regulated in suppressed cells at 36 h. The aborted activation pattern was also seen by real-time RT-PCR analysis of expression of a number of immune activation and differentiation genes (Fig. 3f). Thus, our gene profiling approach confirms the initial functional data, suggesting that early T cell activation proceeds largely unperturbed in the presence of Tregs, but that Tregs disrupt the maintenance/progression of the activation program.

Many of the genes expressed at lower levels in suppressed cells at 36 h reflected a general down-regulation of metabolic pathways: DNA/RNA binding, transcription, translation, and protein modifications (Table I). A large group (n = 36; Table I) of these genes expressed at lower levels in suppressed cells have been designated as Myc target genes (<www.myccancergene.org/site/mycTargetDB.asp>), Myc also being down-regulated in suppressed cells. In addition, all differentially expressed mRNAs for secreted proteins were down-regulated including immune effector molecules such as IL-4, Ccl5 (RANTES), and granzymes (Table I). Down-regulated cell surface proteins included a number of cytokine receptor chains (IL-2R{alpha}, IL-12Rβ2, and IL-7r) and molecules associated with T cell activation (Gp49a, Ly6c, CD24a, and CD62P) consistent with the aborted activation phenotype. In contrast, a number of genes encoding cell surface molecules associated with down-regulation of proliferation were more highly expressed in suppressed cells (Tspan32, TGF-βr2, and Timp2). Interestingly, we found little evidence for Treg targeting of transcription of genes involved in regulation of the cell cycle or apoptotic pathways at 36 h.

Confirmation by quantitative RT-PCR

We designed custom low-density arrays (Applied Biosystems) for real-time PCR analysis of expression of 71 of the most differentially expressed genes identified by microarray at 36 h. This selected 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).


Figure 4
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  		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 = 23) or a lower level (n = 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.

 

Figure 5
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  		FIGURE 5. Tregs induce a distinct gene program in target T cells. CD4+CD25– T cells were stimulated with and without CD4+CD25+ T cells, or under other inhibitory conditions: IL-2 blockade, TGF-β exposure, or ionomycin-induced anergy. Expression of genes differentially expressed in target T cells following CD4+CD25+ 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-β-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.

 
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 nonsuppressed cells at 36 h and remained differentially expressed in suppressed 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 the induction point of suppression. The observed variability probably reflected the asynchrony of the activation and/or suppression events. To identify likely gene candidates that are causative, rather than a consequence, of suppression, we analyzed our 36-h gene program for early expression at 12 h by real-time RT-PCR (Fig. 4). The higher resolution afforded by real-time RT-PCR revealed that many of the genes confirmed as differentially expressed at 36 h were already differentially expressed by 12 h. It is likely that these expression differences were not identified by our microarray analysis given the use of a strict 2-fold cutoff and the selection of genes from the intersection of three separate algorithms. The genesexpressed more highly in suppressed compared with nonsuppressed T cells at 36 h (Fig. 4, a–d) could be subdivided into genes up-regulated or sustained only in suppressed cells early at 12 h relative to nonsuppressed cells (Fig. 4, a and b) or genes up-regulated in suppressed cells late at 36 h (Fig. 4, c and d). The genes differentially expressed early in suppressed cells through up-regulation or maintenance of gene expression represented potential initiators of suppression and contained a number of transcriptional regulators, such as Pou2af1 (also known as BOB.1, OCA-B) and Id3, and signal transduction modifiers, such as S100a10 and Sbk. In contrast to the microarray data (Fig. 3a), Tspan 32 (also known as Tssc6 or Phemx) was up-regulated at 12 h by quantitative RT-PCR and has been implicated in the negative regulation of T cell proliferation. In contrast, genes up-regulated late (Fig. 4, c and d) may be required to maintain the suppressed state, such as the quiescence-associated genes Klf2 and Ramp1, a component of the immunosuppressive calcitonin receptor.

Kinetic analysis of the genes expressed at lower levels in suppressed cells at 36 h (Fig. 4, e–h) revealed some interesting patterns of expression. Half of the genes appeared to be of the aborted activation group, where genes were similarly regulated in both suppressed and nonsuppressed cells at 12 h but diverged at 36 h due to enhanced expression in the nonsuppressed cells, as shown for CD25 (Fig. 4e). Very few genes were specifically down-regulated at 12 h in suppressed cells (Fig. 4f), perhaps suggestive of this time point being at the early stages of Treg suppression. Interestingly, nearly half of the genes down-regulated at 36 h in suppressed cells were, in contrast, more highly expressed (through induction or maintenance) in suppressed cells compared with nonsuppressed cells at 12 h (Fig. 4, g and h). These genes may also be important initial regulators of the suppressed state and include the transcriptional regulator Cdk8 and the suppressor of cytokinesignaling Socs2 (Fig. 4g).

Little overlap with the gene expression profiles known for other nonproliferative states

Although little is known about the mechanisms by which Tregs inhibit an immune response, it has been hypothesized that they may deprive cells of IL-2 (9), induce anergy (20), or act through the action of cell surface-associated TGF-β (30). Indeed, these manipulations all lead to a common cellular outcome, that of a nonproliferative state. Comparison of the genes reported to be associated with anergy, IL-2-deprivation, and TGF-β treatment with our own for Treg encounter at 36 h highlighted a striking disparity between gene expression in suppressed cells and published genes associated with IL-2 deprivation, anergy, or TGF-β exposure (Table II) (31, 32, 33, 34, 35). The expression of many anergy-associated genes remained unchanged in target T cells cultured with Tregs, relative to target T cells in the absence of Tregs, including the ubiquitin ligases, Itch, Cbl-b, and Rnf128 (also known as GRAIL) (27, 36). We found little correlation between suppression and changes in IL-2-deprivation-associated genes including those that control cell cycle and cell death, such as Cdkn1b (also known as p27kip), Bcl2, and Bim. Overlap was seen however with those genes down-regulated on TGF-β exposure including the immune-response molecules granzyme B, IL-4, and the IL-12Rβ2 chain. In addition, subsequent real-time PCR analysis revealed decreases in IL-2, GATA3, and T-bet in suppressed cells (Fig. 3f) as also reported for TGF-β-exposed T cells (35). In contrast, there was little correlation between genes associated with suppression and those genes reported to be induced by TGF-β treatment, such as Smad7 and Foxp3 (37, 38), neither of which was differentially up-regulated in T cell targets following Treg encounter (microarray data).


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

 
A distinct molecular signature in target cells following Treg encounter

It is not clear whether Treg encounter simply terminates target T cell activation or whether Tregs induce a distinct change in the molecular program of the target T cells. Comparison to genes associated with nonproliferating cells from the current literature (Table II) suggested that Tregs did indeed impose a unique gene program in target cells. However, such cross-laboratory comparison suffers from variability between studies with respect to cell type and experimental protocol. Therefore, to directly compare gene profiles of our suppressed cells with other nonproliferative states we isolated mRNA from CD4+CD25– T cell cultures where T cell proliferation had been blocked by Treg encounter or via treatment with TGF-β, deprivation of IL-2, or induction of anergy (by ionomycin treatment as described by Rao (27)). These treated cells were analyzed by real-time RT-PCR for appropriate gene expression patterns. Our anergic cells had up-regulated expression of Fas ligand, Itch, GRAIL, PD-1, and IL-10 and down-regulated Myc, whereas our TGF-β-treated cells had up-regulated expression of Smad7 and Foxp3 and down-regulated Myc, IL-2, Statb1, GATA3, and T-bet. We performed real-time RT-PCR to compare the effect of these treatments to that of Treg encounter using our molecular signature of Treg suppression: the panel of the 57 genes confirmed to be differentially expressed in target T cells on Treg encounter at 36 h (Fig. 4).

Surprisingly, all genes expressed at lower levels in suppressed T cells (n = 34) were shared with one or all of the nonproliferative states (Fig. 5a; see Fig. 4 for genes analyzed). Within this group, the majority of genes (71%) were down-regulated following Treg encounter, IL-2 deprivation, and TGF-β treatment, whereas 26% of genes were similarly regulated in all nonproliferative states. The majority of these genes were of the aborted activation group 1B (Fig. 5d). This notable commonality suggests that Tregs do not regulate target T cell activity by the suppression or down-modulation of distinct genes.

In striking contrast, a distinct set of genes was expressed at higher levels in T cells following Treg encounter (Fig. 5) with 61% of genes uniquely modulated by Treg encounter (14 of 23) (Fig. 5b). Moreover, in many cases, the elevated gene expression in suppressed cells was countered by a concomitant decrease in gene expression in target cells deprived of IL-2 or exposed to TGF-β. Thus, Treg encounter appears to lead to the elevated expression of a distinct set of genes that distinguishes the Treg-induced nonproliferative state from that of IL-2 deprivation, TGF-β exposure, or anergy. Those genes at higher levels in suppressed cells only were predominantly of the aborted activation group, group 1A (Fig. 5d). Noticeably, many of the more highly expressed genes restricted to Treg encounter have been reported to negatively regulate cellular proliferation or differentiation (9 of 14 genes) (Fig 5b, bold, and see Discussion). Therefore, we suggest that Tregs disable their target T cells in part by the induction or maintenance of expression of a unique set of genes that inhibit progression toward the proliferative state.

Of the remaining genes that were more highly expressed in suppressed cells, 35% (8 of 23) were affected in a similar fashion only in anergized cells (Fig. 5c), perhaps reflecting similarities in the pathways for ionomycin-induced anergy and Treg suppression. Interestingly, the genes expressed more highly in suppressed and anergized cells contained a higher number of genes uniquely up-regulated under these conditions (group 2, Fig. 5d), again suggesting shared components in the pathways to these two nonprofilerative states.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well established that the CD4+CD25+Foxp3+ population of regulatory T cells plays an important role in modulating immune responses to both self and foreign Ag. However, their mechanism of action remains elusive. Our analysis of transcriptional changes in target CD4+CD25– T cells following activation in the presence of CD4+CD25+ T cells provides the first insight into molecular events in the target T cells following Treg encounter. The outcome of Treg action appears to manifest at the transcriptional level at a relatively late stage in T cell activation. Kinetic analysis revealed substantial changes in gene expression in target T cells after stimulation for 12 h, and these new activation-induced patterns were similar in the presence or absence of CD4+CD25+ T cells. However, by 36 h, a large group of genes was identified as being differentially expressed following Treg encounter. Examination of changes in individual genes over time revealed a general pattern of aborted activation whereby Tregs did not markedly prevent the normal activation program of CD4+ T cells during the first 12 h but thereafter reversed many of the changes induced by activation. Our previous kinetic analysis of Treg action at the functional level (IL-2 production) suggested a similar pattern of initial activation followed by Treg-mediated shutdown of target T cell activation (12). The kinetics of Treg action could be driven by the time needed for Tregs to acquire activity or by the time required for target T cells to reach a suppressible state. We favor the latter scenario, since preactivation of the CD4+CD25+ population did not change the timing of suppression, at least at the level of IL-2 modulation (12). The time point at which Tregs appear to act is an interesting one, in agreement with recent studies looking at the length of TCR-signaling time that is required for T cell proliferation and function in vitro and in vivo (39, 40). Indeed, premature termination of signaling in the first 12 h in vitro (39) led to the blockade of proliferation and cytokine production, a functional state that may resemble that of Treg encounter. Recent in vivo observations that Tregs may limit the number or duration of stable target T cell-APC interactions (18, 19) correspond well with our gene profile of aborted activation.

There was a remarkable concordance between genes expressed at a reduced level in our Treg-suppressed cells and genes down-regulated under each of the other nonproliferative states analyzed (Fig. 5a). Indeed, real-time PCR analysis of 34 genes expressed at lower levels in suppressed T cells failed to find a single gene that was uniquely decreased following Treg encounter. Given that one of the functional outcomes of Treg suppression is an IL-2 deficit, it was not surprising to find that all genes examined that were down-regulated on Treg encounter were also down-regulated on IL-2 deprivation in our hands. Therefore, the molecular profile identified here for genes expressed at lower levels in suppressed cells appears to represent a common end stage for cells driven into a nonproliferative state under the conditions tested.

However, our data support the idea that Treg encounter does not simply terminate target T cell activation via common pathways of proliferative inhibition, such as IL-2 deprivation or TGF-β exposure. In stark contrast to the genes inhibited after Treg encounter, 61% (14 of 23) of genes expressed at a higher level in suppressed cells were unique to Treg encounter. Notably, this group was enriched (9 of 14) for gene products that have been associated with growth arrest and/or inhibition of proliferation in lymphocytes or other cell types (Fig. 5b, genes in bold). A smaller fraction of genes (3 of 8) was associated with inhibition of proliferative cells in the suppressed and anergized group (Fig. 5c, genes in bold). The most differentially expressed gene was the neuroendocrine protein Sgne1 (also known as 7B2) whose expression has been associated with the inhibition of pituitary cell proliferation following TGF-β treatment, but its role in lymphocytes is not known (41). Although we did not see TGF-β induction of Sgne1 expression in CD4 T cells, Sgne1 was highly expressed in both nonproliferating, unstimulated and Treg-suppressed CD4+ T cells. Id3 was induced by 12 h in suppressed cells and is an inhibitor of basic-helix-loop-helix protein transcription factors important for cellular proliferation and differentiation (42). With relevance to immune regulation, Id3 deficiency in T cells led to the development of a Sjögren-like syndrome (43).

Several of the genes unique to Treg encounter have functions in lymphocyte growth arrest or immunosuppression. Both Tspan32 (also known as Tssc6 or Phemx) and Ramp1 have been directly linked with the inhibition of IL-2 production by T cells (29, 44) and are found more highly expressed in suppressed cells but not other nonproliferative states here. Interestingly, Tspan32 is induced early in the suppression process whereas Ramp1 comes up later, suggesting possible inductive and maintenance roles in suppression, respectively (Fig. 4, a and c). Two members of the Ms4a family of receptors (Ms4a4b and Ms4a4c) are more highly expressed in suppressed cells. Interestingly, the best characterized Ms4a member is CD20, cross-linking of which can also lead to growth arrest (45). Ramp1, which is uniquely increased in Treg-suppressed cells at 36 h compared with the other nonproliferative states tested, is a component of the calcitonin receptor that responds to the neuropeptide calcitonin gene-related peptide that has immunosuppressive activity (46). Calcitonin gene-related peptide inhibits T cell production of IL-2 and other cytokines, such as TNF-{alpha}, TNF-β, and IFN-{gamma} (44), possibly via blockade of NF-{kappa}B activity (47). Also linked to CD4 T cell responses is the general receptor of phosphoinositides 1 (GRP1, also known as Pscd3). GRP1 is rapidly down-regulated in nonsuppressed cells on activation but uniquely maintained in Treg-suppressed cells (Figs. 4b and 5). GRP1 was found differentially expressed in CD4+ T cell clones made anergic by activation in the absence of costimulation and was implicated in the survival of anergized cells (48).

Kinetic analysis revealed a number of genes that were induced at 12 h in suppressed cells but then down-regulated by 36 h (Fig. 4g). Cyclin-dependent kinase 8 and its yeast homologue Srb10 are components of the RNA polymerase II holoenzyme complex and have been implicated in transcriptional repression (49, 50). Its transient up-regulation at 12 h in suppressed cells coincides with the kinetics of aborted activation and may thus play a role in the down-regulation of immune-activating genes. Similarly, the transient induction of Socs2 at 12 h suggests a role in the down-regulation of cytokine signaling via Stat5a (51 , 52) at around the time of the first cell division.

As shown in Fig. 5c, approximately one-third of the genes expressed more highly in suppressed cells were similarly regulated in cells made anergic via stimulation with ionomycin (27). Anergy induced through ionomycin stimulation leads to T cell activation in the presence of unopposed calcium signaling and was found to induce a distinct genetic program that was largely NFAT dependent (32). This similarity between T cells modified by ionomycin and Treg encounter is intriguing given the observation that target T cells are resistant to Treg suppression if they fail to express certain NFAT members (25). Therefore, Tregs may, in part, induce suppression via modulating T cell-signaling pathways in such a way as to leave NFAT signaling unopposed. Notably however, the Treg mechanism does not appear to involve sustained expression of the ubiquitin ligase pathway as suggested for anergy induction via ionomycin (27) or lack of costimulation (36).

It is not clear at this stage whether the genes identified here as being positively regulated in target T cells following Treg encounter are important in establishing or maintaining the suppressed state. Kinetic analysis of gene expression has helped to categorize the signature genes into potential regulators vs upholders of the suppressed state, and we are currently determining their functional relationships. Nonetheless, the identification of a molecular signature of Treg suppression is an important step in the understanding of Treg action and could serve as an important diagnostic for T cells that have been rendered unresponsive specifically via encounter with naturally occurring Tregs. Our data suggest that rather than down-regulating a unique group of activation-associated genes, Tregs work through the specific induction or maintenance of a number of negative regulatory factors that may actively engage the nonproliferative state.


   Acknowledgments
 
We thank the members of the Fowell laboratory for helpful discussion and Dr. Welle of the University of Rochester Functional Genomics Center for help with microarray analysis. Special thanks to Tim Mosmann, Jim Miller, Nick Crispe, and Ben Segal for insightful comments on this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Juvenile Diabetes Research Foundation, Research Grant 1-2000-609 (to D.J.F.) and National Institutes of Health Training Grant AI-07169 (to T.L.S.). Back

2 Address correspondence and reprint requests to Dr. Deborah J. Fowell, David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue, Box 609, Rochester, NY 14642. E-mail address: deborah_fowell{at}urmc.rochester.edu Back

3 Abbreviations used in this paper: Treg, regulatory T cell; 7-AAD, 7-aminoactinomycin D; KLF, Kruppel family; GRP1, general receptor of phosphoinositide 1. Back

Received for publication April 21, 2006. Accepted for publication August 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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