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The Journal of Immunology, 2006, 176: 880-888.
Copyright © 2006 by The American Association of Immunologists

Regulation of Th2 Cytokine Expression in NKT Cells: Unconventional Use of Stat6, GATA-3, and NFAT21

Zheng-Yu Wang*, Saritha Kusam*, Veerendra Munugalavadla{dagger}, Reuben Kapur{dagger}, Randy R. Brutkiewicz* and Alexander L. Dent2,*

* Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202, and Walther Cancer Institute, Indianapolis, IN 46208; {dagger} Department of Pediatrics and the Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NKT cells are unique in that they can produce high levels of both Th1 and Th2 cytokines, yet little is known about how NKT cells control the transcription of Th2 cytokines. The expression of IL-4 by NKT cells is independent of the Th2-associated transcription factor Stat6. We have found that Stat6 is critical for the expression of IL-5, IL-10, and IL-13 by NKT cells. However, the Th2 cell-associated transcription factor GATA-3, normally induced by Stat6 activation, is expressed at low levels in NKT cells. CD4+ NKT cells are highly enriched for Th2 cytokine expression compared with CD4– NKT cells, and we searched for transcription factors that are up-regulated in CD4+ NKT cells that could control Th2 cytokine expression. We found that the NFAT family member NFAT2 is selectively increased in CD4+ NKT cells. We tested the roles of NFAT2 and also GATA-3 in Th2 cytokine expression by retrovirus-mediated gene transduction into NKT cells and nonpolarized conventional T cells. Expression of NFAT2 increased the expression of IL-4 in both NKT cells and conventional T cells, and NFAT2 activated IL-10 in conventional T cells but not in NKT cells. GATA-3 strongly activated IL-4, IL-5, and IL-13 expression in conventional T cells but had comparatively weak effects on these cytokines in NKT cells. Thus, NFAT2, GATA-3, and Stat6 have surprisingly different roles in NKT cells than in conventional T cells. We propose that one mechanism by which CD4+ NKT cells express IL-4 independent of Stat6 is via increased NFAT2 activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer T cells, designated NKT cells, are a unique subpopulation of T cells with powerful immunoregulatory function. NKT cells derive their name because they express cell surface proteins that also are expressed on NK cells (1, 2, 3). Unlike NK cells, NKT cells express a TCR and are dependent on the thymus for their development (1, 2, 3). A large fraction of mouse NKT cells recognize Ag in the context of the MHC class I-like molecule CD1d and also use the V{alpha}14 and J{alpha}18 gene segments for the {alpha}-chain of their TCR (1, 2, 3). CD1d molecules are thought to present lipid and glycolipid Ags to T cells (1, 4, 5). Recently, several new glycolipid Ags able to activate NKT cells via CD1 have been described (6, 7, 8, 9, 10, 11). Unlike conventional T cells, NKT cells are enriched in atypical lymphoid organs, such as the liver and bone marrow (1, 2). The reason for the unusual physiological locations of NKT cells is not known, although presumably NKT cells play a specialized surveillance role at those sites.

NKT cells were initially characterized as cells that produced large amounts of cytokines, particularly IFN-{gamma} and IL-4, immediately after T cell stimulation in vivo (1, 2). NKT cells can promote the polarization of conventional CD4 T cells into Th1 and Th2 cells, and NKT cells have been found to play an extremely important role in modulating different types of immune responses. The expression of specific Th1 and Th2 cytokines by NKT cells is critical to these immunomodulatory roles. Thus NKT cells are important for a wide variety of immune processes, such as: 1) tumor rejection (12, 13); 2) suppression of antitumor responses (14); 3) down-regulation of autoimmune inflammatory diseases, such as experimental allergic encephalomyelitis (15); 4) immune privilege in the eye (16); 5) tolerance to allografts and xenografts (17, 18, 19); 6) fetal-maternal tolerance (20); 7) protection against graft-vs-host disease (21); and 8) protection against pathogens such as Cryptococcus (22) and hepatitis B virus (23). In general, rejection responses, such as tumor rejection and elimination of pathogens, correlate with Th1 responses by NKT cells (where IFN-{gamma} is produced), whereas tolerogenic responses, such as graft acceptance and suppression of tumor responses, generally correlate with Th2 responses by NKT cells (where IL-4, IL-10, and or IL-13 are produced). Additionally, down-regulatory responses, such as inhibition of experimental allergic encephalomyelitis, correlates with the production of Th2 cytokines by NT cells. Therefore, a major question is how NKT cells regulate the expression of Th1 and Th2 cytokines to control specific immune responses.

A large amount of work has uncovered the mechanisms used by conventional Th1 and Th2 cells to express specific cytokines (reviwed in Ref.24), yet relatively little is known about the regulation of cytokine expression by NKT cells. Human NKT cells can be divided into two major subsets of cells, depending on CD4 expression: CD4+ NKT cells and CD4– NKT cells. Whereas both classes of human NKT cells produce high levels of Th1 cytokines, CD4+ NKT cells are greatly enriched for cells that express Th2 cytokines, such as IL-4 (25, 26). CD4 expression in mouse NKT cells also correlates with increased expression of Th2 cytokines (27), although as in the human, CD4+ NKT cells also produce IFN-{gamma} (28). Intracellular cytokine staining experiments demonstrate that individual NKT cells can express both IL-4 and IFN-{gamma}. Interestingly, the vast majority of NKT cells appear to express IL-4 and IFN-{gamma} mRNA constitutively, and therefore part of the regulation of Th1 and Th2 cytokine expression by NKT cells may be regulated at the posttranscriptional stage (29). However, this mode of cytokine regulation by NKT cells is not well understood.

The activation of specific transcription factors in T cells during an immune response is critical for the development of polarized Th1 and Th2 cells (reviewed in Ref.24). The transcription factor T-bet is critical for the development of Th1 cells, and interestingly, mature NKT cells cannot be formed in the absence of T-bet (30). The transcription factor Stat6, which is activated by IL-4 signaling, is vital for the development of a normal Th2 response. Yet NKT cells apparently do not require Stat6 for IL-4 production (29, 31). Mast cells also produce IL-4 independently of Stat6 (32), further showing that the same cytokine can be regulated differently within the hemopoietic system. Mast cells also express different isoforms of the transcription factor NFAT, which also is involved in IL-4 transcription (33). Besides NFAT, GATA-3 is a key transcription factor that controls Th2 cytokine production in conventional T cells (24). Although little is known about the role of GATA-3 in Th2 cytokine expression by NKT cells, increased GATA-3 mRNA correlated with increased IL-4 expression by NKT cells in one study (34). In this study, we analyze in more detail the roles of Stat6, NFAT2, and GATA-3 in Th2 cytokine production by NKT cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Animals were maintained under specific pathogen-free conditions in animal facilities certified by the American Association of Laboratory Animal Care; 6- to 10-wk-old wild-type (WT)3 mice and Stat6–/– mice on a mixed C57BL/6-129 background were used to isolate NKT cells. C57BL/6J mice (The Jackson Laboratory) also were used to obtain NKT cells in several experiments.

T cell isolation and culture

Primary NKT cells were obtained by sorting NK1.1+TCR{alpha}{beta}+ cells from liver mononuclear cells as described previously (35). Freshly isolated NKT cells were stimulated with anti-CD3 Abs and expanded in RPMI 1640 medium containing 10% FCS plus IL-2 as described previously (28). CD4+NK1.1+TCR{alpha}{beta}+ and CD4–NK1.1+TCR{alpha}{beta}+ NKT cells were obtained by cell sorting following three-color staining of liver mononuclear cells. Fluorescently labeled Abs to NK1.1, TCR{alpha}{beta}, and CD4 and purified anti-CD3 Abs were obtained from BD Pharmingen. Primary Th1 and Th2 cells were isolated as described previously (36).

ELISA

Cells were stimulated for 24 h with plate-bound anti-CD3 at 10 µg/ml at 1 x 106 cells per ml, and supernatants were collected and analyzed by ELISA. All ELISA reagents were obtained from BD Pharmingen.

Western blots

Whole-cell lysates and nuclear extracts were analyzed by Western blot using standard conditions. Blots were probed with anti-GATA-3 mAb (clone HG3-31), anti-NFAT1 (4G6-G5), or anti-NFAT2 (7A6), all from Santa Cruz Biotechnology. GATA-3 blots were developed with ECL reagents (Amersham Biosciences). NFAT1 and NFAT2 blots were developed with SuperSignal West Femto maximum sensitivity substrate (Pierce). After anti-GATA-3 or anti-NFAT1/2 probing, blots were probed with Ab to {beta}-actin (Sigma-Aldrich) to demonstrate equivalent protein loading.

TranSignal array analysis

Nuclear extracts from CD4– and CD4+ stimulated with 20 ng/ml PMA and 0.3 µM ionomycin for 6 h were tested in the assay. Thirty micrograms of each nuclear extract was used for each analysis. The TranSignal arrays were performed according to the manufacturer’s protocols (Panomics).

Gel shift assay

Nuclear extracts were prepared, and gel shift assays for NFATs were performed as described previously (37). Binding reactions were performed with labeled probe and 10 µg of nuclear extract from CD4– and CD4+ NKT and Th2 cells stimulated with 20 ng/ml PMA and 0.3 µM ionomycin for 6 h. For NFAT binding, a double-stranded oligonucleotide from the murine IL-4 promoter (–88 to –60) was used: 5'-CTGGTGTAATAAAATTTTCCAATGTAAAC-3' (top strand shown only) (38). The supershift Ab for NFAT2 (7A6) was obtained from Santa Cruz Biotechnology.

RT-PCR

RNA isolation, cDNA synthesis, and RT-PCR were performed by standard procedures. Total RNA was isolated from CD4– and CD4+ NKT cells stimulated with or without anti-CD3 Ab at 10 µg/ml for 3 h. The following primers were used: NFAT2{alpha}, 5'-GAGCGGAGAAACTTTGCG-3' and 5'-CAGAGGACAGGAAGTATCCCG-3'; NFAT2{beta}, 5'-GGAGTTCGACTTCGATTTCCTC-3' and 5'-CAGAGGACAGGAAGTATCCCG-3'; and NFAT1, 5'-GATGGAGTGATCTCGATCCGA-3' and 5'-TATCGAAGAAGAACCGATCGC-3'. Primers for {beta}-tubulin were published previously (39). PCR reactions were run under conditions where amplification was linear, generally 28–30 cycles.

Retroviral infection

Amphotropic retroviruses were prepared with the Phoenix-GP system obtained from G. Nolan (Stanford University, Stanford, CA) and used as described (40, 41). The control human CD4 and the GATA-3-human CD4 retroviral vectors were obtained from M. Kaplan (Indiana University School of Medicine) (42). The NFAT2-expressing retroviruses were constructed by inserting blunt-ended NFAT2 cDNA from pSH160C (43) into the retroviral vector pMIEG3 (37) via the blunt-ended XhoI site. Sorted NKT cells were activated with anti-CD3 Abs as described above, then spin-infected with retroviral supernatant on day 5 of growth, put at 32°C overnight, and moved to 37°C for 24 h. Control CD4 and CD4-GATA-3 virus-infected cells were then sorted for either human CD4 expression following staining with PE-labeled anti-CD4 Ab (BD Pharmingen). Cells infected with pMIEG3 alone or pMIEG3-NFAT2 were sorted for GFP expression. Following sorting, cells were put into wells containing immobilized anti-CD3 Abs (10 µg/ml) and stimulated overnight at 37°C. Supernatants were then collected for ELISA analysis. Nonpolarized conventional T cells were obtained by activating lymph node T cells with Con A at 2 µg/ml before infection without the addition of any cytokine except IL-2. Retrovirus infection of nonpolarized conventional T cells followed a similar protocol, except that the cells were infected at day 3 following activation. In general, NKT cells were infected at a much lower rate than conventional T cells by the retroviruses: 2–8% for the NKT cells vs 30–40% for conventional T cells. In pilot experiments, conventional T cells expressing control CD4 or control GFP retroviruses behaved similarly; due to limitations in numbers of NKT cells, only the GFP control retrovirus was used as a control for the GATA-3 and NFAT2 retroviruses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Stat6 regulates IL-5, IL-10, and IL-13 but not IL-4 expression by NKT cells

Previous published data on the role of Stat6 in NKT cells suggested that IL-4 expression by NKT cells was independent of Stat6 activity (31). To investigate in more detail how Th2 cytokine expression was regulated in NKT cells, we analyzed the role of Stat6 in the expression of other Th2 cytokines (Fig. 1). We found that, consistent with previously published data, Stat6-deficient NKT cells produced similar levels of IL-4 as WT NKT cells. Strikingly, other Th2 cytokines such as IL-5, IL-10, and IL-13, were strongly dependent on Stat6 function in NKT cells. Not surprisingly, IFN-{gamma} expression by NKT cells was unaffected by the loss of Stat6. These data indicate that there are different levels of regulation of Th2 cytokine expression in NKT cells, and that, although IL-4 expression is independent of Stat6 activity, other Th2 cytokines are Stat6 dependent.



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  		FIGURE 1. IL-4, but not other Th2 cytokines, is expressed independently of Stat6 in NKT cells. NKT cells were isolated from the livers of WT and Stat6-deficient mice, activated with anti-CD3, and expanded in IL-2 for 1 wk. The cells were then restimulated with anti-CD3 Abs for 24 h, and cytokine secretion was measured by ELISA. This experiment was repeated once with identical results. Error bars represent SE.

 
Different layers of regulation of Th2 cytokine expression by NKT cells

Previously published data with both human and mouse NKT cells showed that CD4 expression correlates with IL-4 expression, although both CD4+ and CD4– NKT cells produce high levels of IFN-{gamma} (25, 26, 27, 28). Consistent with the findings of Gumperz et al. (26) on human NKT cells, we found that 40–50% of mouse NKT cells were CD4+ (data not shown). We first investigated whether CD4 expression on mouse NKT cells correlates with expression of Th2 cytokines besides IL-4. We found that, with WT CD4+ NKT cells, IL-4, IL-5, and IL-13 are expressed at greatly increased levels, compared with CD4– NKT cells (Fig. 2A). However, both types of NKT cells produced similar levels of IL-10 and IFN-{gamma}. These data indicate several levels of regulation of Th2 cytokines by NKT cells. First, IL-4 is expressed along with CD4 but independent of Stat6. Second, IL-5 and IL-13 are expressed along with CD4 but are dependent on Stat6. Finally, IL-10 is expressed independently of CD4 but dependent on Stat6. We next asked whether Stat6 affected the development of CD4+ NKT cells or the development of Th2 cytokine expression by CD4+ NKT cells. We found that CD4+ NKT cells from Stat6-deficient mice were found at percentages comparable to that of WT (data not shown). Surprisingly, we observed that, like WT CD4+ NKT cells, CD4+ NKT cells from Stat6-deficient mice are enriched for the expression of IL-4, IL-5, and IL-13 (Fig. 2B). However, if we compare the ratio of ng/ml IL-4 to ng/ml IL-5 secreted by CD4+ NKT cells from WT and Stat6-deficient mice, we find that Stat6-deficient NKT cells express relatively less IL-5, compared with IL-4, than WT NKT cells (Fig. 2C). This pattern also holds true for IL-4/IL-10 and IL-4/IL-13 ratios. These data tell us that 1) the correlation between IL-4, IL-5, and IL-13 expression and CD4 expression is independent of Stat6, and 2) the primary function of Stat6 in NKT cells is to amplify the expression of IL-5, IL-10, and IL-13.



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  		FIGURE 2. CD4+ NKT cells produce most of the Th2 cytokines made by NKT cells in both WT and Stat6-deficient mice. NKT cells were isolated from the livers of WT and Stat6-deficient mice, separated into CD4+ and CD4– populations, then grown as described in Fig. 1. The cells were then restimulated with anti-CD3 Abs for 24 h. Cytokine secretion from WT cells (A) and Stat6–/– cells (B) was measured by ELISA. C, The ratios of IL-4 levels to IL-5, IL-10, and IL-13 levels secreted by CD4+ NKT cells from WT and Stat6–/– mice. The data shown are representative of four different experiments for A, and the results of two separate experiments were averaged for B. The graphs shown in C were derived from the data shown in A and B. Error bars represent SE.

 
NKT cells express very little GATA-3

Because a key downstream target of Stat6 signaling is GATA-3 (24), we next investigated whether the altered expression of Th2 cytokines in Stat6-deficient NKT cells correlated with changes in GATA-3 expression. We used Western blots of whole-cell lysates to assess GATA-3 protein levels in NKT cells from WT and Stat6-deficient mice, as well as conventional Th1 and Th2 cells and T cells from Stat6-deficient mice cultured under Th2 conditions. GATA-3 was expressed at very low levels in NKT cells, compared with conventional Th2 cells, and there was little difference in GATA-3 expression between resting or stimulated WT and Stat6-deficient NKT cells (Fig. 3A). As expected, GATA-3 expression was low in T cells from Stat6-deficient mice cultured under Th2 conditions. These data indicate that, in NKT cells, unlike conventional T cells, Stat6 does not up-regulate GATA-3 expression. We next tested whether GATA-3 is up-regulated specifically in CD4+ NKT cells, compared with CD4– NKT cells (Fig. 3B). We found that, before CD3 stimulation, CD4+ NKT cells had more GATA-3 than CD4– NKT cells, although the levels were still low. Following CD3 stimulation, GATA-3 levels were comparable between CD4+ and CD4– NKT cells. Although the increased GATA-3 in CD4+ NKT cells may account for the increase in Th2 cytokine production by these cells, the overall low levels of GATA-3 in CD4+ NKT cells suggests that these cells regulate Th2 cytokine transcription by a very different mechanism than conventional Th2 cells.



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  		FIGURE 3. GATA-3 is expressed at low levels by NKT cells, compared with conventional Th2 cells. NKT cell populations were isolated and grown as described in Fig. 1. Primary T cells from WT and Stat6-deficient (SKO) mice were cultured under standard Th1 or Th2 conditions to obtain the indicated populations. Cells were activated with or without anti-CD3 Ab for 3 h. Whole-cell lysates were prepared and GATA-3, and {beta}-actin levels were analyzed by Western blot. The data shown are representative of three different experiments for A and two different experiments for B.

 
Identification of NFAT2 as a factor up-regulated in CD4+ NKT cells

We next wondered whether other factors besides Stat6 and GATA-3 accounted for the increased Th2 cytokine expression in CD4+ NKT cells. We therefore used a TranSignal protein/DNA array kit to search for transcription factors up-regulated specifically in CD4+ NKT cells, compared with CD4– NKT cells. In this assay, nuclear extracts from two different cell types are incubated with mixtures of DNA probes representing known transcription factor binding sites. The protein-DNA complexes are then purified, the DNA extracted, and then this bound DNA is hybridized to an array containing all the complementary oligonucleotides for the different transcription factor binding sites. The oligonucleotides that bind can then be visualized, and transcription factors expressed in a given cell type can then be identified. We used this procedure to identify that at least one of the NFAT family members was specifically up-regulated in CD4+ NKT cells (Fig. 4). Other transcription factors, such as NF-{kappa}B, the vitamin D receptor, and the thyroid hormone receptor, were present at similar amounts in both types of NKT cells.



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  		FIGURE 4. Array showing differences in transcription factor expression between CD4+ and CD4– NKT cells. TranSignal arrays were performed according to the manufacturer’s protocol using nuclear extracts from P/I-stimulated CD4+ and CD4– NKT cells. Spots represent hybridization of an oligonucleotide (oligo) containing a transcription factor binding site to an array containing complementary oligos for the input oligos. Each spot is in duplicate. TR, thyroid hormone receptor; VDR, vitamin D receptor. Weaker spots below some of the boxed spots on the array represent a 10-fold lower dilution of the complementary oligo to the membrane. Other faint spots not labeled in the figure represent retinoic acid receptor X, NFE2, and Stat4.

 
We next wanted to confirm that NFAT proteins were increased in CD4+ NKT cells. Gel shifts with nuclear extracts from CD4+ NKT cells and CD4– NKT cells showed increased total NFAT DNA binding (data not shown), consistent with the TranSignal array results. Because NFAT can be composed of one of five different family members (44), we wanted to determine which NFAT family member is up-regulated in CD4+ NKT cells. The regulation of IL-4 by NFAT is complex, and of the three NFAT family members expressed in T cells, NFAT1 (also known as NFATp or NFATc2), NFAT2, (also known as NFATc or NFATc1), and NFAT4 (also known as NFATc3), only NFAT2 can clearly activate IL-4 expression (24, 45). Mice deficient in NFAT1 and NFAT4 express more IL-4 than normal mice, suggesting that these proteins repress IL-4 (24). In contrast, NFAT2-deficient mice are defective in expressing normal levels of IL-4 (24). Moreover, other studies have suggested a positive role for NFAT2 in IL-4 transcription (38). We therefore initially examined the expression of NFAT1 and NFAT2 proteins in nuclear extracts from CD4– NKT cells and CD4+ NKT cells (Fig. 5A). We found that NFAT2 was significantly up-regulated in nuclear extracts from PMA plus ionomycin (P/I)-stimulated CD4+ NKT cells, compared with nuclear extracts from P/I-stimulated CD4– NKT cells. At the same time, little difference was seen in the expression of NFAT1 in these nuclear extracts. We next examined NFAT2 expression in total cell extracts and found that NFAT2 protein expression was up-regulated at the whole-cell level in CD4+ NKT cells, not just up-regulated in the nucleus. Normal Th2 cells also express very high levels of NFAT2, which correlates with the fact that these cells also produce high levels of IL-4. We next addressed whether NFAT2 also was up-regulated in CD4+ NKT cells from Stat6-deficient mice (Fig. 5B). NFAT2 was expressed at much higher levels in Stat6-deficient CD4+ NKT cells, compared with Stat6-deficient CD4– NKT cells, suggesting that the increased NFAT2 could promote the Stat6-independent IL-4 expression seen in these cells. We also wanted to test whether NFAT2 contributed to NFAT DNA binding in CD4+ NKT cells. Experiments with gel shifts testing NFAT binding and Ab to NFAT2 showed that overall NFAT binding was up-regulated in CD4+ NKT cells, compared with CD4– NKT cells, and that a significant amount of NFAT binding was shifted with Ab to NFAT2 (Fig. 5C). Overall, CD4+ NKT cells displayed an NFAT DNA binding pattern similar to that of normal Th2 cells. We then tested the expression of the two isoforms of NFAT that have been described, NFAT2{alpha} and NFAT2{beta} (33), to determine whether there was differential expression of these two gene products between CD4+ and CD4– NKT cells (Fig. 5D). Using RT-PCR and primers specific for the two differently spliced forms of NFAT2, we found that mRNA for the common isoform, NFAT2{alpha}, was expressed at about 2-fold higher levels in CD4+ NKT cells than CD4– NKT cells before stimulation, and that after stimulation, both cell types up-regulated NFAT2{alpha} mRNA by 3-fold. After stimulation, CD4+ NKT cells expressed NFAT2{alpha} mRNA at slightly less than 2-fold higher levels than the CD4– NKT cells. The mRNA for the NFAT2{beta} isoform was expressed at higher levels in resting CD4+ NKT cells, compared with resting CD4– NKT cells. Similar to previously published data, expression of NFAT2{beta} mRNA was not altered by stimulation of CD4– NKT cells (33), although NFAT2{beta} mRNA was decreased by stimulation of CD4+ NKT cells. NFAT1 mRNA was expressed at slightly higher levels in CD4– NKT cells than in CD4+ NKT cells. These data show that increased NFAT2{alpha} and NFAT2{beta} expression correlates with IL-4 expression in CD4+ NKT cells.



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  		FIGURE 5. Up-regulation of NFAT2 in CD4+ NKT cells. NKT and Th2 cell populations were isolated and grown as described above. Cells were then left untreated, treated with P/I for 6 h, treated with ionomycin alone for 3 h, or stimulated with anti-CD3 Abs for 3 h. Whole-cell lysates, nuclear extracts, or RNA were then prepared. A, Analysis of NFAT2 and NFAT1 expression by Western blot, either in nuclear extracts or whole-cell extracts. {beta}-actin is a loading control. B, Up-regulated NFAT2 in nuclear extracts from CD4+ NKT cells of both WT and Stat6-deficient mice. C, Analysis of NFAT DNA binding by gel shift using nuclear extracts. Cells were stimulated with P/I for 6 h before preparation of the nuclear extracts. D, Analysis of NFAT2 isoform and NFAT1 mRNA expression in CD4+ and CD4– NKT cells. Cells were activated with or without anti-CD3 Ab for 3 h. Semiquantitative RT-PCR was used to analyze mRNA levels for the indicated genes. Gene expression was analyzed by 3-fold serial dilutions of each input cDNA.

 
Specific inhibition of IL-4 expression in NKT cells by cyclosporin A

The immunosuppressive drug cyclosporin A is known to inhibit activation of the NFAT transcription factor (46), and thus we tested whether cyclosporin A could affect IL-4 expression by CD4+ NKT cells. As shown in Fig. 6, cyclosporin A strongly inhibited IL-4 expression from CD4+ NKT cells in a dose-dependent manner but did not significantly affect IFN-{gamma} expression from the same cells. Although cyclopsorin A can inhibit other transcription factors besides NFAT, such as NF-{kappa}B, our data show that IL-4 expression by NKT cells is preferentially inhibited by cyclosporin A and support a model wherein NFAT activity is critical for IL-4 expression by NKT cells.



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  		FIGURE 6. IL-4 secretion by CD4+ NKT cells is sensitive to cyclosporin A treatment. CD4+ NKT cells isolated and grown as described above were treated with different doses of cyclosporin A for 4 h. These cells were then stimulated with anti-CD3 Ab for 24 h, and ELISAs were performed on the collected supernatants. The data shown are representative of two different experiments. Error bars represent SE.

 
A direct role for NFAT2 in IL-4 expression by NKT cells

We next wanted to test whether NFAT2 overexpression in total NKT cells and in CD4– NKT cells could result in increased Th2 cytokine expression. We therefore used a high-titer retrovirus system to infect NKT cells with a retrovirus construct expressing NFAT2. We also tested the role of GATA-3 in NKT cells by use of a GATA-3-expressing retrovirus (42). After sorting marker-positive cells (GFP in the case of NFAT2 and human CD4 in the case of GATA-3), we stimulated the NKT cells with anti-CD3 Abs and tested cytokine expression by ELISA (Fig. 7). We found that NFAT2 was able to increase IL-4 expression by roughly 50% in CD4– NKT cells, but that NFAT2 did not markedly affect IL-5, IL-10, IL-13, or IFN-{gamma}. These data support the idea that increased expression of NFAT2 in CD4+ NKT cells can promote Stat6-independent IL-4 transcription in these cells. Because other Th2 cytokines were not significantly affected by NFAT2, the role of NFAT2 in Th2 cytokine expression by NKT cells may be limited to IL-4. Overexpression of GATA-3 in CD4– NKT cells by retrovirus led to a similar increase in IL-4 production as NFAT2. GATA-3 also promoted IL-5 expression in CD4– NKT cells, which supports the idea that NFAT2 and GATA-3 have different roles in Th2 cytokine expression. Like NFAT2, GATA-3 did not alter IL-10, IL-13, or IFN-{gamma} production.



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  		FIGURE 7. NFAT2 and GATA-3 induce IL-4 but only weakly induce other Th2 cytokines in CD4– NKT cells. CD4– NKT cells were isolated and grown as described above. On day 5 of culture, the cells were infected with either control retrovirus or retroviruses encoding NFAT2 or GATA-3. Control, NFAT2-, and GATA-3-retrovirus expressing cells were isolated by cell sorting, then restimulated with anti-CD3 Ab for 24 h. ELISAs were performed on the collected supernatants. The cytokine values shown are averages from three experiments, where the cytokine amounts were normalized to the amount produced by control retrovirus-positive cells from each individual experiment. Cytokine values for control retrovirus-positive cells were set at 100. NFAT2 induced at significant increase in IL-4, whereas GATA-3 induced a significant increase in IL-4 and IL-5. Expression of IL-10, IL-13, and IFN-{gamma} was not affected by NFAT2 or GATA-3 retroviruses. Error bars denote SE.

 
Lastly, we tested the effects of NFAT2 and GATA-3 overexpression on conventional T cells. We infected nonpolarized lymph node T cells with our NFAT2 and GATA-3 retroviruses, sorted out marker-positive cells, stimulated the infected cells with anti-CD3 Abs, and again tested cytokine secretion by ELISA. Strikingly, we found that the effects of NFAT2 and GATA-3 were different in conventional T cells, compared with NKT cells (Fig. 8). In conventional T cells, GATA-3 very strongly promoted IL-4, IL-5 and IL-13 expression, but it did not affect IL-10 expression. NFAT2 also was able to activate IL-4 in conventional T cells but not as strongly as GATA-3. In contrast to what was seen in NKT cells, NFAT2 increased IL-10 expression in conventional T cells. As in NKT cells, neither NFAT2 nor GATA-3 affected IFN-{gamma} in conventional T cells. These data show that NFAT2 and GATA-3 play markedly different roles in Th2 cytokine expression, and that the functions of these two transcription factors vary between T cell types.



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  		FIGURE 8. GATA-3 and NFAT2 induce high levels of Th2 cytokines in nonpolarized conventional T cells. Lymph node T cells from WT mice were stimulated with Con A and grown in IL-2 for 3 days. On day 3, the cells were infected with retroviruses as described in Fig. 7. The cells were sorted for either GFP expression (control and NFAT2) or human CD4+ expression (GATA-3) and then restimulated with anti-CD3 Ab for 24 h. ELISAs were performed on the collected supernatants. The experiment shown was repeated once and gave essentially identical results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The differential expression of Th2 cytokine expression by NKT cells is critical for their function in many different disease states, and very little is known about this process. There is evidence that cytokine mRNA expression by NKT cells is constitutive and controlled at the posttranscriptional level, yet very little is known about this mode of regulation (29). In this study, we investigated how Th2 cytokine expression was controlled in NKT cells at the transcriptional level. We focused on three major transcription factors known to be involved in Th2 cytokine expression: Stat6, GATA-3, and NFAT2. Stat6 and GATA-3 were analyzed because of their known role in Th2 cytokine transcription, whereas we identified NFAT2 in this study as a unique transcription factor that is up-regulated in Th2 cytokine-expressing CD4+ NKT cells. We have found that Stat6, GATA-3, and NFAT2 all behave very differently in NKT cells vs conventional T cells.

Stat6, which is activated in response to IL-4 signaling, is a central transcriptional mediator of Th2 differentiation (24). We have found that, although NKT cells require Stat6 for normal expression of Th2 cytokines, NKT cells can produce high levels of IL-4 independent of Stat6. Thus, NKT cells use an alternative pathway from conventional T cells for IL-4 expression. As a major mechanism by which Stat6 activates Th2 cytokine expression is the up-regulation of GATA-3, we wondered whether NKT cells might induce GATA-3 by a different mechanism than Stat6. However, we found that GATA-3 is expressed at extremely low levels in NKT cells, compared with Th2 cells, suggesting that NKT cells do not rely on GATA-3 to the same extent as conventional Th2 cells. Moreover, there was little difference in GATA-3 expression between WT and Stat6-deficient NKT cells, indicating that Stat6 did not induce GATA-3 as in conventional T cells. These results prompted us to search for other transcription factors that could promote Th2 cytokine transcription in NKT cells. Using a transcription factor array system, we identified NFAT2 as being selectively up-regulated in Th2 cytokine-expressing CD4+ NKT cells.

A direct test of the effects of NFAT2 on cytokine expression in NKT cells showed that it could up-regulate IL-4 but not other Th2 cytokines. GATA-3 gave a similar result to NFAT2 when overexpressed in NKT cells, although GATA-3 was also able to weakly up-regulate another Th2 cytokine, IL-5. Tests with NFAT2 and GATA-3 expression in conventional T cells showed clearly divergent results from that obtained in NKT cells: NFAT2 selectively increased IL-4 and IL-10 expression in conventional T cells, and GATA-3 very strongly activated expression of IL-4, IL-5 and IL-13 in conventional T cells. Thus, Stat6, GATA-3, and NFAT2 all behave very differently in NKT cells than in conventional T cells. Taken together, our data support a model wherein the Stat6 signaling pathway is important for the expression of IL-5, IL-10, and IL-13 in NKT cells and NFAT2 can promote IL-4 expression by a Stat6-independent pathway. Although GATA-3 can activate IL-4 and IL-5 in NKT cells, the precise role for GATA-3 in NKT cell cytokine expression is somewhat unclear.

One very striking finding from our work is that GATA-3 can activate IL-4, IL-5, and IL-13 strongly in conventional T cells but not in NKT cells. These data support a model where, in conventional T cells, GATA-3 is critical for opening up the chromatin on the chromosomal locus containing the IL-4, IL-5, and IL-13 genes as well as activating the promoters of IL-5 and IL-13 (47, 48, 49, 50). An important question is why overexpression of GATA-3 was not able to activate IL-4, IL-5, and IL-13 expression to a significant extent in NKT cells, particularly in light of the fact that NKT cells express low levels of GATA-3. One possibility is that the chromatin of the IL-4-IL-5-IL-13 locus is already open in NKT cells, consistent with Stetson et al. (29), and thus overexpression of GATA-3 has no extra effect on chromatin remodeling. If the IL-4-IL-5-IL-13 locus is already open in NKT cells, a further question would be what keeps Th2 cytokine expression off in CD4– NKT cells. One possibility is that Th2 cytokine expression is regulated at the posttranscriptional level in NKT cells (29). This idea also may explain why overexpression of NFAT2 and GATA-3 by retrovirus had only a minor effect on IL-4, IL-5, and IL-13 expression in NKT cells. Another possibility is that low levels of GATA-3 in CD4– NKT cells are important to shut off Th2 cytokine expression, and that other transcription factors besides GATA-3 turn on Th2 cytokine expression in CD4+ NKT cells. Yet another possibility is that NKT cells express a gene, such as repressor of GATA (ROG) (51), that inhibits activation of Th2 cytokine expression by GATA-3 in these cells. Because GATA-3 is an autoactivator of its own expression, a GATA-3 inhibitor may explain the relatively low levels of GATA-3 in NKT cells. Strikingly, we found that Stat6 function did not affect GATA-3 expression levels in NKT cells, and that CD4+ NKT cells, which transcribe high levels of Th2 cytokines, had relatively little expression of GATA-3. These data indicate that GATA-3 is regulated very differently in NKT cells from conventional T cells, and that CD4+ NKT cells use another mechanism to express high levels of Th2 cytokines. Because full expression of IL-5, IL-10, and IL-13 is Stat6 dependent in NKT cells, our data suggest that another factor that is regulated by Stat6 besides GATA-3 may regulate these cytokines in NKT cells.

Our data suggest that NFAT2 may be a key regulator of IL-4 transcription in NKT cells, and this findings fits with other studies correlating increased NFAT2 and IL-4 expression (38, 52, 53). Unexpectedly, we found that NFAT2 is specifically up-regulated in CD4+ NKT cells. This finding is reminiscent of the up-regulation of NFAT2 (NFATc) observed in activated Th2 cells (54). Whereas much of the up-regulation of NFAT2 can be accounted for by increased mRNA expression and protein in the cells, we cannot rule out that NFAT2 is preferentially activated to enter the nucleus over other NFAT family members following cell stimulation. Activation of NFAT proteins is regulated by the strength of T cell stimulation, and NFAT2 appears to be induced by weaker signaling than NFAT1. Thus NFAT2-activated IL-4 transcription is associated with weaker TCR signaling in conventional T cells (38). It is unknown whether this increased sensitivity of NFAT2 to T cell activation is conserved in NKT cells. A key question is what up-regulates overall NFAT2 expression in CD4+ NKT cells and Th2 cells. Currently, very little is known about what regulates expression of the NFAT genes themselves. Interestingly, NFAT1 up-regulates NFAT2 during T cell activation (55), and NFAT2 expression also is regulated by mRNA splicing (56), indicating an intricate regulation of NFAT2 activity. Future experiments will address whether these pathways affect the up-regulation of NFAT2 seen in CD4+ NKT cells.

During the course of these studies, we found that CD4+ NKT cells are much more difficult to expand in vitro than CD4– NKT cells. Although CD4+ NKT cells were transduced at a similar frequency as CD4– NKT cells, it was difficult to isolate significant numbers of transduced CD4+ NKT cells for cytokine expression analysis. However, in two experiments, we found that CD4+ NKT cells transduced with NFAT2 showed a greater induction of IL-4 expression than CD4– NKT cells transduced with NFAT2 (data not shown). These data indicate that CD4+ NKT cells are more receptive to the effects of NFAT2 than CD4– NKT cells. Experiments with transduction of total NKT cells also supported this idea (data not shown). One explanation for this finding may be that CD4– NKT cells express higher levels of inhibitory NFAT proteins, such as NFAT1 and NFAT4, and that more NFAT2 is required to overcome this inhibitory effect. Our data in Fig. 5D show somewhat more NFAT1 in CD4– NKT cells than in CD4+ NKT cells, which is consistent with this idea. Other explanations are that CD4– NKT cells have other inhibitors of NFAT function or that CD4+ NKT cells express proteins that cooperate with NFAT2 to induce IL-4 expression.

One caveat to our experiments is that, to have enough NKT cells for biochemical and molecular analysis, as well as for retroviral infection, we had to expand the NKT cells in vitro in IL-2-containing medium for several days, typically for 7 days. In vitro expansion of NKT cells with IL-2 has been used in other studies with NKT cells (28), and our NKT cells, in general, behave as typical NKT cells. However, it is possible that in vitro expansion in IL-2 alters the expression of and/or dependence on certain transcription factors that regulate cytokine expression in NKT cells. Nonetheless, our results clearly show that NKT cells regulate cytokine gene expression differently than conventional T cells, and it was already established that NKT cells do not require Stat6 for high-level IL-4 production (29, 31), showing that NKT cells have unusual mechanisms for cytokine regulation.

In conclusion, we have found that Th2 cytokine expression in NKT cells is regulated by strikingly different mechanisms than in conventional T cells, and this information is useful for understanding how NKT cells modulate Th1 vs Th2 responses. However, much more work is required to fully understand how NKT cells control Th2 cytokine expression.


   Acknowledgments
 
We are very grateful to Dr. Hiroshi Sato (Indiana University School of Medicine) for protocols and guidance in preparing NKT cells, Dr. Cheong-Hee Chang (Indiana University School of Medicine) for the NFAT2 vector pSH160C, and Dr. David Wilkes (Indiana University School of Medicine) for providing cyclosporin A.


   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 Institutes of Health Grants AI46410 (to A.L.D.) and AI46455 and CA89026 (to R.R.B.). A.L.D. also receives support from the Indiana Genomics Initiative (INGEN) of Indiana University. INGEN is supported in part by Lilly Endowment. R.R.B. is a Scholar of the Leukemia and Lymphoma Society. Back

2 Address correspondence and reprint requests to Dr. Alexander L. Dent, Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Building R2, Room 302, 950 West Walnut Street, Indianapolis, IN 46202. E-mail address: adent2{at}iupui.edu Back

3 Abbreviations used in this paper: WT, wild-type; P/I, PMA plus ionomycin. Back

Received for publication July 22, 2005. Accepted for publication October 20, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kronenberg, M., L. Gapin. 2002. The unconventional lifestyle of NKT cells. Nat. Rev. Immunol. 2: 557-568. [Medline]
  2. Bendelac, A., M. N. Rivera, S. H. Park, J. H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15: 535-562. [Medline]
  3. Godfrey, D. I., H. R. MacDonald, M. Kronenberg, M. J. Smyth, L. Van Kaer. 2004. NKT cells: what’s in a name?. Nat. Rev. Immunol. 4: 231-237. [Medline]
  4. Porcelli, S. A., R. L. Modlin. 1999. The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids. Annu. Rev. Immunol. 17: 297-329. [Medline]
  5. Brutkiewicz, R. R., Y. Lin, S. Cho, Y. K. Hwang, V. Sriram, T. J. Roberts. 2003. CD1d-mediated antigen presentation to natural killer T (NKT) cells. Crit. Rev. Immunol. 23: 403-419. [Medline]
  6. Fischer, K., E. Scotet, M. Niemeyer, H. Koebernick, J. Zerrahn, S. Maillet, R. Hurwitz, M. Kursar, M. Bonneville, S. H. Kaufmann, U. E. Schaible. 2004. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc. Natl. Acad. Sci. USA 101: 10685-10690. [Abstract/Free Full Text]
  7. Zhou, D., J. Mattner, C. Cantu, III, N. Schrantz, N. Yin, Y. Gao, Y. Sagiv, K. Hudspeth, Y. P. Wu, T. Yamashita, et al 2004. Lysosomal glycosphingolipid recognition by NKT cells. Science 306: 1786-1789. [Abstract/Free Full Text]
  8. Wu, D., G. W. Xing, M. A. Poles, A. Horowitz, Y. Kinjo, B. Sullivan, V. Bodmer-Narkevitch, O. Plettenburg, M. Kronenberg, M. Tsuji, et al 2005. Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells. Proc. Natl. Acad. Sci. USA 102: 1351-1356. [Abstract/Free Full Text]
  9. Kinjo, Y., D. Wu, G. Kim, G. W. Xing, M. A. Poles, D. D. Ho, M. Tsuji, K. Kawahara, C. H. Wong, M. Kronenberg. 2005. Recognition of bacterial glycosphingolipids by natural killer T cells. Nature 434: 520-525. [Medline]
  10. Mattner, J., K. L. Debord, N. Ismail, R. D. Goff, C. Cantu, III, D. Zhou, P. Saint-Mezard, V. Wang, Y. Gao, N. Yin, et al 2005. Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434: 525-529. [Medline]
  11. Sriram, V., W. Du, J. Gervay-Hague, R. R. Brutkiewicz. 2005. Cell wall glycosphingolipids of Sphingomonas paucimobilis are CD1d-specific ligands for NKT cells. Eur. J. Immunol. 35: 1692-1701. [Medline]
  12. Brutkiewicz, R. R., V. Sriram. 2002. Natural killer T (NKT) cells and their role in antitumor immunity. Crit. Rev. Oncol. Hematol. 41: 287-298. [Medline]
  13. Smyth, M. J., N. Y. Crowe, Y. Hayakawa, K. Takeda, H. Yagita, D. I. Godfrey. 2002. NKT cells - conductors of tumor immunity?. Curr. Opin. Immunol. 14: 165-171. [Medline]
  14. Terabe, M., S. Matsui, N. Noben-Trauth, H. Chen, C. Watson, D. D. Donaldson, D. P. Carbone, W. E. Paul, J. A. Berzofsky. 2000. NKT cell-mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nat. Immunol. 1: 515-520. [Medline]
  15. Miyamoto, K., S. Miyake, T. Yamamura. 2001. A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells. Nature 413: 531-534. [Medline]
  16. Sonoda, K. H., D. E. Faunce, M. Taniguchi, M. Exley, S. Balk, J. Stein-Streilein. 2001. NK T cell-derived IL-10 is essential for the differentiation of antigen-specific T regulatory cells in systemic tolerance. J. Immunol. 166: 42-50. [Abstract/Free Full Text]
  17. Seino, K. I., K. Fukao, K. Muramoto, K. Yanagisawa, Y. Takada, S. Kakuta, Y. Iwakura, L. Van Kaer, K. Takeda, T. Nakayama, et al 2001. Requirement for natural killer T (NKT) cells in the induction of allograft tolerance. Proc. Natl. Acad. Sci. USA 98: 2577-2581. [Abstract/Free Full Text]
  18. Chargui, J., T. Hase, S. Wada, T. Naganuma, R. Yoshimura. 2001. NKT cells as nonspecific immune-regulator inducing tolerance in mouse model transplantation. Transplant Proc. 33: 3833-3834. [Medline]
  19. Ikehara, Y., Y. Yasunami, S. Kodama, T. Maki, M. Nakano, T. Nakayama, M. Taniguchi, S. Ikeda. 2000. CD4+ V{alpha}14 natural killer T cells are essential for acceptance of rat islet xenografts in mice. J. Clin. Invest. 105: 1761-1767. [Medline]
  20. Ito, K., M. Karasawa, T. Kawano, T. Akasaka, H. Koseki, Y. Akutsu, E. Kondo, S. Sekiya, K. Sekikawa, M. Harada, et al 2000. Involvement of decidual V{alpha}14 NKT cells in abortion. Proc. Natl. Acad. Sci. USA 97: 740-744. [Abstract/Free Full Text]
  21. Zeng, D., D. Lewis, S. Dejbakhsh-Jones, F. Lan, M. Garcia-Ojeda, R. Sibley, S. Strober. 1999. Bone marrow NK1.1– and NK1.1+ T cells reciprocally regulate acute graft versus host disease. J. Exp. Med. 189: 1073-1081. [Abstract/Free Full Text]
  22. Kawakami, K., Y. Kinjo, K. Uezu, S. Yara, K. Miyagi, Y. Koguchi, T. Nakayama, M. Taniguchi, A. Saito. 2001. Monocyte chemoattractant protein-1-dependent increase of V{alpha}14 NKT cells in lungs and their roles in Th1 response and host defense in cryptococcal infection. J. Immunol. 167: 6525-6532. [Abstract/Free Full Text]
  23. Kakimi, K., L. G. Guidotti, Y. Koezuka, F. V. Chisari. 2000. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J. Exp. Med. 192: 921-930. [Abstract/Free Full Text]
  24. Ho, I. C., L. H. Glimcher. 2002. Transcription: tantalizing times for T cells. Cell 109: (Suppl):S109-S120. [Medline]
  25. Lee, P. T., K. Benlagha, L. Teyton, A. Bendelac. 2002. Distinct functional lineages of human V{alpha}24 natural killer T cells. J. Exp. Med. 195: 637-641. [Abstract/Free Full Text]
  26. Gumperz, J. E., S. Miyake, T. Yamamura, M. B. Brenner. 2002. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med. 195: 625-636. [Abstract/Free Full Text]
  27. Yoshimoto, T., W. E. Paul. 1994. CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J. Exp. Med. 179: 1285-1295. [Abstract/Free Full Text]
  28. Chen, H., W. E. Paul. 1997. Cultured NK1.1+ CD4+ T cells produce large amounts of IL-4 and IFN-{gamma} upon activation by anti-CD3 or CD1. J. Immunol. 159: 2240-2249. [Abstract/Free Full Text]
  29. Stetson, D. B., M. Mohrs, R. L. Reinhardt, J. L. Baron, Z. E. Wang, L. Gapin, M. Kronenberg, R. M. Locksley. 2003. Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function. J. Exp. Med. 198: 1069-1076. [Abstract/Free Full Text]
  30. Townsend, M. J., A. S. Weinmann, J. L. Matsuda, R. Salomon, P. J. Farnham, C. A. Biron, L. Gapin, L. H. Glimcher. 2004. T-bet regulates the terminal maturation and homeostasis of NK and V{alpha}14i NKT cells. Immunity 20: 477-494. [Medline]
  31. Kaplan, M. H., A. L. Wurster, S. T. Smiley, M. J. Grusby. 1999. Stat6-dependent and -independent pathways for IL-4 production. J. Immunol. 163: 6536-6540. [Abstract/Free Full Text]
  32. Sherman, M. A., V. H. Secor, S. K. Lee, R. D. Lopez, M. A. Brown. 1999. STAT6-independent production of IL-4 by mast cells. Eur. J. Immunol. 29: 1235-1242. [Medline]
  33. Sherman, M. A., D. R. Powell, D. L. Weiss, M. A. Brown. 1999. NF-ATc isoforms are differentially expressed and regulated in murine T and mast cells. J. Immunol. 162: 2820-2828. [Abstract/Free Full Text]
  34. Matsumoto, G., E. Kubota, Y. Omi, U. Lee, J. M. Penninger. 2004. Essential role of LFA-1 in activating Th2-like responses by {alpha}-galactosylceramide-activated NKT cells. J. Immunol. 173: 4976-4984. [Abstract/Free Full Text]
  35. Takahashi, M., K. Ogasawara, K. Takeda, W. Hashimoto, H. Sakihara, K. Kumagai, R. Anzai, M. Satoh, S. Seki. 1996. LPS induces NK1.1+ {alpha}{beta} T cells with potent cytotoxicity in the liver of mice via production of IL-12 from Kupffer cells. J. Immunol. 156: 2436-2442. [Abstract]
  36. Dent, A. L., J. Hu-Li, W. E. Paul, L. M. Staudt. 1998. T helper type 2 inflammatory disease in the absence of interleukin 4 and transcription factor STAT6. Proc. Natl. Acad. Sci. USA 95: 13823-13828. [Abstract/Free Full Text]
  37. Toney, L. M., G. Cattoretti, J. A. Graf, T. Merghoub, P. P. Pandolfi, R. Dalla-Favera, B. H. Ye, A. L. Dent. 2000. BCL-6 regulates chemokine gene transcription in macrophages. Nat. Immunol. 1: 214-220. [Medline]
  38. Brogdon, J. L., D. Leitenberg, K. Bottomly. 2002. The potency of TCR signaling differentially regulates NFATc/p activity and early IL-4 transcription in naive CD4+ T cells. J. Immunol. 168: 3825-3832. [Abstract/Free Full Text]
  39. Kusam, S., L. M. Toney, H. Sato, A. L. Dent. 2003. Inhibition of Th2 Differentiation and GATA-3 Expression by BCL-6. J. Immunol. 170: 2435-2441. [Abstract/Free Full Text]
  40. Gu, Y., A. C. Jasti, M. Jansen, J. E. Siefring. 2005. RhoH, a hematopoietic-specific Rho GTPase, regulates proliferation, survival, migration, and engraftment of hematopoietic progenitor cells. Blood 105: 1467-1475. [Abstract/Free Full Text]
  41. Wahlers, A., M. Schwieger, Z. Li, D. Meier-Tackmann, C. Lindemann, H. G. Eckert, D. von Laer, C. Baum. 2001. Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells. Gene Ther. 8: 477-486. [Medline]
  42. Chang, H. C., S. Zhang, V. T. Thieu, R. B. Slee, H. A. Bruns, R. N. Laribee, M. J. Klemsz, M. H. Kaplan. 2005. PU1 expression delineates heterogeneity in primary Th2 cells. Immunity 22: 693-703. [Medline]
  43. Ho, S. N., D. J. Thomas, L. A. Timmerman, X. Li, U. Francke, G. R. Crabtree. 1995. NFATc3, a lymphoid-specific NFATc family member that is calcium-regulated and exhibits distinct DNA binding specificity. J. Biol. Chem. 270: 19898-19907. [Abstract/Free Full Text]
  44. Macian, F., C. Lopez-Rodriguez, A. Rao. 2001. Partners in transcription: NFAT and AP-1. Oncogene 20: 2476-2489. [Medline]
  45. Macian, F.. 2005. NFAT proteins: key regulators of T-cell development and function. Nat. Rev. Immunol. 5: 472-484. [Medline]
  46. Rao, A., C. Luo, P. G. Hogan. 1997. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15: 707-747. [Medline]
  47. Lee, H. J., N. Takemoto, H. Kurata, Y. Kamogawa, S. Miyatake, A. O’Garra, N. Arai. 2000. GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells. J. Exp. Med. 192: 105-115. [Abstract/Free Full Text]
  48. Lee, G. R., P. E. Fields, R. A. Flavell. 2001. Regulation of IL-4 gene expression by distal regulatory elements and GATA-3 at the chromatin level. Immunity 14: 447-459. [Medline]
  49. Takemoto, N., Y. Kamogawa, H. Jun Lee, H. Kurata, K. I. Arai, A. O’Garra, N. Arai, S. Miyatake. 2000. Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster. J. Immunol. 165: 6687-6691. [Abstract/Free Full Text]
  50. Zhou, M., W. Ouyang. 2003. The function role of GATA-3 in Th1 and Th2 differentiation. Immunol. Res. 28: 25-37. [Medline]
  51. Miaw, S. C., A. Choi, E. Yu, H. Kishikawa, I. C. Ho. 2000. ROG, repressor of GATA, regulates the expression of cytokine genes. Immunity 12: 323-333. [Medline]
  52. Ranger, A. M., M. Oukka, J. Rengarajan, L. H. Glimcher. 1998. Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development. Immunity 9: 627-635. [Medline]
  53. Fowell, D. J., K. Shinkai, X. C. Liao, A. M. Beebe, R. L. Coffman, D. R. Littman, R. M. Locksley. 1999. Impaired NFATc translocation and failure of Th2 development in Itk-deficient CD4+ T cells. Immunity 11: 399-409. [Medline]
  54. Rincon, M., R. A. Flavell. 1997. Transcription mediated by NFAT is highly inducible in effector CD4+ T helper 2 (Th2) cells but not in Th1 cells. Mol. Cell. Biol. 17: 1522-1534. [Abstract]
  55. Zhou, B., R. Q. Cron, B. Wu, A. Genin, Z. Wang, S. Liu, P. Robson, H. S. Baldwin. 2002. Regulation of the murine Nfatc1 gene by NFATc2. J. Biol. Chem. 277: 10704-10711. [Abstract/Free Full Text]
  56. Chuvpilo, S., M. Zimmer, A. Kerstan, J. Glockner, A. Avots, C. Escher, C. Fischer, I. Inashkina, E. Jankevics, F. Berberich-Siebelt, et al 1999. Alternative polyadenylation events contribute to the induction of NF-ATc in effector T cells. Immunity 10: 261-269. [Medline]



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