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The Journal of Immunology, 2007, 179, 3734 -3741
Copyright © 2007 by The American Association of Immunologists, Inc.
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Reciprocal NFAT1 and NFAT2 Nuclear Localization in CD8+ Anergic T Cells Is Regulated by Suboptimal Calcium Signaling1

Mathangi Srinivasan and Kenneth A. Frauwirth2

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Anergy is an important mechanism of maintaining peripheral immune tolerance. T cells rendered anergic are refractory to further stimulation and are characterized by defective proliferation and IL-2 production. We used a model of in vivo anergy induction in murine CD8+ T cells to analyze the initial signaling events in anergic T cells. Tolerant T cells displayed reduced phospholipase C{gamma} activation and calcium mobilization, indicating a defect in calcium signaling. This correlated with a block in nuclear localization of NFAT1 in anergic cells. However, we found that stimulation of anergic, but not naive T cells induced nuclear translocation of NFAT2. This suggested that NFAT2 is activated preferentially by reduced calcium signaling, and we confirmed this hypothesis by stimulating naive T cells under conditions of calcium limitation or partial calcineurin inhibition. Thus, our work provides new insight into how T cell stimulation conditions might dictate specific NFAT isoform activation and implicates NFAT2 involvement in the expression of anergy-related genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The process of V(D)J recombination generates a tremendously diverse repertoire of T cell Ag receptors (TCRs) capable of binding both pathogenic and nonpathogenic Ags. Tolerance to most, but not all self-Ags is accomplished by thymic deletion of self-reactive T cells (1, 2, 3). However, tolerance to innocuous foreign Ags, and at least some tissue-specific self-Ags, occurs in the periphery after T cells exit the thymus (4). Peripheral tolerance mechanisms use a combination of deletion, suppression by regulatory cells and T cell anergy (5).

Anergy is induced hyporesponsiveness and has been shown to contribute to tolerance to ingested, inhaled, and injected Ags (1, 5, 6). T cell anergy may also contribute to the escape of tumors from immunosurveillance (7, 8). Although anergy likely represents a collection of phenomena induced by incomplete T cell activation, common characteristics are blocks in proliferation and IL-2 secretion in response to subsequent stimulation (9, 10).

Anergic T cells have been reported to have multiple defects in the TCR signaling cascade, including defective tyrosine phosphorylation immediately downstream of TCR recruitment (11, 12), MAPK signaling defects (5, 13, 14, 15), and reduced calcium flux (16, 17, 18). However, these defects have not been reported universally and are influenced by factors such as the mode of introduction of the stimulating Ag, the subset of T cells involved, and the time and strength of the TCR-Ag interaction. Broadly, the anergic phenotype can be classified into being either MAPK limited or calcium limited, with most in vivo anergy models displaying calcium signaling defects (5).

The mobilization of calcium within T cells is a biphasic event divided into the initial intracellular store release of calcium and the subsequent global calcium influx from the plasma membrane channels, referred to as the store-operated calcium channels. In T cells, an initial peak in cytosolic calcium levels is followed by a sustained high concentration of calcium. This sustained increase in calcium levels is essential for downstream signaling events (19, 20, 21, 22). Cytosolic calcium binds calmodulin, an ubiquitously expressed calcium-binding protein in the cytosol. Ca2+/calmodulin activates numerous signaling molecules, including the serine/threonine phosphatase calcineurin (23). Active calcineurin dephosphorylates proteins of the NFAT family of transcription factors, which are key players in T cell activation (24, 25). Dephosphorylation of these proteins causes the exposure of the nuclear localization signal and subsequent nuclear translocation (26, 27, 28). Further phosphorylation events in the nucleus cause activation of the NFAT proteins (29) and induce interaction with binding partners (30, 31), resulting in transcription of important effector genes, including IL-2.

We have previously described a model of in vivo induction of anergy in CD8+ TCR-transgenic mice using i.p. injection of antigenic peptide in the absence of adjuvants to tolerize the T cells (32). When compared with T cells from control mice, anergic T cells showed a decrease in calcium flux upon in vitro stimulation. Consistent with a calcium limitation phenotype, NFAT1 translocation into the nucleus was blocked in anergic T cells. Unlike NFAT1, NFAT2 was rapidly translocated into the nuclei of anergic, but not responsive T cells. We also found that directly limiting extracellular calcium or calcineurin activity with normal T cells was sufficient to replicate the anergic T cell pattern of NFAT localization. This indicates that NFAT isoforms are responsive to different cytosolic calcium levels, an observation we believe to be previously unreported. The reciprocal regulation of NFAT1 and NFAT2 suggests that although NFAT1 is the predominant isoform involved in a productive immune response, NFAT2 might be important for controlling genes involved in T cell tolerance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

Anti-CD3 (mAb 145-2C11), anti-CD28 (mAb 37.51), control hamster IgG, PE-labeled anti-Thy1.2, and PE-labeled anti-CD8 Abs were purchased from eBioscience. Goat anti-hamster IgG was purchased from Pierce. Anti-NFAT1 and anti-NFAT2 Abs were obtained from Affinity Bioreagents. Anti-phospholipase C{gamma} (PLC{gamma})3 and anti-phospho-PLC{gamma} (Y783) Abs were purchased from Cell Signaling Technology. HRP-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from Bio-Rad. The H-2Kb-restricted 2C TCR-reactive peptide SIYRYYGL was purchased from NeoMPS. JNK inhibitor (SP600125) and p38 inhibitor (SB203580) were purchased from BIOMOL International. Leptomycin B was purchased from Sigma-Aldrich.

Animals

2C TCR-Tg/RAG2–/– mice have been described previously (32). C57BL/6J mice (6–8 wk old) were purchased from The Jackson Laboratory. All mice were maintained in ventilated M.I.C.E. microisolator cages (Animal Care Systems) at the University of Maryland Animal facility (College Park, MD). Animals received humane care in compliance with the National Institutes of Health’s "Guide for the Care and Use of Laboratory Animals." Animal studies were reviewed and approved by the University of Maryland Institutional Animal Care and Use Committee.

T cell purification

Murine T cells were purified from the spleens using the SpinSep negative-selection system (Stem Cell Technologies) according to the manufacturer’s protocol. Purified T cells were usually >95% Thy1 positive, as determined by flow cytometry.

Cell culture

All cells were maintained in RPMI 1640 medium (Mediatech) supplemented with 10% FBS (HyClone), 200 µM glutamine, penicillin/streptomycin, 10 mM HEPES buffer, MEM nonessential amino acids, and 55 µM 2-ME at 37°C in a 5% CO2 atmosphere.

In vivo anergy induction

Mice were injected i.p. with 25–50 nmol of the 2C peptide in sterile PBS or PBS alone. Spleens were harvested 7 days after administration. T cells were purified and tested for proliferation and cytokine secretion upon stimulation in vitro with C57BL/6J splenocytes plus titrated doses of peptide.

Proliferation and cytokine assays

Proliferation after 3 days of stimulation in vitro was determined by [3H]thymidine incorporation. Cells were pulsed for 6–8 h with 1 µCi/well [3H]thymidine (MP Biomedicals), transferred to glass fiber filters with a 96-well cell harvester (Tomtec), and analyzed by liquid scintillation using a 1450 Microbeta Trilux scintillation counter (Wallac). IL-2 and IFN-{gamma} levels in 24-h stimulation supernatants were determined using a sandwich ELISA. Primary and biotinylated secondary anti-cytokine Abs and recombinant cytokine standards were purchased from eBioscience and used at the concentrations recommended by the manufacturer. Alkaline phosphatase-conjugated avidin was purchased from Jackson ImmunoResearch Laboratories and used at a 1/3000 dilution. Colorimetric alkaline phosphatase substrate was purchased from Sigma-Aldrich and used at 1 mg/ml in 10% diethanolamine buffer, and quantification was performed on a Versamax spectrophotometer (Molecular Devices). Data were analyzed using Softmax Pro software (Molecular Devices). Data points for all analyses are presented as the mean of triplicate wells ± SD.

T cell stimulation

T cells were restimulated with anti-CD3 and anti-CD28 Abs. The cells were first incubated with anti-CD3 and anti-CD28 Abs (10 µg/ml) on ice for 30 min and then stimulated for appropriate time points with goat anti-hamster IgG as a secondary cross-linking Ab (10 µg/ml) at 37°C. The stimulation was stopped at the different time points by addition of ice-cold 1x PBS to the samples.

Immunofluorescence microscopy

T cells were stimulated with anti-CD3 and anti-CD28 Abs. Briefly, 1 x 106 cells/sample were incubated with anti-CD3 plus anti-CD28 (10 µg/ml) in complete RPMI 1640 medium on ice for 30 min. Goat anti-hamster IgG (secondary cross-linking Ab) was added and samples were transferred to 37°C for appropriate times. Stimulations were stopped by addition of 10 volumes of ice-cold PBS and samples were resuspended in 100 µl of RPMI/BSA (RPMI 1640, 10 mM HEPES, penicillin/streptomycin, and 0.6% BSA). Cells were plated onto poly-L-lysine-coated microscope slides (Polysciences) and incubated on ice for 40 min. Cells were fixed with ice-cold methanol for 15 min and washed once with cold PBS. Cells were then blocked and permeabilized with PB buffer (RPMI 1640, 10% FBS, 0.05% saponin, 10 mM glycine, and 10 mM HEPES) for 20 min. Cells were incubated with anti-NFAT1 or anti-NFAT2 Ab at 2 µg/ml for 1 h. Cells were washed with PB buffer and incubated with 2 µg/ml Alexa Fluor 594-linked anti-IgG (Molecular Probes) in PB buffer for 1 h in the dark. After three washes in PB, cells were incubated in 1 µM SYTO13 (Molecular Probes) in PB buffer for 20 min in the dark. Cells were postfixed in 4% paraformaldehyde and coverslips were then fixed onto the slides. The cells were viewed using a LSM 510 microscope (Zeiss Microimaging). Quantification of nuclear localization was done with the NIH ImageJ software package using the colocalization macro. For leptomycin treatment experiments, cells were incubated with 200 nM leptomycin for 30 min at 4°C before addition of anti-CD3 and anti-CD28. Leptomycin (200 nM) was also added to the RPMI-BSA during cross-linking and stimulation. For the MAPK inhibition assays, cells were incubated with 250 µM of the JNK inhibitor (SP600125) or 20 µM of the p38 inhibitor (SB203580) along with anti-CD3 and anti-CD28 Abs. The inhibitors were also added to the complete RPMI 1640 medium during secondary cross-linking and stimulation. The concentrations used for these pharmacological inhibitors were obtained by either titration experiments (JNK inhibitor; data not shown) or from established protocols (p38 inhibitor (33) and leptomycin (34)).

Western blots

T cells were stimulated as above and lysed in radioimmunoprecipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, and 1 mM Na3VO4 in PBS) The lysates were resolved on a 8% SDS-PAGE gel (~1.25 x 106) cells/sample) and transferred onto nitrocellulose membrane. The blots were blocked with 5% nonfat dry milk in PBS/0.1% Tween 20 (PBS-T) and probed with primary Ab (1/1000 in PBS-T) followed by HRP-conjugated anti-rabbit and anti-mouse IgG, respectively. The bands were visualized with SuperSignal West Pico Chemiluminescent substrate (Pierce).

Measurement of intracellular calcium

The intracellular calcium flux was measured by flow cytometry using the calcium-sensitive dyes Fluo3 and Fura Red (Molecular Probes). Fluo3 fluorescence at 530 nm increases with increasing Ca+2 binding, whereas Fura Red fluorescence at 670 nm decreases with increasing Ca+2 binding, allowing ratiometric measurement of calcium (35). Purified T cells were resuspended in RPMI 1640 medium supplemented with 1% FBS. Cells were stained with 4 µM Fluo3 and 10 µM Fura Red and coincubated with anti-CD3 Ab and PE-labeled anti-CD8 for 30 min at 30°C. The Fluo3/Fura Red fluorescence values were then read on a FACSCalibur flow cytometer (BD Biosciences). Basal Fluo3 and Fura Red fluorescence levels were measured for 60 s, after which the cells were transferred to prewarmed (37°C) RPMI 1640 containing 7.5 µg/ml goat anti-hamster IgG to cross-link the TCR-CD3 complex. The increase (Fluo3) and decrease (Fura Red) in fluorescence levels were then recorded for at least 500 s, collecting ~100 events per data point using CellQuest software (BD Biosciences) The relative calcium concentration inside the cell was then plotted as a ratio of Fluo3 to Fura Red emission values using FlowJo software (Tree Star).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Anergic T cells display a defective calcium response

Intraperitoneal injection of 2C TCR-transgenic mice with antigenic peptide resulted in T cell anergy, characterized by strongly reduced proliferation (Fig. 1A) and IL-2 secretion (Fig. 1B), as described previously (32). Unlike IL-2, IFN-{gamma} production was only modestly reduced in anergic T cells (Fig. 1C). This indicates that anergy induced by i.p. injection of peptide differs from both the three-signal and the adaptive tolerance models, in which T cells show markedly impaired IFN-{gamma} responses (5, 36).


Figure 1
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  		FIGURE 1. T cells from peptide-injected mice are anergic. T cells were purified from PBS- and peptide-injected mice and stimulated in vitro. A, In vitro proliferation measurements for splenic T cells (5 x 104) purified from PBS- and peptide-injected mice. T cells were stimulated with graded concentrations of peptide in the presence of splenic APCs for 72 h. Proliferation was assayed by [3H]thymidine uptake. B and C, ELISA for IL-2 secretion (B) and IFN-{gamma} secretion (C) in 24-h stimulation supernatants. All results are representative of more than three individual experiments.

 
Anergic T cells have been found to display significant signaling defects, including impaired calcium signals. Calcium flux in anergic 2C T cells was clearly reduced compared with that of naive T cells, although calcium signaling was not completely abrogated (Fig. 2A). This is consistent with the observation of Dubois et al. (16) in a similar peptide injection system, but using the F5 (influenza NP366-374/Db) TCR transgene. Thus, defective calcium signaling may be an important component of peptide-induced in vivo anergy in CD8+ T cells.


Figure 2
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  		FIGURE 2. Anergic CD8+ T cells display a calcium signaling defect. A, Anergic and naive T cells were loaded with Fluo3 and Fura Red, and intracellular calcium responses to anti-CD3 stimulation were determined by flow cytometry, using the ratio of Fluo3/Fura Red fluorescence. B, ER store release of calcium in anergic T cells was measured by stimulation in RPMI 1640 containing 2 mM EGTA. After ~200 s of stimulation, 3 mM CaCl2 was added to replenish the extracellular calcium source. Calcium flux was measured as in A. C, In vitro-stimulated anergic and naive T cell lysates were assayed for activation of PLC{gamma} by Western blot for Y783 phosphorylation.

 
Calcium signaling in T cells is biphasic. Release of endoplasmic reticulum (ER) calcium activates surface calcium release-activated calcium channels, causing a large influx of extracellular calcium. These phases occur in rapid succession, but can be resolved by chelating extracellular calcium with EGTA. Cross-linking the TCR of naive T cells in the presence of EGTA produced a small rise in intracellular calcium (Fig. 2B), a result of ER calcium release. The rise in anergic T cells was comparable to that in naive cells, but occurred more slowly. When extracellular calcium was restored, we still saw a reduced calcium influx in anergic T cells, possibly due to the delayed kinetics in ER release (Fig. 2B).

Because ER calcium release was altered in anergic T cells, we examined the activation of PLC{gamma}, an upstream inducer of calcium release. Complete activation of PLC{gamma} requires phosphorylation at Y783, which was detected using a phospho-specific Ab. As shown in Fig. 2C, TCR-induced phosphorylation of PLC{gamma} in anergic T cells was impaired compared with naive T cells. This was not due to a delay in activation, since phosphorylation in anergic cells peaked by 2 min and remained below naive T cell levels for at least 10 min, by which time the cytosolic calcium levels had peaked and were returning to baseline in both naive and anergic T cells (see Fig. 2A). Total PLC{gamma} levels remained stable for at least 30 min in both responsive and anergic cell lysates, indicating that the impaired phosphorylation is not a result of proteolytic degradation of PLC{gamma} (Fig. 2C and data not shown).

NFAT1 translocation into the nucleus is inhibited in tolerant cells

Calcium entry into T cells activates the calcium/calmodulin-dependent phosphatase calcineurin, leading to NFAT dephosphorylation and translocation into the nucleus. Because anergic cells displayed a defective calcium response, we investigated whether there was a corresponding defect in the movement of NFAT into the nucleus. After stimulating T cells for different time points, the localization of NFAT1 in anergic cells was analyzed by immunofluorescence microscopy. In nonanergic cells, NFAT1 could be seen as a cytosolic ring at 2 min after stimulation, and translocation into the nucleus was detected within 5 min of stimulation (Fig. 3A). By 30 min, NFAT1 had largely returned to the cytosol. In anergic T cells, NFAT1 was also detected in the cytosol after 2 min of stimulation, but translocation into the nucleus was strongly inhibited compared with naive cells (Fig. 3B). The level of NFAT1 nuclear localization was quantified and is represented in Fig. 3C. This indicates that the reduced calcium flux in anergic T cells prevents proper NFAT1 localization.


Figure 3
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  		FIGURE 3. NFAT1 translocation into the nucleus is impaired in anergic T cells. A, Naive T cells were stimulated for the indicated times with anti-CD3 and anti-CD28 Abs. Nuclei were stained with SYTO13 (green, top panels) and NFAT1 nuclear localization (red, middle panels) was measured by immunofluorescence microscopy. Colocalization is shown in yellow in the merge images (yellow, bottom panels). B, Anergic T cells were stimulated and NFAT1 nuclear translocation was measured as in B. C, Quantification of NFAT1 nuclear localization. Graphs were generated with data from 25 cells/sample. Results are representative of three independent experiments. D, Anergic T cells were stimulated in the presence or absence of leptomycin B and NFAT1 nuclear localization was determined by immunofluorescence microscopy. Colocalization was quantified for 20 cells/sample as in C. Naive 5 min: Naive T cells stimulated in the absence of leptomycin B. E, Western blot of NFAT1 in whole-cell lysates from resting (lane 1) and 5-min stimulated (lane 2) naive T cells.

 
The reduction, but not absence, of calcium flux in anergic T cells suggested that NFAT1 might still be imported into the nucleus after stimulation, but at a rate that has fallen below the rate of export. To address this possibility, tolerized T cells were stimulated in the presence or absence of leptomycin B, a nuclear export inhibitor (34). As shown in Fig. 3D, leptomycin-treated anergic T cells have increased NFAT1 nuclear localization after stimulation, compared with leptomycin-untreated cells. Although this result indicates that some NFAT1 is getting into the nuclei of anergic T cells, only to be quickly exported, the degree of nuclear localization in leptomycin-treated anergic cells after 30 min of stimulation is still lower than that of control naive T cells after 5 min of stimulation. This suggests that a lowered rate of import plays a significant role in the reduced nuclear localization of NFAT1 in anergic cells.

Surprisingly, we were unable to detect NFAT1 in resting naive or anergic T cells, although the same Ab was able to detect NFAT1 in lysates from both resting and stimulated naive T cells by Western blots (Fig. 3E). This suggests that the epitope (which is distinct from the nuclear localization signal) is hidden before stimulation, either by protein conformation or by a binding partner. Stimulation presumably induces a conformational change in the protein that precedes nuclear translocation by minutes, allowing its detection in the cytosol.

NFAT2 is regulated differently from NFAT1 in anergic cells

NFAT2 is another isoform of the NFAT family of proteins that is expressed in peripheral T lymphocytes. NFAT1 and NFAT2 share >70% sequence identity in the Rel homology region, which is the primary DNA-binding domain of these proteins. Furthermore, NFAT1 and NFAT2 both bind to the basic ARRE-2 IL-2 promoter site in vitro (25). Although sequence identity in the other domains is much lower, both isoforms appear able to activate transcription of many of the same genes (reviewed in Ref. 37). However, most previous studies on NFAT localization have focused only on NFAT1. We therefore investigated whether NFAT2 displayed a similar pattern of nuclear translocation. In resting naive cells, we could readily detect cytosolic NFAT2 (Fig. 4A). However, unlike NFAT1, there was little translocation to the nucleus after stimulation, even after 30 min. Resting anergic T cells also showed cytosolic NFAT2, but displayed NFAT2 localization into the nucleus within 5 min of stimulation (Fig. 4B), when NFAT1 in these cells was largely cytosolic. We could detect nuclear localization of NFAT2 for at least 30 min after stimulation. Similar to Fig. 3, NFAT2 nuclear colocalization was quantified and represented in Fig. 4C. Thus, NFAT1 and NFAT2 are reciprocally regulated in naive and anergic T cells. In naive T cells, NFAT1, but not NFAT2, responds to TCR ligation by rapidly translocating to the nucleus, and this pattern is reversed in anergic T cells.


Figure 4
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  		FIGURE 4. NFAT2 nuclear translocation is enhanced in anergic T cells. A, Naive T cells were stimulated as in Fig. 3, and NFAT2 nuclear localization was determined by immunofluorescence microscopy. Nuclei (green) are shown in the top panels, NFAT2 (red) in the middle panels, and colocalization (yellow) is shown in the bottom panels. B, Anergic T cells were treated and NFAT2 localization was measured as in A. C, Quantification of NFAT2 nuclear localization. Results are representative of three independent experiments.

 
NFAT1/2 translocation is controlled by extracellular calcium availability

The correlation between the defective calcium signaling and the switch to NFAT2 nuclear translocation in anergic cells suggested a causal connection. To determine whether reduced calcium flux could account for the pattern of NFAT localization in anergic cells, we examined the translocation patterns of NFAT1 and NFAT2 in naive T cells under conditions of calcium limitation. The calcium available to T cells was varied by using EGTA to chelate extracellular calcium. By titrating EGTA and measuring calcium flux by flow cytometry, we determined that 600–700 µM EGTA produced calcium response curves similar to those seen in anergic T cells (data not shown). Cells stimulated in medium with no added EGTA displayed NFAT1 nuclear localization (Fig. 5, A and C) while NFAT2 remained cytosolic (Fig. 5, B and C), as previously observed. When 600 µM EGTA was added to the medium, this pattern was reversed, with NFAT1 cytosolic and NFAT2 in the nucleus (Fig. 5). In excess EGTA there was no NFAT nuclear localization (Fig. 5C), confirming that both NFAT1 and NFAT2 are completely dependent on the influx of extracellular calcium for activation. Thus, decreasing calcium signaling in T cells is sufficient to account for the altered NFAT1/2 regulation observed in anergic cells.


Figure 5
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  		FIGURE 5. Responsive T cells display the anergic NFAT1/2 translocation pattern when extracellular calcium availability is limited. A and B, NFAT1 (A) and NFAT2 (B) localization was visualized using immunofluorescence microscopy in resting cells and in cells stimulated for 5 min in the absence or presence of 600 µM EGTA. C, Quantification of NFAT1 and NFAT2 nuclear localization. Graphs were generated with data from 25 cells/sample. Results are representative of more than three experiments.

 
NFAT2 localization in T cells is controlled at the level of nuclear import

The results described above led us to hypothesize that the altered pattern of NFAT1 and NFAT2 localization in anergic and calcium-limited cells might be due to differential sensitivities to calcineurin. To confirm this hypothesis, we treated cells with cyclosporin A to probe the effects of partial inhibition of calcineurin on nuclear localization of the two isoforms. Naive T cells were stimulated in the presence of titrated concentrations of cyclosporin A, and the cellular localization patterns were determined by immunofluorescence. As shown in Fig. 6, partial inhibition of calcineurin with 300 nM cyclosporin A blocked NFAT1 translocation but induced NFAT2 translocation, similar to the effects of 600 µM EGTA. Complete inhibition of calcineurin with 750 nM cyclosporin caused cytosolic localization of both isoforms. Thus, a partial reduction in calcium signaling, by reducing either calcium influx or calcineurin activity, mimics the effects of T cell anergy on NFAT1 and NFAT2 localization patterns.


Figure 6
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  		FIGURE 6. NFAT1 and NFAT2 reciprocal nuclear translocation is regulated by calcineurin activity. Purified T cells were treated with different concentrations of cyclosporin A and stimulated for 5 min. NFAT1 (A) and NFAT2 (B) localization was visualized by immunofluorescence and quantified as in Fig. 3 (C).

 
The nuclear localization of NFAT1/2 is determined by a balance in the rates of dephosphorylation by calcineurin and rephosphorylation of the same serine residues by NFAT kinases which promote nuclear export. Some of these kinases, such as glycogen synthase kinase 3 (38) and casein kinase 1 (39), act on both isoforms. Other kinases show specificity for one isoform, with p38 MAPK acting on NFAT1 (40) and JNK acting on NFAT2 (41). To discern the effect of these proteins on the nuclear residence of NFAT1 and NFAT2, we stimulated T cells in the presence of pharmacological inhibitors of p38 and JNK and determined translocation patterns. The p38 inhibitor SB203580 slowed export of NFAT1 from the nucleus, as seen by the increased nuclear NFAT1 (relative to untreated cells) after 15 min of stimulation, but had no effect on NFAT2 (Fig. 7A). This is consistent with a role for p38 in rephosphorylating NFAT1. However, inhibition of JNK did not lead to increased nuclear NFAT2 out to 10 min of stimulation (Fig. 7B), by which time JNK activity had peaked and returned to baseline in untreated cells (data not shown). We inferred from these results that regulation of NFAT2 in naive T cells is upstream of nuclear entry.


Figure 7
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  		FIGURE 7. NFAT2 localization is controlled at the level of nuclear import. Cells were treated with 20 µM of the p38 inhibitor SB203580 (A), 250 µM of the JNK inhibitor SP600125 (B), or 200 nM of the nuclear export inhibitor leptomycin (C) during stimulation. NFAT1 and NFAT2 nuclear translocation were measured as in Fig. 3.

 
To confirm that the apparent failure of NFAT2 to enter the nucleus in stimulated nonanergic T cells was not actually due to rapid export, we stimulated T cells in the presence of leptomycin B and examined the pattern of NFAT1 and NFAT2 localization. As shown in Fig. 7C, treatment with 200 nM leptomycin retained NFAT1 in the nucleus out to 30 min after stimulation. However, NFAT2 remained cytosolic even in the presence of leptomycin. Thus, the cellular localization NFAT2 is controlled at the level of nuclear import. Together, these results indicate that the altered regulation of NFAT translocation in anergic T cells is due to the reduced calcium flux and resulting reduction in calcineurin activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
To better understand the induction of hyporesponsiveness in anergic T cells, we studied the signaling alterations of in vivo-tolerized CD8+ 2C TCR-transgenic T cells. Two major signaling pathways frequently found to be altered in anergic T cells are the ras/MAPK cascade and calcium signaling (5). Although we could not detect consistent defects in MAPK activation in our model of in vivo anergy induction (data not shown), we found that calcium responses in anergic cells were blunted relative to naive cells. This is consistent with multiple other in vivo anergy models, spanning different induction methods and both CD4+ and CD8+ T cells (12, 16, 17), suggesting that the calcium limitation is a common feature of in vivo T cell anergy.

An important initiating event of T cell calcium signaling is the activation of PLC{gamma} (42, 43). In anergic 2C T cells, phosphorylation of PLC{gamma} was partially inhibited, indicating reduced activation. This correlated with a slower release of ER calcium, and we are currently investigating whether the reduced PLC{gamma} activation is sufficient to account for the inhibition of calcium signaling. It has been reported that degradation of PLC{gamma} mediated by ubiquitin ligases, such as Cbl-b and gene related to anergy in lymphocytes (GRAIL), accounts for the reduction of calcium flux and subsequent functional defects in anergic T cells (44). Examination of total PLC{gamma} in anergic T cells from peptide-injected 2C TCR-transgenic mice did not show degradation for at least 30 min after restimulation. Thus, reduction in PLC{gamma} protein levels does not appear to be a significant contributing factor to the reduced calcium flux in this system. Although calcium flux in anergic cells was inhibited, it was not completely abrogated. We therefore compared the downstream effects of calcium signaling in naive and anergic T cells. We focused on the NFAT transcription factor family members NFAT1/NFATc2 and NFAT2/NFATc1, since nuclear translocation of these isoforms requires the activation of the calcium/calmodulin-dependent phosphatase calcineurin and these isoforms are expressed in peripheral T cells (25, 28).

NFAT1 distribution was nuclear within 5 min of stimulation in naive T cells, but NFAT1 failed to translocate in anergic T cells, as predicted. The failure of NFAT1 to accumulate in the nucleus of anergic T cells could be explained by a decrease in nuclear import (due to reduced calcineurin activity) or an increase in nuclear export (via activation of nuclear NFAT kinases). To distinguish between these options, we examined the pattern of NFAT1 localization in anergic T cells treated with the nuclear export inhibitor leptomycin. Treated cells showed an increase in nuclear NFAT1, but required prolonged TCR ligation to approach the level seen in stimulated naive T cells. Thus, although there may be some increase in NFAT kinase activity in anergic cells, this is unlikely to account fully for the blockade in nuclear NFAT1 accumulation. We hypothesize that, in concert with any increased NFAT kinase activity, the reduced calcium flux lowers calcineurin activity enough to shift the balance from import to export.

Interestingly, NFAT1 was undetectable in resting cells, but was detected in the cytosol after 2 min of stimulation in both naive and anergic T cells. Because we could detect NFAT1 in Western blots of lysates from resting cells, this result suggests that nuclear localization of NFAT1 occurs in at least two distinct steps. First, TCR ligation causes a conformational shift in NFAT1, or induces release of a binding partner, which reveals the Ab epitope. This event appears to be independent of calcium flux or has a relatively low threshold, because it occurs in both naive and anergic T cells. The second step is the dephosphorylation and translocation of NFAT1, which is sensitive to calcium flux and does not occur in anergic cells. Although we hypothesize that these two steps are independently regulated by multiple pathways, they might both be mediated by calcineurin. Calcineurin dephosphorylates up to 20 serine residues on the serine-rich region and the serine proline xx-repeat motif 1 domains of NFAT1 in a specific order in a concerted fashion (45). Dephosphorylation of the first residues may have a lower calcium requirement, whereas the final steps in uncovering the nuclear localization signal require higher calcium concentrations. Further characterization of this process may reveal novel drug targets for the regulation of T cell activation.

We also determined the cellular distribution of NFAT2 in anergic T cells. Although NFAT1 and NFAT2 show a high degree of homology in their DNA-binding Rel similarity domain (>70%) (23, 25), their translocation patterns after stimulation differed in naive and anergic T cells. To our knowledge, this observation has not previously been reported. Unlike NFAT1, there was no nuclear translocation of NFAT2 for at least 30 min after stimulation of naive T cells. Furthermore, stimulation of anergic cells did lead to nuclear localization of NFAT2 within 5 min. Thus, NFAT1 and NFAT2 seem to be reciprocally regulated in naive and anergic T cells. By limiting calcium in the culture medium, we were also able to demonstrate that the reduction in calcium influx in anergic T cells is sufficient to account for the switch from NFAT1 to NFAT2 as the predominant translocating isoform. Analysis of the nuclear translocation pattern of NFAT1 and 2 using the pharmacological inhibitors cyclosporin A (inhibits calcineurin) and leptomycin B (inhibits nuclear export) has led us to conclude that the reciprocal regulation takes place at the level of dephosphorylation before nuclear import. The involvement of nuclear NFAT kinases, which rephosphorylate the serine residues targeted by calcineurin, in controlling NFAT2 localization in T cells seems to be limited, since stimulating naive T cells in the presence of a JNK inhibitor during stimulation did not retain NFAT2 in the nucleus. Together, these results support a model in which localization of NFAT2 is controlled mainly by calcium-dependent calcineurin activity, rather than rephosphorylation and nuclear export by JNK.

We are currently investigating the significance of this difference in NFAT use between responsive and anergic T cells. One intriguing hypothesis is that NFAT1 is responsible for the regulation of activation-induced genes (e.g., IL-2), whereas NFAT2 is important for the expression of anergy-related genes. Identification of NFAT2-controlled genes may therefore shed light on the factors responsible for maintaining the hyporesponsiveness of anergic T cells. It is important to distinguish this from the events involved in establishing the anergic state, which occur during the initial exposure of T cells to Ag and are likely to involve NFAT1-regulated genes (46). We also recognize that the reduced calcium flux and resultant switch in NFAT isoform usage are unlikely to be the only signaling alterations in anergic T cells. We are currently investigating the involvement of other signal transduction pathways, including JNK and NF-{kappa}B, as well as the potential role of reduced metabolic activity in the hyporesponsive state.

Despite the high degree of homology between NFAT1 and NFAT2, they have previously been shown to be nonredundant in T cells (28, 39, 40, 41). Notably, NFAT1 and NFAT2 knockouts systems have different immunological phenotypes. NFAT1-deficient T cells are skewed toward a Th2 response, while NFAT2 knockout cells have a Th1 bias (26, 47, 48, 49). Although it is not clear how this may relate to T cell anergy, particularly in CD8+ T cells, our observations suggest that regulation of calcium flux may also play a previously unsuspected role in T cell differentiation. Strong calcium signaling during stimulation may bias T cells toward a Th1 program by activating NFAT1, whereas suboptimal calcium signals would preferentially activate NFAT2, directing cells toward a Th2 bias. Investigation of the factors involved in the fine control of cellular calcium levels may therefore uncover novel targets for immunomodulation.


   Acknowledgments
 
We thank Nandini Arunkumar and Amy Beaven for assistance with fluorescence microscopy. We are grateful to Drs. Craig Thompson and Wenxia Song for reagents and advice and to Dr. David Mosser, Segun Oonabajo, and the members of the Frauwirth laboratory for helpful discussions and critical reading of this manuscript.


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


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

1 This work was supported by National Cancer Institute Grant K01 CA092156 (to K.A.F.). Back

2 Address correspondence and reprint requests to Dr. Kenneth Frauwirth, University of Maryland, 2113 Microbiology Building, College Park, MD 20742. E-mail address: kfrauwir{at}umd.edu Back

3 Abbreviations used in this paper: PLC{gamma}, phospholipase C{gamma}; ER, endoplasmic reticulum. Back

Received for publication July 26, 2006. Accepted for publication July 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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