PeproTech Inc.
Originally published online as doi:10.1189/jlb.0507321 on November 21, 2007

Published online before print November 21, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures and Table
Right arrow All Versions of this Article:
jlb.0507321v1
83/3/708    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sumpter, T. L.
Right arrow Articles by Wilkes, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sumpter, T. L.
Right arrow Articles by Wilkes, D. S.
(Journal of Leukocyte Biology. 2008;83:708-717.)
© 2008 by Society for Leukocyte Biology

Regulation of the NFAT pathway discriminates CD4+CD25+ regulatory T cells from CD4+CD25– helper T cells

Tina L. Sumpter*, Kyle K. Payne{dagger} and David S. Wilkes*,{dagger},{ddagger},1

* Departments of Microbiology and Immunology and
{dagger} Medicine,
{ddagger} Center for Immunobiology, Indiana University School of Medicine, Indianapolis, Indiana, USA

1Correspondence: Center for Immunobiology, Indiana University School of Medicine, 635 Barnhill Dr., Rm. 224, Indianapolis, IN 46202, USA. E-mail: dwilkes{at}iupui.edu


arrow
ABSTRACT
 
CD4+CD25+ regulatory T cells (Tregs) are potent modulators of immune responses. The transcriptional program distinguishing Tregs from the CD4+CD25– Th cells is unclear. NFAT, a key transcription factor, is reported to interact with forkhead box p3, allowing inhibitory and activating signals in T cells. In the current study, we hypothesize that distinctive NFAT regulation in Tregs as compared with Th cells, may contribute to specific functions of these cells. Tregs express basal levels of cytoplasmic NFATc1 and NFATc2. In contrast to Th cells, anti-CD3-mediated T cell activation did not induce nuclear translocation of NFATc1 or NFATc2 in Tregs. This effect was associated with altered regulation for NFAT in Tregs that included reduced calcium flux, diminished calcineurin activation, and increased activity of glycogen synthase kinase-3β, a negative regulatory kinase for NFAT in Tregs relative to Th cells. These data suggested that NFAT inhibition in Th cells may induce regulatory function. Indeed, pharmacologically mediated NFAT inhibition induced Th cells to function as Tregs, an effect that was mediated by induction of membrane-bound TGF-β on Th cells. Collectively, these data suggest that maintaining NFAT at basal levels is a part of the transcriptional program required for Tregs.

Key Words: Gsk3β • calcineurin • cyclosporine A • TGF-β


arrow
INTRODUCTION
 
Regulatory T cells (Tregs; CD4+CD25+) modulate diverse immune responses in vitro and in vivo. Treg-induced suppression is accomplished through myriad mechanisms, including, at least in part, membrane-bound TGF-β [1 2 3 ]. Forkhead box p3 (foxp3), a transcription factor, is expressed in CD4+CD25+ Tregs and is used to identify T cells with regulatory function [4 , 5 ]. The transcriptional program directed by foxp3 in regulatory cells may involve direct interactions with NFATc2 [6 ]. Intriguingly, NFATc2 induces foxp3 expression de novo in CD4+CD25– Th cells, which are suppressive in vitro [7 ], suggesting that NFAT and foxp3 may act in a negative-feedback loop in naturally occurring Tregs. In spite of this link between NFAT and foxp3, mice deficient in NFATc2 and NFATc3 have functional glucocorticoid-induced TNFR (GITR)+CD4+CD25+ Tregs [8 ]. Evidence from other studies suggests that expression or activity of NFAT proteins may be diminished in different subsets of Tregs [9 10 11 ].

The disparate results from the previous studies may reflect the activation status of NFAT in Tregs compared with conventional Th cells reflective of differences in NFAT regulation. The regulation of the NFAT family of transcription factors (NFATc1-c4 or NFAT5) has not been defined clearly in natural Tregs and may be different in Tregs when compared with that of naïve CD4+CD25– Th cells. Cellular localization and subsequent transcriptional activity of NFAT are dependent on its phosphorylation state [12]. During steady-state conditions, NFAT is maintained in an inactive, heavily phosphorylated state. Following ligation of the TCR and increases in free intracellular calcium levels, NFAT is dephosphorylated by the phosphatase, calcineurin. Dephosphorylated NFAT interacts with nuclear importins, resulting in nuclear translocation and subsequent transcriptional activity. The cellular localization and activation of NFAT are altered by a diverse group of kinases. Glycogen synthase kinase 3β (Gsk3β) [13 ] rephosphorylates NFATc1 [13 ], resulting in cytoplasmic retention. Other kinases, such as JNK, phosphorylate NFATc2, causing nuclear retention [14 ]. Interactions with nuclear importins and exportins have also been described, which alter NFAT localization [15 ].

Previous studies show that the signaling events most proximal to TCR ligation are down-regulated in Tregs, relative to Th cells [10 , 16 ]. In this study, we further tested the hypothesis that the regulation of NFAT is different in Tregs compared with that found in Th cells. Our data show that Tregs and Th cells differ in NFAT compartmentalization, paralleling differences in intracellular calcium flux, calcineurin activity, and expression and activity of the NFAT regulatory kinase, Gsk3β. Our data also show that NFAT inhibition induces up-regulation of membrane-bound TGF-β, which converts CD4+CD25– Th cells into cells with regulatory function.


arrow
MATERIALS AND METHODS
 
Animals
Female BALB/c mice, 6–10 weeks old, were purchased from Harlan Sprague Dawley (Indianapolis, IN, USA) or bred in the Laboratory Animal Resource Center at Indiana University School of Medicine (Indianapolis, IN, USA). Animal procedures were performed in accordance with institutional guidelines.

Preparation of cells
Cell suspensions were prepared from the spleens of five to 20 mice. RBCs were lysed with an NH4Cl lysis buffer followed by removal of dead cells and neutrophils with Lympholyte-M (Cedar Lane Laboratories, Hornsby, Ontario, Canada). Tregs were isolated through a series of magnetic selection steps using the mouse CD4+CD25+ isolation kit (Miltenyi Biotech, Auburn, CA, USA), per the manufacturer’s instructions. Where indicated, the CD4– fraction was {gamma}-irradiated (2000 rads) and used as APCs. The CD4-enriched CD25– fraction was purified using CD4 magnetic beads. The purity of CD4+CD25+ Tregs and CD4+CD25– Th cells determined by flow cytometry ranged from 90% to 97%.

Immunoblotting for NFATc1 and NFATc2
Purified Tregs or Th cells (a minimum of 2x106) in RPMI were incubated on ice for 30 min prior to stimulation with 1.0 µg per 5 x 105 cells of anti-CD3 (clone 2C11, BD Biosciences, San Diego, CA, USA) at 37ºC for the indicated time. Following stimulation, cells were centrifuged and washed in ice-cold 1x PBS. After washing, cell pellets were stored at –80ºC until cell lysis. Cell lysates were fractionated with NE-PER nuclear cytoplasmic reagent kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Protein concentrations were determined using the BioRad protein assay (BioRad, Hercules, CA, USA). Nuclear (2.5 or 5.0 µg) or cytoplasmic (10 µg) lysates were denatured with 2x Laemmli buffer (BioRad) at 95ºC for 5 min. Lysates were resolved on 8.0% acrylamide gels (Pierce Biotechnology, Inc.), transferred to nitrocellulose membranes, and then blocked with a 5.0% milk solution in TBS with 0.1% Tween. Membranes were then probed with anti-mouse NFAT1/c2 and NFAT2/c1 (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mouse GADPH (BioDesign, Sako, ME, USA), or anti-mouse TATA-binding protein (TBP; Abcam, Cambridge, MA, USA). Rabbit anti-mouse and goat anti-rabbit secondary antibodies were used as directed with the SuperSignal West Dura extended duration substrate (Pierce Biotechnology, Inc.). After exposing to film, blots were stripped with Restore Western stripping buffer (Pierce Biotechnology, Inc.) at 37ºC for 30 min, washed, and reprobed.

Measurement of intracellular calcium flux
Calcium flux was measured in purified Tregs and Th cells using the Fluo-4 NW calcium assay kit (Invitrogen, Carlsbad, CA, USA). In brief, 2.5 x 106 Tregs or Th cells were resuspended in 1.0 ml assay buffer (1x HBSS, 20 mM HEPES) and then aliquoted in 50 µl (1.25x105 cells) in a 96-well plate and incubated for 1 h at 37ºC. The cells were then loaded with the 50 µl Fluo-4 dye diluted in assay buffer with 2.5 mM probenecid acid for 30 min at 37ºC, followed by a 30-min incubation at room temperature. Cells were then added directly to polypropolynene tubes containing stimulant as indicated and read on a Becton Dickenson FC 5000 flow cytometer for 300 s (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using FCSExpress v3 (De Novo Software, Thornhill, Ontario, Canada).

Calcineurin activity assays
To equilibrate cells, purified Th cells or Tregs (5x105) in RPMI were incubated on ice for 30 min prior to the addition of anti-mouse CD3 (1.0 µg/5x105 cells). Cells were then incubated for the indicated times at 37ºC, washed two times with ice-cold 1x PBS, pelleted, and stored at –80ºC. Calcineurin activity was measured with the colormetric calcineurin cellular activity kit (Calbiochem, San Diego, CA, USA). Cells were lysed by high-speed centrifugation in the presence of phosphatase inhibitors in 75 µl of the provided lysis buffer. Free phosphates were removed from cellular lysates using a spin column. Lysate (5.0 µl) was then incubated in duplicate with a calcineurin-specific phosphopeptide in the presence of okadaic acid (OA), which inhibits other phosphatases but not calcineurin or OA with EGTA for 30 min at 30ºC. The reaction was stopped by adding Green reagent to each well. Absorbance was read at 620 nm with the SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Total calcineurin activity was calculated for each sample as a function of OA-(OA+EGTA) and then quantified by extrapolation from a standard curve.

SuperArray profiling of NFAT signaling molecules and RT-PCR
RNA was extracted from a minimum of 1 x 106-purified Tregs and Th cells with TriReagent plus a polyacryl carrier (Molecular Research Center, Cincinnati, OH, USA). For SuperArray, RNA (2.0 µg) was reversed-transcribed and biotinylated with the AmpoLabeling LRP kit (SuperArray, Frederick, MD, USA) and biotin-UTP (Roche, Indianapolis, IN, USA). Biotinylated cDNA was denatured and used to probe GEArray Q series mouse Ca2+/NFAT signaling pathway gene array membranes (SuperArray) that had been prehybridized with sheared salmon sperm DNA (SuperArray). Following hybridization, membranes were washed and then developed using the chemiluminescent detection kit (also from SuperArray). Autoradiographs were scanned and up-loaded into the GEAnalysis suite (SuperArray) for analysis. Data were normalized to expression of ribosomal L13 by subtracting background binding as a function of intraquartile density. For RT-PCR analysis, RNA (1.0 µg) was reverse-transcribed with the iScript RT kit (BioRad). cDNA (4.0 µl) was amplified for 35 cycles with TaqMasterMix (Qiagen, Valencia, CA, USA) using primers for foxp3 (forward 5'-CTTACACTGAGAGGGGTGC-3', reverse 5'-CCAGATGTTGTGGGTGAGTG-3'), Gsk3β (forward 5'-GTGGCAGAAGAAAGATGAGG-3', reverse 5'-GCAGGCGGTGAAGCA-3'), or β-actin (forward 5'-ATGGATGACGATATCGCT-3', reverse 5'-ATGAGGTAGTCTGTCAGGT-3') with a 55ºC annealing temperature.

Flow cytometry
Th cells or Tregs were cocultured with anti-CD3 (0.5 µg/ml) and {gamma}-irradiated APCs (two T cell:one APC) for 68–72 h. Cells were collected and washed two times in staining buffer (1x HBSS with 0.5% BSA). Nonspecific binding was blocked with staining buffer supplemented with 10% mouse serum and anti-CD16/anti-CD32 (0.5 µg/tube, eBioscience, San Diego, CA, USA). Cells were then stained with anti-mouse CD4 PerCP, anti-mouse CD25 PE (both from eBioscience), and anti-human TGF-β (clone TB21, Serotec, Raleigh, NC, USA). Cells were incubated with goat anti-mouse FITC secondary antibody (Jackson ImmunoResearch Laboratories, Inc., New Grove, PA). After staining, cells were fixed in a 1.0% paraformaldehyde solution. Intracellular staining for foxp3 was done using the rat/mouse foxp3 staining set (eBioscience). Cells were stained with annexin V FITC (BD Bioscience, San Jose, CA, USA), CD4 PerCP, and CD25 PE and read immediately on the flow cytometer. The data from 20,000 cells in the live gate were analyzed with a FACScan flow cytometer (Becton Dickinson). WinMDI Software v 2.8 (Scripps Research Institute, San Diego, CA, USA) was used for further analysis.

ELISAs for JNK, Gsk3β, and active TGF-β
To measure active JNK1/2, purified Tregs and Th cells (1x105) were stimulated as indicated with anti-CD3 (1.0 µg/well) in duplicate in a 96-well flat-bottom plate for the indicated times. Stimulation ceased when 100 µl of 8.0% paraformaldehyde was added to each well. Plates were stored at 4ºC and then assayed for phospho-JNK T183/Y185 or total JNK with the CASE JNK ELISA (SuperArray). Total Gsk3{alpha}/β and phospho-S9 Gsk3β were measured from cell lysates of 2.5 x 105-purified Tregs and Th cells rested for 30 min and then stimulated as indicated with anti-CD3 (1.0 µg/5x105 cells). Cells were lysed using 1x cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Sandwich ELISAs were performed in duplicate in accord with the manufacturer’s instructions (R & D Systems, Minneapolis, MN, USA). Active TGF-β was measured in duplicate from cell-free supernatants using the Emax immunoassay system (Promega, Madison, WI) per the manufacturer’s protocol.

Proliferation assays and treatment of cells
Purified Th cells or Tregs (1x106 cells/ml) were incubated with the indicated concentration of cyclosporine A (CsA; LC Laboratories, Woburn, MA, USA) diluted in RPMI 1640 (Gibco, Carlsbad, CA, USA) or the appropriate vehicle [0.2% ethyl alcohol (EtOH)] for 4 h and then washed four times with RPMI 1640. Drug or vehicle-treated Th cells or Tregs (5x104) were cocultured as indicated with APCs (5x104), anti-mouse CD3 antibody (0.5 µg/ml), and untreated Th cells (5x104) in complete RPMI 1640 for 70–72 h in a final volume of 200 µl. Cocultures were pulsed with 3[H]-thymidine (0.5 µCi/well) for the final 6 h and then harvested with a Brandel cell harvestor (Brandel, Gaithersburg, MD, USA) or a Skatron Basic 96 harvestor (Molecular Devices, Sunnyvale, CA, USA). 3[H]-Thymidine incorporation was measured using a liquid scintillation counter (Beckman Coulter, Fullerton, CA, USA) or Wallac 1450 Microbeta Plus (Perkin Elmer, Boston MA, USA). Where indicated, neutralizing TGF-β antibody (clone A411, a generous gift from Dr. P. Heeger) or mouse IgG1 isotype control (BD Biosciences) was added to cocultures. Cell culture supernatants were analyzed by HPLC for CsA by Indiana University Hospital Department of Pathology and Laboratory Medicine Reference Laboratory.

Statistical analysis
Differences between groups were determined by t-tests, and data were reported by mean ± SEM unless stated otherwise. P values <0.05 were considered significant.


arrow
RESULTS
 
Induction of nuclear NFAT is reduced in Tregs
Regulation of NFAT activation can be assessed as a function of NFAT nuclear localization. Immunofluorescent microscopy was used to determine if NFAT activation differed in Tregs compared with Th cells. Following 30 min of stimulation with anti-CD3 and IL-2, nuclear localization of NFATc2 was strongly induced in Th cells in accord with the established paradigm for NFAT activation [12]. Nuclear and cytoplasmic NFATc2 could be seen in Tregs in the absence of stimulation. However, levels of nuclear NFAT in Tregs remained constant following stimulation with IL-2 and anti-CD3 at 30 and 60 min following stimulation. Tregs failed to demonstrate strong nuclear induction of NFAT as seen in Th cells (Supplemental Fig. 1).

To further test the hypothesis that NFAT activation is different in Th cells and Tregs, immunoblotting was performed in fractionated nuclear and cytoplasmic lysates from Tregs and Th cells stimulated with anti-CD3 for increasing time periods. In the absence of stimulation, only low levels of nuclear NFATc1 are found in Th cells, and more NFATc1 is detected in Tregs. In Th cells (CD25–) and Tregs (CD25+), a constitutive level of NFATc2 was seen in nuclear lysates. Following stimulation with anti-CD3, nuclear NFATc1 and NFATc2 were strongly induced in Th cells. Nuclear NFATc1 and NFATc2 remained fairly constant in Tregs (Fig. 1A ).


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		Figure 1. Induction of NFATc1 and NFATc2 is reduced in Tregs. Expression of (A) nuclear or (B) cytoplasmic NFATc1 and -c2 by Western blot in Tregs (CD25+) and Th cells (CD25–) following incubation with anti-CD3 (1.0 µg/5x105 cells) for the indicated times. TBP or GAPDH was used as loading controls for nuclear or cytoplasmic protein, respectively. To visualize cytoplasmic proteins, blots were exposed for 90 min, reflected by the GAPDH control. Data are representative of three experiments with similar results. (C) Purified CD4+CD25– and CD4+CD25+ T cells were stimulated with anti-CD3 for the indicated times. Calcineurin activity was measured from cell lysates. Data represent mean ± SEM from three independent experiments; *, P < 0.05. (D) Calcium flux was measured for 300 s in resting CD4+CD25– or CD4+CD25+ T cells immediately following stimulation with anti-CD3 (1.0 µg/ml). In the density plots shown, blue is representative of the greatest number of events positive for a particular level of Fluo-4; red is indicative of the fewest number of events positive for that level of Fluo-4. Data are representative of three experiments with similar results.

In the absence of stimulation, NFATc1 is detectable in the cytoplasmic fraction of Th cell and Treg lysates (Fig. 1B , Time 0). Following 30 min of stimulation with anti-CD3, two bands are detected for NFATc1 in Th cells reflecting dephosphorylation or activation. After 60 min of stimulation in Th cells, a lower level of NFATc1 is seen in the cytoplasmic fraction, corresponding to the influx of NFATc1 into the nucleus seen in Figure 1A . In Tregs, only a single band of high molecular weight is detected for cytoplasmic NFATc1 at all time-points evaluated, suggesting that NFATc1 is being held in its heavily phosphorylated, inactive state. Likewise, in the cytoplasmic protein from Th cells, NFATc2 has two bands at all time-points considered, suggesting that it is found in the cytoplasm in its active (low molecular weight) and inactive (higher molecular weight) states. Cytoplasmic NFATc2 in Tregs also has two bands in the absence of stimulation (Fig. 1B , Time 0). Following stimulation with anti-CD3, however, the only band detected for NFATc2 is of the high molecular weight or is inactive. Collectively, these data illustrate differential regulation of NFAT activation in Tregs compared with Th cells.

Calcineurin activity and calcium flux are blunted in Tregs following ligation of the TCR
The proximal event upstream of NFAT activation is activation of the phosphatase, calcineurin. Evaluation of calcineurin activity in Tregs and Th cells following stimulation with anti-CD3 showed increased calcineurin activity over time in both cell types (Fig. 1B) . However, a more substantial increase in calcineurin activity was seen in Th cells following 15 min of stimulation with anti-CD3 compared with Tregs (P<0.05). Further evaluation of upstream signaling events confirmed that in Tregs, intracellular calcium flux was deficient following ligation of the TCR (Fig. 1C) , accounting for the diminished calcineurin activity. Increasing the concentration of anti-CD3 or further stimulation with anti-CD28 or mitogenic stimulation with PMA and ionomycin failed to increase intracellular calcium levels in Tregs (data not shown). These data show that calcineurin activity and calcium flux, events upstream of NFAT activation, are differentially regulated in Tregs compared with Th cells.

RNA profiling for NFAT regulatory molecules is altered in Tregs
Many intracellular mediators interact with NFAT. Therefore, an RNA-based array was used to determine if the reduced nuclear NFAT induction in Tregs involved molecules in addition to calcineurin. In these experiments, RNA from purified Tregs and Th cells was hybridized to a membrane containing cDNA for housekeeping genes, for NFAT, for genes regulated by NFAT, and for NFAT regulatory genes. As described in Table 1 , many genes were up-regulated in the Treg population, including those for known Treg markers, such as CD25 and CTLA-4. Expression of RNA for the NFAT regulatory kinases, JNK and Gsk3β, as well as casein kinase (CK), which primes substrates for Gsk3β activity, was moderately up-regulated in Tregs compared with Th cells when evaluated by array (Fig. 2A ).


View this table:
[in this window]
[in a new window]

  		Table 1. Mean Expression of NFAT-Associated Genes by SuperArray in Tregs and Th Cells


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		Figure 2. SuperArray profiling for NFAT regulatory molecules in Tregs and Th cells. The SuperArray membrane was hybridized with RNA from freshly isolated Tregs or Th cells. Expression of RNA for NFAT regulatory kinases (A) or for nuclear importins and exportins (B), depicted graphically, was normalized by subtracting the lowest interquartile of density and then comparing with the expression of the housekeeping gene, ribosomal L13A. Bars represent the mean expression of genes ± SEM from Treg cells relative to Th cells from three sets of arrays.

Interactions between nuclear localization signals on proteins in the nucleus and nuclear exportins are an important means for regulation of transcription factors. Nuclear import of NFAT involves interactions with importin β1 [17 ], and nuclear export involves exportin 1 [15 ]. There was no difference in mRNA expression for importin β1 or exportin 1 between Tregs and Th cells (Fig. 2B) .

Gsk3β, not JNK, is up-regulated in Tregs
From the SuperArray data, the NFAT regulatory kinases JNK and Gsk3β were elevated in Tregs, suggesting a role for these kinases in NFAT regulation. To evaluate this question, we used RT-PCR to assess kinase expression and ELISA to quantitate total and active JNK and Gsk3β in Tregs and Th cells after anti-CD3-induced activation. JNK transcripts, reported as increased by SuperArray data in Figure 2 , were not increased in Tregs when examined by RT-PCR (data not shown). However, ELISA revealed that basal levels of active JNK were slightly higher in Tregs compared with Th cells (P=0.08) but did not change after anti-CD3 stimulation (Fig. 3 ). In contrast, anti-CD3 induced a trend toward greater levels of JNK in Th cells compared with Tregs at 15 min but returned to baseline by 30 min poststimulation (Fig. 3) . These data derived from natural Tregs are consistent with JNK activity reported for antigen-specific Tregs [18 ] and potentially illustrate a functional link between natural and antigen-specific Treg populations.


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		Figure 3. Ligation of the TCR does not alter JNK activity in CD4+CD25+ T cells. Purified Tregs or Th cells were incubated with anti-CD3 (1.0 µg/5x105 cells) for the indicated times. Cells were lysed and evaluated for phospho-JNK1/2 and total JNK1/2/3 by ELISA. Data shown depict the average ± SEM of the ratio of phosphor-JNK1/2 relative to total JNK1/2/3, detected from three independent experiments.

RT-PCR confirmed increased Gsk3β mRNA expression in Tregs compared with Th (Fig. 4B , 0 h). To further delinate the role of Gsk3β in Tregs compared with Th cells, Gsk3β protein was measured by ELISA in cells stimulated with anti-CD3 for 0, 15, and 30 min. Gsk3{alpha}/β protein was found initially at lower levels in Tregs compared with Th cells. Stimulation with anti-CD3 increased total Gsk3{alpha}/β protein in Tregs but decreased total Gsk3{alpha}/β protein in Th cells (data not shown). In parallel, ELISAs were performed evaluating phospho-S9 Gsk3β, indicative of autoinhibition of the Gsk3β substrate-binding pocket [19 ]. The amount of inactive Gsk3β relative to total Gsk3{alpha}/β in Tregs is lower than the amount seen in Th cells (Fig. 4A) following ligation of the TCR. These data suggest that Gsk3β, a NFAT inhibitory kinase, may inhibit nuclear localization of NFAT in Tregs following stimulation with anti-CD3.


Figure 4
View larger version (30K):
[in this window]
[in a new window]

  		Figure 4. Gsk3β is up-regulated in Tregs. (A) Purified Tregs or Th cells were incubated with anti-CD3 (1.0 µg/5x105 cells) for the indicated times. Cells were lysed, and total and phospho-S9 Gsk3β was evaluated by ELISA. Data show the ratio of inactive phospho-Gsk3β relative to total Gsk3β detected. Points represent mean ± SEM from three independent experiments; *, P = 0.053. (B) Purified Tregs or Th cells were cocultured with {gamma}-irradiated APCs (1:1) and anti-CD3 (0.5 µg/ml) for the indicated time-points. RT-PCR was performed on CD4+ cells magnetically isolated from cocultures at the indicated time-points. Data are representative of three independent experiments.

Gsk3β mRNA was further evaluated following coculture with APCs and anti-CD3. In CD4+CD25+ Tregs, expression of Gsk3β mRNA was elevated in freshly isolated Tregs and after 24 h of stimulation. Following longer durations in coculture (48 and 72 h), expression of Gsk3β mRNA was diminished in Tregs. In freshly isolated Th cells, expression of Gsk3β was lower compared with that seen in Tregs. In Th cells, expression of Gsk3β mRNA increased following coculture (24 h) with APCs and anti-CD3 and remained elevated (Fig. 4B) .

Inhibition of calcineurin generates regulatory cells from naïve CD4+CD25– T cells
From these data, we hypothesized that early NFAT inhibition may prime Tregs to be suppressive. Indeed, suppressor function in T cells was noted in secondary MLRs in classical studies evaluating the calcineurin inhibitor CsA [20 ], although the mechanism through which this occurred was not reported. To assess the functional consequences of lower NFAT induction, purified CD4+CD25– T cells were incubated for 4 h with increasing concentrations of CsA, which inhibits NFAT via blocking calcineurin activity. After washing to remove CsA from culture media, cells were cocultured with {gamma}-irradiated APCs, anti-CD3, and equal numbers of nontreated Th cells. All concentrations of CsA used in these conditions were able to inhibit the proliferation of treated cells when measured by 3[H]-thymidine incorporation (Fig. 5A ). When CsA-treated cells were added into cocultures with untreated cells, the proliferation of cocultures decreased (Fig. 5B) . If diminished proliferation was caused by loss of proliferation in the treated group, the predicted proliferation of these cocultures would be equal to that of the untreated cells alone. However, CsA (1.0 µg/ml)-treated Th cells inhibited proliferation in these cocultures 42 ± 8.8% (mean±SEM of three independent experiments) when compared with the proliferation of untreated Th cells alone (Fig. 5B) , suggesting that active suppression was occurring. To delineate the relationship between natural Tregs and CsA-treated Th cells, Tregs were treated in parallel with CsA-treated Th cells, which suppressed the proliferation of untreated cells 55.0 ± 9.9% (mean±SEM of three independent experiments), and natural Tregs suppressed the proliferation of untreated cells 82.2 ± 5.9% (Fig. 5C) at 1:1 ratios. Treating natural Tregs with CsA had no effect on suppressive function in these in vitro suppression assays.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Figure 5. Calcineurin inhibition generates regulatory cells from nonregulatory CD4+CD25– Th cells. (A) CD4+CD25– T cells were incubated with CsA for 4 h at the indicated concentrations and then washed and cocultured with anti-CD3 and {gamma}-irradiated CD4– splenocytes as APCs. (B) CD4+CD25– cells were treated as in A with the indicated concentrations of CsA and then cocultured with equal numbers of untreated Th cells for 72h. (C) Th cells or Tregs were incubated with CsA (1.0 µg/ml) or vehicle control (0.2% EtOH) and then added to suppression assays as described in A. Points represent the mean ± SD of triplicate values; representative of three independent experiments.

To verify that suppressed proliferation was not a result of residual CsA in culture supernatants, HPLC for CsA was performed on the medium of treated Th cells after the third wash and confirmed to be below the limit of detection (less than 25 ng/ml). To determine if increased cell death in the CsA-treated population caused the decline in proliferation seen in Figure 5 , annexin V staining was done on Th cells treated with CsA or its vehicle. No difference in apoptosis was noted (Fig. 6A ).


Figure 6
View larger version (31K):
[in this window]
[in a new window]

  		Figure 6. CsA does not induce expression of foxp3 nor increase apoptotic cell death in CD4+CD25– cells. CD4+ cells incubated with CsA (1.0 µg/ml) or vehicle control and then cocultured with anti-CD3 and APCs. (A) Cocultures were stained for annexin V. Data shown are gating on live CD4+ cells. foxp3 mRNA (B) and protein (C) were evaluated in CD4+ cells from cocultures with {gamma}-irradiated APCs and anti-CD3 (0.5 µg/ml). (B) CD4+ cells were magnetically isolated following 72 h in culture, and then RT-PCR was performed for fopx3. (+) Control was purified CD4+CD25+ T cells. Data shown are gating on live CD4+ cells. Upper panels depict foxp3 expression in magnetically purified CD4+CD25– cells treated for 4 h with CsA and then cocultured with APCs and anti-CD3 for 72 h. Lower panels depict expression of foxp3 in purified CD4+CD25+ cells treated in parallel to CD4+CD25– cells. Also shown as a (+) control is foxp3, detected in freshly isolated CD4+CD25+ cells stained in parallel. All data are representative of three independent experiments.

CsA-treated regulators function independent of foxp3 and do not express other Treg markers
Suppression seen in cocultures could be accounted for if this CsA treatment expanded contaminating foxp3+ natural Tregs or induced expression of foxp3 in CD4+CD25– T cells. No increase in foxp3 expression was seen in CsA-treated Th cells or Tregs by RT-PCR (Fig. 6A) . Further evaluation of foxp3 expression in CsA-treated Th cells caused a modest decrease in the basal levels of foxp3 found in Th cells (Fig. 6B) . foxp3 expression also decreased modestly in purified Tregs treated with CsA and then cocultured with syngenic APCs and anti-CD3 in parallel. CD25 expression was also evaluated in CsA-treated cells. CD25 is up-regulated in vehicle control-treated cells following coculture with anti-CD3 and APCs. Compared with control cells, CD25 expression is inhibited by 62% ± 17.5 in CsA-treated cells. This was not unexpected, as NFAT regulates CD25 transcription [21 ]. Other Treg markers, GITR [22 ] and CD103 [23 ], were also not up-regulated in CsA-treated Th cells (data not shown).

Inhibition of calcineurin increases expression of surface-bound TGF-β on CD4+CD25– T cells
Suppression mediated by natural Tregs is contact-driven [24 ] and involves membrane-bound TGF-β [1 2 3 ], which may be induced by CsA in T cells, although this is controversial as a result of experimental differences between prior studies [25 26 27 ]. ELISAs were performed on the supernatant of CsA-treated Th cells to determine if secreted TGF-β increased following calcineurin inhibition. No difference was detected by ELISA in secreted, active TGF-β in coculture supernatants (Fig. 7A ), which is consistent with the inability of conditioned media from CsA-treated cells to suppress proliferation in secondary cocultures (data not shown). To further examine the relationship between CsA and TGF-β, CsA-treated and vehicle-treated Th cells and Tregs cells were analyzed by flow cytometry for expression of membrane-bound TGF-β using an antibody (clone TB21) that is specific for active (secreted) and membrane-bound TGF-β, detectable on 14.5% ± 1.54 of CD4+ cells treated with vehicle prior to coculture. In comparison, the percentage of cells expressing membrane TGF-β increased significantly to 22.2% ±1.97 from cocultures initially treated with CsA (Fig. 7B ; P<0.05). Using a second antibody specific for the latency associated peptide (LAP), which tethers TGF-β to the cell surface, similar results were obtained (data not shown). The TGF-β+ cells from the CsA-treated group were found primarily in the CD25– compartment (data not shown).


Figure 7
View larger version (19K):
[in this window]
[in a new window]

  		Figure 7. CsA treatment increases cell-bound TGF-β on CD4+CD25– cells. (A) ELISA for active TGF-β was performed on the supernatant of cocultures from CsA or vehicle-treated Th cells, APCs, and anti-CD3 collected after 24 h. (B) Purified CD4+CD25– or CD4+CD25+ cells were incubated with CsA (1.0 µg/ml) and cocultured with anti-CD3 for 68–72 h and then stained for surface TGF-β (clone TB21) and a secondary anti-mouse FITC antibody and CD25 PE and CD4 PerCP. Data shown are gating on live CD4+ cells. Numbers represent the percentage of live CD4+ cells in quadrants. (C) Coculture experiments were performed as described in A with neutralizing TGF-β antibody at the indicated concentrations or isotype control (30 µg/ml). Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01.

The relative expression of membrane-bound TGF-β, although up-regulated by CsA, was fairly low. However, the percentage of cells expressing membrane-bound TGF-β from CsA-treated CD4+CD25– Th cells rivaled that detected on purified CD4+CD25+ Tregs cultured in identical conditions (Fig. 7B) . Surface TGF-β is one mechanism through which Tregs are known to suppress other cells in vitro [1 ]. To determine the functional significance of TGF-β on CsA-treated Th cells, experiments were done in which CsA-treated Th cells were added to cocultures with untreated cells containing neutralizing TGF-β antibody. Significantly, the addition of neutralizing TGF-β antibody into the cocultures reversed the suppression mediated by CsA-treated Th cells (P<0.01). Specifically, blocking TGF-β (15 µg/ml) restored proliferation to 95.5 ± 10.7% of control (untreated cells plus isotype control antibody; mean±SEM n=3). These data demonstrate that inhibition of calcineurin induces Th cells with a TGF-β-mediated regulatory function.


arrow
DISCUSSION
 
The key findings in this study are that nuclear NFATc1 and NFATc2 were found in Tregs in the absence of stimulation and that following ligation of the TCR, nuclear NFAT was not induced further in Tregs as it was in Th cells. The differential nuclear induction of NFAT in Tregs could be accounted for by diminished intracellular calcium flux, decreased calcineurin levels, and increased activity of Gsk3β, an inhibitory NFAT kinase. Finally, inhibiting calcineurin activity in Th cells increased expression of TGF-β on the cell surface to levels equivalent to that detected on natural Tregs. These Th cells, expressing surface TGF-β, behaved as suppressor cells in vitro.

These data support a model in which the regulation of NFAT is important for CD4+ T cells with regulatory function. NFATc2 induces expression of foxp3 [7 ], and NFATc2 interacts with foxp3 as part of the transcriptional program required for Tregs [6 ]. Th cells, however, also express NFATc2. The pattern of nuclear NFAT localization is quite different in Th cells compared with Tregs. In Tregs, NFAT levels remain consistently low, and nuclear NFAT in Th cells increases. The level of NFAT available to interact with binding partners, such as foxp3 or AP-1, may determine the balance between initiation of an inflammatory or Treg response [28 ]. Active maintenance of a low level of nuclear NFAT in Tregs may reflect such a system.

In a model for active regulation of NFAT in Tregs, redundant checkpoints may be essential, including diminished calcineurin activity and increased activity of NFAT regulatory kinases, such as Gsk3β (Supplemental Fig. 2). Interestingly, when calcineurin activity increases in Th cells (Fig. 1C) , Gsk3β activity decreases (Fig. 4A) , creating an intracellular environment permissible for active NFAT to be maintained in the nucleus. In Tregs, however, when calcineurin activity is elevated, Gsk3β activity also increases, suggesting that Gsk3β may be a compensatory mechanism for inactivating nuclear NFAT. Inactivated nuclear NFAT would then be shuttled back to the cytoplasm in its heavily phosphorylated form (Fig. 1B) .

Gsk3β is best known for its role in the glycogen metabolism and is also involved in cell division and apoptosis [19 ]. Prior to this study, there are no reports of a role for Gsk3β in Tregs. Here, it is shown that Gsk3β activity is higher in Tregs compared with Th cells. PI-3K negatively regulates Gsk3β [19 ], and PI-3K activity is deficient in Tregs [29 ], suggesting a mechanism for the increase in Gsk3β activity identified herein. Additionally, these data show that transcriptional regulation of Gsk3β differs between Tregs and Th cells. In Tregs, Gsk3β may act on many substrates including cyclin D1, c-myc, cyclin-dependent protein kinases 4 and 6, c-jun, as well as NFAT [19 ]. As such, further clarification for the role of Gsk3β in Tregs may shed light on multiple signaling pathways, in addition to the NFAT signaling pathway, which are involved in regulation of the regulators.

Inhibition of NFAT induction is a hallmark of Tregs as described. To determine the relationship between NFAT signaling and Treg function in vitro, NFAT dephosphorylation was pharmacologically inhibited in CD4+CD25– nonregulatory Th cells using calcineurin inhibitors. Inhibition of NFAT in Th cells generated TGF-β-dependent, foxp3-independent regulators. In vitro, these regulators were not as efficacious as natural Tregs, suggesting that natural Tregs act through multiple mechanisms and may require foxp3 for full potency. Many studies have reported in vitro generation of regulators from nonregulatory precursors using TGF-β [30 31 32 33 ]. In these studies, however, foxp3 is also induced, suggesting that Smad signaling, initiated by TGF-β, may be important for foxp3 expression. TGF-β may intersect with the NFAT regulatory pathway, independent of foxp3. TGF-β also inhibits intracellular calcium flux and nuclear translocation of NFATc1 [34 , 35 ]. A facet of TGF-β-induced regulatory cells may involve NFAT inhibition.

Other studies, evaluating the effects of calcineurin inhibitors in vivo and in vitro, have shown conflicting results. In vivo, calcineurin inhibitors inhibit and generate Tregs, depending on the dose and treatment regiment [36 ]. Treating mice in vivo with a dose of CsA described previously to generate Tregs increases the level of LAP detectable on total CD4+ cells when stimulated with anti-CD3 and syngeneic APCs ex vivo (Supplemental Fig. 2). In vitro, many studies evaluating the effects of calcineurin inhibitors on Tregs have done so by directly adding CsA or FK506 to allogeneic MLRs [37 , 38 ]. In a murine system, Tregs cultured with allogeneic DCs and CsA were less able to suppress the proliferation of cells in a secondary, allogeneic MLR compared with Tregs cultured without CsA or with rapamycin in the primary culture [38 ]. This was coupled with a decline in foxp3 expression. In similar experiments using human CD25bright Tregs, foxp3 mRNA was inhibited by CsA and FK506 [37 ].

These experiments provide valuable insight into the effects of calcineurin inhibition on many cell types but do not specifically address the effects of calcineurin inhibition on Tregs alone. Expression of foxp3 RNA is not affected in human Tregs treated with CsA without other cells present [7 ]. In the experimental system used in the present study, CsA was added to Tregs independently, prior to coculture with other cells. CsA moderately diminished foxp3 expression and had no effect on TGF-β expression. CsA-treated Tregs remained suppressive in vitro.

The concept that calcineurin inhibitors may generate regulatory cells was described originally in classical studies characterizing the mechanism of action for CsA [39 ], although a mechanism through which this occurred had not been defined prior to the present study. CD4+CD25+-suppressive cells were described in rat allograft recipients following long-term CsA treatments [40 , 41 ], supporting a role for calcineurin inhibition in the generation of suppressive cells. In a nonhuman primate model of kidney rejection, expression of surface-bound TGF-β correlated with prolonged graft acceptance following weaning from immunsuppressants, including CsA [42 ]. Expression of surface-bound TGF-β in this model may reflect inhibition of calcineurin activity, similar to the data shown here.

These data suggest that multiple mechanisms are important for generation of regulatory cells and that the regulation of NFAT may have unique and nonoverlapping functions in Tregs. Lower levels of NFAT may be involved in maintaining surface TGF-β. However, a low level of nuclear NFAT may be important for the maintenance of foxp3 in Tregs. It is this balance of NFAT activity that may be important for the transcriptional program of regulatory cells. Further studies evaluating this pathway are necessary to better understand the widespread clinical use of NFAT inhibitors and their direct effects on the cells required for immune homeostasis.


arrow
ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Institutes of Health (NIH) HL60797 and HL/Al67177 to D. S. W. The Center for Immunobiology is in part supported by the Indiana Genomics Initiative (INGEN©) of Indiana University, which is supported in part by Lilly Endowment Inc. T. L. S. was supported by a fellowship from the Immunology and Infectious Diseases Training Program NIH T32 AI060519.

Received May 23, 2007; revised October 25, 2007; accepted October 31, 2007.


arrow
REFERENCES
 
    1
  1. Kitani, A., Fuss, I., Nakamura, K., Kumaki, F., Usui, T., Strober, W. (2003) Transforming growth factor (TGF)-β1-producing regulatory T cells induce smad-mediated interleukin 10 secretion that facilitates coordinated immunoregulatory activity and amelioration of TGF-β1-mediated fibrosis J. Exp. Med. 198,1179-1188[Abstract/Free Full Text]
    2. 2
  2. Nakamura, K., Kitani, A., Strober, W. (2001) Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells is mediated by cell surface-bound transforming growth factor β J. Exp. Med. 194,629-644[Abstract/Free Full Text]
    3. 3
  3. Nakamura, K., Kitani, A., Fuss, I., Pedersen, A., Harada, N., Nawata, H., Strober, W. (2004) TGF-β1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice J. Immunol. 172,834-842[Abstract/Free Full Text]
    4. 4
  4. Fontenot, J. D., Gavin, M. A., Rudensky, A. Y. (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells Nat. Immunol. 4,330-336[CrossRef][Medline]
    5. 5
  5. Hori, S., Nomura, T., Sakaguchi, S. (2003) Control of regulatory T cell development by the transcription factor foxp3 Science 299,1057-1061[Abstract/Free Full Text]
    6. 6
  6. Wu, Y., Borde, M., Heissmeyer, V., Feuerer, M., Lapan, A., Stroud, J., Bates, D., Guo, L., Han, A., Ziegler, S. F., Mathis, D., Beoist, C., Chen, L., Rao, A. (2006) Foxp3 controls regulatory T cell function through cooperation with NFAT Cell 126,375-387[CrossRef][Medline]
    7. 7
  7. Mantel, P-Y., Ouaked, N., Ruckert, B., Karagiannidis, C., Welz, R., Blaser, K., Schmidt-Weber, C. B. (2006) Molecular mechanisms underlying FOXP3 induction in human T cells J. Immunol. 176,3593-3602[Abstract/Free Full Text]
    8. 8
  8. Bopp, T., Palmetshofer, A., Serfling, E., Heib, V., Schmitt, S., Richter, C., Klein, M., Schild, H., Schmitt, E., Stassen, M. (2005) NFATc2 and NFATc3 transcription factors play a crucial role in suppression of CD4+ T lymphocytes by CD4+ CD25+ regulatory T cells J. Exp. Med. 201,181-187[Abstract/Free Full Text]
    9. 9
  9. Anderson, P. O., Sundstedt, A., Yazici, Z., Minaee, S., Woolf, R., Nicolson, K., Whitley, N., Li, L., Li, S., Wraith, D. C., Wang, P. (2005) IL-2 overcomes the unresponsiveness but fails to reverse the regulatory function of antigen-induced T regulatory cells J. Immunol. 174,310-319[Abstract/Free Full Text]
    10. 10
  10. Hickman, S. P., Yang, J., Thomas, R. M., Wells, A. D., Turka, L. A. (2006) Defective activation of protein kinase C and Ras-ERK pathways limits IL-2 production and proliferation by CD4+CD25+ regulatory T cells J. Immunol. 177,2186-2194[Abstract/Free Full Text]
    11. 11
  11. Ostroukhova, M., Seguin-Devaux, C., Oriss, T. B., Dixon-McCarthy, B., Yang, L., Ameredes, B. T., Corcoran, T. E., Ray, A. (2004) Tolerance induced by inhaled antigen involves CD4+ T cells expressing membrane-bound TGF-β and FOXP3 J. Clin. Invest. 114,28-38[CrossRef][Medline]
    12. 12
  12. Macian, F. (2005) NFAT proteins: key regulators of T-cell development and function Nat. Rev. Immunol. 5,472-484[CrossRef][Medline]
    13. 13
  13. Neal, J.W., Clipstone, N. (2001) Glycogen synthase kinase-3 inhibits the DNA binding activity of NFATc J. Biol. Chem. 276,3666-3673[Abstract/Free Full Text]
    14. 14
  14. Martinez-Martinez, S., Rodriguez, A., Lopez-Maderuelo, M. D., Ortega-Perez, I., Vazquez, J., Redondo, J. M. (2006) Blockade of NFAT activation by the second calcineurin binding site J. Biol. Chem. 281,6227-6235[Abstract/Free Full Text]
    15. 15
  15. Kehlenbach, R. H., Dickmanns, A., Gerace, L. (1998) Nucleocytoplasmic shuttling factors including Ran and CRM1 mediate nuclear export of NFAT in vitro J. Cell Biol. 141,863-874[Abstract/Free Full Text]
    16. 16
  16. Tsang, J. Y-S., Camara, N. O. S., Eren, E., Schneider, H., Rudd, C., Lombardi, G., Lechler, R. (2006) Altered proximal T cell receptor (TCR) signaling in human CD4+CD25+ regulatory T cells J. Leukoc. Biol. 80,145-151[Abstract/Free Full Text]
    17. 17
  17. Ishiguro, K., Ando, T., Maeda, O., Ohmiya, N., Niwa, Y., Goto, H. (2007) Acetate inhibits NFAT activation in T cells via importin β1 interference Eur. J. Immunol. 37,2309-2316[CrossRef][Medline]
    18. 18
  18. Anderson, P. O., Manzo, B. A., Sundstedt, A., Minaee, S., Symonds, A., Khalid, S., Rodriguez-Cabezas, M. E., Nicolson, K., Li, S., Wraith, D. C., Wang, P. (2006) Persistent antigenic stimulation alters the transcription program in T cells, resulting in antigen-specific tolerance Eur. J. Immunol. 36,1374-1385[CrossRef][Medline]
    19. 19
  19. Cohen, P., Frame, S. (2001) The renaissance of GSK3 Nat. Rev. Mol. Cell Biol. 2,769-776[CrossRef][Medline]
    20. 20
  20. Hess, A. D., Colombani, P. M., Esa, A. H. (1986) Cyclosporine and the immune response: basic aspects Crit. Rev. Immunol. 6,123-149[Medline]
    21. 21
  21. Schuh, K., Twardzik, T., Kneitz, B., Heyer, J., Schimpl, A., Serfling, E. (1998) The interleukin 2 receptor {alpha} chain/CD25 promoter is a target for nuclear factor of activated T cells J. Exp. Med. 188,1369-1373[Abstract/Free Full Text]
    22. 22
  22. McHugh, R.S., Whitters, M., Piccirillo, C., Young, D., Shevach, E., Collins, M., Byrne, M. (2002) CD4+CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor Immunity 16,311-323[CrossRef][Medline]
    23. 23
  23. Lehmann, J., Huehn, J., de la Rosa, M., Maszyna, F., Kretschmer, U., Krenn, V., Brunner, M., Scheffold, A., Hamann, A. (2002) Expression of the integrin {alpha} E β 7 identifies unique subsets of CD25+ as well as CD25– regulatory T cells Proc. Natl. Acad. Sci. USA 99,13031-13036[Abstract/Free Full Text]
    24. 24
  24. Thornton, A. M., Shevach, E. M. (1998) CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production J. Exp. Med. 188,287-296[Abstract/Free Full Text]
    25. 25
  25. Shin, G. T., Khanna, A., Ding, R., Sharma, V., Lagman, M., Li, B., Suthanthiran, M. (1998) In vivo expression of transforming growth factor-β1 in humans: stimulation by cyclosporine Transplantation 65,313-318[CrossRef][Medline]
    26. 26
  26. Minguillon, J., Morancho, B., Kim, S-J., Lopez-Botet, M., Aramburu, J. (2005) Concentrations of cyclosporin A and FK506 that inhibit IL-2 induction in human T cells do not affect TGF-β1 biosynthesis, whereas higher doses of cyclosporin A trigger apoptosis and release of preformed TGF-β1 J. Leukoc. Biol. 77,748-758[Abstract/Free Full Text]
    27. 27
  27. Goppelt-Struebe, M., Esslinger, B., Kunzendorf, U. (2003) Failure of cyclosporin A to induce transforming growth factor β TGF-β synthesis in activated peripheral blood lymphocytes Clin. Transplant. 17,20-25[Medline]
    28. 28
  28. Rudensky, A. Y., Gavin, M., Zheng, Y. (2006) FOXP3 and NFAT: partners in tolerance Cell 126,253-256[CrossRef][Medline]
    29. 29
  29. Bensinger, S. J., Walsh, P. T., Zhang, J., Carroll, M., Parsons, R., Rathmell, J. C., Thompson, C. B., Burnhill, M. A., Farrar, M. A., Turka, L. A. (2004) Distinct IL-2 receptor signaling pattern in CD4+CD25+ regulatory T cells J. Immunol. 172,5287-5296[Abstract/Free Full Text]
    30. 30
  30. Zheng, S. G., Gray, J. D., Ohtsuka, K., Yamagiwa, S., Horwitz, D. A. (2002) Generation ex vivo of TGF-{β}-producing regulatory T cells from CD4+CD25– precursors J. Immunol. 169,4183-4189[Abstract/Free Full Text]
    31. 31
  31. Zheng, S. G., Wang, J. H., Koss, M. N., Quismorio, F., Jr, Gray, J. D., Horwitz, D. A. (2004) CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-{β} suppress a stimulatory graft-versus-host disease with a lupus-like syndrome J. Immunol. 172,1531-1539[Abstract/Free Full Text]
    32. 32
  32. Yamagiwa, S., Gray, J. D., Hashimoto, S., Horwitz, D. A. (2001) A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood J. Immunol. 166,7282-7289[Abstract/Free Full Text]
    33. 33
  33. Chen, W., Jin, W., Hardegen, N., Lei, K-J., Li, L., Marinos, N., McGrady, G., Wahl, S. M. (2003) Conversion of peripheral CD4+CD25– naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor foxp3 J. Exp. Med. 198,1875-1886[Abstract/Free Full Text]
    34. 34
  34. Bommireddy, R., Ormsby, I., Yin, M., Boivin, G. P., Babcock, G. F., Doetschman, T. (2003) TGF{β}1 inhibits Ca2+-calcineurin-mediated activation in thymocytes J. Immunol. 170,3645-3652[Abstract/Free Full Text]
    35. 35
  35. Chen, C-H., Seguin-Devaux, C., Burke, N. A., Oriss, T. B., Watkins, S. C., Clipstone, N., Ray, A. (2003) Transforming growth factor {β} blocks tec kinase phosphorylation, Ca2+ influx, and NFATc translocation causing inhibition of T cell differentiation J. Exp. Med. 197,1689-1699[Abstract/Free Full Text]
    36. 36
  36. Kawai, M., Hiroaki, K., Mathieu, C., Waer, M., Pirenne, J. (2005) Inhibitory and stimulatory effects of cyclosporine A on the development of regulatory T cells Transplantation 79,1073-1077[CrossRef][Medline]
    37. 37
  37. Baan, C. C., van der Mast, B. J., Klepper, M., Mol, W. M., Peeters, A. M. A., Korevaar, S. S., Balk, A. H. M. M., Weimar, W. (2005) Differential effect of calcineurin inhibitors, anti-CD25 antibodies and rapamycin on the induction of FOXP3 in human T cells Transplantation 80,110-117[CrossRef][Medline]
    38. 38
  38. Zeiser, R., Nguyen, V. H., Beilhack, A., Buess, M., Schulz, S., Baker, J., Contag, C. H., Negrin, R. S. (2006) Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production Blood 108,390-399[Abstract/Free Full Text]
    39. 39
  39. Hess, A. D., Tutschka, P. J. (1980) Effect of cyclosporin A on human lymphocyte responses in vitro. I. CsA allows for the expression of alloantigen activated suppressor cells while preferentially inhibiting the induction of cytolytic effector lymphocytes in MLR J. Immunol. 124,2601-2608[Abstract]
    40. 40
  40. Hall, B. M., Pearce, N., Gurley, K., Dorsch, S. (1990) Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. III. Further characterization of the CD4+ suppressor cell and its mechanisms of action J. Exp. Med. 171,141-157[Abstract/Free Full Text]
    41. 41
  41. Hall, B. M., Jelbart, M. E., Gurley, K. E., Dorsch, S. E. (1985) Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. Mediation of specific suppression by T helper/inducer cells J. Exp. Med. 162,1683-1694[Abstract/Free Full Text]
    42. 42
  42. Torrealba, J. R., Katayama, M., Fechner, J. H., Jr, Jankowska-Gan, E., Kusaka, S., Xu, Q., Schultz, J. M., Oberley, T. D., Hu, H., Hamawy, M. M., et al (2004) Metastable tolerance to rhesus monkey renal transplants is correlated with allograft TGF-{β}1+CD4+ T regulatory cell infiltrates J. Immunol. 172,5753-5764[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures and Table
Right arrow All Versions of this Article:
jlb.0507321v1
83/3/708    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sumpter, T. L.
Right arrow Articles by Wilkes, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sumpter, T. L.
Right arrow Articles by Wilkes, D. S.