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International Immunology Advance Access published online on March 15, 2007
International Immunology, doi:10.1093/intimm/dxm021
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

CD4+CD25+ regulatory T cells are activated in vivo by recognition of self

John Andersson, Irena Stefanova, Geoffrey L. Stephens and Ethan M. Shevach

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institute of Health, 10 Center Drive, MSC-1892, Bethesda, MD 20892-1892, USA

Correspondence to: Correspondence to: E. M. Shevach; E-mail: eshevach{at}niaid.nih.gov


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Naturally occurring CD4+CD25+ regulatory T cells (nTR) comprise a separate lineage of T cells that are essential for maintaining immunological tolerance to self. Here we demonstrate that the level of phosphorylation of the TCR {zeta}-chain is ~1.5- to 4-fold higher in nTR as compared with CD4+CD25– T cells. The increased level of TCR {zeta}-chain phosphorylation is presumably secondary to their higher affinity for self, resulting in a stronger TCR signal as it was completely blocked by treatment with anti-MHC class II. The enhanced level of TCR {zeta}-chain phosphorylation was correlated with the capacity of nTR to develop non-specific suppressor effector function following culture with IL-2 or IL-4 in the absence of TCR stimulus. Thus, a sub-population of nTR is activated by recognition of self-peptide–MHC class II ligands in vivo, resulting in their capacity to be induced to mediate suppressor function in vitro in the absence of TCR stimulation.

Keywords: IL-2, IL-4, regulatory T cells, TCR, tolerance


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
Immunological tolerance to self is maintained by several distinct mechanisms including the deletion of potentially harmful self-reactive lymphocytes during development as well as suppression of pathogenic autoreactive T cells in the periphery by naturally occurring CD4+CD25+ regulatory T cells (nTR). nTR comprise a distinct subset of CD4+ T cells that are dependent on the transcription factor FoxP3 for their development (1–3) and in most cases are characterized by expression of CD25, the IL-2 receptor {alpha}-subunit (4). The importance of nTR in immune homeostasis is best illustrated by the development of severe autoimmune diseases in both mice and humans with genetic deficiencies of Foxp3 (5–7). The TCR repertoire of nTR is formed in the thymus and appears to be as diverse as, but only partially overlapping, with the TCR repertoire of CD4+CD25– T cells (8). CD4+CD25+ T cells are thought to be generated in the thymus from precursors with a higher overall affinity for self than the majority of CD4+CD25– T cells, but one that is still low enough for these cells to escape negative selection (8–10). It has been hypothesized that because of this enhanced affinity for self, nTR are constantly being activated in vivo and thereby prevent the host from generating a pathogenic immune response to autoantigens or an immune response to tumor antigens (8, 11). However, little direct experimental evidence is available to support this concept.

Although nTR may have a higher affinity for self, it has also been proposed that conventional CD4+CD25– T cells have their sensitivity to foreign antigen stimulation maintained by recognition of self-MHC in the periphery (12). Such continuous stimulation has biochemical consequences, with T cells isolated from lymph nodes or spleen exhibiting partial phosphorylation of the TCR {zeta}-chain (12, 13). It has been assumed that nTR must be stimulated via their TCR to exert their suppressive effects. In the present report, we demonstrate that nTR have enhanced TCR {zeta}-chain phosphorylation secondary to recognition of self-MHC class II ligands in vivo as compared with non-regulatory T cells. Furthermore, the enhanced TCR {zeta}-chain phosphorylation correlates with their capacity to suppress in vitro in the absence of stimulation via their TCR.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
Mice
Female mice aged between 6 and 12 weeks were used in this study. BALB/c and C57BL/6 mice were obtained from the National Cancer Institute (Frederick, MD, USA). OT-I (specific for OVA257–264 peptide) CD8+ TCR transgenic mice and hemagglutinin (HA) (specific for HA110–119) CD4+ TCR transgenic mice were obtained from Taconic (Germantown, NY, USA).

Media, cytokines and peptides
Cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U ml–1 penicillin, 100 µg ml–1 streptomycin, 2 mM L-glutamine, 10 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate (all from Biofluids, Rockville, MD, USA) and 50 µM 2-mercaptoethanol (Sigma, St Louis, MO, USA). Recombinant mouse IL-2, human IL-13 and IL-15 were purchased from Peprotech (Rocky Hill, NJ, USA). Recombinant mouse IL-4, IL-7, IL-9 and IL-21 were purchased from R&D Systems (Minneapolis, MN, USA). OVA257–264 and HA110–119 peptides were synthesized and purified by the Laboratory of Molecular Structure, Peptide Synthesis Laboratory (National Institute of Allergy and Infectious Diseases, National Institutes of Health).

Cell separation
Axillary, inguinal, cervical, mandibular and mesenteric lymph nodes were harvested and passed over a cell strainer to prepare single-cell suspensions, followed by lysis of RBCs using ACK lysis buffer. The cells were then incubated with a cocktail of FITC-labeled antibodies ({alpha}-CD11b, {alpha}-CD11c, {alpha}-CD16/32, {alpha}-CD19 and {alpha}-B220), PE-labeled {alpha}-CD25 (all from PharMingen) and tri-color-labeled {alpha}-CD4 for 15 min at 4°C. FITC–CD4+CD25+ and FITC–CD4+CD25– single cells were isolated using a FACStar® Cell Sorter (Becton Dickinson). CD4+ and CD8+ responder T cells and T-depleted spleen cells were isolated using autoMACS from HA and OT-I transgenic mice.

Processing of T cells for analysis of signaling or immunoblotting
T cells (1 x 105) were washed once with PBS and placed in NP-40 lysis buffer for 30 min on ice. After removal of nuclear debris by centrifugation, the resultant supernatants were analyzed by SDS–PAGE and immunoblotting. The following antibodies were used in these experiments: 4G10, a mouse mAb to phosphotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY, USA); rabbit antiserum to {zeta}2; K-23 and C-14, rabbit polyclonal antibodies to extracellular signal-regulated kinase (ERK)-1 and -2, respectively (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); C-19 rabbit polyclonal antibodies to SHP-1 (Santa Cruz Biotechnology); C-23 rabbit polyclonal antibodies to Grb-2 (Santa Cruz Biotechnology); mouse mAb 3A5 to Lck (Santa Cruz Biotechnology) and peroxidase-linked goat antibodies to mouse and rabbit Ig (Bio-Rad Laboratories, Richmond, CA, USA). Quantitative data were obtained from multiple film exposures using a Kodak ImageStation 440CF and Kodak Digital Sciences 1D software.

Suppression assays
CD4+CD25+ T cells cultured with 5-µg ml–1 plate-bound {alpha}-CD3 and 100 U IL-2 for 3 days were used as a positive control for suppression. CD4+CD25+ T cells were also pre-incubated with 5–20 ng ml–1 IL-4, IL-7 IL-9, IL-13, IL-15 or IL-21 for 3 days and tested for suppressive ability. Non-specific suppressor effector function was measured by culturing CD4+ T cells (5 x 104) from HA TCR transgenic mice with T-depleted spleen cells (5 x 104) and 8 µM HA peptide for 3 days in the presence of varying numbers of pre-treated BALB/c-derived CD4+CD25+ T cells. Proliferation was measured in triplicates by the incorporation of [3H]thymidine ([3H]TdR) over the last 6–8 h of the co-culture or assessed by CFSE dilution using flow cytometry. OT-I transgenic CD8+ T cells were cultured in the presence of 0.1 mg ml–1 SIINFEKL-H-2Kb tetramers and varying numbers of pre-treated C57BL/6-derived CD4+CD25+ T cells. Proliferation was measured in triplicates by the incorporation of [3H]TdR over the last 6–8 h of the co-culture.

IL-2 secretion assay
The percentage of responder cells that produce IL-2 was determined using an IL-2 secretion assay (Miltenyi Biotech) according to the manufacturer’s instructions. Briefly, CFSE-labeled CD4+ HA transgenic T cells (5 x 104) were cultured in the presence of bone marrow-derived dendritic cells (104), 8 µM HA peptide and pre-treated BALB/c-derived CD4+CD25+ T cells (5 x 104) for 48 h. The cells were then harvested, incubated for 10 min with IL-2 catch reagent, washed and then incubated in 50 ml complete media at 37°C for 45 min. Cells that had secreted IL-2 during the incubation period were detected with an APC-labeled anti-IL-2 antibody and analyzed using flow cytometry.

MHC class II blocking in vivo
C57BL/6 mice received 2.5 mg day–1 of either mouse IgG or Y3P, an {alpha}-I-Ab antibody, over a period of 2 days. Cellular composition after antibody treatment was analyzed using flow cytometry and the efficiency of blocking was verified by assessing TCR {zeta}-chain phosphorylation as described above.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
nTR cells have enhanced TCR {zeta}-chain phosphorylation
To address whether the recognition of self by CD4+CD25+ T cells differs qualitatively or quantitatively from CD4+CD25– T cells, we examined the level of phosphorylation of the TCR {zeta}-chain as a measure of the intensity of TCR signaling resulting from encounters with self-peptide:MHC class II complexes in vivo. The level of phosphorylation of the TCR {zeta}-chain as measured by phosphotyrosine immunoblotting was consistently ~1.5- to 4-fold higher in CD25+ as compared with CD25–CD4+ T cells, isolated from either lymph nodes or thymus, while the expression of various signaling molecules (SHP-1, Lck, ERK-1/2, TCR {zeta}-chain and Grb-2) was comparable between CD25+ and CD25–CD4+ T cells (Fig. 1). The increased level of TCR {zeta}-chain phosphorylation in the CD25+ T cells is presumably secondary to their higher affinity for self, resulting in a stronger TCR signal. The fact that the difference in TCR {zeta}-chain phosphorylation was seen both in lymph nodes and in thymus suggests that it indeed is a feature associated with nTR rather than activated CD25+ effector cells, as the number of activated CD25+ effectors is very low in the thymus.


Figure 1
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  		Fig. 1. Freshly isolated CD4+CD25+ T cells have enhanced TCR {zeta}-chain phosphorylation. CD4+CD25+ and CD4+CD25– T cells (105) from lymph nodes or thymus were analyzed for total content of SHP-1, Lck, ERK-1/2, TCR {zeta}-chain, Grb-2 (upper panel) and phosphorylated TCR {zeta}-chain (lower panel) by immunoblotting as described in (13). The relative levels of phosphorylated TCR {zeta}-chain was analyzed using image analysis, normalized to TCR {zeta}-chain expression and shown as the ratio between CD25– and CD25+CD4+ T cells in either thymus or lymph nodes. One representative experiment out of five experiments is shown.

 
IL-2/IL-4 alone activates the suppressor effector function of CD4+CD25+ T cells
One important correlation of the in vivo suppressive capacity of nTR is their ability to suppress TCR-induced proliferative responses of CD4+CD25– T cells in vitro by inhibiting the capacity of the latter to transcribe IL-2 mRNA (14, 15). However, the TCR signal nTR receive in vivo, as reflected by their enhanced level of {zeta}-chain phosphorylation, does not appear to be sufficient to activate them to a state able to mediate suppression, because freshly isolated CD4+CD25+ T cells do not inhibit T cell proliferation in vitro in the absence of additional signals (14–16). We have recently shown that activation of the suppressive function of nTR required not only a TCR-derived signal but also a signal provided by IL-2 and/or IL-4 (17). Activated nTR exhibit non-specific suppressor effector function, seen as inhibition of the activation of both CD4+ and CD8+ T cells irrespective of their antigen specificity (16, 18). To test the possibility that high concentrations of cytokines alone might be sufficient to complement an in vivo derived TCR signal to induce suppressor function, highly purified CD4+CD25+ T cells from lymph node of BALB/c mice were cultured in medium containing IL-2 for 3 days, washed and mixed with T cells expressing a TCR transgene specific for an influenza HA peptide. Culture in IL-2 resulted in markedly enhanced survival of the nTR compared with culture in media alone (Andersson, J, unpublished data). In addition, FoxP3 expression levels were unchanged after cytokine stimulation (Supplementary Figure S1, available at International Immunology Online). Interestingly, nTR activated with only IL-2 markedly suppressed the response of the HA-specific T cells (Fig. 2A), while freshly isolated CD4+CD25+ (Fig. 2A) or IL-2-stimulated CD4+CD25– T cells did not manifest suppressive function (Fig. 2B). It should be noted that the suppressive capacity of the nTR activated by exposure to IL-2 alone was usually less than that of the same cells pre-stimulated with IL-2 and plate-bound anti-CD3 (Fig. 2A). Similar results were obtained when CD4+CD25+ T cells isolated from the thymus were pre-cultured with IL-2 alone (Fig. 2C).


Figure 2
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  		Fig. 2. IL-2 alone activates the suppressor effector function of CD4+CD25+ T cells. (A) CD4+CD25+ T cells from lymph nodes from BALB/c mice, (B) CD4+CD25– T cells from lymph nodes or (C) CD4+CD25+ T cells from thymus were used immediately after isolation (open square), or cultured in IL-2 (open diamond) or cultured with IL-2 and plate-bound anti-CD3 (open circle) for 72 h. The cells were then assayed for their ability to suppress proliferative responses of HA TCR transgenic responders (from B10.D2 mice) stimulated with APC and HA peptide. (D) CFSE-labeled HA transgenic T cells were stimulated for 48 h with bone marrow-derived dendritic cells (104) and HA peptide in the presence of medium (left), CD4+CD25+ T cells that had been cultured in IL-2 (center) or CD4+CD25+ T cells that had been cultured in IL-2 and plate-bound anti-CD3 (right). IL-2 production was measured using an IL-2 secretion assay as described in (13) which allows the identification of IL-2-secreting cells on a per-cell basis. The percentage of IL-2-secreting cells indicated. One representative experiment out of at least three experiments is shown for (A–D).

 
One trivial explanation for the capacity of the IL-2-only activated nTR to inhibit T cell proliferation was that the activated cells were binding IL-2 produced by the responders, effectively lowering the available concentration of IL-2, and functioning as ‘IL-2 sinks’. We have previously shown that the suppression of proliferation by CD4+CD25+ T cells is secondary to a blockade of IL-2 production and not IL-2 consumption (17). In accordance with these previous results, IL-2 absorption does not appear to play a role in the present study, because both IL-2 and IL-2 plus anti-CD3 pre-activated nTR markedly inhibited IL-2 production as measured with an IL-2 secretion assay by the responder HA TCR CD25– T cells when they were stimulated in the presence of peptide and mature bone marrow-derived dendritic cells (Fig. 2D). When dendritic cells are used as stimulators, proliferation of the responders is not inhibited (19–21).

It was also possible that the suppressive capacity of the IL-2-only activated nTR was secondary to a TCR-derived signal delivered by contaminating antigen-presenting cells (APCs) in the first culture or the APC presenting the peptide antigen (or minor histocompatibility antigens) in the second culture. To examine this possibility, we cultured CD4+CD25+ T cells from the lymph nodes of C57BL/6 mice in IL-2 for 72 h. We then tested the capacity of these IL-2-activated nTR to inhibit the proliferative response of CD8+ T cells from the OT-I TCR transgenic mouse stimulated with H-2Kb tetramers containing the SIINFEKL peptide in the absence of APC. In addition, we added Y3P, an anti-I-Ab mAb to block any MHC class II dependent signals from potentially contaminating APC to both the first and second cultures. nTR activated by IL-2 under these conditions were almost as potent as nTR pre-activated with anti-CD3 and IL-2 in inhibiting the response of OT-I cells to stimulation by antigen-specific tetramers (Fig. 3A), thereby excluding the possibility of TCR-induced activation of the nTR suppressor function in vitro. It is unlikely that the capacity of the IL-2 pre-incubated cells to exhibit suppressor function was secondary to their enhanced viability as following removal from IL-2, the survival of the IL-2 pre-treated cells was comparable to freshly explanted cells (Fig. 3B).


Figure 3
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  		Fig. 3. IL-2-induced suppression is not dependent on MHC class II re-stimulation in vitro or increased survival. (A) CD4+CD25+ T cells from C57BL/6 mice were pre-cultured with IL-2 and the anti-I-Ab mAb, Y3P (10 µg ml–1, open square) or IL-2 and plate-bound anti-CD3 (open circle) for 72 h, and then co-cultured with OT-I transgenic T cells in the presence of SIINFEKL-H-2Kb tetramers and Y3P (10 µg ml–1). (B) CD4+CD25+ T cells were pre-cultured with either IL-2 for 3 days (open square) or used immediately after isolation (open circle) and the survival of these cells in complete media was measured for 3 days. Values are expressed as mean and standard deviation.

 
It has previously been demonstrated that both IL-2 and IL-4 were capable of producing expansion of nTR in the presence of a TCR stimulus (22). Similarly, IL-4, and to a much lesser extent IL-7, could induce suppressor effector function in nTR in the absence of a TCR stimulus (Fig. 2D). Culture of nTR with other cytokines that used the {gamma}c-chain as part of their receptor failed to induce suppressor activity (Fig. 4).


Figure 4
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  		Fig. 4. IL-4 can also induce the suppressor effector function of CD4+CD25+ T cells. CD4+CD25+ T cells were cultured for 3 days with the 5–20 ng ml–1 of the indicated cytokines and thereafter assayed for suppressive ability by co-culturing them with HA responders, APC and HA peptide. One representative experiment out of three experiments is shown.

 
Engagement of the GITR enhances the induction of suppression by IL-2
The studies above indicate that the suppressive capacity of nTR activated by TCR and IL-2 stimulation was greater than that seen with IL-2 alone. As we had previously demonstrated (23) that nTR will proliferate when stimulated with IL-2 in the presence of an agonistic antibody to the GITR, we evaluated whether GITR-mediated co-stimulation could further improve the suppressive effects induced by IL-2. Culture of nTR in the presence of IL-2 and anti-GITR, but in the absence of TCR stimulation, resulted in both increased proliferation (Fig. 5A) and increased suppressive function of the CD4+CD25+ T cells (Fig. 5B). The enhancement of suppression by GITR stimulation strongly favors the view that the abrogation of suppression by anti-GITR in co-cultures of nTR and CD4+CD25– T cells is secondary to the effects of the anti-GITR in rendering the responder cells resistant to suppression (24). As nTR stimulated with anti-GITR and IL-2 demonstrates both increased proliferation and suppressive activity, we wanted to know if there was any correlation between proliferation and suppression. Although most nTR do not proliferate in response to exogenous IL-2 (25), suppression may have been mediated by a minor sub-population of nTR that divided in response to IL-2. Indeed, after 6 days of culture in IL-2, a very small number of nTR had proliferated as measured by dilution of CFSE (Fig. 5A). However, both nTR that had divided in response to IL-2 and the major sub-population that remained undivided exhibited suppressor activity when mixed with CD25– responders (Fig. 5D).


Figure 5
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  		Fig. 5. GITR functions as a co-stimulatory molecule for CD4+CD25+ T cells. (A) CFSE-labeled CD4+CD25+ T cells were cultured in complete media supplemented in IL-2 or IL-2 (shaded area) and 5 µg ml–1 of the agonistic {alpha}-GITR antibody DTA-1 (solid line). Proliferation was measured after 6 days using FACS. (B) The suppressive ability of CD4+CD25+ T cells cultured in IL-2 (open square) or IL-2 and 5 µg ml–1 DTA-1 (open circle) for 3 days were assayed by co-culture with HA responders, APC and HA peptide. Values are expressed as mean and standard deviation. (C) CFSE-labeled CD4+CD25+ T cells were cultured in IL-2 for 6 days, sorted according to whether they had diluted their CFSE (open square) or not (cross symbol) or used non-sorted (open circle) and assayed for their suppressive ability by co-culturing them with HA responders, APC and HA peptide (D). One representative experiment out of at least three experiments is shown for (A–D). Values are expressed as mean and standard deviation.

 
Recognition of MHC class II is required for the activation of CD4+CD25+ T cells in vivo
We have thus far assumed that the capacity of IL-2 alone to induce the suppressor effector function of nTR was secondary to an MHC class II-derived TCR signal that the nTR had received in vivo. To directly test this hypothesis, we administered large quantities of an anti-I-Ab Y3P mAb (Y3P) to naive mice for 48 h to block all TCR–MHC class II interactions in vivo prior to the isolation of CD4+CD25+ T cells. Mice treated with Y3P had substantially reduced levels of APC in their lymph nodes, when compared with mice that had been treated with control IgG, which strongly suggests that the Y3P antibody in addition to blocking was also leading to the depletion of some MHC class II-expressing cells (Fig. 6A). Nevertheless, Y3P pre-treatment had no effect on the total number of CD4+ or CD4+CD25+ T cells recovered from the treated mice. Both CD25+ and CD25–CD4+ T cells from mice treated with Y3P showed a marked decrease in phosphorylation of the TCR {zeta}-chain (Fig. 6B) suggesting that the treatment regimen had blocked TCR–MHC class II interactions as previously reported (12). When CD4+CD25+ T cells were isolated from both the treated and control groups and cultured in IL-2, the cell recovery after 3 days was slightly lower for CD4+CD25+ T cells isolated from the Y3P (~60%) treated than from the control mice (~80%). Most importantly, the suppressive potency of the IL-2-only cultured CD4+CD25+ T cells was dramatically reduced when they came from mice that had received Y3P (Fig. 6C). Thus, it would appear that IL-2 is not sufficient to induce nTR to mediate their suppressor effector function, but that IL-2 complements the self-peptide–MHC class II signal delivered to nTR in vivo. Because treatment with Y3P also induced significant depletion of APC, it was still possible that cell-surface antigens in addition to MHC class II are involved in activation of the CD4+CD25+ T cells in vivo. To test this, we repeated the experiment above and used Y3P to block MHC class II interactions for 48 h using Fcer1{gamma}-deficient mice, which lack the {gamma}-chain subunit of the Fc{gamma}RI, Fc{gamma}RIII and Fc{epsilon}RI receptors. Treatment of these mice with Y3P did not result in depletion of MHC class II-positive cells (Fig. 6A). nTR from the treated mice still demonstrated a marked decrease in phosphorylation of the TCR {zeta}-chain (Fig. 6B) and suppressive potency in response to IL-2 (Fig. 6C). Thus, it would appear that IL-2 is not sufficient to induce nTR to mediate their suppressor effector function, but that IL-2 complements the self-peptide–MHC class II signal delivered to nTR in vivo.


Figure 6
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  		Fig. 6. Recognition of MHC class II is required for the activation of CD4+CD25+ T cells in vivo. C57BL/6 and Fcer1{gamma}-deficient mice were treated with either mouse IgG or Y3P (2.5 mg) at 0 and 24 h and sacrificed at 48 h. (A) Single-cell suspensions from harvested lymph nodes were stained for APCs (cells expressing CD11b, CD11c, CD16/32, CD19 or B220), CD4+ cells and CD25+ cells. (B) Immunoblotting was used to determine the total content of TCR {zeta}-chain and phospho {zeta}-chain in CD25+ and CD25–CD4+ T cells from both Y3P-treated and control mice. (C) CD4+CD25+ T cells from Y3P-treated (open square) and IgG-treated (open circle) mice were cultured in IL-2 for 72 h and their suppressive activity then assayed by co-culturing them with HA transgenic responders. One representative experiment out of at least three experiments is shown.

 
IL-2 signaling is intact in Y3P-treated CD4+CD25+ T cells
CD4+ T cells isolated from lymph nodes exhibit an asymmetric distribution of TCRs (12). This polarization of TCRs is dependent on MHC class II interactions as T cells isolated from mice that had received blocking anti-MHC class II antibodies exhibit diminished polarization (12). This redistribution of TCRs after MHC class II mAb treatment might be the reason why self-recognition results in increased T cell responses as clustering of receptors can result in enhanced signaling. In a similar manner, it is possible that Y3P treatment could affect IL-2 signaling since CD25 can at times be found in association with the TCR complex (I. Stefanova, unpublished observations). To test if Y3P treatment affects IL-2 signaling, we isolated CD4+CD25+ T cells and treated these cells with varying amounts of IL-2. STAT5 phosphorylation was measured by immunoblotting and used as a measure of the IL-2 signal strength. The degree of STAT5 phosphorylation was comparable between Y3P-treated and non-treated cells (Fig. 7) suggesting that Y3P treatment did not affect the ability of CD4+CD25+ T cells to respond to IL-2.


Figure 7
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  		Fig. 7. Intact IL-2 signaling after Y3P treatment. CD4+CD25+ T cells were isolated from C57BL/6 that had been treated with either mouse IgG or Y3P (2.5 mg) at 0 and 24 h and sacrificed at 48 h. Immunoblotting was used to quantify STAT5 phosphorylation. One representative experiment out of at least two experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
Self-recognition is one of the hallmarks of the T lymphocyte lineage. nTR are thought to have TCRs with intermediate to high affinity for self, below the threshold for negative selection, but sufficient to induce a differentiation signal, perhaps involving the induction of Foxp3, that results in their unique phenotype (8–10). Conventional CD4+CD25– T cells appear to be stimulated by self in vivo in the lymph nodes where they are in close contact with APC as reflected by partial phosphorylation of their TCR {zeta}-chain (26, 27). We demonstrate here that nTR freshly isolated from lymph nodes have an even higher level of constitutive TCR {zeta}-chain phosphorylation than non-nTR CD4+ T cells that is consistent with their enhanced reactivity with self. While enhanced {zeta}-chain phosphorylation facilitates the recognition of foreign antigen-derived peptides by CD4+CD25– T cells (12), the signal delivered to nTR that results in {zeta}-chain phosphorylation initiates or sustains a program where the regulatory T cells can be activated to mediate T suppressor function in vitro in the presence of IL-2/IL-4 without further stimulation via their TCR.

Previous studies of the requirements for the induction of suppressor function by nTR in vitro strongly suggested that nTR must first be activated via the TCR for suppression to be manifest (15, 16). Although TCR stimulation was necessary for suppression to be observed in vitro, it was not sufficient as the capacity of nTR to suppress IL-2 mRNA synthesis in responder cells was abrogated by the addition of anti-IL-2 to the co-cultures (17). This study indicated that nTR required an IL-2 signal in addition to a TCR signal to develop suppressor effector function. The role of IL-2 in the function of nTR has recently been questioned, as it has been shown that Foxp3+CD25– T cells from CD25-deficient mice are fully capable of suppressing CD4+CD25– T cells in vitro (28). One possible explanation for these apparently contradictory results is that the Foxp3+CD25– T cells may have been activated by IL-4 that is also capable of activating suppressor nTR function (17). Alternatively, the Foxp3+CD25– nTR may have been activated by IL-2 signals delivered via the ß and {gamma} subunits of the IL-2R complex that are constitutively expressed on nTR. In any case, the potential involvement of IL-2 in the induction of suppression, together with our data that nTR exist in a partially activated state in vivo, prompted us to re-examine the capacity of cytokines to induce nTR suppressor function in the absence of a TCR signal. Our experiments clearly demonstrate that pre-culture of nTR in either IL-2 or IL-4, and to a much lesser extent IL-7, is capable of inducing their suppressive functions. The role of IL-2 in this process is to complement a TCR-derived activation signal delivered in vivo as treatment of mice with anti-class II not only abolished the enhancement of TCR {zeta}-chain phosphorylation but also inhibited the ability to induce suppressor function in response to stimulation with IL-2. We have not as yet determined whether the capacity to develop suppressor function in response to cytokine stimulation is a property of all nTR or a sub-population of nTR. It has previously been reported (23, 29) that the CD103+ subset of nTR exhibits more potent suppressor activity when activated via the TCR, but both CD103+ and CD103– nTR develop suppressor function when pre-cultured in IL-2 (data not shown).

One characteristic feature of nTR is their inability to produce IL-2 under conditions that normally would induce substantial levels of IL-2 in conventional CD4+ T cells. The lack of IL-2 production from nTR appears to be dependent on a failure to undergo chromatin remodeling of the promoter region of the IL-2 gene (30). The inability of nTR to produce IL-2 following TCR stimulation may restrict their ability to expand in vivo and thereby prevent the induction of a long-lasting state of generalized immunosuppression. nTR differ from conventional CD4+ T cells not only in their inability to produce IL-2 but also in the signal transduction pathways that are stimulated in response to IL-2. Although IL-2 induced STAT5 phosphorylation in nTR is similar to that observed in conventional CD4+ T cells, nTR neither phosphorylate STAT1 and STAT3 nor appear to activate targets downstream of phosphatidylinositol-3-kinase after IL-2 stimulation (25, 31). The unique IL-2-signaling pattern exhibited by nTR could potentially play an important role in the development and maintenance of their suppressive phenotype. Treatment of mice with anti-class II abolished the ability of nTR to respond to IL-2 and develop suppressor function, but had no effect on their capacity to phosphorylate STAT5.

Our blocking studies with anti-MHC class II under non-depleting conditions in vivo suggest that recognition of self-peptide–MHC class II complexes is critical for activating the responsiveness of the nTR for stimulation by IL-2/IL-4 in vitro. We cannot exclude the potential contribution of co-stimulatory molecules such as CD80/CD86 in this process. One co-stimulatory molecule that involved in the activation of nTR is the GITR, a member of the tumor necrosis factor receptor superfamily. GITR is constitutively expressed at high levels on CD4+CD25+ T cells and at low levels on CD4+CD25– and CD8+ T cells. Expression of GITR is up-regulated after TCR stimulation on both CD4+CD25– and CD8+ T cells. Previous studies have demonstrated that nTR-mediated suppression was not seen when the co-cultures were performed in the presence of an agonistic anti-GITR (23). Although these studies were originally interpreted as demonstrating that anti-GITR acted on the nTR to reverse their suppressive ability, more recent experiments using combinations of suppressor and responder T cells from GITR–/– and GITR+/+ mice indicated that the anti-GITR acted on the responder cells to increase their resistance to suppression (24). It is therefore interesting that the agonistic anti-GITR in combination with IL-2 increased the suppressive ability of nTR (Fig. 3) suggesting that the GITR-L on APC can provide a co-stimulatory signal to the nTR during an inflammatory response. Thus, it would appear that GITR–GITR-L interactions create a delicate balance between augmentation and suppression of immune responses.

Our findings have important implications for the activation of nTR function in vivo. First, they raise the possibility that some nTR are continuously activated by recognition of self on APC and by cytokines (IL-2/IL-4) produced by effectors as part of normal T cell homeostasis. Second, in an inflammatory environment in which cytokines are produced in abundance, nTR could be activated to mediate potent suppressor functions in the absence of triggering by the cognate antigen. Thus, early in the course of suppression of the immune response to autoantigens, tumor antigens or pathogen-derived antigens, there would be no need to generate a population of antigen-specific nTR. During the more chronic phases of a response, the polyclonal population of nTR may convert to one with an increased frequency of antigen-specific nTR (32–34). The concept that nTR can be activated in a paracrine fashion is supported by the observation that nTR isolated from HSV-infected mice were suppressive in vitro without TCR re-stimulation (35). It would appear unlikely that majority of nTR in HSV-infected mice would have received a TCR signal, as HSV is not known to encode for any superantigens and only a minor population of the CD4+CD25+ T cells are likely to be HSV specific. Collectively, these results and other studies suggest that nTR represent a highly efficient immunologic ‘rapid-response force’ that exists in a partially primed, activated state that is capable of responding to signals received from activated effector cells. Although suppression of the immune response to autoantigens and modulation of the immune response to pathogen-derived antigens represent beneficial consequences of the rapid capability of nTR to modulate immune responses, the predominance of nTR in tumor-derived T cell infiltrates suggests that this rapid induction of suppression may also inhibit the capacity of the host to irradicate poorly immunogenic tumors (36).


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary figure S1 is available at International Immunology Online.


   Abbreviations
 
APC, antigen-presenting cell
ERK, extracellular signal-regulated kinase
HA, hemagglutinin
[3H]TdR, [3H]thymidine
nTR, naturally occurring CD4+CD25+ regulatory T cell

   Notes
 
Transmitting editor: J. Allison

Received 17 August 2006, accepted 29 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

  1. Hori S, Nomura T, Sakaguchi S. (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057.[Abstract/Free Full Text]
  2. Fontenot JD, Gavin MA, Rudensky AY. (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4:330.[CrossRef][Web of Science][Medline]
  3. Khattri R, Cox T, Yasayko SA, Ramsdell F. (2003) An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4:337.[CrossRef][Web of Science][Medline]
  4. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
  5. Wildin RS, Ramsdell F, Peake J, et al. (2001) X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 27:18.[CrossRef][Web of Science][Medline]
  6. Brunkow ME, Jeffery EW, Hjerrild KA, et al. (2001) Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27:68.[Web of Science][Medline]
  7. Bennett CL, Christie J, Ramsdell F, et al. (2001) The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27:20.[CrossRef][Web of Science][Medline]
  8. Hsieh CS, Liang Y, Tyznik AJ, Self SG, Liggitt D, Rudensky AY. (2004) Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors. Immunity 21:267.[CrossRef][Web of Science][Medline]
  9. Jordan MS, Boesteanu A, Reed AJ, et al. (2001) Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2:301.[CrossRef][Web of Science][Medline]
  10. Romagnoli P, Hudrisier D, van Meerwijk JP. (2002) Preferential recognition of self antigens despite normal thymic deletion of CD4(+)CD25(+) regulatory T cells. J. Immunol. 168:1644.[Abstract/Free Full Text]
  11. Fisson S, Darrasse-Jeze G, Litvinova E, et al. (2003) Continuous activation of autoreactive CD4+ CD25+ regulatory T cells in the steady state. J. Exp. Med. 198:737.[Abstract/Free Full Text]
  12. Stefanova I, Dorfman JR, Germain RN. (2002) Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes. Nature 420:429.[CrossRef][Medline]
  13. van Oers NS, Love PE, Shores EW, Weiss A. (1998) Regulation of TCR signal transduction in murine thymocytes by multiple TCR zeta-chain signaling motifs. J. Immunol. 160:163.[Abstract/Free Full Text]
  14. Thornton AM and Shevach EM. (1998) CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
  15. Takahashi T, Kuniyasu Y, Toda M, et al. (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.[Abstract/Free Full Text]
  16. Thornton AM and Shevach EM. (2000) Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164:183.[Abstract/Free Full Text]
  17. Thornton AM, Donovan EE, Piccirillo CA, Shevach EM. (2004) Cutting edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function. J. Immunol. 172:6519.[Abstract/Free Full Text]
  18. Piccirillo CA and Shevach EM. (2001) Cutting edge: control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells. J. Immunol. 167:1137.[Abstract/Free Full Text]
  19. Fehervari Z and Sakaguchi S. (2004) Control of Foxp3+ CD25+CD4+ regulatory cell activation and function by dendritic cells. Int. Immunol. 16:1769.[Abstract/Free Full Text]
  20. Pasare C and Medzhitov R. (2003) Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299:1033.[Abstract/Free Full Text]
  21. Brinster C and Shevach EM. (2005) Bone marrow derived dendritic cells reverse the "anergic" state of CD4+CD25+ T cells without reversing their suppressive function. J. Immunol. 175:7332.[Abstract/Free Full Text]
  22. Thornton AM, Piccirillo CA, Shevach EM. (2004) Activation requirements for the induction of CD4+CD25+ T cell suppressor function. Eur. J. Immunol. 34:366.[CrossRef][Web of Science][Medline]
  23. McHugh RS, Whitters MJ, Piccirillo CA, et al. (2002) CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:311.[CrossRef][Web of Science][Medline]
  24. Stephens GL, McHugh RS, Whitters MJ, et al. (2004) Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J. Immunol. 173:5008.[Abstract/Free Full Text]
  25. Bensinger SJ, Walsh PT, Zhang J, et al. (2004) Distinct IL-2 receptor signaling pattern in CD4+CD25+ regulatory T cells. J. Immunol. 172:5287.[Abstract/Free Full Text]
  26. Witherden D, van Oers N, Waltzinger C, Weiss A, Benoist C, Mathis D. (2000) Tetracycline-controllable selection of CD4(+) T cells: half-life and survival signals in the absence of major histocompatibility complex class II molecules. J. Exp. Med. 191:355.[Abstract/Free Full Text]
  27. Dorfman JR, Stefanova I, Yasutomo K, Germain RN. (2000) CD4+ T cell survival is not directly linked to self-MHC-induced TCR signaling. Nat. Immunol. 1:329.[CrossRef][Web of Science][Medline]
  28. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. (2005) A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. 6:1142.[CrossRef][Web of Science][Medline]
  29. Lehmann J, Huehn J, de la Rosa M, et al. (2002) Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25+ as well as CD25– regulatory T cells. Proc. Natl Acad. Sci. USA 99:13031.[Abstract/Free Full Text]
  30. Su L, Creusot R, Gallo E, et al. (2004) Murine CD4+CD25+ regulatory T cells fail to undergo chromatin remodeling across the proximal promoter region of the IL-2 gene. J. Immunol. 173:4994.[Abstract/Free Full Text]
  31. Chan SM, Ermann J, Su L, Fathman CG, Utz PJ. (2004) Protein microarrays for multiplex analysis of signal transduction pathways. Nat. Med. 12:1390.
  32. Mendez S, Reckling SK, Piccirillo CA, Sacks D, Belkaid Y. (2004) Role for CD4(+) CD25(+) regulatory T cells in reactivation of persistent leishmaniasis and control of concomitant immunity. J. Exp. Med. 200:201.[Abstract/Free Full Text]
  33. Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL. (2002) CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420:502.[CrossRef][Medline]
  34. Wang HY, Lee DA, Peng G, et al. (2004) Tumor-specific human CD4+ regulatory T cells and their ligands: implications for immunotherapy. Immunity 20:107.[CrossRef][Web of Science][Medline]
  35. Suvas S, Kumaraguru U, Pack CD, Lee S, Rouse BT. (2003) CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses. J. Exp. Med. 198:889.[Abstract/Free Full Text]
  36. Curiel TJ, Coukos G, Zou L, et al. (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 10:942.[CrossRef][Web of Science][Medline]

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