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The Journal of Immunology, 2007, 178: 58-66.
Copyright © 2007 by The American Association of Immunologists, Inc.

Sensitivity of NK1.1-Negative NKT Cells to Transgenic BATF Defines a Role for Activator Protein-1 in the Expansion and Maturation of Immature NKT Cells in the Thymus1

Alfred J. Zullo*, Kamel Benlagha2,{dagger}, Albert Bendelac{dagger} and Elizabeth J. Taparowsky3,*

* Department of Biological Sciences and Purdue Cancer Center, Purdue University, West Lafayette, IN 47907; and {dagger} Department of Pathology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NKT cells are glycolipid-reactive lymphocytes that express markers and perform functions common to both T lymphocytes and NK cells. Although the genetic events controlling conventional T cell development are well defined, the transcription factors and genetic programs regulating NKT cell development are only beginning to be elucidated. Previously, we described the NKT cell-deficient phenotype of transgenic (Tg) mice constitutively expressing B cell-activating transcription factor (BATF), a basic leucine zipper protein and inhibitor of AP-1. In this study, we show that Tg BATF targets the majority of V{alpha}14J{alpha}281 (V{alpha}14i7) NKT cells, regardless of CD4 expression and Vbeta gene usage. The residual NKT cells in the thymus of BATF-Tg mice are CD44+, yet are slow to display the NK1.1 marker characteristic of mature cells. As a population, BATF-expressing NKT cells are TCRbeta/CD3{epsilon}low, but express normal levels of CD69, suggesting a failure to expand appropriately following selection. Consistent with the sensitivity of NKT cells to BATF-induced changes in AP-1 activity, we detect a full complement of AP-1 basic leucine zipper proteins in wild-type NKT cells isolated from the thymus, spleen, and liver, and show that AP-1 DNA-binding activity and cytokine gene transcription are induced in NKT cells within a few hours of glycolipid Ag exposure. This study is the first to characterize AP-1 activity in NKT cells and implicates the integrity of this transcription factor complex in developmental events essential to the establishment of this unique T cell subset in the thymus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The V{alpha}14i NKT cells are an atypical lymphocyte subset that expresses markers and performs functions common to both conventional T lymphocytes (TCRbeta+) and innate NK cells (NK1.1+). In contrast to conventional T cells, which are MHC restricted and respond to peptide ligands, V{alpha}14i NKT cells interact with glycolipid ligands such as synthetic {alpha}-galactosylceramide ({alpha}-GalCer),4 endogenous iGB3, and bacterially derived glycolipids presented by CD1d (1). Although NKT cells impact a myriad of immune system functions in response to glycolipid ligands (2), V{alpha}14i NKT cells are best known for their rapid and robust production of the Th1 and Th2 cytokines, IFN-{gamma} and IL-4, respectively (2). Consistent with this unique pattern of dual cytokine production, there is evidence supporting a role for NKT cells in the defense against pathogens, the prevention of autoimmune diseases, and the hypersensitivity to airway allergens (3, 4).

The discovery of {alpha}-GalCer (5) and the development of fluorescent {alpha}-GalCer multimers (6, 7, 8) as a tool to detect, stimulate, and purify NKT cells have permitted many laboratories to identify over two dozen genes whose products are essential for the development and function of V{alpha}14i NKT cells. Included in this group are a number of genes encoding transcription factors, including members of the NF-{kappa}B family (9, 10, 11, 12), T-bet (13, 14), Ets-1 (15), Mef (16), Irf-1 (17), Runx (18), and ROR{gamma}t (18, 19). Previously, our laboratory described the NKT cell-deficient phenotype of transgenic (Tg) mice expressing a T cell-specific, p56lck transgene encoding a hemagglutinin (HA) Ag-tagged B cell-activating transcription factor (BATF) (20), an inhibitory component of the AP-1 family of basic leucine zipper (bZIP) transcription factors (21). As predicted, the constitutive expression of BATF in thymocytes results in altered AP-1 DNA-binding activity, reduced expression of a stably integrated AP-1 reporter gene, and the absence of a mitogenic response following stimulation in vitro (20, 22, 23). Interestingly, whereas conventional T cell development remains intact in these animals, thymic and peripheral TCRbeta+NK1.1+ NKT lymphocytes are dramatically underrepresented in BATF-Tg mice (20). The selective sensitivity of the NKT cell lineage to the perturbation of AP-1 activity by BATF was an intriguing result that clearly warranted further investigation.

In this study, we extend our analysis of BATF-Tg mice to show that expression of the p56lck-HA-BATF transgene influences the majority of {alpha}-GalCer-reactive V{alpha}14i NKT cells, including the predominant Vbeta8 and Vbeta7 subsets and both CD4+ and double-negative NKT cells. Maturation analysis using CD1d-{alpha}-GalCer tetramers with anti-CD44 and anti-NK1.1 Abs revealed that as early as 3 wk of age, BATF-Tg NKT cells are reluctant to adopt a mature, NK1.1-positive phenotype. In addition, the NKT cells populating in the thymus of BATF-Tg mice show a bias toward being TCRlow, express reduced levels of CD3{epsilon}, but express normal levels of CD69. Undoubtedly, the lack of NKT cells in the thymus of BATF-Tg mice most likely reflects the ability of BATF, as an AP-1 inhibitor, to restrict cell growth and precursor expansion. Interestingly, the Tg NKT cells that do complete maturation in the thymus persist throughout the life of the animals as stable, BATF-expressing populations in the spleen and the liver. These observations prompted us to characterize the AP-1 activity that functions downstream of V{alpha}14i TCR signaling in wild-type NKT cells and NKT cell hybridomas. We show that NKT cells possess transcripts for the major Jun and Fos family proteins, and that, within hours of stimulation, they display the characteristic induction of sequence-specific DNA binding associated with AP-1 transcriptional activity. Furthermore, this induction of AP-1 DNA binding is correlated temporally with both the rate-limiting presentation of glycolipid Ag and the enhanced transcription of IL-4, a well-established AP-1 target gene (24, 25). Taken together, these experiments are the first to demonstrate that the AP-1 transcription complex is an endogenous regulator of NKT cell gene expression, and that the proper in vivo regulation of this activity is critical during NKT cell development within the thymus.


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

The generation of p56lck-HA-BATF (BATF-Tg) mice has been described previously (20). BATF-Tg mice were backcrossed eight generations and were maintained on the C57BL/6 background. CD1d–/– and J{alpha}18–/– mice (26, 27, 28, 29) were obtained from R. Brutkiewicz (Indiana University School of Medicine, Indianapolis, IN). V{alpha}14-Tg mice, on a C57BL/6 genetic background (30), were obtained from L. Van Kaer (Vanderbilt University, Nashville, TN). All mice were housed in the pathogen-free conditions of the Purdue University Transgenic Mouse Core Facility, according to institutional guidelines. All procedures involving these animals have been reviewed and approved by the Purdue University Animal Care and Use Committee. Unless otherwise noted in the figure legends, all experiments were performed using age- and sex-matched animals between 6 and 12 wk of age.

Cell culture

The isolation and culturing of primary T lymphocytes have been described previously (22). The DN32.D3 (V{alpha}14+) and 431.A11 (V{alpha}14–) NKT cell hybridomas have been described (31, 32) and were maintained at a density no greater than 1 x 106 cells/ml in IMDM, 10% heat-inactivated FBS, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

Antibodies

The following Abs used in this study were purchased from BD Biosciences: anti-mouse IgG PE (A85-1), Fc block (2.4G2), anti-Vbeta8.1/8.2 FITC (MR5-2), anti-Vbeta7 FITC (TR310), anti-HSA FITC (M1/69), anti-CD4 FITC, anti-CD4 PE (RM45), anti-CD8 FITC, anti-CD8 PerCP (53.6.7), anti-TCRbeta FITC, anti-PE-Cy5 (H57-597), anti-NK1.1 PE, anti-NK1.1 PE-Cy7 (PK136), anti-CD3{epsilon} PE-Cy5 (145-2C11), anti-CD44 PE- Cy5 (IM7), and anti-CD69 PE Cy7 (H1.2F3).

Staining with CD1d-Ig-{alpha}-GalCer dimers and tetramers

Dimeric CD1d-Ig (BD Biosciences) was loaded at neutral pH, according to manufacturers’ instructions, using a 40-fold molar excess of {alpha}-GalCer (Axxora). The loaded dimers were labeled by incubating for 1 h at room temperature with PE anti-mouse IgG, followed by unlabeled mouse IgG. The amount used for staining was determined experimentally for each batch of dimers prepared. For staining, 1 x 106 cells were resuspended in 50 µl of PBS, 2% FBS, and were incubated for 15 min at 4°C with 1 µg of Fc block, followed by 1.5 h at 4°C with prepared dimers. For costaining, Abs were added for another 30-min incubation, after which time the cells were washed twice in staining buffer, once in PBS, and resuspended in PBS for analysis by flow cytometry using a Coulter FC500 (Beckman Coulter). PE-CD1d-{alpha}-GalCer tetramers were prepared and used to stain thymocytes and splenocytes, according to the procedures described by Benlagha et al. (6, 33).

Analysis of gene expression in NKT cells

Thymic (CD1d-Ig-{alpha}-GalCer+TCRbeta+), splenic (TCRbeta+NK1.1+), and liver (CD4+NK1.1+) NKT cells from C57BL/6 mice were isolated using a FACS. Cells were recovered in T cell medium, and RNA prepared using TRIzol with 3 µg of yeast tRNA (BD Biosciences) was added as a carrier. As a nonsorted control, RNA was prepared from an equal number of total thymocytes, splenocytes, or liver lymphocytes. One microgram of each RNA sample was converted to cDNA, and the levels of C{alpha} and V{alpha}14J{alpha}281 transcripts were determined by PCR, as described (27). Fifteen microliters of the same cDNA prep was used with a custom-designed SuperArray PCR kit (SuperArray) to detect the transcripts for the indicated AP-1 components, Gapdh, and the NKT cell-enriched T-bet and NK1.1 transcripts. For the analysis of {alpha}-GalCer-induced IL-4 expression, 1 x 107 thymocytes and splenocytes from non-Tg, CD1d–/–, and BATF-Tg mice were cultured for 18 h at 37°C, 5% CO2 in the presence of 1 µg of {alpha}-GalCer. RNA was prepared, converted to cDNA, and analyzed for HA-BATF and C{alpha} transcripts by PCR. The following primers were used to detect IL-4: forward, 5'-GATGGATGTGCCAAACGTCC-3'; reverse, 5'-CATGATGCTCTTTAGGCTTTCC-3'. For the time course experiments, parallel cultures of 1 x 107 thymocytes or splenocytes were rested overnight and then left unstimulated, or treated with 1 µg of {alpha}-GalCer or vehicle as a control. RNA harvested at the indicated time points was analyzed for IL-4 and C{alpha} transcripts by PCR. The number of cycles used was adjusted to allow inducible IL-4 gene expression to be distinguished from the basal transcript levels that are characteristic of resting NKT cells (34, 35, 36). All PCR-generated DNA products were resolved by 3% agarose gel electrophoresis and visualized with ethidium bromide.

Intracellular staining of HA-BATF protein

Splenocytes from non-Tg and BATF-Tg mice were isolated and stained, as described in Thornton et al. (23). Briefly, splenocytes were blocked with 2.4G2, surface stained with anti-TCRbeta PE-Cy5 and anti-NK1.1 PE-Cy7, and fixed, as recommended by eBioscience. The cells were permeabilized using eBioscience permeabilization buffer and were incubated with anti-HA FITC clone 3F10 (Roche) to detect intracellular HA-BATF. After extensive washing in PBS, the cells were analyzed by flow cytometry. NKT cells (TCR+NK1.1+), NK cells (TCR–NK1.1+), and conventional T cells (TCR+NK1.1–) were identified, and levels of intracellular HA-BATF (anti-HA FITC) fluorescence were measured for each population.

Analysis of V{alpha}14i-induced AP-1 DNA-binding activity

CD1d-Ig-{alpha}-GalCer dimers or anti-CD3{epsilon} at a concentration of 2 µg/ml in PBS were added to 60-mm tissue culture dishes and incubated at 37°C for 2 h (dimers) or overnight (CD3) to allow plate binding. Plates were washed three times with PBS before adding 1 x 107 hybridoma or primary cells in 5 ml of their respective medium. Cultures were incubated for 6 h, after which nuclear extracts were prepared using the NE-PER Kit (Pierce) and protein was quantified using the bicinchoninic acid protein assay (Bio-Rad). The procedure for EMSA using a 32P-labeled AP-1 oligonucleotide probe has been described previously (23). To assay APC-induced, AP-1 activity, 5 x 106 thymocytes prepared from C57BL/6 mice were incubated overnight at 37°C with 1 µg of {alpha}-GalCer or vehicle control (0.5% Tween 20, PBS). On the following day, 5.0 x 106 DN32.D3 (V{alpha}14i+) or 431.A11 (V{alpha}14i–) cells were added, and after 6 h, nuclear extracts were prepared and EMSA was performed, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HA-BATF targets all classes of V{alpha}14i NKT cells

p56lck-HA-BATF-Tg mice express an HA-tagged, human BATF protein from the constitutive, T cell-specific p56lck proximal promoter (20, 22). Previous studies have shown that all T cell subsets within the thymus of BATF-Tg mice express the transgene (23), yet the only thymic T cell population that is underrepresented in these animals is the TCR+NK1.1+ (NKT) cells (20). To firmly establish that the thymus of BATF-Tg mice contains reduced numbers of glycolipid-reactive V{alpha}14i NKT cells, primary thymocytes were stained with either anti-CD8, anti-TCRbeta, or anti-CD44, and dimeric CD1d loaded with the synthetic NKT cell Ag, {alpha}-GalCer. As shown in Fig. 1A, thymocytes from BATF-Tg mice exhibited a marked reduction in {alpha}-GalCer-reactive NKT cells compared with non-Tg control animals. Thymocytes from J{alpha}18–/– mice, which lack the J{alpha}18 cassette needed to form the V{alpha}14i NKT cell TCR, and CD1d–/– mice, which lack the ability to positively select NKT cell development in the thymus, were included as controls. Although BATF-Tg mice possess reduced numbers of thymic NKT cells, the NKT cell deficiency is not as severe as in J{alpha}18–/– or CD1d–/– mice, in which the NKT cell lineage fails to develop (Fig. 1A and data not shown). Consistent with the observation that residual NKT cells are present in the thymus of BATF-Tg mice, semiquantitative RT-PCR on total RNA purified from thymocytes stimulated in vitro with {alpha}-GalCer revealed a level of IL-4 transcription that is dramatically less than non-Tg, but above the level in CD1d–/– mice (Fig. 1B). We conclude that Tg expression of BATF, an in vivo inhibitor of AP-1 transcriptional activity (21, 22), profoundly impacts the population of {alpha}-GalCer-reactive NKT cells in the thymus.


Figure 1
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  		FIGURE 1. HA-BATF impacts all classes of thymic V{alpha}14i NKT cells. A, Thymocytes were isolated from two non-Tg, two BATF-Tg, and two J{alpha}18–/– mice; stained with CD1d-Ig-{alpha}-GalCer dimers, anti-CD8, anti-TCR, and anti-CD44; and analyzed by flow cytometry to quantify the percentage of NKT cells. Representative plots identifying dimer+CD44+ NKT cells (inscribed circles) in each sample are shown. The percentages quantifying NKT cells (±SD, where possible) represent the averages from three independent experiments performed in duplicate, with the exception of the J{alpha}18–/– control, which was performed once. B, Thymocytes from non-Tg, CD1d–/–, and BATF-Tg mice were cultured for 18 h in the presence of {alpha}-GalCer. RT-PCR was used to detect IL-4, HA-BATF, and C{alpha} transcripts. Parallel reactions without RNA (no RNA) are included as controls. The experiment was repeated a second time, and similar results were obtained. C, Thymocytes from one non-Tg and one BATF-Tg mice were stained with dimers and anti-Vbeta8.1/8.2 or anti-Vbeta7 Abs. Dimer+ Vbeta8.1/2+ (upper) and dimer+ Vbeta7+ (lower) cells are identified by the inscribed squares on the representative plots. Similar results were obtained using two additional mice and anti-Vbeta7, anti-Vbeta8, and anti-NK1.1 Abs. D, Thymocytes from one non-Tg and one BATF-Tg mouse were stained with dimers, anti-CD4, and anti-CD8. After gating on CD8low/negative cells, the indicated percentages of double-negative (left inscribed square) and CD4 single-positive (right inscribed square) NKT cells were determined.

 
Although {alpha}-GalCer-reactive NKT cells show invariant expression of the V{alpha}14J{alpha}281 (V{alpha}14i) TCR{alpha} chain, NKT cells pair this {alpha}-chain predominantly with either Vbeta8 or Vbeta7. Additionally, some V{alpha}14i cells are CD4+, whereas others are CD4–CD8–. Recently, it has been shown that these V{alpha}14i subtypes possess different patterns of cytokine expression (37, 38) and, upon migration to the periphery, orchestrate different immune system functions (39). To establish whether constitutive BATF expression targets discrete NKT cell subsets within the thymus, T cells from non-Tg and BATF-Tg mice were stained with dimeric CD1d-Ig-{alpha}-GalCer and Abs against Vbeta8.1/8.2, Vbeta7, CD4, and CD8. Results show that the Vbeta8+, Vbeta7+, CD4+, and CD4–CD8– NKT subtypes are all decreased in BATF-Tg mice (Fig. 1, C and D).

We extended our examination of V{alpha}14i NKT cells in BATF-Tg and non-Tg mice using anti-CD8 and {alpha}-GalCer tetramers (Fig. 2A) and anti-Vbeta8 or anti-Vbeta7 Abs with anti-NK1.1 (Fig. 2B) to detect NKT cells in the spleen. The results parallel what was observed in the thymus, leading us to conclude that BATF acts globally to impact the development of all classes of V{alpha}14i NKT cells in the thymus and in the periphery.


Figure 2
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  		FIGURE 2. Splenic V{alpha}14i NKT cells are decreased in BATF-Tg mice. A, Splenocytes from two non-Tg and two BATF-Tg mice were stained with {alpha}-GalCer tetramers and anti-CD8 and analyzed by flow cytometry. NKT cells are identified by the inscribed squares. The average percentage (±SD) was calculated from three independent experiments. B, Splenocytes from two non-Tg and two BATF-Tg mice were stained with anti-Vbeta7, anti-Vbeta8.1/2, and anti-NK1.1 Abs, and analyzed by flow cytometry. NKT cells were identified (inscribed squares) with the percentages representing the average obtained from two, independent experiments.

 
NKT cell maturation is delayed in BATF-Tg mice

In response to positive selection by CD1d and glycolipid ligands (18, 19, 40, 41, 42), V{alpha}14i NKT cells undergo a series of expansion and maturation events that correlate first with the expression of the activation/memory marker, CD44, and later with the coexpression of NK markers such as NK1.1 (33, 43, 44). To determine how HA-BATF might impact NKT cell maturation, flow cytometry with CD1d-{alpha}-GalCer tetramers was used to track the emergence of CD44–NK1.1–, CD44+NK1.1–, and CD44+NK1.1+ NKT cells over time. Consistent with published reports (33), 70% of the tetramer-positive cells in control, non-Tg mice coexpress CD44 and NK1.1 as early as 3 wk of age, and this percentage increases to 85% by 12 wk of age (Fig. 3). In contrast, thymic NKT cells from BATF-Tg mice express CD44+, but do not transition efficiently to a CD44+NK1.1+ phenotype, even by 12 wk of age (Fig. 3). As a result, the maturation profile of BATF-Tg NKT cells undergoes minimal change between 3 and 12 wk of age. The reduced numbers of all classes of NKT cells in BATF-Tg mice, coupled with the relative inability of the BATF-expressing cells to adopt a CD44+NK1.1+ profile, demonstrate that both NKT cell expansion and maturation are impaired in these animals.


Figure 3
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  		FIGURE 3. NKT cell expansion and maturation are impaired in BATF-Tg mice. Thymocytes from non-Tg and BATF-Tg mice at 3 wk (A), 6 wk (B), and 12 wk (C) of age were stained with CD1d-{alpha}-GalCer tetramers, anti-CD8, anti-CD44, anti-HSA, and anti-NK1.1. NKT cells were identified as tetramer+CD8low/negative or tetramer+HSAlow/negative cells (inscribed squares, left panels) and then were reanalyzed for expression of CD44 and NK1.1 (right panels). This experiment was performed three times for A, two times for B, and a single time for C. For each age and genotype, the average percentages (±SD, where possible) are noted for total NKT cells (left), and for the CD44–NK1.1–, CD44+NK1.1–, and CD44+ NK1.1+ NKT cell subsets (right).

 
The residual V{alpha}14i NKT population in BATF-Tg mice is TCR/CD3low and CD69+

It was noted during our analysis of V{alpha}14i NKT cells from BATF-Tg mice using CD1d-Ig-{alpha}-GalCer dimers and tetramers that the level of staining displayed by these cells was consistently less intense than that of control cells. This observation suggested that BATF-Tg NKT cells express reduced quantities of the {alpha}-GalCer-reactive TCR and its associated proteins. To investigate this further, NKT cells (dimer+ CD8low/negative) from non-Tg and BATF-Tg mice were identified and then quantified based on staining intensity as dimerlow, dimermedium, or dimerhigh. Results show that 56% of the residual NKT cells present in the thymus of BATF-Tg mice are dimerlow and only 7% of the cells are dimerhigh (Fig. 4A). This contrasts with the profile of normal NKT cells, which show that 44% are dimerlow and 14% are dimerhigh. To validate these results, thymocytes from non-Tg and BATF-Tg mice were stained with CD1d-Ig-{alpha}-GalCer, anti-TCRbeta, and anti-CD3{epsilon}, and the V{alpha}14i NKT cells (dimer+TCRbeta+) were reanalyzed for CD3{epsilon} staining intensity (Fig. 4B). Consistent with reduced levels of the {alpha}-GalCer-reactive TCR, BATF-Tg NKT cells show a 28% reduction in CD3{epsilon} mean fluorescence intensity compared with control cells. The reduced level of surface TCR expression on these cells does not correlate with the inability of cells to receive, or to interpret, positive selection signals. As reported previously, BATF-Tg thymocytes express normal levels of CD1d (20), and the cells were responsive initially to Ag presentation, as CD69 expression is equivalent to that of non-Tg controls (Fig. 4C). These findings support the previous observation that BATF-Tg NKT cells acquire expression of CD44 and thus are committed to the NKT cell lineage (Fig. 2).


Figure 4
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  		FIGURE 4. Residual NKT cells in the thymus of BATF-Tg mice are TCR/CD3low and CD69 positive. A, Thymocytes from two non-Tg (black) and two BATF-Tg (red) mice were stained with CD1d-Ig-{alpha}-GalCer dimers and anti-CD8. Dimer+CD8low/negative were replotted to compare the distribution of NKT cells exhibiting low (lo), medium (med), and high (hi) staining intensity. Brackets denote the range of cells counted. The percentages indicated for non-Tg and BATF-Tg NKT cells falling within each range represent the average of results obtained from two, independent experiments. B, Thymocytes from two non-Tg (black) and two BATF-Tg (red) mice were stained with dimers, anti-TCRbeta, and anti-CD3{epsilon}. Dimer–TCRbeta+ T cells (right panel) and dimer+TCRbeta+ NKT cells (left panel) were reevaluated for CD3{epsilon} expression. Mean fluorescence intensity (MFI) was determined, and the results are presented as the average MFI. C, Thymocytes from one non-Tg and two BATF-Tg mice were stained with dimers, anti-CD8, and anti-CD69. The percentage of CD69+ Tg NKT cells is indicated on each plot.

 
NKT cells in the spleen of BATF-Tg mice are stable over time

To examine whether peripheral NKT cells in BATF-Tg mice are persistently low, decline further, or perhaps recover over time, flow cytometry with NK1.1 and TCRbeta Abs was used to establish the percentages of NKT cells in the spleens of BATF-Tg and non-Tg mice at 12 wk, 8 mo, 10 mo, and 1 year of age. As controls, the profiles of splenic NKT cells from CD1d–/– (26, 27, 28) mice and Tg mice biased for overproduction of NK1.1+ V{alpha}14i NKT cells (V{alpha}14-Tg) (30) were used. As shown in Fig. 5A, there is no significant change in the NKT cell-deficient phenotype of BATF-Tg mice over time. This result appears to conflict with a previous study in which it was shown that NKT cells will divide to fill a deficient niche (45). However, using the same Abs to purify peripheral NK (NK1.1+TCRbeta–), NKT (NK1.1+TCRbeta+), and T (NK1.1–TCRbeta+) cells and an anti-HA Ab to detect HA-BATF protein, we show that the p56lck-HA-BATF transgene continues to be expressed in >95% of peripheral T and NKT cells (Fig. 5B), suggesting that BATF continues to exert its negative influence on the dynamics of NKT cell expansion in the periphery. Additional factors possibly contributing to the maintenance of a stable NKT cell deficiency in BATF-Tg animals will be considered in Discussion.


Figure 5
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  		FIGURE 5. Peripheral NKT cells in BATF-Tg mice express HA-BATF and are stable over time. A, Splenocytes from non-Tg and BATF-Tg animals of the indicated ages, as well as splenocytes from control CD1d–/– and V{alpha}14-Tg animals at 12 wk of age, were stained with anti-TCRbeta and anti-NK1.1. TCRbeta+ NK1.1+ cells were identified by flow cytometry, and the average percentages for each genotype at each age are presented as a bar graph. B, Splenocytes from non-Tg and BATF-Tg mice (left panels) were stained with anti-TCRbeta and anti-NK1.1. After washing, fixation, and permeabilization, the cells were stained with anti-HA FITC. After identifying NK cells (NK1.1+ TCRbeta–), NKT cells (TCRbeta+ NK1.1+), and T cells (TCRbeta+ NK1.1–), the level of anti-HA fluorescence (right panels) was determined. The percentages represent the number of cells expressing HA-BATF obtained from the analysis of five BATF-Tg and five non-Tg mice.

 
AP-1 components and activity are present in NKT cells

BATF inhibits AP-1 activity by forming heterodimers with the Jun proteins and displacing the canonical Jun partners (i.e., Jun or Fos proteins) associated with the transcriptional activation of AP-1 target genes (21, 46, 47). Because constitutive expression of BATF disrupts the development of NKT cells, the mechanism underlying this defect most likely involves the Jun and Fos family members that are expressed in NKT cells. To characterize the AP-1 factors that are expressed in NKT cells, a FACS was used to purify NKT cells from the thymus, spleen, and liver of C57BL/6 mice using marker combinations (dimer+TCR+; TCR+NK1.1+; CD4+NK1.1+) validated previously for the isolation of NKT cells from these tissues (6, 7, 48). RNA was prepared from the sorted cells and from equivalent numbers of unsorted cells prepared from the same tissues. To manipulate the low RNA yield from small numbers of purified NKT cell samples without significant losses, 3 µg of carrier yeast tRNA was added to each sample and subsequently used in all assays as a control (data not shown). RT-PCR with primers specific for the transcripts of six major Jun and Fos family genes was performed initially with RNA prepared from PMA- and ionomycin-treated mouse EL-4 cells, in which all six genes are expressed (49) (data not shown), and then on each of the NKT cell samples. Detection of transcripts representing the V{alpha}14J{alpha}281 TCR{alpha} chain, the T-bet transcription factor, and the NK1.1 surface marker was used as NKT cell expression controls. GAPDH, C{alpha}, or beta-actin primers were used as a control for RNA integrity. As shown in Fig. 6, all three Jun and two of the three Fos genes are expressed in NKT cells.


Figure 6
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  		FIGURE 6. AP-1 factors are present in wild-type NKT cells. A, 1 x 104 dimer+TCRbeta+ cells from thymus; B, 7 x 104 TCRbeta+NK1.1+cells from spleen; and C, 2 x 104 CD4+NK1.1+ cells from liver were identified (left flow cytometry plots, inscribed circles) and purified using a FACS. RNA isolated from these purified NKT cells and from an equivalent number of nonsorted cells from the same tissues (total) was used with a custom SuperArray PCR kit to detect transcription of the indicated AP-1 family member genes and the T-bet, NK1.1, and GAPDH genes as controls. Standard, semiquantitative RT-PCR was used with the same RNA samples to detect transcripts from the V{alpha}14i TCR gene, as a unique identifier of NKT cells, and from either the C{alpha} or beta-actin genes as a control for sample integrity. The gels shown in each panel are independent experiments, with NKT cells (in each experiment) purified from pools of 5–10 C57BL/6 mice.

 
The presence of AP-1 component transcripts in purified NKT cells should correlate with the production of Jun and Fos proteins and with the presence of AP-1 DNA-binding activity. Because the phenotype of BATF-Tg mice suggests that the proper activation of AP-1 target genes is required for NKT cell responses, we predict that NKT cells stimulated through the V{alpha}14i TCR should have enhanced AP-1 activity, which is most reliably assessed by detecting an increase in AP-1 DNA binding. To test this prediction, EMSA were performed with nuclear extracts and a radiolabeled, double-stranded oligonucleotide containing an AP-1 consensus site. A pair of NKT cell hybridomas, DN32.D3 (V{alpha}14i+) and 431.A11 (V{alpha}14i–), was used as the source of nuclear extracts following a 6-h exposure to plate-bound CD1d-Ig-{alpha}-GalCer. As controls, the hybridomas were cultured on plates coated with anti-CD3{epsilon} Ab (positive) or empty CD1d-Ig (negative). Resolution of the DNA:protein complexes demonstrates a clear increase in AP-1 DNA-binding activity in DN32.D3 cells (Fig. 7A), but not in 431.A11 cells, indicating that AP-1 induction by {alpha}-GalCer depends on the V{alpha}14i TCR. The experiment also was performed with nuclear extracts prepared from total splenocytes stimulated in the same manner (Fig. 7B). As expected, due to the reduced target cell number, {alpha}-GalCer-stimulated AP-1 activity is not as prominent as in the DN32 cell line, but is increased compared with the empty CD1d-Ig control. These results clearly indicate that AP-1 DNA-binding activity is present in NKT cells and is induced by signaling downstream of the glycolipid-reactive V{alpha}14i TCR.


Figure 7
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  		FIGURE 7. AP-1 DNA-binding activity and IL-4 gene expression in NKT cells. A, DN32.D3 and A431.A11 cells were cultured without stimulation (non) or with plate-bound anti-CD3{epsilon} (CD3), dimeric CD1d-Ig (empty), or dimeric CD1d-Ig-{alpha}-GalCer ({alpha}-GalCer). After 6 h, nuclear extracts were prepared and used in EMSA with 32P-labeled AP-1 DNA (see Materials and Methods for details). B, Nuclear extracts prepared from total splenocytes exposed for 6 h to the indicated stimuli were used for EMSA, as described in A. C, DN32.D3 and 431.A11 cells were cultured for 6 h together with wild-type C57BL/6 thymocytes that had been stimulated overnight with 1 µg of {alpha}-GalCer or solvent (vehicle) as a control. Nuclear extracts were prepared and analyzed for AP-1 DNA-binding activity, as described in A. D, Thymocytes and splenocytes from wild-type C57BL/6 mice were cultured in the absence (non) or presence of solvent (vehicle) or with {alpha}-GalCer for the indicated times (hr). RNA was prepared from the cultures and used in standard semiquantitative RT-PCR to evaluate levels of IL-4 gene expression (see Materials and Methods for details). RT-PCR to detect C{alpha} transcripts are included as the controls. The gels shown are representative of results obtained from two, independent experiments.

 
The ability of TCR signaling to efficiently activate a transcription factor within a given time frame is dependent upon the kinetics of Ag presentation by APCs. To confirm that thymocytes, an APC population relevant to the development of V{alpha}14i NKT cells in vivo, can present glycolipid that induces AP-1 activity in NKT cells within 6 h, thymocytes from wild-type mice were incubated with {alpha}-GalCer overnight. The following day, either DN32.D3 (V{alpha}14i+) or 431.A11 (V{alpha}14i–) cells were added to the cultures for 6 h and nuclear extracts were prepared for use in EMSA. As shown in Fig. 7C, {alpha}-GalCer-pulsed thymocytes caused a significant increase in the AP-1 DNA-binding activity in DN32.D3 cells. In contrast, the AP-1 activity in 431.A11 cells was unchanged following exposure to {alpha}-GalCer-pulsed thymocytes. As predicted, the presentation of {alpha}-GalCer by thymocytes isolated from CD1d–/– mice had no effect on the AP-1-binding activity in V{alpha}14i+ DN32.D3 cells (data not shown).

As a demonstration that these EMSA results correlate with the kinetics of V{alpha}14i NKT cell activation, thymocytes and splenocytes were treated with {alpha}-GalCer. At various time points following treatment, the cells were harvested and RNA was prepared. RT-PCR was used to track changes in the expression of IL-4, a well-characterized, AP-1 target gene (24, 25) that encodes a cytokine that is produced by NKT cells within 90 min of stimulation (50). For these experiments, PCR cycle number was reduced to distinguish induced IL-4 mRNA expression from the basal levels of IL-4 mRNA expressed by resting NKT cells (34, 35, 36). As shown in Fig. 7D, after only 1 h of exposure to {alpha}-GalCer, both splenocytes and thymocytes show a marked increase in IL-4 transcription. This induced expression peaked between 2 and 4 h and remained high throughout the 6-h duration of the experiment. These results are consistent with what is known about cytokine production by NKT cells and correlate well with the induction of AP-1 DNA binding downstream of the V{alpha}14i TCR (Fig. 7, A–C). Therefore, we conclude that TCR-induced AP-1 activity is linked temporally to NKT cell gene expression. Furthermore, perturbation of AP-1 activity (through the constitutive expression of BATF) identifies AP-1 as a critical regulator of the earliest stages of NKT cell development within the thymus.


   Discussion
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 Abstract
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 Materials and Methods
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 Discussion
Disclosures
 References
 
Previously, our laboratory reported the NKT cell-deficient phenotype of Tg mice expressing a T cell-restricted transgene encoding BATF, a negative regulator of the AP-1 family of bZIP transcription factors. BATF dimerizes with the Jun proteins to generate sequence-specific DNA-binding complexes with little to no trans- activation potential and promotes a slow growth phenotype that has been linked to the inhibition of mitogenic signaling (21, 23) and to the initiation of cellular differentiation (51). Our observation that constitutive expression of BATF in vivo results in a NKT-specific phenotype, rather than the anticipated, broad impact on proliferation and signaling in all T lymphocytes, warranted further investigation.

In the current work, we extend our analysis of BATF-Tg mice to show that BATF expression results in reduced numbers of all {alpha}-GalCer-reactive NKT cells, regardless of Vbeta gene usage or CD4 coexpression. This indicates that the effects of BATF are not dependent on particular ligand affinities or on the specific functionalities described recently for distinct NKT cell subsets (37, 38, 39). Fortunately, the V{alpha}14i NKT cell-deficient phenotype of BATF-Tg mice is not completely penetrant, thus providing us with an opportunity to examine the properties of the residual NKT cells found in the thymus and periphery.

Using the CD44 and NK1.1 markers to follow the developmental profile of thymic NKT cells in BATF-Tg mice, we found that maturation of these cells was delayed. In addition, the residual NKT cell population in the thymus of BATF-Tg mice had a clear bias toward reduced surface expression of both the V{alpha}14i TCR and the associated signaling subunit CD3{epsilon}. Following normal activation, NKT cells naturally lack surface TCR expression for a period of time (52, 53, 54, 55). However, a reduction in surface TCR expression during T cell development also can be diagnostic of aberrant signal transduction during positive selection (56). In this regard, our observation that BATF-Tg NKT cells express normal levels of CD69 and then, ultimately, CD44, indicates that the earliest presentation and signaling events are taking place. Therefore, it appears that expression of BATF skews the NKT cells that eventually emerge toward a population with a reduced avidity for ligand. Although these cells do develop over time, they progress with delayed kinetics, as might be predicted for cells expressing an AP-1 inhibitor. The mechanism underlying this skewing is unknown, although recent data from our lab show that {alpha}-Gal-Cer-reactive thymic NKT cells from BATF-Tg mice display increased staining with annexin V, an indication that apoptosis might play a role (A. Zullo and E. Taparowsky, unpublished data).

There is a clear, negative effect of HA-BATF expression on the development of the NKT cell lineage in the thymus. In addition, we have shown that the BATF transgene continues to be expressed in the periphery, where the NKT cells that complete maturation are maintained as a small, but stable, population of cells. The observation that the NKT cells in the spleens of BATF-Tg mice fail to expand over time is consistent with the documented negative influence of BATF on cell growth (21, 23). However, we have recent evidence that BATF-Tg splenocytes contain twice the number of CD8+ lymphocytes coexpressing the activation/memory marker, CD44 (A. Zullo and E. Taparowsky, unpublished data). These peripheral memory-type CD8+ cells have been shown by Matsuda et al. (45) to engage in direct and rigorous competition with both NK and NKT cells for the cytokine IL-15, which is required for both NK and NKT cells to expand following stimulation. In support of this, we have noted in all of our studies that NK cells (TCR–NK1.1+), which are negative for expression of the HA-BATF transgene (Fig. 5B), are less abundant in BATF-Tg mice than in control mice (20) (Figs. 2B and 5B). Clearly, understanding why the inhibition of AP-1 activity by BATF leads to an increase in memory-type, CD8+CD44+ cells is an interesting future avenue of research. Additionally, future studies using BATF-Tg animals to examine the responsiveness of peripheral V{alpha}14i NKT cells to glycolipid Ag must take into account potential effects generated from the increased presence of these potent IL-15 consumers.

Based upon the phenotype of BATF-Tg mice, we predicted that AP-1 family bZIP proteins would be expressed in NKT cells and that increased DNA binding by AP-1 complexes would be observed in response to V{alpha}14i TCR signaling. Indeed, purified NKT cells possess transcripts for five of the six AP-1 factors examined, including the c-Fos and c-Jun transcripts that were detected in a recent profiling of NKT cell transcripts using microarrays (14). Most importantly, we detect expression of all three Jun genes, indicating that NKT cells possess sufficient quantities of the bZIP proteins that are the preferred dimerization partners for BATF (46, 57, 58). In this regard, endogenous BATF mRNA is expressed in purified NKT cells (data not shown), although at much reduced levels compared with the level expressed from the p56lck-HA-BATF transgene (23). Within 6 h of exposure of NKT cells to {alpha}-GalCer presented either by dimers or by primary APCs, there is a substantial increase in the binding of AP-1 complexes to DNA. The timing of this induction correlates well with increased expression of IL-4, a well-established AP-1 target gene (24), providing solid evidence to link signaling through the V{alpha}14i TCR with AP-1 activity and a relevant, NKT cell response.

The role of AP-1 in conventional T lymphocyte function has been the focus of intense research for many years. In conventional T cells, signals transduced through both the TCR and CD28 coreceptor collaborate to generate maximum AP-1 transcriptional activity in response to Ag (59). In that way, complete AP-1 activity is a tangible demonstration of the two-signal hypothesis. Although our data demonstrate biochemically that signaling through the V{alpha}14i TCR induces AP-1 activity, recent studies from other groups have shown that NKT cells, like other T cells, rely on signals from additional receptors and pathways (60, 61, 62, 63). The extent to which these other pathways activate AP-1 is not known. Although we plan to continue to use experimental approaches with AP-1 inhibitors to further explore this question, establishing the dependence of any one set of signaling events on AP-1 will be a challenge given the modular nature of the multiple bZIP proteins that comprise this dimeric transcription factor (64) and the fact that vectors designed to target gene knockouts exclusively to the NKT cell lineage are not yet readily available.

In recent years, a number of studies have been focused on NKT cell transcription factor networks to further understand the unique features associated with signaling through the V{alpha}14i TCR. These studies have shown that Runx and ROR{gamma}t function are required for the earliest stages of NKT selection (18, 19), that members of the NF-{kappa}B family are required extrinsically (and in a cell autonomous fashion), throughout NKT cell maturation (9, 10, 11, 12), and that T-bet is essential for production of mature, NK1.1-expressing NKT cells in thymus (13, 14). Although not as precisely defined, the NKT compartment does not develop properly in mice lacking the Ets-1 (15), Mef (16), or Irf-1 (17) transcription factors. In addition, the unique and plastic properties of NKT cell transcription have been exemplified by recent work showing differential use of GATA-3, Stat6, and NF-AT2 by TCR+NK1.1+ NKT cells (48). This further supports the idea that broadly used transcription factor networks, such as AP-1, can be reconfigured in NKT cells to generate new models of gene regulation. As a result of our studies, we now know that the positive action of AP-1 complexes is critical to the establishment and maturation of thymic NKT cells. Ultimately, with continued effort, a complete map of the signal transduction cascades active in all NKT cell subsets will be generated and provide new opportunities to predict the transcriptional output of this important class of T lymphocyte.


   Acknowledgments
 
We thank Mark Kaplan and Randy Brutkiewicz for helpful discussions, and Wendy Garrett for commenting on the manuscript. Additional thanks to J. Hallett and A. Kaufmann of the Purdue Transgenic Mouse Core Facility for coordinating the care and maintenance of the mice used in these studies, and to J. P. Robinson, K. Ragheb, and C. Holdmann of the Purdue Analytical Cytology Resource for their expert technical assistance with flow cytometry and sorting.


   Disclosures
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 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 Public Health Service Award CA78264 (to E.J.T.) and by a special fellowship from the Leukemia and Lymphoma Society (to K.B.). A.J.Z. was a predoctoral fellow supported by Public Health Service Award T32 GM08737 and was a recipient of a Sigma Xi Grant in Aid of Research. Back

2 Current address: Groupe Avenir, Institut National de la Sante et de la Recherche Medicale-U561, Hopital St.-Vincent de Paul, 75014 Paris-France. Back

3 Address correspondence and reprint requests to Dr. Elizabeth J. Taparowsky, Department of Biological Sciences, Hansen Life Science Research Building, 201 South University Street, Purdue University, West Lafayette, IN 47907-2064. E-mail address: ejt{at}bilbo.bio.purdue.edu Back

4 Abbreviations used in this paper: {alpha}-GalCer, {alpha}-galacotsylceramide; BATF, B cell-activating transcription factor; bZIP, basic leucine zipper; HA, hemagglutinin; Tg, transgenic. Back

Received for publication April 24, 2006. Accepted for publication October 18, 2006.


   References
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 Abstract
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 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Brutkiewicz, R. R.. 2006. CD1d ligands: the good, the bad, and the ugly. J. Immunol. 177: 769-775. [Abstract/Free Full Text]
  2. Van Kaer, L.. 2005. {alpha}-Galactosylceramide therapy for autoimmune diseases: prospects and obstacles. Nat. Rev. Immunol. 5: 31-42. [Medline]
  3. Kronenberg, M.. 2005. Toward an understanding of NKT cell biology: progress and paradoxes. Annu. Rev. Immunol. 23: 877-900. [Medline]
  4. Akbari, O., P. Stock, E. Meyer, M. Kronenberg, S. Sidobre, T. Nakayama, M. Taniguchi, M. J. Grusby, R. H. DeKruyff, D. T. Umetsu. 2003. Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity. Nat. Med. 9: 582-588. [Medline]
  5. Natori, T., K. Akimoto, K. Motoki, Y. Koezuka, T. Higa. 1997. Development of KRN7000, derived from agelasphin produced by Okinawan sponge. Nippon Yakurigaku Zasshi 110: (Suppl. 1):63P-68P. [Medline]
  6. Benlagha, K., A. Weiss, A. Beavis, L. Teyton, A. Bendelac. 2000. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J. Exp. Med. 191: 1895-1903. [Medline]
  7. Matsuda, J. L., O. V. Naidenko, L. Gapin, T. Nakayama, M. Taniguchi, C. R. Wang, Y. Koezuka, M. Kronenberg. 2000. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J. Exp. Med. 192: 741-754. [Abstract/Free Full Text]
  8. Schumann, J., R. B. Voyle, B. Y. Wei, H. R. MacDonald. 2003. Cutting edge: influence of the TCR Vbeta domain on the avidity of CD1d:{alpha}-galactosylceramide binding by invariant V{alpha}14 NKT cells. J. Immunol. 170: 5815-5819. [Abstract/Free Full Text]
  9. Nakagawa, K., K. Iwabuchi, K. Ogasawara, M. Ato, M. Kajiwara, H. Nishihori, C. Iwabuchi, H. Ishikura, R. A. Good, K. Onoe. 1997. Generation of NK1.1+ T cell antigen receptor {alpha}beta+ thymocytes associated with intact thymic structure. Proc. Natl. Acad. Sci. USA 94: 2472-2477. [Abstract/Free Full Text]
  10. Sivakumar, V., K. J. Hammond, N. Howells, K. Pfeffer, F. Weih. 2003. Differential requirement for Rel/nuclear factor {kappa}B family members in natural killer T cell development. J. Exp. Med. 197: 1613-1621. [Abstract/Free Full Text]
  11. Stanic, A. K., J. S. Bezbradica, J. J. Park, L. Van Kaer, M. R. Boothby, S. Joyce. 2004. Cutting edge: the ontogeny and function of V{alpha}14J{alpha}18 natural T lymphocytes require signal processing by protein kinase C{theta} and NF-{kappa}B. J. Immunol. 172: 4667-4671. [Abstract/Free Full Text]
  12. Elewaut, D., R. B. Shaikh, K. J. Hammond, H. De Winter, A. J. Leishman, S. Sidobre, O. Turovskaya, T. I. Prigozy, L. Ma, T. A. Banks, et al 2003. NIK-dependent RelB activation defines a unique signaling pathway for the development of V{alpha}14i NKT cells. J. Exp. Med. 197: 1623-1633. [Abstract/Free Full Text]
  13. 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]
  14. Matsuda, J. L., Q. Zhang, R. Ndonye, S. K. Richardson, A. R. Howell, L. Gapin. 2005. T-bet concomitantly controls migration, survival and effector functions during the development of V{alpha}14i NKT cells. Blood 107: 2797-2805. [Medline]
  15. Walunas, T. L., B. Wang, C. R. Wang, J. M. Leiden. 2000. Cutting edge: the Ets1 transcription factor is required for the development of NK T cells in mice. J. Immunol. 164: 2857-2860. [Abstract/Free Full Text]
  16. Lacorazza, H. D., Y. Miyazaki, A. Di Cristofano, A. Deblasio, C. Hedvat, J. Zhang, C. Cordon-Cardo, S. Mao, P. P. Pandolfi, S. D. Nimer. 2002. The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells. Immunity 17: 437-449. [Medline]
  17. Ohteki, T., H. Yoshida, T. Matsuyama, G. S. Duncan, T. W. Mak, P. S. Ohashi. 1998. The transcription factor interferon regulatory factor 1 (IRF-1) is important during the maturation of natural killer 1.1+ T cell receptor-{alpha}beta+ (NK1+ T) cells, natural killer cells, and intestinal intraepithelial T cells. J. Exp. Med. 187: 967-972. [Abstract/Free Full Text]
  18. Egawa, T., G. Eberl, I. Taniuchi, K. Benlagha, F. Geissmann, L. Hennighausen, A. Bendelac, D. R. Littman. 2005. Genetic evidence supporting selection of the V{alpha}14i NKT cell lineage from double-positive thymocyte precursors. Immunity 22: 705-716. [Medline]
  19. Bezbradica, J. S., T. Hill, A. K. Stanic, L. Van Kaer, S. Joyce. 2005. Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+ stage of thymic ontogeny. Proc. Natl. Acad. Sci. USA 102: 5114-5119. [Abstract/Free Full Text]
  20. Williams, K. L., A. J. Zullo, M. H. Kaplan, R. R. Brutkiewicz, C. D. Deppmann, C. Vinson, E. J. Taparowsky. 2003. BATF transgenic mice reveal a role for activator protein-1 in NKT cell development. J. Immunol. 170: 2417-2426. [Abstract/Free Full Text]
  21. Echlin, D. R., H. J. Tae, N. Mitin, E. J. Taparowsky. 2000. B-ATF functions as a negative regulator of AP-1 mediated transcription and blocks cellular transformation by Ras and Fos. Oncogene 19: 1752-1763. [Medline]
  22. Williams, K. L., I. Nanda, G. E. Lyons, C. T. Kuo, M. Schmid, J. M. Leiden, M. H. Kaplan, E. J. Taparowsky. 2001. Characterization of murine BATF: a negative regulator of activator protein-1 activity in the thymus. Eur. J. Immunol. 31: 1620-1627. [Medline]
  23. Thornton, T. M., A. J. Zullo, K. L. Williams, E. J. Taparowsky. 2006. Direct manipulation of activator protein-1 controls thymocyte proliferation in vitro. Eur. J. Immunol. 36: 160-169. [Medline]
  24. Rooney, J. W., T. Hoey, L. H. Glimcher. 1995. Coordinate and cooperative roles for NF-AT and AP-1 in the regulation of the murine IL-4 gene. Immunity 2: 473-483. [Medline]
  25. Macian, F., C. Garcia-Rodriguez, A. Rao. 2000. Gene expression elicited by NFAT in the presence or absence of cooperative recruitment of Fos and Jun. EMBO J. 19: 4783-4795. [Medline]
  26. Smiley, S. T., M. H. Kaplan, M. J. Grusby. 1997. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells. Science 275: 977-979. [Abstract/Free Full Text]
  27. Chen, Y. H., N. M. Chiu, M. Mandal, N. Wang, C. R. Wang. 1997. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity 6: 459-467. [Medline]
  28. Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, L. Van Kaer. 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity 6: 469-477. [Medline]
  29. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, M. Taniguchi. 1997. Requirement for V{alpha}14 NKT cells in IL-12-mediated rejection of tumors. Science 278: 1623-1626. [Abstract/Free Full Text]
  30. Bendelac, A., R. D. Hunziker, O. Lantz. 1996. Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells. J. Exp. Med. 184: 1285-1293. [Abstract/Free Full Text]
  31. Lantz, O., A. Bendelac. 1994. An invariant T cell receptor {alpha} chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4–8– T cells in mice and humans. J. Exp. Med. 180: 1097-1106. [Abstract/Free Full Text]
  32. Bendelac, A., O. Lantz, M. E. Quimby, J. W. Yewdell, J. R. Bennink, R. R. Brutkiewicz. 1995. CD1 recognition by mouse NK1+ T lymphocytes. Science 268: 863-865. [Abstract/Free Full Text]
  33. Benlagha, K., T. Kyin, A. Beavis, L. Teyton, A. Bendelac. 2002. A thymic precursor to the NK T cell lineage. Science 296: 553-555. [Abstract/Free Full Text]
  34. Tanaka, S., J. Tsukada, W. Suzuki, K. Hayashi, K. Tanigaki, M. Tsuji, H. Inoue, T. Honjo, M. Kubo. 2006. The interleukin-4 enhancer CNS-2 is regulated by Notch signals and controls initial expression in NKT cells and memory-type CD4 T cells. Immunity 24: 689-701. [Medline]
  35. Matsuda, J. L., L. Gapin, J. L. Baron, S. Sidobre, D. B. Stetson, M. Mohrs, R. M. Locksley, M. Kronenberg. 2003. Mouse V{alpha}14i natural killer T cells are resistant to cytokine polarization in vivo. Proc. Natl. Acad. Sci. USA 100: 8395-8400. [Abstract/Free Full Text]
  36. 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]
  37. 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]
  38. 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]
  39. Crowe, N. Y., J. M. Coquet, S. P. Berzins, K. Kyparissoudis, R. Keating, D. G. Pellicci, Y. Hayakawa, D. I. Godfrey, M. J. Smyth. 2005. Differential antitumor immunity mediated by NKT cell subsets in vivo. J. Exp. Med. 202: 1279-1288. [Abstract/Free Full Text]
  40. Benlagha, K., D. G. Wei, J. Veiga, L. Teyton, A. Bendelac. 2005. Characterization of the early stages of thymic NKT cell development. J. Exp. Med. 202: 485-492. [Abstract/Free Full Text]
  41. 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]
  42. Gapin, L., J. L. Matsuda, C. D. Surh, M. Kronenberg. 2001. NKT cells derive from double-positive thymocytes that are positively selected by CD1d. Nat. Immunol. 2: 971-978. [Medline]
  43. Pellicci, D. G., K. J. Hammond, A. P. Uldrich, A. G. Baxter, M. J. Smyth, D. I. Godfrey. 2002. A natural killer T (NKT) cell developmental pathway involving a thymus-dependent NK1.1–CD4+ CD1d-dependent precursor stage. J. Exp. Med. 195: 835-844. [Abstract/Free Full Text]
  44. Gadue, P., P. L. Stein. 2002. NK T cell precursors exhibit differential cytokine regulation and require Itk for efficient maturation. J. Immunol. 169: 2397-2406. [Abstract/Free Full Text]
  45. Matsuda, J. L., L. Gapin, S. Sidobre, W. C. Kieper, J. T. Tan, R. Ceredig, C. D. Surh, M. Kronenberg. 2002. Homeostasis of V{alpha}14i NKT cells. Nat. Immunol. 3: 966-974. [Medline]
  46. Dorsey, M. J., H. J. Tae, K. G. Sollenberger, N. T. Mascarenhas, L. M. Johansen, E. J. Taparowsky. 1995. B-ATF: a novel human bZIP protein that associates with members of the AP-1 transcription factor family. Oncogene 11: 2255-2265. [Medline]
  47. Foletta, V. C., D. H. Segal, D. R. Cohen. 1998. Transcriptional regulation in the immune system: all roads lead to AP-1. J. Leukocyte Biol. 63: 139-152. [Abstract]
  48. Wang, Z. Y., S. Kusam, V. Munugalavadla, R. Kapur, R. R. Brutkiewicz, A. L. Dent. 2006. Regulation of Th2 cytokine expression in NKT cells: unconventional use of Stat6, GATA-3, and NFAT2. J. Immunol. 176: 880-888. [Abstract/Free Full Text]
  49. Kim, H. P., W. J. Leonard. 2002. The basis for TCR-mediated regulation of the IL-2 receptor {alpha} chain gene: role of widely separated regulatory elements. EMBO J. 21: 3051-3059. [Medline]
  50. Yoshimoto, T., A. Bendelac, J. Hu-Li, W. E. Paul. 1995. Defective IgE production by SJL mice is linked to the absence of CD4+, NK1.1+ T cells that promptly produce interleukin 4. Proc. Natl. Acad. Sci. USA 92: 11931-11934. [Abstract/Free Full Text]
  51. Senga, T., T. Iwamoto, S. E. Humphrey, T. Yokota, E. J. Taparowsky, M. Hamaguchi. 2002. Stat3-dependent induction of BATF in M1 mouse myeloid leukemia cells. Oncogene 21: 8186-8191. [Medline]
  52. Crowe, N. Y., A. P. Uldrich, K. Kyparissoudis, K. J. Hammond, Y. Hayakawa, S. Sidobre, R. Keating, M. Kronenberg, M. J. Smyth, D. I. Godfrey. 2003. Glycolipid antigen drives rapid expansion and sustained cytokine production by NK T cells. J. Immunol. 171: 4020-4027. [Abstract/Free Full Text]
  53. Wilson, M. T., C. Johansson, D. Olivares-Villagomez, A. K. Singh, A. K. Stanic, C. R. Wang, S. Joyce, M. J. Wick, L. Van Kaer. 2003. The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion. Proc. Natl. Acad. Sci. USA 100: 10913-10918. [Abstract/Free Full Text]
  54. Harada, M., K. Seino, H. Wakao, S. Sakata, Y. Ishizuka, T. Ito, S. Kojo, T. Nakayama, M. Taniguchi. 2004. Down-regulation of the invariant V{alpha}14 antigen receptor in NKT cells upon activation. Int. Immunol. 16: 241-247. [Abstract/Free Full Text]
  55. Uldrich, A. P., N. Y. Crowe, K. Kyparissoudis, D. G. Pellicci, Y. Zhan, A. M. Lew, P. Bouillet, A. Strasser, M. J. Smyth, D. I. Godfrey. 2005. NKT cell stimulation with glycolipid antigen in vivo: costimulation-dependent expansion, Bim-dependent contraction, and hyporesponsiveness to further antigenic challenge. J. Immunol. 175: 3092-3101. [Abstract/Free Full Text]
  56. Alberola-Ila, J., G. Hernandez-Hoyos. 2003. The Ras/MAPK cascade and the control of positive selection. Immunol. Rev. 191: 79-96. [Medline]
  57. Newman, J. R., A. E. Keating. 2003. Comprehensive identification of human bZIP interactions with coiled-coil arrays. Science 300: 2097-2101. [Abstract/Free Full Text]
  58. Vinson, C., M. Myakishev, A. Acharya, A. A. Mir, J. R. Moll, M. Bonovich. 2002. Classification of human B-ZIP proteins based on dimerization properties. Mol. Cell. Biol. 22: 6321-6335. [Free Full Text]
  59. Rincon, M., R. A. Flavell. 1994. AP-1 transcriptional activity requires both T-cell receptor-mediated and co-stimulatory signals in primary T lymphocytes. EMBO J. 13: 4370-4381. [Medline]
  60. Nichols, K. E., J. Hom, S. Y. Gong, A. Ganguly, C. S. Ma, J. L. Cannons, S. G. Tangye, P. L. Schwartzberg, G. A. Koretzky, P. L. Stein. 2005. Regulation of NKT cell development by SAP, the protein defective in XLP. Nat. Med. 11: 340-345. [Medline]
  61. Pasquier, B., L. Yin, M. C. Fondaneche, F. Relouzat, C. Bloch-Queyrat, N. Lambert, A. Fischer, G. de Saint-Basile, S. Latour. 2005. Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product. J. Exp. Med. 201: 695-701. [Abstract/Free Full Text]
  62. Chung, B., A. Aoukaty, J. Dutz, C. Terhorst, R. Tan. 2005. Signaling lymphocytic activation molecule-associated protein controls NKT cell functions. J. Immunol. 174: 3153-3157. [Abstract/Free Full Text]
  63. Larkin, J., G. J. Renukaradhya, V. Sriram, W. Du, J. Gervay-Hague, R. R. Brutkiewicz. 2006. CD44 differentially activates mouse NK T cells and conventional T cells. J. Immunol. 177: 268-279. [Abstract/Free Full Text]
  64. Deppmann, C. D., R. S. Alvania, E. J. Taparowsky. 2006. Cross-species annotation of basic leucine zipper factor interactions: insight into the evolution of closed interaction networks. Mol. Biol. Evol. 23: 1480-1492. [Abstract/Free Full Text]



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