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International Immunology Advance Access originally published online on June 1, 2007
International Immunology 2007 19(6):785-799; doi:10.1093/intimm/dxm047
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

The maintenance of human CD4+CD25+ regulatory T cell function: IL-2, IL-4, IL-7 and IL-15 preserve optimal suppressive potency in vitro

John Yates1, Flavia Rovis1, Peter Mitchell2, Behdad Afzali2, JY-S Tsang2, Marina Garin2, RI Lechler2, Giovanna Lombardi2 and OA Garden1,3

1 Regulatory T Cell Laboratory, Department of Immunology, Division of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK
2 Immunoregulation Laboratory, Department of Nephrology and Transplantation, Fifth Floor, Thomas Guy House, Guy's Hospital Campus, King's College London School of Medicine at Guy's, King's College and St Thomas' Hospitals, London SE1 9RT, UK
3 Department of Veterinary Clinical Sciences, Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK

Correspondence to: O. A. Garden; E-mail: ogarden{at}rvc.ac.uk or o.garden{at}imperial.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
CD4+CD25+ regulatory T cells (Tregs) have far-reaching immunotherapeutic applications, the realization of which will require a greater understanding of the factors influencing their function and phenotype during ex vivo manipulation. In murine models, IL-2 plays an important role in both the maintenance of a functional Treg population in vivo and the activation of suppression in vitro. We have found that IL-2 maintains optimal function of human CD4+CD25+ Tregs in vitro and increases expression of both forkhead box protein 3, human nomenclature (FOXP3) and the distinctive markers CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor superfamily member number 18 (GITR). Although IL-2 reduced spontaneous apoptosis of Tregs, this property alone could not account for the optimal maintenance of the regulatory phenotype. The inhibition of phosphatidylinositol 3-kinase (PI3K) signaling by LY294002, a chemical inhibitor of PI3K, abolished the maintenance of maximal suppressive potency by IL-2, yet had no effect on the up-regulation of FOXP3, CD25, CTLA-4 and GITR. Other common gamma chain ({gamma}c) cytokines—IL-4, IL-7 and IL-15—had similar properties, although IL-4 showed a unique lack of effect on the expression of FOXP3 or Treg markers despite maintaining maximal regulatory function. Taken together, our data suggest a model in which the {gamma}c cytokines IL-2, IL-4, IL-7 and IL-15 maintain the optimal regulatory function of human CD4+CD25+ T cells in a PI3K-dependent manner, offering new insight into the effective manipulation of Tregs ex vivo.

Keywords: common gamma chain, FOXP3, LY294002, phosphatidylinositol 3-kinase


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
CD4+CD25+ regulatory T cells (Tregs) serve an important suppressive function, limiting potentially pathogenic immune responses to self- and foreign antigens (1, 2). Their ex vivo manipulation and reintroduction is likely to become an important immunotherapy in clinical practice (3, 4), necessitating an understanding of the factors required to maintain the regulatory phenotype in vitro.

Since the identification of the IL-2R {alpha} chain (CD25) as a marker for this regulatory population (5), the role of IL-2 in their normal homeostasis and function has become increasingly clear (6). Mice genetically engineered to be deficient in lymphocyte IL-2 signaling develop lethal autoimmune disease (7–10), attributed to a failure in Treg development (11). Subsequent work has revealed that transfer of CD4+CD25+ Tregs from wild-type mice prevents disease in this model and that restoration of IL-2 signaling in CD4+ T cells allows the normal development of Tregs (12–15). This work has been confirmed by the treatment of normal mice with anti-IL-2 antibodies, which deplete CD4+CD25+ Tregs (16) and induce autoimmune disease (17) in a similar fashion to the genetic studies. IL-2 is also a survival factor that limits the apoptosis of Tregs in vitro (18) and—together with TCR stimulation—has been implicated in the suppressive activity of murine CD4+CD25+ Tregs (19–23).

CD25 is a unique component of the high-affinity IL-2R, which also includes a ß chain (CD122)—shared by the IL-15R—and a common gamma chain ({gamma}c) (CD132)—shared by the receptors for IL-4, IL-7, IL-9, IL-15 and IL-21. The cytoplasmic domains of the ß and {gamma} chains are responsible for signaling, which occurs by the recruitment and activation of Janus-activated kinases 1 and 3, with activation of both the signal transducer and activator of transcription (STAT) factor 5 and the dual phosphatidylinositol 3-kinase (PI3K) and Ras mitogen-activated protein kinase pathways (24, 25). The shared receptor components allow elements of IL-2 signaling to be induced by the other {gamma}c cytokines, though IL-4 stands out as being a somewhat unique member of this family (24, 26). For example, with the exception of IL-4, all the {gamma}c cytokines activate STAT5 (24). Furthermore, IL-2, IL-7 and IL-15 regulate virtually identical sets of genes in T cells, whereas IL-4 induces only a partially overlapping—and largely distinct—profile of genes (27).

The role of cytokines other than IL-2 in CD4+CD25+ Treg homeostasis and activation is being increasingly recognized. Although reduced in number, CD4+CD25+Foxp3+ cells are nevertheless present in IL-2–/– mice (28), and at higher frequency than in STAT5–/– mice (29); similarly, a human patient with a mis-sense A630P STAT5b mutation showed decreased numbers, forkhead box protein 3, human nomenclature (FOXP3) expression and suppressor function of CD4+CD25HI Tregs (30). Moreover, constitutive activation of STAT5 within lymphocytes results in an expanded population of highly potent CD4+CD25+ Tregs, suggesting that cytokines other than IL-2 able to activate STAT5 may also support Treg expansion and maintenance (29). Indeed, the role of IL-2 in Treg homeostasis has recently been re-evaluated (31, 32) with evidence that signaling through the {gamma}c, rather than the IL-2R {alpha} chain, is crucial for the thymic ontogeny of CD4+CD25+ Tregs (33). Importantly, IL-2 signaling per se appears to be essential only for the peripheral survival and competitive ‘fitness’ of Tregs in these models (33, 34). Experiments performed in vitro have also supported a role for IL-4 and IL-7 in the survival and suppressive activity of murine CD4+CD25+ Tregs (19, 20, 35–37). Furthermore, both IL-4 and IL-13 induce the differentiation of murine CD4+CD25+Foxp3+ Tregs from CD4+CD25– precursors (38).

As a preliminary step toward the optimization of expansion protocols for clinical practice, we have examined the effect of IL-2 on human CD4+CD25+ Tregs. We have established that this cytokine maintains the regulatory phenotype in vitro, preserving maximal suppressive potency and increasing the expression of FOXP3 and CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) (CD152) and glucocorticoid-induced tumor necrosis factor receptor superfamily member number 18 (GITR). IL-2 also prevents the apoptosis of Tregs, though this did not appear to be an important mechanism underlying the preservation of maximal regulatory function, which required the presence of an intact PI3K-signaling pathway. The individual {gamma}c cytokines IL-4, IL-7 and IL-15 were also able to maintain maximal regulatory function, suggesting a degree of redundancy of the cytokines required to maintain Treg function in vitro that may recapitulate the key role of the {gamma}c in the thymic ontogeny of these cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Source of cells
Human CD4+ T cells were purified from either buffy coat preparations (Blood Transfusion Service, London, UK) or blood, immediately following venesection of healthy volunteers.

Culture medium and cytokines
Human CD4+ T cells were cultured in Rosswell Park Memorial Institute 1640 medium, supplemented with penicillin (100 IU ml–1), streptomycin (100 mg ml–1) and L-glutamine (2 mM) (RPMI–PSG; all from GIBCO, Paisley, UK) and 10% v/v human AB serum (HS; Biowest, Ringmer, UK). Recombinant human IL-2 (Roche, Lewes, UK), IL-4 (Biosource, Nivelles, Belgium), IL-7 (R&D Systems, Abingdon, UK) and IL-15 (Peprotech, Northampton, UK) were used at the concentrations indicated in figure legends.

Purification of T cell subsets
Human PBMCs were isolated from blood samples by density gradient centrifugation over LymphoprepTM (Axis-Shield, Oslo, Norway). Washed PBMCs were re-suspended in ice-cold RPMI–PSG supplemented with 2% v/v HS, before gentle rolling at 4°C for 25 min with murine mAbs against CD8 (B-H7), CD14 (B-A8), CD16 (B-E16) (all from Diaclone, Boldon, UK), CD19 (HD37), CD56 (MEM188), glycophorin A (BRIC 256) (all from Chemicon, Chandlers Ford, UK), CD33 (P67.6) and TCR {gamma}{delta} (11F2) [both from Becton Dickinson (BD), Cowley, UK]. After washing, antibody-coated PBMCs were incubated with goat anti-mouse Fc-coated Biomag® beads (Qiagen; Metachem Diagnostics, Northampton, UK) for 25 min and non-CD4+ T cells depleted by magnetic separation. Isolated CD4+ T cells were washed and then mixed with 500 µl anti-CD25-coated beads (Dynal, Wirral, UK) by gentle rolling at 4°C for 20 min. CD25-depleted cells (referred to as CD25–) were removed in the supernatant and the beads were washed four times by gentle pipetting in RPMI–PSG/2% v/v HS. After washing, the beads were re-suspended in RPMI–PSG/10% v/v HS with DETACHabead® (Dynal) and shaken at room temperature for 45 min to release CD25+ T cells; the beads were removed by magnetic separation, leaving CD25+ cells in the supernatant. Purity ranged from 92 to 98% for CD25-depleted CD4+ T cells and 90 to 97% for CD4+CD25+ T cells. The number of CD4+CD25+ T cells isolated was typically 1–3% of total CD4+ cells.

Pre-incubation with cytokines
Isolated human CD4+ T cells were suspended at a density of 5 x 105–1 x 106 cells ml–1 in RPMI–PSG/10% v/v HS, before aliquoting volumes of 400 µl into 48-well plates. Cytokines were added at the concentrations indicated and the volumes made up to 500 µl. LY294002 (LY), a chemical inhibitor of PI3K (Calbiochem; Merck, Beeston, UK), was used at 10 µM and ciclosporin A (CSA) (Sigma, Poole, UK) at 0.5 µM during pre-incubations. Where cells were pre-incubated with cytokines and either of the drugs, the appropriate volumes of the diluents dimethylsulfoxide (for LY: Sigma) and ethanol (for CSA: VWR, Lutterworth, UK) were added to the medium or cytokine controls. After overnight incubation (18 h), cells were washed three times in RPMI–PSG before use in proliferation assays or surface phenotyping.

Proliferation assays
T cell proliferation assays were set-up in triplicate in 96-well, round-bottomed plates, suspending both CD25– and CD25+ CD4+ T cells in RPMI–PSG with 10% v/v HS at 1 x 105 cells ml–1, from which serial dilutions were prepared. The T cells were added to the plate in 50 µl aliquots (5 x 103 T cells per well) in the combinations described in the figures. Anti-CD3/anti-CD28-coated beads (Dynal) were added at 1000 beads per well and the final volume in each well adjusted to 250 µl. On day 5, the plate was pulsed with 1 µCi of [3H]-labeled thymidine per well (Amersham Biosciences, Chalfont St Giles, UK). Preliminary experiments revealed that proportional suppression of proliferation increased with time in this assay system, independently of the prior IL-2 history of the CD4+CD25+ T cells; identical conclusions were derived when shorter cultures were examined, though proportional suppression was lower, hence our choice of 5 days. After 16–20 h, cells were harvested in routine fashion for liquid scintillation counting. The percentage suppression was determined using the formula {1 – [counts per minute (c.p.m.) CD25–/+/c.p.m. CD25– alone]} x 100.

Flow cytometry
T cell subsets were stained with anti-CD4–allophycocyanin (APC) (S3.5), CD25–PE (CD25-3G10) (both from Caltag Medsystems Ltd, Towcester, UK) and CD3–FITC (UCHT-1) (Sigma). Surface phenotype was analyzed with anti-HLA-DR–FITC (L243), CD45RA–PE, CD45RO–FITC, CD127–FITC (all from BD PharMingen, Cowley, UK) and GITR–PE (110416) (R&D systems). Intracellular CTLA-4 was detected using anti-CTLA-4–PE (BN123) (BD) after fixation with 4% w/v paraformaldehyde (PFA) (Sigma) and permeabilization with 0.5% w/v saponin (Sigma). Intracellular FOXP3 was detected using anti-FOXP3–APC (PCH101; eBioscience, San Diego, CA, USA) after fixation and permeabilization with Fix/Perm solutionTM (eBioscience). Isotype controls included IgG1–FITC (MOPC-21), IgG2a–FITC (UPC-10), IgG1–PE (MOPC-21) (all from Sigma), IgG2a–PE (G155-178) (BD) and IgG2a–APC (MG2a) (Caltag). Events were acquired using a FACSCaliburTM with CellQuestTM software (BD). Both CellQuestTM and FlowJo© version 6 (Tree Star Inc., San Carlos, CA, USA) were used for analysis. Fluorescence-activated sorting of CD4+ T cells from fresh blood was performed with a FACSDivaTM after staining with anti-CD4–FITC (Q1420) (Sigma) and anti-CD25–PE (M-A251) (PharMingen). The IgG1–PE isotype control was used to delineate gates for the CD25– and CD25INT populations; the gate for CD25HI cells was drawn to collect the 2% of CD4+ T cells showing the highest expression of CD25. All isolations were >98% pure.

Determination of viable cell numbers
Equal volumes (200 µl) of a CD4+CD25+ T cell suspension were incubated overnight under the conditions indicated in figures, within triplicate wells of 96-well, round-bottomed plates. After overnight incubation, 150 µl of re-suspended cells were transferred from the 96-well plate to FACSTM tubes (BD) using a reverse pipetting technique, before washing and then re-suspending in 100 µl Annexin-binding buffer (BD PharMingen). Cells were stained with Annexin V (conjugated to PE for bead-separated populations and APC for flow-sorted CD25 subsets) and 7-aminoactinomycin-D (7-AAD) (BD PharMingen) at room temperature. A volume of 20 µl of Perfect-Count® microspheres (Cytognos, Santander, Spain) was added by reverse pipetting, before re-suspension in a total volume of 250 µl binding buffer. Acquisition was limited by gating on Perfect-Count® microspheres, collecting 2500 events for each sample. Viable cell number was determined by counting Annexin V–FITC/7-AAD double-negative events in a lymphocyte gate. Relative cell viability was determined using the formula: (viable cell number acquired under condition ‘x’)/(viable cell number acquired after incubation in medium alone).

Western blots
Isolated CD4+ T cells were suspended (1 x 106 cells ml–1) in RPMI–PSG/10% v/v HS, before being dispensed into 48-well plates. During pre-incubations, IL-2 was used at 0.5 ng ml–1 and LY at 10 µM in a final volume of 500 µl. After overnight incubation (18 h), cells were washed three times in phosphate-buffered saline (PBS) (Oxoid, Hampshire, UK). The cells were treated with ice-cold lysis buffer containing 1% (v/v) Triton-X 100, 1 mM Na3VO4, 1 mM EDTA and a protease inhibitor cocktail (Sigma) for 30 minutes. The lysate was cleared by centrifugation at 13 000 x g for 20 minutes at 4°C, before measurement of protein concentration with the Bio-Rad Protein Quantitation KitTM (Bio-Rad Laboratories, Hemel Hempstead, UK). Cell lysate (10 µg of protein) was then separated by 10% w/v SDS–PAGE and transferred onto a polyvinylidene fluoride membrane. Membranes were blocked for 1 h with 5% w/v skimmed milk in PBS with 0.01% v/v. Tween 20, before probing with the anti-phospho-serine/threonine kinase (pAKT) antibody (193H12) (Cell Signaling Technology, Beverly, MA, USA). Bound antibody was revealed with HRP-conjugated anti-rabbit antibody, using enhanced chemiluminescence (Amersham Biosciences). The intensity of bands was determined with the GelDoc-It Imaging System® using LabWorks software (UVP Ltd, Cambridge, UK).

Statistical analysis
Statistical analyses were performed using GraphPadTM Prism, version 4.00 for WindowsTM (GraphPadTM Software, San Diego, USA). Paired t-tests were applied for two columns of data; one-way analysis of variances with Tukey's adjustment for multiple comparisons were applied for three or more columns.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Incubation with IL-2 preserves human CD4+CD25+ Treg function in vitro
IL-2 is necessary for the maintenance of a functional murine CD4+CD25+ Treg population in vivo (17) and freshly isolated cells express a number of IL-2–inducible genes, indicating a degree of tonic cytokine stimulation (39, 40). In vitro data suggest that IL-2 is necessary to activate suppression (20–22), implying that constitutive IL-2 signaling may maintain CD4+CD25+ Tregs in a ‘primed’ state. We hypothesized that incubation of human CD4+CD25+ Tregs with IL-2 would pre-activate these cells, enhancing their suppressive potency. Using anti-CD25 antibody-coated Dynabeads®, we purified CD4+CD25+ T cells from buffy coats and incubated the cells overnight with IL-2 or medium alone. Using a polyclonal bead stimulus, we then measured the ability of these pre-incubated cells to suppress the proliferation of CD4+CD25– T cells in co-culture. CD4+CD25+ T cells pre-incubated with IL-2 more potently suppressed CD4+CD25– cell proliferation than CD25+ cells pre-incubated in medium alone. In some, though not all, donors, this phenomenon was apparent at IL-2 concentrations as low as 0.05 ng ml–1 (Fig. 1A). Maximal enhancement was usually observed at 0.5 ng ml–1: using this concentration, the mean enhancement of suppression in 1:1 co-cultures in 15 experiments was 15.4% (P < 0.0001; Fig. 1B). These concentrations of IL-2 did not lead to proliferation of CD4+CD25+ T cells without TCR stimulation (data not shown). Although modest in absolute terms, this difference represented an increase of 36% in suppression observed when CD4+CD25+ T cells were incubated in medium alone (42.5 versus 57.9%).


Figure 1
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  		Fig. 1. IL-2 maintains maximal suppressive potency of human CD4+CD25+ T cells. (A) CD4+CD25+ T cells, immunomagnetically selected from buffy coats, were incubated overnight in medium or medium with recombinant IL-2 at the concentrations indicated. Washed CD4+CD25+ T cells were mixed with CD4+CD25– T cells at the ratios indicated and the co-cultures stimulated with anti-CD3/CD28-coated Dynabeads®. [3H]-thymidine was added on day 5. Results for an individual donor are shown. Error bars represent standard deviations. *P < 0.05, **P < 0.01, ***P < 0.001 (analysis of variance). (B) Both the percentage suppression by human CD4+CD25+ T cells at a 1:1 ratio with CD4+CD25– T cells and the augmentation achieved by pre-incubating with IL-2 varied between individuals. IL-2 pre-incubation (0.5 ng ml–1) increased suppression by a mean of 15.4% (P < 0.0001; n = 15, Student's paired t-test). (C) In five experiments, CD4+CD25+ T cells were selected from fresh blood. Some of them were used in a suppression assay (day 0), while the remainder were incubated overnight with medium or medium and IL-2 (0.5 ng ml–1). Fresh CD4+CD25+ T cells selected from the same donor the following day were used in a proliferation assay alongside the CD4+CD25+ T cells that were incubated overnight. Proportional suppression was calculated for the cells isolated on day 0, freshly isolated cells and the cells incubated overnight in medium or medium and IL-2, incubated at a 1:1 ratio with CD4+CD25– T cells. Error bars represent standard deviations.

 
These results suggested that pre-incubation in IL-2 either enhanced the suppressive potency of human CD4+CD25+ Tregs or prevented deterioration in potency that occurred with overnight incubation in medium alone. To determine whether enhancement or maintenance of maximal suppressive potency was predominant, we isolated CD4+CD25+ T cells on consecutive days from single donors. We compared the suppression observed in an assay when first isolated (day 0) with that seen after overnight incubation in medium, in medium with IL-2 (0.5 ng ml–1) and with freshly isolated CD4+CD25+ T cells from the same donor. Serial comparisons of cells from individual donors controlled for differences in purity between selections—though these were likely to be small, since all separations were 90–98% CD4+CD25+. Pre-incubation with IL-2 did not enhance suppression beyond that seen either with freshly isolated cells or cells used in proliferation assays on day 0 (Fig. 1C). Cells incubated in medium alone were less suppressive, suggesting that IL-2 acted to maintain, rather than to enhance, optimal suppressive potency.

IL-2 is a survival factor for CD4+CD25+ Tregs, but prevention of apoptosis is not sufficient to maintain maximal suppression
A trivial explanation for our results was that IL-2 simply acted as a survival factor for Tregs (18). Although equal numbers of viable cells were used in each proliferation assay, we sought to rule out an anti-apoptotic effect as an explanation for the observed phenomenon by comparing the ability of IL-2 to maintain CD4+CD25+ T function with another pro-survival cytokine, IL-6. Isolated CD4+CD25+ T cells were incubated overnight with medium or medium containing IL-2 (0.5 ng ml–1) or IL-6 (50 ng ml–1). Viable cells were counted using a bead-based flow cytometric method and Annexin V/7-AAD staining to exclude dead cells or those undergoing apoptosis. In a similar manner to IL-2, IL-6 was able to reduce CD4+CD25+ T cell apoptosis, with a mean increase in survival of 19 versus 21% for IL-2 (Fig. 2A). Despite protecting from apoptosis, IL-6 had no effect on the suppressive potency of the CD4+CD25+ T cells (Fig. 2B and C).


Figure 2
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  		Fig. 2. Both IL-2 and IL-6 reduce apoptosis of human CD4+CD25+ T cells, but only IL-2 maintains maximal suppressive potency. (A) IL-2 and IL-6 protect CD4+CD25+ T cells from apoptosis. Selected CD4+CD25+ T cells were incubated overnight in medium, medium with IL-2 (0.5 ng ml–1) or medium with IL-6 (50 ng ml–1). Total viable cells recovered were counted by flow cytometry, making reference to Perfect-Count® beads and excluding dead and dying cells with Annexin V–PE and 7-AAD. Cell recovery was calculated from triplicate wells. Relative cell viability was calculated by means of the formula: (number recovered with condition ‘x’)/(number recovered with medium alone). Mean increase in survival: 19% for IL-6 versus 21% for IL-2 (n = 5 different donors). Error bars represent standard deviations. *P < 0.05 (analysis of variance). (B) IL-6 does not maintain maximal suppressive potency of human CD4+CD25+ T cells. Selected CD4+CD25+ T cells were incubated overnight in medium, medium with IL-2 (0.5 ng ml–1) or medium with IL-6 (50 ng ml–1). After washing, the CD4+CD25+ T cells were cultured with CD4+CD25– T cells (1:1) for 5 days, before pulsing with [3H]-thymidine. Error bars represent standard deviations from a single experiment. ns P > 0.05, **P < 0.01 (analysis of variance). (C) IL-6 does not maintain maximal suppressive potency by human CD4+CD25+ T cells. Percentage suppression was calculated for three experiments with different donors [including donor shown in (B)]. Mean increase in suppression: 10% for IL-2 (n = 3). Error bars represent standard deviations. ns P > 0.05, **P < 0.01 (analysis of variance).

 
We next determined whether the pro-survival effect of IL-2 could be dissociated from the ability to maintain maximal suppression. IL-2 is thought to prevent apoptosis through the PI3K-dependent activation of protein kinase B/serine/threonine kinase (AKT) (41). However, work with murine CD4+CD25+ Tregs and human CD4+CD25+ Treg lines has shown that inhibition of PI3K with LY does not abolish IL-2 pro-survival effects in these cells (42, 43). IL-2R signaling is incompletely understood and effects such as cell-cycle progression and increased survival are thought to occur through integration of distinct downstream pathways (24). We surmised that inhibition of PI3K, although unlikely to inhibit pro-survival effects, may interfere with the maintenance of maximal suppression by IL-2. We first sought to demonstrate that IL-2 activates PI3K within human CD4+CD25+ T cells, before showing that LY is able to inhibit this rise in activity. Overnight incubation with IL-2 increased AKT activation in CD4+CD25+ T cells, and this phenomenon could be inhibited by co-incubation with LY (Fig. 3). We next examined the functional consequences of LY during pre-incubation of the CD4+CD25+ T cells, before testing their suppressive potency within our assay system. While LY did not inhibit IL-2 pro-survival effects (mean increase in survival of 19% IL-2 versus 23% IL-2 + LY, P > 0.05; Fig. 4A), it nevertheless abolished the maintenance of maximal suppression (Fig. 4B and C). In contrast, LY pre-incubation had no effect on suppression mediated by cells incubated in medium alone (Fig. 4C).


Figure 3
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  		Fig. 3. IL-2 stimulates the activation of AKT in CD4+CD25+ T cells. CD4+CD25+ T cells were immunomagnetically selected, before overnight (18 h) incubation in complete medium with or without IL-2 (0.5 ng ml–1). The PI3K inhibitor LY (10 µM) was included in some of the wells containing IL-2. Cell lysates (10 µg protein per lane) were prepared in routine fashion for Western blotting, before probing the membranes with anti-pAKT (clone 193H12). Bands were visualized with HRP-conjugated anti-rabbit antibody, using enhanced chemiluminescence. The ratios of the intensities (total OD/area) of the pAKT to ß-actin bands are noted. A representative of two independent experiments is shown.

 

Figure 4
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  		Fig. 4. Inhibition of PI3K abolishes the maintenance of maximal CD4+CD25+ T cell-suppressive potency by IL-2. (A) Inhibition of PI3K does not inhibit the anti-apoptotic effect of IL-2. Selected CD4+CD25+ T cells were incubated with IL-2 or IL-2 and LY (10 µM in all experiments). Relative cell viability was calculated as in Fig. 2(A) for four experiments with different donors. Mean increase in survival: 19% for IL-2 versus 23% for IL-2 + LY, P > 0.05 (n = 4; analysis of variance). Error bars represent standard deviations. (B) Inhibition of PI3K abolishes the maintenance of maximal suppressive potency by IL-2. Pre-incubated human CD4+CD25+ T cells were washed, mixed with CD4+CD25– T cells (1:1) and stimulated with anti-CD3/CD28-coated Dynabeads®. Proliferation was measured by [3H]-thymidine incorporation at 5 days. A representative experiment is shown. Error bars represent standard deviations. *P < 0.05, **P < 0.01, ns P > 0.05 (analysis of variance). (C) Inhibition of PI3K abolishes the maintenance of maximal suppressive potency by IL-2. In a series of experiments with different donors, LY inhibited the maintenance of maximal suppressive potency by IL-2. Mean increase in suppression: 11% for IL-2 (P < 0.05, n = 6 different donors; analysis of variance). LY had no effect on suppressive potency of cells incubated in medium alone. Error bars represent standard deviations.

 
Maintenance of maximal CD4+CD25+ Treg suppression by IL-2 is not dependent on simultaneous TCR signaling
A possible explanation for the maintenance of suppression with pre-incubation of CD4+CD25+ T cells with IL-2 was the receipt of signals delivered by the TCR, by virtue of interactions with MHC class II expressed by other CD4+CD25+ T cells. Exogenous IL-2 combined with TCR signaling is known to enhance suppression (19); furthermore, CD4+CD25+ Tregs may respond to epitopes derived from self-TCRs (44). IL-2 had a variable effect on MHC class II expression by CD4+CD25+ T cells: in some experiments, a small increase in the percentage of cells expressing HLA-DR was observed, though this was not a consistent finding (Fig. 5A). To prevent TCR signaling, we added CSA during pre-incubation with IL-2 or medium alone, thereby inhibiting calcineurin and Jun N-terminal kinase/p38 activation and proliferation without influencing IL-2 signaling (45, 46). At a concentration that yielded 90% inhibition of T cell proliferation when added during stimulation with Dynabeads® coated with antibodies against CD3 and CD28 (data not shown), CSA had no effect on the ability of IL-2 to maintain maximal suppressive potency (Fig. 5B).


Figure 5
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  		Fig. 5. Inhibition of TCR signaling has no effect on the maintenance of maximal CD4+CD25+ T cell-suppressive potency by IL-2. (A) Human CD4+CD25+ T cells express HLA-DR. CD4+CD25+ T cells were incubated overnight in medium or medium and IL-2 (0.5 ng ml–1), before measurement of HLA-DR expression by flow cytometry. Percentage expressing HLA-DR: 20% medium and 29% IL-2. (B) Pre-incubation with CSA does not inhibit the maintenance of maximal suppressive potency by IL-2. Either CSA (0.5 µM) or an equal volume of diluent (ethanol) was added at the onset of pre-incubation in medium or medium with IL-2 (0.5 ng ml–1). Proliferation assays were set-up as in Fig. 2. A representative experiment is shown, of three performed with different donors. Error bars represent standard deviations. ns P >0.05, **P < 0.01 (analysis of variance).

 
IL-2 increases the expression of CD4+CD25+ Treg markers
We had not expected to see functional changes in CD4+CD25+ Tregs at such low concentrations of IL-2, since these cells do not proliferate in response to IL-2 without concomitant TCR (47) or GITR stimulation (40) and constitutively express several proteins involved in down-regulation of cytokine signaling (39, 40, 48). To examine further the effect of IL-2 on the phenotype of human CD4+CD25+ Tregs, we assessed the expression of CD25, which is up-regulated by IL-2 via STAT5 (49, 50). IL-2 increased CD25 expression at concentrations as low as 0.05 ng ml–1 (data not shown); this effect appeared to be maximal at 0.5 ng ml–1 (Fig. 6A), with no higher expression observed at 5 ng ml–1 (data not shown). We also examined CTLA-4 (CD152) and GITR, two other distinctive markers of CD4+CD25+ Tregs (40, 51–53). IL-2 up-regulated intracellular CTLA-4 expression by CD4+CD25+ T cells at 0.5 ng ml–1 (Fig. 6B). Overnight incubation induced CD25 and CTLA-4 expression to higher levels than those seen in freshly isolated cells (Fig. 6A and B). Neither up-regulation of CD25 nor CTLA-4 was inhibited by LY (Fig. 6A; data for CTLA-4 not shown), suggesting that either the up-regulation of these molecules and the maintenance of maximal suppressive potency were unrelated phenomena or up-regulation was necessary but not sufficient to maintain optimal suppressor function. Increased CTLA-4 expression by CD4+CD25+ Tregs after incubation with IL-2 has been noted previously (54). In contrast to murine CD4+CD25+ Tregs, only 5–10% of freshly isolated CD4+CD25+ T cells expressed GITR in our hands, using a protocol that yielded >70% cellular expression with activation (data not shown). Pre-incubation with IL-2 increased the percentage of CD4+CD25+ T cells expressing GITR, but the effect was marginal (Fig. 6C). In all cases, we assumed that differences in expression reflected increases in protein expression per unit cell volume rather than increases in the size of the CD4+CD25+ Tregs (Fig. 6D).


Figure 6
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  		Fig. 6. IL-2 increases the expression of CD4+CD25+ Treg markers. (A) IL-2 up-regulates the expression of CD25 by CD4+CD25+ T cells. Selected CD4+CD25+ T cells were incubated overnight with medium, medium with IL-2 (0.5 ng ml–1) or IL-2 (0.5 ng ml–1) and LY (10 µM) and staining compared with freshly isolated cells. Cells were stained with anti-CD25–PE. (B) IL-2 up-regulates intracellular CTLA-4 expression by CD4+CD25+ T cells. Selected CD4+CD25+ T cells were incubated overnight with medium or IL-2 (0.5 ng ml–1) and staining compared with freshly isolated cells. Cells were fixed in paraformaldehyde, permeabilized with saponin and stained with anti-CD152–PE. (C) IL-2 up-regulates GITR expression by CD4+CD25+ T cells. Selected CD4+CD25+ T cells were incubated overnight with medium or medium and IL-2 (0.5 ng ml–1). Cells were stained with anti-GITR–PE. The mean fluorescence intensity and proportion of positive cells are displayed in the inset. (D) IL-2 has a minimal effect on CD4+CD25+ T cell size. Selected human CD4+CD25+ T cells were incubated overnight with medium or medium and IL-2 (0.5 ng ml–1); forward scatter was assessed by flow cytometry. (E) IL-2 has no effect on CD4+CD25+ T cell expression of CD45RA/RO with overnight culture, suggesting that gross changes in the naive and memory Treg sub-populations are unlikely to be responsible for the maintenance of optimal suppressive potency.

 
We considered that the bead-selected CD4+CD25+ T cells were likely to represent a heterogeneous population, comprising both CD45RA+ and CD45RO+ regulatory and conventional T cells. Human Tregs of both naive and memory phenotypes have been characterized (55, 56), either of which could have contributed to the phenomenon observed in the current study. Thus, one explanation for the maintenance of optimal suppressive potency by IL-2 was the selective survival of a more potent regulatory sub-population in our overnight cultures—one study, for example, suggests that memory Tregs are more potent suppressors than their naive CD4+CD25+ counterparts (55). However, the composition of the CD4+CD25+ T cell population, as documented by CD45RA/RO staining, remained unchanged with overnight culture in medium containing IL-2 (Fig. 6E), suggesting that gross changes in the Treg sub-populations were unlikely to be responsible for the maintenance of optimal suppressive potency. Nevertheless, a more subtle selective effect of IL-2 on one or other of the Treg sub-populations, operating at the level of their suppressor effector function, could not be excluded.

IL-2 pre-incubation influences only the CD4+CD25HI T cells
Approximately 30% of human CD4+ T cells express CD25 [our data and (57, 58)], but only the CD25HI population shows regulatory function (59). The majority of human CD4+CD25+ T cells express intermediate levels of CD25 (CD25INT), are predominantly CD45RO+, CD69–, HLA-DR– and CTLA-4–, and do not show anergic or regulatory properties [our data and (59)]. Using magnetic bead separation techniques, a variable percentage of CD25HI and CD25INT CD4+ T cells were isolated within the CD25+ population, which partly accounted for the variable suppression seen between CD25+ preparations (Fig. 1B). Although the majority of CD4+CD25INT T cells do not express the IL-2R ß chain (CD122) and thus lack a functional high-affinity IL-2R, the possibility remained that they were the specific target of IL-2 in our experiments: IL-2 may have conferred a regulatory phenotype on the CD25INT T cells, thereby increasing the regulatory population in the CD4+CD25+ fraction. To exclude this possibility, we used FACSTM to separate the CD25HI and CD25INT populations, pre-incubating these cells separately with IL-2. IL-2 had no effect on the ability of the CD25INT population to suppress, but augmented suppression by the CD25HI T cells (Fig. 7A). As would be expected, IL-2 had no effect on the apoptosis of the CD25INT population (Fig. 7B). A small sub-population of CD25INT T cells up-regulated CD25 expression in response to IL-2 (Fig. 7C), thought likely to represent the minority expressing CD122, but there was no change in the overall population; in contrast, the CD25HI T cells almost universally up-regulated CD25 (Fig. 7D). However, the expression of other phenotypic markers, such as CD45RA and CD45RO, was not appreciably altered by overnight exposure to IL-2, confirming the specificity of the CD25 response (Fig. 7E). Furthermore, the expression of CD127—the IL-7R {alpha} chain expressed at low levels by CD4+CD25HI T cells (60, 61)—showed no significant change with overnight exposure to IL-2, but could be abolished with exposure to IL-7, attributed to activation and internalization of the IL-7R (62) (Fig. 7F).


Figure 7
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  		Fig. 7. IL-2 targets predominantly the CD4+CD25HI Treg population. (A) Pre-incubation with IL-2 maintains maximal suppressive potency of CD4+CD25HI Tregs, but has no effect on the CD4+CD25INT subset. Freshly isolated CD4+ T cells were separated by FACSTM into CD25–, CD25INT and CD25HI subsets. After overnight incubation in medium or medium and IL-2 (0.5 ng ml–1), the cells were washed and proliferation assays set-up as before, except that the CD25HI subset were co-cultured with the CD25– cells at a 1:2 ratio. A representative of two independent experiments is shown. Error bars represent standard deviations. ns P > 0.05, *P < 0.05 (analysis of variance). (B) IL-2 reduces apoptosis of the CD25HI subset, but has no effect on the CD25INT. After overnight incubation, viable cell recovery was assessed as in Fig. 2. Error bars represent standard deviations of triplicate wells. ns P > 0.05, *P < 0.05 (Student's t-test). (C) IL-2 does not up-regulate CD25 expression by the CD25INT subset. After overnight incubation, CD25INT cells were re-stained with anti-CD25–PE. CD25 was up-regulated in only a small proportion of the total CD25INT cells. The mean fluorescence intensity (MFI) is shown in the inset. (D) IL-2 up-regulates CD25 expression by the CD25HI subset. After overnight incubation, CD25HI cells were re-stained with anti-CD25–PE. CD25 expression had increased in the majority of cells. MFI is shown in the inset. (E) IL-2 has no appreciable effect on CD4+CD25HI T cell expression of CD45RA/RO with overnight culture. In this experiment, the majority of the Tregs were CD45RO+ and only a minority were CD45RA+; overnight exposure to IL-2 had little impact on the expression of either marker. (F) Similarly, IL-2 had no significant impact on the pattern of expression of CD127, though as anticipated, IL-7 abolished expression of this marker.

 
Pre-incubation with IL-4, IL-7 or IL-15 maintains maximal suppressive potency of CD4+CD25+ T cells
Expansion and maintenance of CD4+CD25+ Tregs in vitro necessarily involves withdrawal from many factors present in vivo, coupled with repetitive high-affinity stimulation. Under these circumstances, IL-2 alone may not be an ideal maintenance cytokine, since pre-incubation with IL-2 can prime for cell death on re-stimulation (63) and other {gamma}c cytokines may have properties that could support optimal expansion (64). Indeed, there is evidence supporting a degree of redundancy in the ability of {gamma}c cytokines to maintain functional CD4+CD25+ Treg populations (6, 19, 35–37, 65). We hypothesized that these other {gamma}c cytokines would maintain human CD4+CD25+ Treg function and phenotype in a manner similar to IL-2. Pre-incubation with IL-4, IL-7 or IL-15 maintained maximal suppression by human CD4+CD25+ Tregs, though the concentrations required were 20- to 50-fold higher than for IL-2 (Fig. 8A), thought to reflect the high density of expression of the IL-2 {alpha}ß{gamma} receptor by CD4+CD25+ Tregs. The {gamma}c cytokines that also activate STAT5 (IL-7 and IL-15) increased CD25 and CTLA-4 expression to levels similar to those seen with IL-2, whereas IL-4 had no effect on the expression of these markers (Fig. 8B). As with IL-2, IL-7 and IL-15 had only a small impact on the expression of GITR, in contrast to IL-4, which made no difference. The resistance of IL-2–induced up-regulation of CTLA-4 to PI3K inhibition and the ability of other cytokines activating STAT5 to increase levels of this marker together suggested that CTLA-4 expression by CD4+CD25+ Tregs may be regulated through STAT5.


Figure 8
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  		Fig. 8. Pre-incubation with individual {gamma}c cytokines IL-4, IL-7 or IL-15 maintains the regulatory phenotype of CD4+CD25+ T cells. (A) Pre-incubation with IL-4, IL-7 or IL-15 maintains maximal suppressive potency by CD4+CD25+ T cells. Selected CD4+CD25+ T cells were incubated in medium or medium supplemented with the individual cytokines IL-2 (0.5 ng ml–1), IL-4 (10 ng ml–1), IL-7 (10 ng ml–1) or IL-15 (25 ng ml–1). Cells were washed and cultured with CD4+CD25– T cells in the presence of anti-CD3/CD28-coated Dynabeads®. Proliferation was measured by [3H]-thymidine incorporation on day 5. Representative examples of three experiments for IL-4 and IL-15, and two for IL-7, are shown. Error bars represent standard deviations. ns P > 0.05, *P < 0.05 (analysis of variance). (B) IL-7 and IL-15 up-regulate the expression of CD25, CTLA-4 and GITR by human CD4+CD25+ T cells, while IL-4 has no effect. Selected CD4+CD25+ T cells were incubated overnight in medium or medium with IL-4 (10 ng ml–1), IL-7 (10 ng ml–1) or IL-15 (25 ng ml–1). Cells were stained for surface CD25 and GITR or permeabilized and stained for intracellular CTLA-4. The proportions of positive cells are displayed in the insets.

 
Pre-incubation of human CD4+CD25+ T cells with IL-2, IL-7 or IL-15, but not IL-4, up-regulates the expression of FOXP3
Transfection of human CD4+CD25– T cells with FOXP3 confers a hypoproliferative, suppressive phenotype on them and induces the expression of CD25 and CTLA-4 (66)—key features of CD4+CD25+ Tregs. We speculated that the {gamma}c cytokines may act ‘upstream’ of FOXP3, functioning to maintain tonic expression of this gene and therefore the regulatory phenotype. While pre-incubation of CD4+CD25+ T cells with IL-2, IL-7 or IL-15 increased the expression of FOXP3 beyond that observed in either freshly isolated T cells or those incubated in medium alone, IL-4 had no impact on its expression (Fig. 9A); furthermore, up-regulation of FOXP3 by IL-2 could not be inhibited with LY at concentrations that abolished the maintenance of maximal suppressive potency (Fig. 9A). FOXP3 expression of CD4+CD25– T cells remained unaltered, regardless of the cytokine examined (Fig. 9B).


Figure 9
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  		Fig. 9. The {gamma}c cytokines IL-2, IL-7 and IL-15 up-regulate the expression of FOXP3 by CD4+CD25+ T cells. CD4+CD25+ T cells were immunomagnetically selected, before overnight (16 h) incubation in complete medium with or without IL-2 (0.5 ng ml–1), IL-4 (10 ng ml–1), IL-7 (10 ng ml–1) or IL-15 (25 ng ml–1). The PI3K inhibitor LY (10 µM) was included in some of the wells containing IL-2. (A) IL-2, IL-7 and IL-15 all up-regulate the expression of FOXP3 by CD4+CD25+ T cells, in contrast to IL-4, which has no impact on its expression. Despite abolishing the maintenance of optimal suppressive potency by IL-2, the PI3K inhibitor LY has no effect on the increased expression of FOXP3 induced by this cytokine (IL-2 and IL-2 + LY responses are superimposable). (B) In contrast to the CD25+ T cells, FOXP3 expression by the CD25–CD4+ T cells is not influenced by {gamma}c cytokines.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The manipulation of human CD4+CD25+ Tregs in vitro is a pivotal first step toward their therapeutic application in autoimmune disease and transplantation. A thorough understanding of the conditions required to maintain CD4+CD25+ Tregs in vitro is required. To our knowledge, this is the first study to demonstrate the role of IL-2 in the maintenance of the regulatory phenotype of human CD4+CD25+ T cells, a phenomenon that appeared to be dependent upon the integrity of the PI3K/AKT-signaling pathway. Key advances offered by the current work that merit special emphasis include our examination of human, rather than murine, CD4+CD25+ T cells and the demonstration of a phenomenon that occurred in the absence of attendant TCR signals. As recently reviewed, there are key differences between the murine and human immune systems (67). Advancement of immunological knowledge of relevance to medicine will only ultimately come from the study of human T cells: murine studies, while interesting and vital, are insufficient alone to recommend novel immunotherapeutic strategies, since therapies that seem to work in the mouse may either fail to be effective or even prove detrimental in higher mammals [e.g. anti-CD28 and anti-CD154 in transplantation tolerance (68)]. IL-2 maintained maximal suppressive potency and prevented apoptosis, while up-regulating the expression of key cellular markers. These effects occurred uniquely in the regulatory CD4+CD25HI population and were not observed in the non-regulatory CD4+CD25INT cells, likely to be due to their well-recognized differential expression of CD122. Using our in vitro system, we also demonstrated a redundancy of IL-2 with other {gamma}c cytokines—IL-4, IL-7 and IL-15—which similarly maintained optimal suppressive potency. Whether these cytokines may show additive or even synergistic effects in vitro was not investigated in the current study, but could be explored in future, pre-clinical work designed to optimize culture conditions for the maintenance and expansion of human Tregs.

Pre-incubation of human CD4+CD25+ T cells with IL-2 initially appeared to enhance their suppressive properties in vitro. IL-2 activates the suppressor effector function of murine Tregs in vitro: the initial production of IL-2 by CD25– responder cells is essential to allow the ultimate suppression of CD25– IL-2 mRNA (20). IL-2 production by responder CD25– cells is thought to promote CD25 expression by CD4+CD25+ Tregs, which facilitates Treg uptake and sequestration of IL-2 (21, 22, 69) and the expression of forkhead box protein 3, murine nomenclature (Foxp3) (70). We hypothesized that incubation with IL-2 may ‘pre-activate’ these regulatory pathways, thus augmenting suppression. However, comparing pre-incubated with freshly isolated cells provided no evidence for the enhancement of suppression with IL-2, but supported a model in which IL-2 protects the cells from deteriorating suppressor function in the presence of medium alone. This led us to determine whether the role of IL-2 was simply the inhibition of apoptosis within the bulk population or the provision of unique signals that maintain suppressive potency of individual cells. We and others (18) have found that IL-2 reduces the rapid decline in viability of freshly isolated human CD4+CD25+ T cells in vitro (20–30% apoptosis in 20 h; data not shown). However, this survival effect was unlikely to be of importance to the current phenomenon, since IL-6 had no effect on suppression despite reducing apoptosis, while blockade of PI3K with LY had no effect on viability despite inhibiting suppression. Previous work has shown that pre-incubation with IL-2 enhances the suppressive function of murine CD4+CD25+ Tregs if they are also stimulated through the TCR (19). Importantly, however, the effect we observed did not depend on TCR signaling—as may occur by T to T cell presentation of self-peptides—since it could not be inhibited with CSA. Taken together, our data favored a model in which cytokine signaling maintains the regulatory phenotype in a primed state beyond anti-apoptotic or suppression-activating effects.

All our initial studies used immunomagnetically selected CD4+CD25+ T cells, which are known to contain a significant population of non-regulatory CD4+CD25INT cells that confound the interpretation of functional assays. Furthermore, the CD4+CD25+ Treg population itself is known to be heterogeneous, comprising both naive and memory sub-populations (55, 56). However, while FACSTM allows the selection of Tregs to high stringency, it suffers the practical limitation of yielding far fewer cells to work with—<50% of the number selected using beads—precluding the parallel examination of multiple culture conditions. In addition, prolonged sorting times for large numbers of cells from buffy coats also reduced the viability of the recovered cells prior to pre-incubation with the cytokines. In contrast, beads yielded a functional population that was more rapidly selected and that displayed the expected properties of Tregs, albeit less potently. Moreover, studies examining the expression of CD45RA and CD45RO by the CD4+CD25+ T cells demonstrated that the observed phenomenon could not be explained simply by a survival advantage of memory Tregs, which are thought to mediate more potent suppression in vitro (55). The recent development of FACSTM protocols involving the marker CD127 are a welcome addition to the field, facilitating human Treg studies by allowing a greater number of viable cells to be sorted in one sitting (71, 72). While the utility of this marker had not been recognized at the time our initial experiments were performed, we were nevertheless able to show in more recent experiments that IL-2 appeared not to modulate the pattern of its expression with overnight culture; in contrast, IL-7 abolished the expression of CD127, suggesting that it activated and caused internalization of the high-affinity IL-7R in these cells (62). However, the possibility of a CD127-independent effect of IL-7 on the CD4+CD25HICD127– Tregs could not be excluded.

The mechanism underlying the ‘protective’ influence of IL-2 and the other {gamma}c cytokines on human CD4+CD25+ T cell function currently remains unclear. Changes in expression of CD25 and CTLA-4 did not correlate with functional potency in our system, although both have been implicated in the function of Tregs in vitro. Thus, high expression of CD25—up-regulated by the early release of IL-2 from responder cells—is thought to allow CD4+CD25+ Tregs to compete with effector T cells for this growth factor (21, 22). Though controversial (19), CTLA-4 may also play a role in suppression in vitro (73). However, up-regulation of these markers was thought to be of questionable importance in our system for three reasons. First, IL-2 increased the expression of CD25 and CTLA-4 to levels beyond those observed in freshly isolated CD4+CD25+ T cells, yet did not truly enhance suppression. Second, the inhibition of PI3K with LY during pre-incubation with IL-2 prevented the maintenance of maximal suppression, yet had no effect on up-regulation of either molecule. Finally, IL-4 failed to up-regulate either marker on human CD4+CD25+ T cells, yet was effective in maintaining optimal suppression. We suggest two potential scenarios: either other unrecognized mechanisms underlie optimal maintenance of the suppressive phenotype in vitro or—in the case of IL-2, IL-7 and IL-15—the up-regulation of these molecules is necessary but not sufficient for the maintenance of Treg function.

Interestingly, the expression patterns of FOXP3 mirrored those of CD25, CTLA-4 and GITR: up-regulation was observed with all cytokines except IL-4. This pattern of parallel expression was consistent with the known up-regulation of Treg phenotypic markers by murine Foxp3 (74) and human FOXP3 (75), taken as evidence that FOXP3 may act as a transcriptional activator—rather than a repressor—in certain contexts. The apparent decoupling of the suppressive potency of CD4+CD25+ T cells from increments in FOXP3 expression observed in the current study is reminiscent of work performed by Allan et al. (75), which suggests that additional factors beyond FOXP3 are required to confer full regulatory function on human CD4+ T cells. Such additional factors, which currently remain uncharacterized, may be the specific target of the {gamma}c cytokines and presumably involve PI3K signaling in this context. Though controversial, these authors suggest that FOXP3 may be viewed as an activation marker in human CD4+ T cells, necessary but not sufficient for the induction of regulatory activity (75). Gavin et al. (76) have advanced this hypothesis by suggesting that sustained, high-level FOXP3 expression is required for the induction of human Tregs and that transient FOXP3 expression with short-term stimulation of CD4+CD25– T cells fails to activate a regulatory developmental program. Moreover, Wang et al. (77) have demonstrated that transient FOXP3 expression in stimulated conventional human T cells distinguishes a non-suppressive population. With these concepts in mind, we were interested to observe that the CD4+CD25– T cells incubated in IL-2, IL-7 and IL-15 failed to up-regulate FOXP3 (Fig. 9B) or other Treg markers (data not shown), suggesting that the increased expression by CD4+CD25+ T cells represented a genuine, differential effect of the {gamma}c cytokines, rather than background TCR activation. Interestingly, a recent study by Zorn et al. (78) concurs with our own data, demonstrating that IL-2 selectively up-regulates the expression of FOXP3 by CD25+—but not CD25–—CD4+ T cells through a STAT-dependent mechanism. Furthermore, the clinical relevance of this phenomenon was highlighted by the expansion of CD4+CD25+ T cells in vivo by low-dose IL-2 treatment in patients with metastatic neoplasia and chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. The upstream pathways of FOXP3 remain unclear (79), but—at least for IL-2—appear not to depend on PI3K-signaling mechanisms (Fig. 9A). Future work will need to dissect the PI3K-independent intracellular pathways mediating the transcription of FOXP3 by some, but not all, of the {gamma}c cytokines, and how they might differ between individual family members.

Recent work by Bensinger et al. (42) has demonstrated constitutive inhibition of early signaling downstream of IL-2-activated PI3K by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in murine CD4+CD25+ Tregs, confirmed in a subsequent study by Walsh et al. (80). This observation seems at odds with the results of the current study, which suggest that IL-2 and other cytokines preserve optimal Treg function by a PI3K-dependent mechanism. There are three possible explanations for this apparent disparity. First, the PI3K-dependent pathway responsible for the maintenance of optimal CD4+CD25+ Treg suppression may be less efficiently inhibited by PTEN than the PDK/AKT pathway examined by Bensinger and others (81). Second, the possibility remains of a late PI3K-dependent mechanism, which escapes the immediate PTEN inhibitory activity described by these authors (42). Indeed, there are examples of such delayed PI3K activation in T cells, including the STAT-5–dependent activation of PI3K/AKT required for IL-2-induced cyclin D2 expression and cell-cycle progression (82, 83). Furthermore, our Western blots confirmed the presence of pAKT in T cells that were incubated with IL-2 overnight, providing direct evidence for late activation of PI3K in these cells. Finally, the role of PI3K in murine and human CD4+CD25+ T cells may show fundamental differences unrelated to pathway specificity or kinetics of activation. Indeed, a recent study provides some evidence for this third hypothesis, showing that downstream signals mediated by the altered activation of AKT were actually required for the suppressive function of human CD4+CD25+ Tregs (84); changes in the activity of PI3K or the basal expression of src homology 2-containing inositol phosphatase or PTEN, which could have accounted for the altered activation status of AKT, were not observed. Taken together, we suggest that these data and our own support a mechanism of delayed, or sustained, activation of PI3K in human CD4+CD25+ T cells by {gamma}c cytokines, possibly involving STAT5—though other STAT proteins are presumably also implicated, since STAT6 is known to mediate IL-4 signaling in T cells (24). Further work is required to explore both the role of STAT5 in the IL-2–induced preservation of human Treg viability versus potency, and the kinetics of its activation by the {gamma}c cytokines in these cells.

In summary, we have demonstrated for the first time that the {gamma}c cytokines IL-2, IL-4, IL-7 and IL-15 maintain optimal suppressive function by human CD4+CD25HI Tregs. While the mechanism of this phenomenon remains to be fully defined, it is at least in part dependent upon the activation of PI3K. We propose that this redundancy of the stimuli governing suppressive function provides a mechanism to ensure maximal flexibility of regulation in various different microenvironments in vivo, where one or more {gamma}c cytokines may predominate. Our data suggest that the use of {gamma}c cytokines in protocols to culture CD4+CD25HI Tregs in vitro may optimize expansion while maintaining maximal suppressive potency. Future work will dissect the mechanisms underlying this ‘priming’ phenomenon, thus offering the potential to augment human CD4+CD25HI Treg function in vivo by the manipulation of key effector pathways.


   Acknowledgements
 
J.Y. and F.R. share first authorship. G.L. and O.A.G. share last authorship. We would like to thank Professor Brian Foxwell for helpful discussion and advice on IL-2 signaling. We would also like to thank Eric O'Connor from the FACSTM Laboratory at the Clinical Sciences Centre, Medical Research Council, Hammersmith Campus, for his expertise in sorting. This work was partly funded by a grant from the UK National Kidney Research Fund. At the time this work was performed, J.Y. was the recipient of a Medical Research Council Clinical Research Fellowship and O.A.G. an Advanced Fellowship from the Wellcome Trust.


   Abbreviations
 
AKT, serine/threonine kinase
APC, allophycocyanin
7-AAD, 7-aminoactinomycin D
BD, Becton Dickinson
{gamma}c, common gamma chain
c.p.m., counts per minute
CSA, ciclosporin A
CTLA-4, cytotoxic T lymphocyte antigen-4
Foxp3, forkhead box protein 3, murine nomenclature
FOXP3, forkhead box protein 3, human nomenclature
GITR, glucocorticoid-induced tumor necrosis factor receptor superfamily member number 18
HS, human serum
LY, LY294002
pAKT, phospho-serine/threonine kinase
PBS, phosphate-buffered saline
PFA, paraformaldehyde
PI3K, phosphatidylinositol 3-kinase
PTEN, phosphatase and tensin homolog deleted on chromosome 10
RPMI 1640–PSG, Rosswell Park Memorial Institute 1640 medium supplemented with penicillin, streptomycin and L-glutamine
STAT, signal transducers and activators of transcription
Treg, regulatory T cell

   Notes
 
Transmitting editor: A. Cooke

Received 13 July 2006, accepted 23 March 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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