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J Exp Med. Sep 1, 2003; 198(5): 737–746.
PMCID: PMC2194185

Continuous Activation of Autoreactive CD4+ CD25+ Regulatory T Cells in the Steady State

Abstract

Despite a growing interest in CD4+ CD25+ regulatory T cells (Treg) that play a major role in self-tolerance and immunoregulation, fundamental parameters of the biology and homeostasis of these cells are poorly known. Here, we show that this population is composed of two Treg subsets that have distinct phenotypes and homeostasis in normal unmanipulated mice. In the steady state, some Treg remain quiescent and have a long lifespan, in the order of months, whereas the other Treg are dividing extensively and express multiple activation markers. After adoptive transfer, tissue-specific Treg rapidly divide and expand preferentially in lymph nodes draining their target self-antigens. These results reveal the existence of a cycling Treg subset composed of autoreactive Treg that are continuously activated by tissue self-antigens.

Keywords: CD4+ T lymphocytes, suppressor cells, homeostasis, immune tolerance, self-antigens

Introduction

CD4+ CD25+ regulatory T cells (Treg) play a major role in the maintenance of self-tolerance and the control of autoimmune diseases (1, 2). They are also involved in the regulation of T cell homeostasis (3, 4) and in the modulation of immune responses to alloantigens, cancer cells, and pathogens (510). These findings have opened new prospects in immunotherapeutic interventions for several diseases. For instance, we and others have shown that transfer of Treg could be used to control autoimmune diabetes (11, 12), solid allograft rejection (1315), or graft versus host disease (7, 9, 16) in mice. In addition, Treg-specific depletion at the time of tumor inoculation dramatically increased immune rejection of some tumors (10). These recent data indicate that the Treg represent a master player in the immune system, and that their manipulation could be used in new therapeutics. It is therefore essential to gain more basic information on the biology of these cells.

The thymic origin of Treg has been established in mice from several observations. Thymectomy of day-3 neonates induced long-term depletion of Treg and a severe autoimmune syndrome (17). The thymus contains cells with similar phenotype and suppressor function as the Treg described in the spleen and LN (18). Finally, early thymocyte precursors have the potential to differentiate in mature Treg after adoptive transfer (18). Importantly, thymic selection of Treg precursors appears to favor the emergence of a repertoire of highly autoreactive Treg. Indeed, Treg specific to self-Ag presented by the thymic epithelium are positively selected in the thymus and then colonize secondary lymphoid organs (1922).

In the periphery, for a given age and genetic background, Treg represent a stable proportion of the CD4+ T cells in the steady state, suggesting that the homeostasis of Treg is tightly regulated. In young adult mice not prone to autoimmune diseases, Treg constitute ~10% of CD4+ T cells. This proportion seems be reduced in genetically autoimmune-prone individuals (11, 12). There is also some preliminary evidence in humans that type 1 diabetic patients may have decreased blood Treg cell numbers (23). Mice of the scurvy mutant strain, which have a deficit of Treg, develop a severe autoimmune syndrome (24, 25). Thus, a decreased proportion of these cells could lead to an augmentation of the risk to develop autoimmune diseases. On the opposite, an increased proportion of Treg may alter antitumor and antiinfectious immunity. Thus, homeostasis of Treg is likely an important process in the proper functioning of the immune system.

Several molecules are involved in regulation of the homeostasis of Treg. IL-2 plays a critical role because IL-2 KO and IL-2Rβ KO mice have a profound deficit of Treg (26, 27). This cytokine might be involved in both their thymic production and their peripheral survival (3, 27, 28). B7/CD28 and CD40/CD40L costimulatory pathways are also involved in the regulation of homeostasis of Treg. Indeed, B7-1 B7-2 double KO mice, CD28 KO mice, and CD40 KO mice all present a severe quantitative deficit of Treg (11, 29). Disruption of the B7/CD28 pathway in nonobese diabetic mice was associated with an exacerbation of autoimmune diabetes due to the defect of Treg (11).

Despite the emerging importance of Treg in the immune system, fundamental parameters of the biology and homeostasis of these cells, such as their lifespan, turnover, and recirculation properties remain poorly known. In this work, we addressed these points using a model of adoptive transfer of highly purified Thy-1 congenic Treg into unmanipulated normal hosts. Because donor cells represented <3% of the endogenous Treg population, their homeostasis should not be modified. Thus, studying the intrinsic behavior of donor cells provided data on the homeostasis of Treg in the steady state. We showed that Treg retain a stable expression of CD25 in vivo and that a Treg subset is composed of quiescent cells with long lifespan, whereas cells of the other Treg fraction have a rapid turnover and express multiple activation markers. This latter subset appears to be composed of autoreactive Treg that are continuously activated by tissue self-antigens.

Materials and Methods

Animals.

6–8-wk-old female BALB/c mice were obtained from Charles River Laboratories. The ins-hemagglutinin (HA) transgenic mice expressing HA of influenza virus in islet β cells (30) were backcrossed >10 generations onto BALB/c genetic background and then intercrossed to generate mice homozygous for the ins-HA transgene. The TCR-HA transgenic mice (31) that express a TCR recognizing I-Ed-restricted HA epitope 110–120 (SFERFEIFPKE) were backcrossed >10 generations onto BALB/c genetic background and then bred with congenic Thy-1.1 BALB/c mice to generate [TCR-HA × Thy-1.1] F1 mice. Congenic Thy-1.1 BALB/c mice, ins-HA mice, and TCR-HA × Thy-1.1 mice were bred in our animal facility. Mice were housed in filter-topped cages under specific pathogen-free conditions. They were manipulated according to European Union guidelines.

Cell Preparation and Adoptive Transfer.

Treg were purified as previously described (7). After a mechanical dissociation, spleen and peripheral LN (inguinal, brachial, axillary, and cervical) cells from Thy-1.1 BALB/c mice were first incubated in PBS 3% fetal calf serum with saturating amounts of biotin-labeled anti-CD25 mAb (7D4; BD Biosciences) and then with anti-biotin–coated microbeads (Miltenyi Biotec), and purified using magnetic cell separation LS columns (Miltenyi Biotec). The CD25-depleted cells (referred to as CD25 cells in the text), harvested from the flow through, were depleted of erythrocytes by ammonium chloride lysis. They contained <0.3% CD4+ CD25+ T cells. The positively selected cells (80% CD25+) were stained with FITC-labeled anti-CD4 (GK1.5 or RM4-5; BD Biosciences), PE-labeled anti-CD62L (MEL-14; BD Biosciences), and CyChrome-streptavidin (BD Biosciences) that bound to free biotin-labeled CD25 molecules, uncoupled to beads. The CD4+ CD25+ CD62Lhigh and CD4+ CD25+ CD62Llow cells were sorted on a FACStar™ (Becton Dickinson), giving a purity of 98–99.5%. Then, purified cells were labeled with 5,6-carboxy-fluorescein succinimidyl ester (CFSE; Sigma-Aldrich) by incubation with 2.5 μM CFSE in protein-free PBS for 10 min at room temperature and 1 min with 1 vol serum. Cells were then washed twice in PBS and 0.55–0.7 × 106 purified Treg or 107 CD25 cells were intravenously transferred to congenic Thy-1.2 BALB/c mice. For the transfer experiments into ins-HA transgenic mice, we injected 0.65–1.25 × 106 purified Treg or 2–3 × 106 CD25 cells from TCR-HA transgenic mice.

Antibodies and Flow Cytometric Analysis.

After a mechanical dissociation, cells from spleen or peripheral LNs were preincubated with 2.4G2 mAb (BD Biosciences) to block nonspecific binding to Fc receptors and then stained in PBS 3% fetal calf serum with saturating amounts of combinations of the following mAbs: FITC-conjugated anti-CD4 (clone GK1.5); CyChrome- and allophycocyanin-labeled anti-CD4 (RM4-5); allophycocyanin-labeled anti-CD25 (PC61); PE-labeled CD45RB (C363.16A); PE- and allophycocyanin-labeled anti-CD62L (MEL-14); and PE-labeled anti–Thy-1.1/CD90.1 (OX-7; all from BD Biosciences). We also used the following biotinylated antibodies: anti-CD5 (53-7.3; eBioscience); anti-CD25 (7D4; BD Biosciences); anti-CD38 (90; eBioscience); anti-CD44 (IM7; Caltag Laboratories); anti-CD54 (KAT-1; Caltag Laboratories); anti-CD69 (H1.2F3; BD Biosciences); anti-CD71 (R17 217.1.4; Caltag Laboratories); anti–Thy-1.1/CD90.1 (OX-7; BD Biosciences); anti-CD103 (M290; BD Biosciences); anti-CD122 (TM-b1; BD Biosciences); anti–OX-40/CD134 (OX-86; BD Biosciences); and anti–glucocorticoid-induced TNF receptor (GITR; goat polyclonal Ab; R&D Systems). The biotinylated mAbs were detected by CyChrome- or allophycocyanin-streptavidin (BD Biosciences). Labeling with the anti-clonotypic mAb (clone 6.5) specific to TCR-HA was revealed by a biotin anti–rat IgG2b Ab (BD Biosciences) and streptavidin-CyChrome (BD Biosciences). Isotype-irrelevant mAbs (BD Biosciences) were used as controls. Lymphocytes were gated according to their forward and side scatter characteristics and four-color FACSCalibur™ analyses were performed either with CELLQuest™ (Becton Dickinson) or FlowJo® (Tree Star) software.

After adoptive transfer in wild-type hosts, donor Treg represented <0.1% of splenocytes or LN cells. Therefore, we acquired 1–2 × 106 events on a flow cytometer to detect significant numbers of donor cells. As controls, untransferred mice were systematically analyzed in the same time to evaluate the level of background. Within the CD4+ Thy-1.1+ gate, the number of events were typically of 500 for mice injected with Treg versus <10 for untransferred mice.

Bromodeoxyuridine (BrdU) Labeling and Cell Cycling Analysis.

Mini osmotic pumps (ALZET2001; Durect Corporation), delivering 1.2 mg per day of BrdU (Sigma-Aldrich) for 7 d, were transplanted subcutaneously under ketamine/xylazine anesthesia to 7–8-wk-old mice (Charles River Laboratories). Then, LN cells and splenocytes were stained with anti-CD4 CyChrome (GK1.5; BD Biosciences), anti–CD25-PE (PC61; BD Biosciences), anti–CD44-biotin (IM7.8.1; Caltag), and streptavidin-allophycocyanin (BD Biosciences). For BrdU detection, triple-stained cells were fixed in 1% paraformaldehyde (Sigma-Aldrich) for 12 h in the dark, permeabilized first in absolute ethanol for 4 min on ice, washed in PBS, incubated for 30 min at room temperature followed by 30 min on ice in PBS containing 1% paraformaldehyde and 0.01% Tween-20 (Sigma-Aldrich). After washing in PBS, cells were incubated for 30 min at room temperature with 1 mg/ml DNase I (Sigma-Aldrich) in a buffer containing 0.15 M NaCl, 4.2 mM MgCl2, and 10 mM HCl, pH 5. After being washed in PBS, cells were incubated for 45 min at 4°C in PBS containing 5% fetal calf serum, 0.5% Tween-20, and FITC-labeled anti-BrdU mAb (B44; Becton Dickinson). Then, cells were washed twice in PBS and analyzed using a FACSCalibur™ flow cytometer and CELLQuest™ software (both from Becton Dickinson).

Results

Long-Term Survival and Stable CD25 Expression of Treg In Vivo.

To evaluate the recirculation properties, lifespan, turnover rate, and CD25 marker expression stability of the natural suppressor Treg, we transferred Treg purified from normal mice, having a diversified T cell repertoire, into unmanipulated nonlymphopenic normal Thy-1 congenic mice. We focused this study on the transfer of the CD62Lhigh Treg subset because we and others have previously demonstrated the high suppressive activity of this population in vivo (7, 32) and also because the CD4+ CD25+ CD62Lhigh cell population should contain low, if any, contamination with activated conventional CD4+ T lymphocytes, which display a CD4+ CD25+ CD62Llow phenotype (33). Experiments described below and shown in Figs. 1, ,2,2, and and33 were all performed in the BALB/c genetic background. For all of these experiments, similar findings were observed in the C57BL/6 genetic background (unpublished data).

Figure 1.Figure 1.Figure 1.
Long-term survival and stable CD25 expression of Treg after adoptive transfer. CD62Lhigh Treg or CD25 cells purified from Thy-1.1 BALB/c mice were injected intravenously into 8–10-wk-old Thy-1.2 congenic BALB/c mice and analyzed by flow ...
Figure 2.
Rapid turnover and acquired activated phenotype of a Treg subset. 2, 9, 35, and 70 d after transfer of CFSE-labeled Thy-1.1 CD62Lhigh Treg into Thy-1.2 BALB/c mice, donor cells (gated on CD4+ Thy1.1+ cells) were analyzed for cell division (A) and activation ...
Figure 3.
Analysis of turnover of Treg by BrdU incorporation. BALB/c mice were treated with BrdU administered continuously for 7 d using osmotic pumps. Then, peripheral LN cells and splenocytes were analyzed for cell surface expression of CD4, CD25, and CD44, and ...

2, 9, 35, or 70 d after transfer of CD62Lhigh Treg, low but reliable numbers of donor cells were found in both the spleen and LN (Fig. 1, A–C) , whereas in mice transferred with the CD62Llow Treg, donor cells were detected in the spleen but hardly in the LN (unpublished data). This result can be explained by the known important role of the CD62L molecule in the migration of lymphocytes from blood into LNs (33). A moderate increase of donor cell numbers was observed between days 2 and 35 after cell transfer, which could be due to a recirculation from the liver where we observed a sizeable proportion of donor cells at an early time point (unpublished data). Remarkably, in mice transferred with CD62Lhigh Treg, the numbers of donor cells at an early time point (day 9) or late time points (35 or 70 d) after transfer remained relatively stable (Fig. 1, B and C). We analyzed in parallel the survival of a population of conventional purified CD25 T cells, similarly injected into normal unmanipulated Thy-1 congenic mice. In contrast to the Treg, the number of transferred CD25 T cells progressively decreased with time in the spleen and LN. 35 and 70 d after transfer their numbers were severely reduced (Fig. 1, B and C).

The CD25 molecule is the classical cell surface marker used to identify the natural suppressor CD4+ T cells, but stability of its expression in the steady state has not been addressed. Up to 70 d after transfer, when the experiment was stopped, most of transferred cells maintained a stable phenotype with high level expression of CD4 and CD25 (Fig. 1 D). Similar findings were observed in the spleen (not depicted). This result shows that contrary to activated conventional T cells, which express CD25 for only a few days, the CD25 marker has a stable expression on Treg in vivo.

Rapid Turnover of a Treg Subset.

The stable numbers of infused Treg observed at various times after cell transfer could be maintained by quiescent cells with long lifespan and/or by a more dynamic process involving a balance of divisions compensated by cell deaths. To explore their division rates, donor CD62Lhigh Treg were labeled with CFSE before transfer. In LNs, cells had not yet divided 2 d after transfer. At day 9, a significant proportion of Treg had gone through at least one division, some having already divided extensively (more than six times). 35 and 70 d after transfer, a majority of the remaining cells had divided, most of them more than six times, whereas ~30% of the remaining cells had never divided. Similar findings were observed in the spleen at days 2 and 9, whereas over time the proportion of cells that had never undergone division dropped to only ±20 and 14% at days 35 and 70, respectively (Fig. 2 A). This result indicates that within the Treg, two subsets exhibit very distinct fates. Some cells are quiescent and have long lifespan, in the order of months. On the opposite, another Treg subset appears to be constituted of cells with rapid turnover and short lifespan.

Treg with a Rapid Turnover Acquire a Phenotype of Activated Cells.

T cell activation is followed by phenotypic changes with up-regulation of CD44, CD69, and OX40/CD134, and down-regulation of CD62L. Therefore, we analyzed these markers at various times after transfer of purified CD62Lhigh Treg. Quiescent Treg predominantly kept a stable CD62Lhigh CD44int CD134int phenotype for at least 2 mo. In contrast, the cells that had divided extensively acquired an activated phenotype with up-regulated expression of CD44, CD69, and CD134, and down-regulated expression of CD62L (Fig. 2, B and C). CD62L down-regulation was observed predominantly after six divisions, suggesting a relative stability of this marker at high level expression even after activation, as previously described in vitro (7). Similar findings were observed in the spleen (not depicted).

Rapid Turnover of CD44high Regulatory T Cells.

To confirm that a fraction of Treg has a rapid turnover at the steady state, we performed a BrdU incorporation assay. Normal mice received continuous administration of BrdU for 7 d. Cells that went through divisions during this period incorporated this nucleoside analogue in their DNA and were quantified by flow cytometry. Because up-regulation of CD44 was observed progressively from the first division of Treg, whereas down-regulation of CD62L happened only after several rounds of divisions (Fig. 2 B), we quantified BrdU incorporation within the CD44high Treg and CD44int Treg subsets. After 1 wk of BrdU administration, 50–60% of CD44high Treg from peripheral LNs and spleen had incorporated the nucleoside analogue. This contrasts with data obtained for the CD44int Treg population, for which only ~5% of the cells had incorporated BrdU during the same period in these compartments (Fig. 3) . Similar findings were observed in short-term BrdU incorporation experiments. After 24 h of BrdU treatment, BrdU+ cells were contained within the CD44high Treg and not the CD44int Treg (not depicted). These experiments confirmed data obtained in the adoptive transfer experiments that a fraction of Treg population has a high turnover rate in the steady state.

Two Treg Subsets with Different Phenotypes.

Our results indicate that Treg are constituted of two subsets with different phenotypes and turnover properties. To further define their phenotype, we performed flow cytometric analyses of these subsets in unmanipulated normal mice. Using cells from mice from clean animal facilities, the CD4+ CD25+ CD62Llow cells are likely composed mostly of suppressor T cells because these cells have comparable suppressive activity in vitro as the CD4+ CD25+ CD62Lhigh cells (34). Cells gated on the CD4+ CD25+ Treg phenotype were analyzed in LNs for CD62L expression and activation markers, such as CD44, OX40/CD134, GITR, CD69, and IL-2Rβ/CD122, all up-regulated upon activation of Treg (35 and unpublished data). Compared with Treg expressing high levels of CD62L, the CD4+ CD25+ CD62Llow cells had increased expression of the activation markers CD44, OX40/CD134, GITR, CD69, and IL-2Rβ/CD122 (Fig. 4 and Table I). Importantly, these last two markers are up-regulated only for a few days upon Treg activation (35 and unpublished data), suggesting that most of the CD4+ CD25+ CD62Llow cells are continuously activated in the steady state. The increased expression of the transferrin receptor CD71 in these cells, up-regulated in dividing cells (36), supports this hypothesis (Fig. 4 and Table I).

Figure 4.
Cell surface phenotype reveals two Treg subsets. Peripheral LN cells of BALB/c adult mice were analyzed for cell surface expression of CD4, CD25, CD62L, and the indicated markers. Gated on CD4+ CD25+ cells, expression of the indicated markers was plotted ...
Table I.
Phenotypic Characterization of the Two Treg Subsets and CD4+ CD25 Cells

Besides its constitutive expression of CD25, natural suppressor CD4+ T cells have been described by various phenotypes such as CD5high (37), GITR+ (35, 38), CD38+ (39), or CD45RBlow (40) cells. Indeed, we observed that both subsets of Treg (CD62Lhigh and CD62Llow) expressed higher levels of CD5, GITR, or CD38 than CD4+ CD25 T cells, and lower levels of CD45RB than CD4+ CD25 T cells (Table I). Again, CD38 and CD45RB were differentially expressed by the CD62Lhigh Treg and CD62Llow Treg subsets. This could be explained by the different activation status of the two subsets because CD38 is up-regulated whereas CD45RB is down-regulated upon T cell activation (39).

Based on data shown in Figs. 2, ,3,3, and and4,4, it can be hypothesized that at a given time, a fraction of the Treg population is chronically activated. First, they up-regulate CD69 while remaining CD62Lhigh, and then, after several divisions they acquire a CD69+ CD62Llow phenotype. In support of this scenario, we found that after 24 h of BrdU incorporation, only 1% of the CD69 CD62Lhigh cells were BrdU+, whereas 6–10% of both CD69+ CD62Lhigh and CD69+ CD62Llow cells were BrdU+ (not depicted). One Treg subset, subsequently referred to as activated Treg, would predominantly have a CD4+ CD25+ CD62Lhigh/low CD44high CD69+ CD122high CD134high CD71high CD54high CD5high GITRhigh CD38high CD45RBlow phenotype. The other subset, subsequently referred to as resting Treg, would predominantly have a CD4+ CD25+ CD62Lhigh CD44int CD69 CD122low CD134int CD71low CD54int CD5int GITRint CD38 CD45RBint phenotype. Interestingly, compared with the CD62Llow Treg, the CD62Lhigh Treg also expressed higher levels of the CCR7 chemokine receptor (32). Importantly, it has been shown that among the CD4+ CD25+ Treg, CD62Lhigh and CD62Llow cells have similar suppressive activity in vitro, as well as CD45RBlow and CD45RBhigh cells, CD69+ and CD69 cells, and CD38+ and CD38 cells (32). From this, it can concluded that the two Treg subsets that we have defined have both potent suppressive activity in vitro. Thus, our data offer a model to explain the described phenotypic heterogeneity of the suppressor Treg for numerous markers, such as CD62L, CD44, CD69, CD38, or CD45RB (1, 2).

Sustained Activation of Autoreactive Treg in the Steady State.

Highly autoreactive Treg precursors are positively selected in the thymus (1921), suggesting that Treg are enriched in autoreactive cells in the periphery (22). Thus, we speculated that the activated Treg subset is constituted mostly of autoreactive cells that respond to self-antigens. To test this hypothesis, we used ins-HA transgenic mice expressing the model HA Ag in pancreatic islets and TCR-HA transgenic mice expressing a T cell receptor transgene specific to an HA peptide. In these mice, HA-specific CD4+ T cells were identified using the anticlonotypic 6.5 mAb. Highly purified CD62Lhigh Treg from TCR-HA transgenic mice were labeled with CFSE and transferred intravenously into ins-HA transgenic mice. At various times after transfer, donor Treg were analyzed by flow cytometry in pancreatic LNs and control peripheral LNs. In the pancreatic LN, the numbers of donor cells significantly increased, with a peak at days 5–7 depending on the experiment. Then, their numbers decreased dramatically by day 11. On the contrary, donor Treg did not expand in peripheral LNs and their numbers decreased progressively up to day 11 (Fig. 5 A). The increased proportion of donor Treg in pancreatic LNs was due to a significant proliferation of HA-specific 6.5+ cells, a phenomenon not observed in peripheral LNs (Fig. 5 B). The proliferating HA-specific 6.5+ Treg acquired a CD44high CD62Lhigh/low activated phenotype (Fig. 5 C), as previously observed for the activated Treg subset derived from nontransgenic mice (Fig. 2 B). 11 d after transfer, we could hardly detect any 6.5+ cells that had divided in various LNs, spleens, or pancreas (not depicted), suggesting that most of the HA-specific cells were deleted shortly after expansion in pancreatic LNs. The preferential expansion of HA-specific Treg in pancreatic LNs of ins-HA transgenic mice indicates that activation of Treg is indeed auto-Ag driven. Thus, the low level physiological presentation of tissue self-Ag in draining LNs is sufficient to induce intense proliferation of specific autoreactive Treg.

Figure 5.
The activated Treg population contains autoreactive cells. CFSE-labeled CD62Lhigh Treg purified from Thy-1.1 TCR-HA transgenic mice were intravenously injected into Thy-1.2 ins-HA transgenic mice. (A) The proportion of CD4+ Thy-1.1+ donor cells was determined ...

Discussion

So far, CD25 has been the best cell surface marker used to identify the natural suppressor CD4+ CD25+ T cells. Recent studies showed that the in vitro suppressive activity of a polyclonal population (41) or even clones (42) of Treg was contained within cells expressing high but not intermediate levels of CD25, emphasizing the importance of this marker for this population. CD25 expression by Treg likely plays a critical role because their proliferation and survival is dependent on IL-2 (3, 26, 43, 44). However, conflicting data have been published on the stability of this marker on Treg. We and others have shown a stable CD25 expression on Treg in vitro that maintained a suppressive activity, even after 4–6 wk of culture (7, 45). On the other side, after transfer of purified Treg into lymphopenic mice, injected cells expanded and most of them lost CD25 expression (28, 46). However, contrary to the cells that maintained CD25 expression, the CD25 cells had also lost their suppressive activity, suggesting that they resulted from the preferential expansion of contaminant cells (being either activated CD25+ conventional CD4+ T cells or CD25 cells) in the injected Treg population (28). To analyze the stability of CD25 expression on Treg in physiological steady-state conditions, we transferred, in nonlymphopenic congenic recipients, highly purified CD4+ CD25+ CD62Lhigh cells (>98% pure), limiting the risk to inject CD25 cells or activated CD25+ conventional CD4+ T cells. Indeed, conventional activated CD4+ T cells are prominently in the CD62Llow population. Our data showed a remarkable in vivo stability of CD25 expression on Treg for at least 2 mo. These data reinforce the hypothesis that these cells belong to a specific lineage, distinct from conventional CD4+ T cells or from other regulatory CD4+ T cell populations secreting IL-10 or TGFβ immunosuppressive cytokines, which display unstable CD25 expression (42). In addition to the Treg-specific transcription factor Foxp3 (47), stable CD25 expression is therefore a hallmark of the natural suppressor Treg.

In vitro data showed that Treg do not proliferate to antigenic or anti-CD3 stimulation, except if IL-2, anti-CD28 antibody, or lipopolysaccharide is added to the culture (43, 44, 48). Their hyporesponsiveness has also been described after adoptive transfer of Treg that proliferated poorly to their cognate Ag administrated subcutaneously in complete Freund's adjuvant (46). Thus, Treg are usually considered anergic cells. This property has been challenged by data aimed at analyzing their turnover in steady-state conditions using BrdU incorporation experiments. After 3 d of BrdU administration, 10.5% of CD4+ CD25+ cells, versus only 4.5% of CD4+ CD25 cells, were BrdU+ (49, 50). However, without knowing the level of possible conversions from CD4+ CD25+ phenotype into CD4+ CD25 phenotype and vice versa, a definitive point could not be made. Indeed, for example, some of the BrdU+ CD4+ CD25+ cells could have incorporated BrdU while they were CD4+ CD25 cells (for instance conventional activated CD4+ T cells). Because our data showed few, if any, conversion from the CD4+ CD25+ phenotype into the CD4+ CD25 phenotype and vice versa in steady-state conditions, we can now conclude from the BrdU incorporation experiments that Treg are cycling in vivo and therefore cannot be considered as “anergic” stricto-sensu. In support of this contention, after transferring CFSE+ Treg, a fraction of them rapidly lost CFSE staining, indicative of cell division.

Importantly, only a subset, and not the whole population of Treg, is cycling in the steady state. When analyzed 1 mo after transferring CFSE+ Treg, about one third in the LN and one fifth in the spleen of the remaining donor cells had not lost CFSE staining and thus had not divided for 1 mo. These cells were mostly CD62Lhigh, CD44int, CD69, and OX40int. In marked contrast, cells that had divided extensively (more than six divisions after 1 mo) displayed an activated phenotype because they were mostly CD62Llow, CD44high, CD69+, and OX40high. BrdU experiments confirmed the existence of two subsets of Treg, one with a rapid turnover, characterized by its CD44high phenotype, and a quiescent subset, characterized by its CD44int phenotype. Phenotypic analyses on nonmanipulated mice also support the existence of a resting Treg subpopulation and a subset displaying a phenotype of activated cells. Indeed, these latter cells expressed higher levels of markers up-regulated upon activation on Treg such as CD44, CD134, and GITR (35). Importantly, they also display increased expression of markers only transiently up-regulated upon cell activation, such as CD69 (unpublished data) and CD122 (35), as well as CD71, a molecule up-regulated in dividing cells (36). Altogether, these data strongly suggest that a Treg subset is composed of cells that are continuously activated.

By analogy with signals driving proliferation of conventional T cells, the signals that drive continuous activation of cycling Treg could be cytokine- and/or TCR-mediated. We show here that Treg expressing a TCR specific to islet-Ag proliferated extensively in pancreatic LNs but not in other LNs. Thus, in addition to the fact that a fraction of the repertoire of Treg is autoreactive (1922), the activated Treg subset is likely composed for a large part of autoreactive Treg. After expansion of activated Treg in the steady state, most of the cells probably die because no accumulation or significant migration into nonlymphoid tissues were observed (not depicted). Some of them recirculated preferentially in the spleen (not depicted), a property explained by their CD62Llow CCR7low phenotype.

Thus, we propose a model that summarizes some of the features of the homeostasis of Treg in adults (Fig. 6) . In permanence, tissue self-Ag, originated from dying cells, molecule shedding, or direct capture from live cells, are presented by Ag-presenting cells in draining LNs (51, 52). From the pool of resting Treg, the highly autoreactive cells that encounter their cognate self-Ag rapidly expand and acquire a phenotype of activated Treg, and then, most of them die or recirculate in the spleen.

Figure 6.
A model describing some of the features of the homeostasis of Treg. In the steady state, tissue self-Ag, originated from dying cells, molecule shedding, or direct capture from live cells, are presented by Ag-presenting cells in draining LNs. This presentation ...

These findings may shed new light on the mode of action of Treg and give plausible explanations on several previous findings. In vitro experiments have shown that switching on suppressor function on Treg is dependent on their activation via TCR engagement, but once activated, they exhibit a nonspecific bystander suppression on other T cells (34, 44). The existence of a pool of activated Treg in lymphoid tissues suggests that this subset exerts a basal and permanent suppression on T cell activation. This would explain why depletion of endogenous Treg in the steady state induced a rapid increased proliferation of memory CD8 T cells (3) and a rapid increase of antitumor responses (10). Our data also support a very dynamic process in which new clones, originated from the pool of resting Treg, are activated when they encounter their cognate self-Ag, leading to their expansion followed by contraction in draining LNs. This phenomenon may have two important implications. In LNs, the pool of activated Treg would be enriched in cells specific to self-Ag originated from drained tissues. This would explain why Treg from pancreatic LNs, but not the ones from other LNs, efficiently regulated autoimmune diabetes (53). The second implication is related to the dynamism of this process. A removal of the source of tissue-Ag may lead to a rapid contraction of the pool of tissue-specific Treg. Experimental findings support this hypothesis. The capacity of Treg from secondary lymphoid tissues to prevent autoimmune thyroiditis was lost in rats whose thyroids were ablated, while they maintained the capacity to prevent autoimmune diabetes (54). In addition, tolerance to ovarian Ag involved in prevention of autoimmune oophoritis was lost as soon as 1 wk after ablation of ovaries (55). Finally, cytotoxic treatments killing cycling cells, such as irradiation or chemotherapy, most likely delete activated Treg. This could explain why these treatments may paradoxically increase severity of some autoimmune diseases (56).

Acknowledgments

We are grateful to Harald von Boehmer for providing the TCR-HA transgenic mice, Abul Abbas, José Cohen, and Olivier Boyer for critical comments on the manuscript, and Jeffrey Bluestone for helpful discussions. We thank Micael Yagello for expert technical assistance with flow cytometry sorting, Fathia Djelti for her contribution to some of the experiments, and Gwenaelle Piriou and Isabelle Raymond for taking care of our animal colony.

S. Fisson is supported by the Association Française contre les Myopathies and E. Litvinova is supported by the Association pour la Recherche contre le Cancer. This work was supported by the Ministère de la Recherche (Action Concertée Incitative Jeunes Chercheurs), the Association Française des Diabétiques (no. 435), the University Pierre and Marie Curie (Paris VI), and the CNRS.

Notes

Abbreviations used in this paper: BrdU, bromodeoxyuridine; CFSE, 5,6-carboxy-fluorescein succinimidyl ester; GITR, glucocorticoid-induced TNF receptor; HA, hemagglutinin; Treg, CD4+ CD25+ regulatory T cells.

S. Fisson and G. Darrasse-Jèze contributed equally to this work.

References

1. Sakaguchi, S., N. Sakaguchi, J. Shimizu, S. Yamazaki, T. Sakihama, M. Itoh, Y. Kuniyasu, T. Nomura, M. Toda, and T. Takahashi. 2001. Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol. Rev. 182:18–32. [PubMed]
2. Shevach, E.M. 2002. CD4+ CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2:389–400. [PubMed]
3. Murakami, M., A. Sakamoto, J. Bender, J. Kappler, and P. Marrack. 2002. CD25+ CD4+ T cells contribute to the control of memory CD8+ T cells. Proc. Natl. Acad. Sci. USA. 99:8832–8837. [PMC free article] [PubMed]
4. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T.C. Barbosa, A. Cumano, and A. Bandeira. 2001. CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10. J. Immunol. 166:3008–3018. [PubMed]
5. Belkaid, Y., C.A. Piccirillo, S. Mendez, E.M. Shevach, and D.L. Sacks. 2002. CD4+ CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature. 420:502–507. [PubMed]
6. Aseffa, A., A. Gumy, P. Launois, H.R. MacDonald, J.A. Louis, and F. Tacchini-Cottier. 2002. The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+ CD25+ T cells. J. Immunol. 169:3232–3241. [PubMed]
7. Cohen, J.L., A. Trenado, D. Vasey, D. Klatzmann, and B.L. Salomon. 2002. CD4+ CD25+ immunoregulatory T cells: new therapeutics for graft-versus-host disease. J. Exp. Med. 196:401–406. [PMC free article] [PubMed]
8. Kursar, M., K. Bonhagen, J. Fensterle, A. Kohler, R. Hurwitz, T. Kamradt, S.H. Kaufmann, and H.W. Mittrucker. 2002. Regulatory CD4+ CD25+ T cells restrict memory CD8+ T cell responses. J. Exp. Med. 196:1585–1592. [PMC free article] [PubMed]
9. Taylor, P.A., C.J. Lees, and B.R. Blazar. 2002. The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality. Blood. 99:3493–3499. [PubMed]
10. Onizuka, S., I. Tawara, J. Shimizu, S. Sakaguchi, T. Fujita, and E. Nakayama. 1999. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res. 59:3128–3133. [PubMed]
11. Salomon, B., D.J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, and J.A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+ CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity. 12:431–440. [PubMed]
12. Wu, A.J., H. Hua, S.H. Munson, and H.O. McDevitt. 2002. Tumor necrosis factor-alpha regulation of CD4+ CD25+ T cell levels in NOD mice. Proc. Natl. Acad. Sci. USA. 99:12287–12292. [PMC free article] [PubMed]
13. Hara, M., C.I. Kingsley, M. Niimi, S. Read, S.E. Turvey, A.R. Bushell, P.J. Morris, F. Powrie, and K.J. Wood. 2001. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J. Immunol. 166:3789–3796. [PubMed]
14. Gregori, S., M. Casorati, S. Amuchastegui, S. Smiroldo, A.M. Davalli, and L. Adorini. 2001. Regulatory T cells induced by 1 alpha,25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance. J. Immunol. 167:1945–1953. [PubMed]
15. Graca, L., S. Thompson, C.Y. Lin, E. Adams, S.P. Cobbold, and H. Waldmann. 2002. Both CD4(+)CD25(+) and CD4(+)CD25(−) regulatory cells mediate dominant transplantation tolerance. J. Immunol. 168:5558–5565. [PubMed]
16. Hoffmann, P., J. Ermann, M. Edinger, C.G. Fathman, and S. Strober. 2002. Donor-type CD4+ CD25+ regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J. Exp. Med. 196:389–399. [PMC free article] [PubMed]
17. Asano, M., M. Toda, N. Sakaguchi, and S. Sakaguchi. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184:387–396. [PMC free article] [PubMed]
18. Itoh, M., T. Takahashi, N. Sakaguchi, Y. Kuniyasu, J. Shimizu, F. Otsuka, and S. Sakaguchi. 1999. Thymus and autoimmunity: production of CD25+ CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162:5317–5326. [PubMed]
19. Jordan, M.S., A. Boesteanu, A.J. Reed, A.L. Petrone, A.E. Holenbeck, M.A. Lerman, A. Naji, and A.J. Caton. 2001. Thymic selection of CD4+ CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2:301–306. [PubMed]
20. Kawahata, K., Y. Misaki, M. Yamauchi, S. Tsunekawa, K. Setoguchi, J. Miyazaki, and K. Yamamoto. 2002. Generation of CD4(+)CD25(+) regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression. J. Immunol. 168:4399–4405. [PubMed]
21. Apostolou, I., A. Sarukhan, L. Klein, and H. von Boehmer. 2002. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3:756–763. [PubMed]
22. Romagnoli, P., D. Hudrisier, and J.P. van Meerwijk. 2002. Preferential recognition of self antigens despite normal thymic deletion of CD4(+)CD25(+) regulatory T cells. J. Immunol. 168:1644–1648. [PubMed]
23. Kukreja, A., G. Cost, J. Marker, C. Zhang, Z. Sun, K. Lin-Su, S. Ten, M. Sanz, M. Exley, B. Wilson, et al. 2002. Multiple immuno-regulatory defects in type-1 diabetes. J. Clin. Invest. 109:131–140. [PMC free article] [PubMed]
24. Fontenot, J.D., M.A. Gavin, and A.Y. Rudensky. 2003. Foxp3 programs the development and function of CD4(+)CD25(+) regulatory T cells. Nat. Immunol. 4:330–336. [PubMed]
25. Khattri, R., T. Cox, S.A. Yasayko, and F. Ramsdell. 2003. An essential role for Scurfin in CD4(+)CD25(+) T regulatory cells. Nat. Immunol. 4:337–342. [PubMed]
26. Papiernik, M., M.L. de Moraes, C. Pontoux, F. Vasseur, and C. Penit. 1998. Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency. Int. Immunol. 10:371–378. [PubMed]
27. Malek, T.R., A. Yu, V. Vincek, P. Scibelli, and L. Kong. 2002. CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2. Immunity. 17:167–178. [PubMed]
28. Almeida, A.R., N. Legrand, M. Papiernik, and A.A. Freitas. 2002. Homeostasis of peripheral CD4+ T cells: IL-2R alpha and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers. J. Immunol. 169:4850–4860. [PubMed]
29. Kumanogoh, A., X. Wang, I. Lee, C. Watanabe, M. Kamanaka, W. Shi, K. Yoshida, T. Sato, S. Habu, M. Itoh, et al. 2001. Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development. J. Immunol. 166:353–360. [PubMed]
30. Lo, D., J. Freedman, S. Hesse, R.D. Palmiter, R.L. Brinster, and L.A. Sherman. 1992. Peripheral tolerance to an islet cell-specific hemagglutinin transgene affects both CD4+ and CD8+ T cells. Eur. J. Immunol. 22:1013–1022. [PubMed]
31. Kirberg, J., A. Baron, S. Jakob, A. Rolink, K. Karjalainen, and H. von Boehmer. 1994. Thymic selection of CD8+ single positive cells with a class II major histocompatibility complex-restricted receptor. J. Exp. Med. 180:25–34. [PMC free article] [PubMed]
32. Szanya, V., J. Ermann, C. Taylor, C. Holness, and C.G. Fathman. 2002. The subpopulation of CD4+ CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7. J. Immunol. 169:2461–2465. [PubMed]
33. Jung, T.M., W.M. Gallatin, I.L. Weissman, and M.O. Dailey. 1988. Down-regulation of homing receptors after T cell activation. J. Immunol. 141:4110–4117. [PubMed]
34. Thornton, A.M., and E.M. Shevach. 2000. Suppressor effector function of CD4+ CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164:183–190. [PubMed]
35. McHugh, R.S., M.J. Whitters, C.A. Piccirillo, D.A. Young, E.M. Shevach, M. Collins, and M.C. Byrne. 2002. CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity. 16:311–323. [PubMed]
36. Bayer, A.L., P. Baliga, and J.E. Woodward. 1998. Transferrin receptor in T cell activation and transplantation. J. Leukoc. Biol. 64:19–24. [PubMed]
37. Sakaguchi, S., K. Fukuma, K. Kuribayashi, and T. Masuda. 1985. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease. J. Exp. Med. 161:72–87. [PMC free article] [PubMed]
38. Shimizu, J., S. Yamazaki, T. Takahashi, Y. Ishida, and S. Sakaguchi. 2002. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3:135–142. [PubMed]
39. Read, S., S. Mauze, C. Asseman, A. Bean, R. Coffman, and F. Powrie. 1998. CD38+ CD45RB(low) CD4+ T cells: a population of T cells with immune regulatory activities in vitro. Eur. J. Immunol. 28:3435–3447. [PubMed]
40. Powrie, F., R. Correa-Oliveira, S. Mauze, and R.L. Coffman. 1994. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J. Exp. Med. 179:589–600. [PMC free article] [PubMed]
41. Baecher-Allan, C., J.A. Brown, G.J. Freeman, and D.A. Hafler. 2001. CD4+ CD25high regulatory cells in human peripheral blood. J. Immunol. 167:1245–1253. [PubMed]
42. Levings, M.K., R. Sangregorio, C. Sartirana, A.L. Moschin, M. Battaglia, P.C. Orban, and M.G. Roncarolo. 2002. Human CD25+ CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells. J. Exp. Med. 196:1335–1346. [PMC free article] [PubMed]
43. Thornton, A.M., and E.M. Shevach. 1998. CD4+ CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287–296. [PMC free article] [PubMed]
44. Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata, J. Shimizu, and S. Sakaguchi. 1998. Immunologic self-tolerance maintained by CD25+ CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10:1969–1980. [PubMed]
45. Kuniyasu, Y., T. Takahashi, M. Itoh, J. Shimizu, G. Toda, and S. Sakaguchi. 2000. Naturally anergic and suppressive CD25(+)CD4(+) T cells as a functionally and phenotypically distinct immunoregulatory T cell subpopulation. Int. Immunol. 12:1145–1155. [PubMed]
46. Gavin, M.A., S.R. Clarke, E. Negrou, A. Gallegos, and A. Rudensky. 2002. Homeostasis and anergy of CD4(+) CD25(+) suppressor T cells in vivo. Nat. Immunol. 3:33–41. [PubMed]
47. Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science. 299:1057–1061. [PubMed]
48. Caramalho, I., T. Lopes-Carvalho, D. Ostler, S. Zelenay, M. Haury, and J. Demengeot. 2003. Regulatory T cells selectively express Toll-like receptors and are activated by lipopolysaccharide. J. Exp. Med. 197:403–411. [PMC free article] [PubMed]
49. Papiernik, M., M. do Carmo Leite-de-Moraes, C. Pontoux, A.M. Joret, B. Rocha, C. Penit, and M. Dy. 1997. T cell deletion induced by chronic infection with mouse mammary tumor virus spares a CD25-positive, IL-10-producing T cell population with infectious capacity. J. Immunol. 158:4642–4653. [PubMed]
50. Hori, S., M. Haury, J.J. Lafaille, J. Demengeot, and A. Coutinho. 2002. Peripheral expansion of thymus-derived regulatory cells in anti-myelin basic protein T cell receptor transgenic mice. Eur. J. Immunol. 32:3729–3735. [PubMed]
51. Heath, W.R., and F.R. Carbone. 2001. Cross-presentation in viral immunity and self-tolerance. Nat. Rev. Immunol. 1:126–134. [PubMed]
52. Steinman, R.M., and M.C. Nussenzweig. 2002. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc. Natl. Acad. Sci. USA. 99:351–358. [PMC free article] [PubMed]
53. Green, E.A., Y. Choi, and R.A. Flavell. 2002. Pancreatic lymph node-derived CD4(+)CD25(+) Treg cells: highly potent regulators of diabetes that require TRANCE-RANK signals. Immunity. 16:183–191. [PubMed]
54. Seddon, B., and D. Mason. 1999. Peripheral autoantigen induces regulatory T cells that prevent autoimmunity. J. Exp. Med. 189:877–882. [PMC free article] [PubMed]
55. Garza, K.M., S.S. Agersborg, E. Baker, and K.S. Tung. 2000. Persistence of physiological self antigen is required for the regulation of self tolerance. J. Immunol. 164:3982–3989. [PubMed]
56. Gleeson, P.A., B.H. Toh, and I.R. van Driel. 1996. Organ-specific autoimmunity induced by lymphopenia. Immunol. Rev. 149:97–125. [PubMed]

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