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The Journal of Immunology, 2007, 179, 111 -119
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Key Role for Itk in Both IFN{gamma} and IL-4 Production by NKT Cells1

Byron B. Au-Yeung and Deborah J. Fowell2

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, University of Rochester, Rochester, NY 14642


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NKT cells rapidly secrete cytokines upon TCR stimulation and thus may modulate the acquired immune response. Recent studies suggest that signaling for development and effector function in NKT cells may differ from conventional T cells. The tyrosine kinase Itk is activated downstream of the TCR, and its absence in CD4+ T cells results in impaired Th2, but not Th1 responses. In this study, we investigated NKT cell function in the absence of Itk as impaired type 2 responses in vivo could be manifest through IL-4 defects in a number of cell types. We show that Itk-deficient NKT cells up-regulate IL-4 mRNA in the thymus and express constitutive IL-4 and IFN-{gamma} transcripts in peripheral organs. Thus, Itk is not required for the developmental activation of cytokine loci in NKT cells. Nevertheless, Itk-deficient NKT cells are severely impaired in IL-4 protein production. Strikingly, unlike conventional CD4+ T cells, Itk-deficient NKT cells also have profound defects in IFN-{gamma} production. Furthermore, both IL-4 and IFN-{gamma} production were markedly impaired following in vivo challenge with {alpha}-galactosyl ceramide. Function can be restored in Itk-deficient NKT cells by provision of calcium signals using ionomycin. These results suggest that NKT cells are highly dependent on Itk for IL-4- and IFN-{gamma}-mediated effector function. Thus, the pattern of cytokine genes that are affected by Itk deficiency appears to be cell lineage-specific, likely reflecting differences in activation threshold between immune effectors. The severe defect in NKT cell function may underlie a number of the Th1 and Th2 immune defects in Itk-deficient mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Coordination between multiple cell types is required for successful immunity. Innate immune cell types, including NKT cells are capable of rapid cytokine production and thus may help to shape an adaptive immune response. NKT cells are lymphocytes that express a TCR along with surface markers expressed on NK cells, such as NKG2D, Ly49G2, Ly49C, Ly49I, and NK1.1 (1). Most NKT cells express a rearranged V{alpha}14-J{alpha}18 semi-invariant TCR, which recognizes the glycolipid {alpha}-galactosyl ceramide ({alpha}GalCer)3 presented in the context of the MHC class I-like molecule CD1d (2). Upon TCR stimulation, NKT cells are able to rapidly secrete both Th1 and Th2 cytokines, possibly due to the presence of constitutive cytokine mRNAs or hyperacetylation of histones associated with the IL-4 and IFN-{gamma} promoters (3, 4). However, the TCR signal requirements for NKT cell activation and cytokine production are not well characterized.

Due to their rapid secretion of immunomodulatory cytokines, NKT cells are thought to influence protective immune responses against some viruses (5, 6) and tumors (7). Recently, it has been shown that NKT cells can recognize glycolipid Ags derived from Sphingomonas and Ehrlichia, two Gram-negative, LPS-negative bacteria (8, 9). Although NKT cells play a beneficial role in several models of Th1 infection, their ability to promote Th2 responses is less clear (10). The role of NKT cells in autoimmunity is also controversial. In type I diabetes, both diabetes-prone NOD mice and human patients have a reduced frequency of NKT cells. Increasing the number of NKT cells in the NOD mouse reduces disease progression and is associated with the induction of a Th2 response (11, 12, 13). In multiple sclerosis, a similar reduction in NKT cells has been observed in multiple sclerosis patients (14). However, in the mouse, activation of NKT cells by injection of {alpha}-GalCer has been reported to either ameliorate or exacerbate the severity of experimental autoimmune encephalomyelitis by shifting the balance of IFN-{gamma} and IL-4 in the CNS (15, 16). Clearly, although the physiological role of NKT cells in immunity is not well understood, NKT cells are capable of influencing immune responses and represent an important immunoregulatory therapeutic target.

Itk is a member of the Tec family of kinases and is expressed by T cells, mast cells, NK cells, and NKT cells (17, 18). In T cells, Itk is activated downstream of TCR stimulation and modulates the magnitude of the calcium flux via phosphorylation of phospholipase C{gamma} (PLC{gamma}) (19). Consistent with its role in modulating TCR signal strength, Itk plays an important role in thymic selection of conventional CD4+ and CD8+ T cells. In itk–/– mice, the ratio of CD4:CD8 cells is reduced in the thymus and, in peripheral organs, there is approximately a 2-fold reduction in the total numbers of CD4+ cells (20). Similarly, in the absence of Itk, T cells bearing a low-avidity, MHC class II-restricted transgenic TCR specific for moth cytochrome c peptide (88–103) were less efficiently selected compared with T cells bearing a high-avidity TCR specific for the same peptide (21). Early studies showed that the frequency of CD8+ cells in the thymus and periphery were not as severely reduced in the absence of Itk (22). However, additional studies revealed that positive selection of HY-specific, MHC class I-restricted transgenic T cells was impaired in the absence of Itk, and that many of the CD8+ cells present in itk–/– mice are nonclassical, innate-like MHC class Ib-restricted T cells with a previously activated phenotype (23, 24, 25). Thus, it appears that Itk-dependent signals are required for efficient thymic selection of both conventional CD4+ and CD8+ T cell lineages.

Functionally, Itk-deficient CD4+ cells are able to differentiate into IFN-{gamma}-secreting Th1 effectors, but Th2 responses are severely impaired (26, 27, 28). Upon further analysis of the itk–/– phenotype, it was shown that under Th2-polarizing conditions, the kinetics and magnitude of IL-4, IL-5, IL-13, and GATA-3 transcript up-regulation during Th2 priming are similar in WT and itk–/– cells, suggesting that Itk is not required for early Th2 differentiation steps and Th2 lineage commitment. However, upon a secondary TCR stimulation, Th2-primed itk–/– cells fail to enhance IL-4 transcript levels, correlating with severe defects in IL-4 protein production both in vitro and in vivo (29). Thus, Itk appears to be required for the implementation, rather than acquisition of Th2 effector function.

Several studies have identified signaling molecules that are required for efficient NKT cell development, such as the Src kinase Fyn, as well as signaling lymphocytic activation molecule-associated protein (SAP), DOCK2, protein kinase C{theta} (PKC{theta}), NF-{kappa}B1, Vav-1, and IKK2 (30, 31, 32, 33, 34, 35, 36). Additionally, the decreased proportion of mature NKT cells, coupled with a decline in total NKT cell frequency with age, suggests that Itk also plays an important role in NKT cell development and/or homeostasis (37). The signals for execution of effector function in NKT cells are less well defined but the PKC{theta}/NF-{kappa}B1 pathway appears important. In addition, an interesting study recently revealed a role for GM-CSF (Csf-2) in the secretion of effector molecules by NKT cells (38).

Given Itk’s essential role in CD4+ Th2 effector function, we asked whether Itk deficiency would also alter the array of cytokines produced by NKT cells. In this study, we show that in addition to a role in NKT cell maturation, Itk was indeed required for mature NKT cell production of IL-4. Surprisingly, NKT cell production of IFN-{gamma} was also severely impaired. Despite a failure to detect cytokine production, analysis of cytokine mRNA revealed that Itk-deficient NKT cells maintained constitutive IL-4 and IFN-{gamma} transcripts similar to wild-type (WT) NKT cells but failed to up-regulate these transcripts on {alpha}GalCer stimulation. Therefore, NKT cells show a striking dependency on Itk-mediated signals for the execution of both Th1- and Th2-like effector function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and {alpha}GalCer treatment

WT B10.D2 mice were purchased from The Jackson Laboratory. Itk–/– B10.D2, WT BALB 4get, itk–/– BALB 4get, and TCR C{alpha}–/– mice were maintained in the pathogen-free animal care facility at the University of Rochester Medical Center (Rochester, NY). Mice were 6–8 wk of age, unless otherwise stated. For in vivo challenge, mice were injected i.v. with 2 µg {alpha}GalCer (Alexis Biochemicals) in a vehicle consisting of 0.9% NaCl and 0.5% Polysorbate-20 (Bio-Rad).

Flow cytometry

Cells were preincubated with blocking anti-FcR mAb clone 2.4G2 for 15 min at 4°C, followed by staining with PBS-57/CD1d tetramers (National Institutes of Health tetramer core facility) at 4°C for 45 min. Abs also used in this study include anti-TCRbeta clone H57-597, anti-CD3{epsilon} clone 145-2C11, anti-CD122 clone TM-beta1, anti-CD69 clone H1.2F3 (BD Pharmingen), and anti-NK1.1 clone PK136, anti-CD44 clone IM7 (eBioscience).

Cell purification and stimulation

CD4+ cells were enriched from spleen and lymph node by Ab/complement-mediated lysis (26) and sorted (FACSAria; BD Biosciences) for naive CD4+ cells, >99% CD62LhighCD44low. For NKT cell purification, spleen cells were depleted of MHC class II+ and CD24+ cells by Ab/complement lysis, followed by sorting for CD1d/PBS-57 tetramer+CD3{epsilon}+ cells. Irradiated (2500 rad) Itk-sufficient APCs were used in all experiments. Splenocytes were either negatively selected from WT mice for TCR-negative CD1d-tetramer-negative cells by FACS (WT APCs) or isolated from TCR C{alpha}–/– mice (TCR C{alpha}–/– APCs). We found no difference in the stimulatory capacity of WT and TCR C{alpha}–/– splenocytes for WT or itk–/– NKT cell cytokine production. Unless otherwise indicated, NKT cells were stimulated with plate-bound anti-TCRbeta coated at a concentration of 0.5 µg/ml or with {alpha}-GalCer at a concentration of 100 ng/ml plus APCs.

Liver lymphocyte isolation

Livers were first perfused with 5 ml of PBS through the portal vein. Each homogenized liver was then incubated at 37°C with 5 mg of collagenase (Sigma-Aldrich) and 1 mg of DNase I (Sigma-Aldrich) for 45 min. Cell suspensions were then centrifuged in a 21.5% Optiprep solution (Accurate) density gradient. Liver lymphocytes were removed from the interface and washed for FACS analysis.

Real-time PCR

Total RNA was extracted from sorted naive CD4+CD62LhighCD44low cells and NKT cells (TCRbeta+ PBS-57/CD1d tetramer+) using TRIzol reagent (Invitrogen Life Technologies) and reverse-transcribed (reverse transcriptase for PCR kit; BD Clontech). RNA was also harvested from NKT cells after stimulation with {alpha}GalCer and TCR C{alpha}–/– APCs for 24 h for analysis of IL-4, IFN-{gamma}, T-bet, and GATA-3 mRNA induction. Real-time PCR was performed using Assays-on-Demand TaqMan primer/probe sets with an Applied Biosystems Prism 7900 sequence detection system (Applied Biosystems). Graphs display transcript levels relative to the endogenous control CD3{delta} and were calibrated relative to unstimulated naive CD4+ cells.

Cytokine measurements

For analysis of IL-4 and IFN-{gamma} production by ELISA, sorted NKT cells were stimulated either with plate-bound anti-TCRbeta mAb or with {alpha}GalCer and WT APCs for 48 h. For analysis of IL-4 and IFN-{gamma} production by ELISPOT, sorted NKT cells were stimulated with either plate-bound anti-TCRbeta mAb or with {alpha}GalCer and WT APCs for 24 h and cytokine-producing cells were detected as described (26). APC cultures with {alpha}GalCer in the absence of NKT cells yielded <5 IFN-{gamma} spots and <4 IL-4 spots. The cytokine secretion assay (Miltenyi Biotec) was performed as previously described (29). Briefly, splenocytes from vehicle or {alpha}GalCer-treated mice were incubated directly ex vivo in the presence or absence of ionomycin (1 µg/ml; Calbiochem) for 4 h. After this incubation period, cells were washed and labeled with a bispecific "catch reagent" and incubated for 45 min at 37°C. Cells were washed and IL-4 and IFN-{gamma} were detected with PE-conjugated Abs. For intracellular cytokine staining, splenocytes from vehicle- or {alpha}GalCer-treated mice were harvested and incubated for 4 h with brefeldin A (BD Pharmingen) in the presence or absence of 1 µg/ml ionomycin at 37°C. Cells were stained with CD1d/PBS-57 tetramer and anti-CD3{epsilon}, followed by permeabilization with Cytofix/Cytoperm and stained with anti-IL-4 clone BVD4-1D11 or anti-IFN-{gamma} clone XMG1.2 (BD Pharmingen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Absence of Itk results in partial block of NKT cell maturation

Given the previously described age-related defect in NKT cell numbers in the absence of Itk (37), we first measured the frequency and number of NKT cells in WT and itk–/– mice. To identify NKT cells, we labeled cells from the thymus, spleen and liver with CD1d tetramers loaded with the {alpha}GalCer analog PBS-57. The frequency of TCRbeta+tetramer+ NKT cells in itk–/– mice was reduced <2-fold in the thymus, whereas the reduction was more severe in the spleen and liver (5- to 6-fold; Fig. 1A). Total numbers of NKT cells were significantly decreased (Mann-Whitney U test; p = 0.02) in all three organs (Fig. 1B), suggesting that NKT cells are highly dependent on Itk expression for development and/or survival. In contrast, the number of tetramer–TCRbeta+ T cells (Fig. 1C) and NK cells (NK1.1+TCR–; Fig. 1D) were not significantly reduced in itk–/– mice. However, recent reports suggest that the "conventional" TCR{alpha}beta+ T cell compartment contains a substantial number of nonconventional innate-like T cells (23, 24).


Figure 1
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  		FIGURE 1. Decreased frequency and number of NKT cells in itk–/– mice. Cells from the thymus, spleen, and liver were harvested from 6- to 8-wk-old WT and itk–/– B10.D2 mice and stained with CD1d/PBS-57 tetramers and anti-TCRbeta mAb for FACS analysis. A, Frequency of NKT cells in WT and itk–/– mice. Dot plots are gated on live lymphocytes, the numbers indicate the percentage of TCRbeta+tetramer+ NKT cells. B, Graphs show total numbers of NKT cells (TCRbeta+tetramer+) and C, conventional T cells (TCRbetahightetramer–) per organ. Itk-deficient mice ({diamond}) have significantly lower NKT cell numbers than WT mice (•); Mann-Whitney U test, *, p < 0.05. Data are representative of at least three independent experiments, four mice per group. Each symbol represents an individual mouse. D, Dot plots (left) of WT and itk–/– B10.D2 splenocytes. Numbers indicate percentage of NK (TCRbeta–NK1.1+) cells. The graph (right) shows total numbers of splenic NK cells in WT (•) and itk–/– ({diamond}) B10.D2 mice. Each symbol represents an individual mouse. Data are representative of two independent experiments.

 
Previous studies have reported that the frequency of mature NK1.1+ and CD69+ NKT cells in itk–/– mice is decreased (37), suggesting that Itk is important for NKT cell maturation. However, it is not clear at which stage itk–/– NKT cells are blocked. To identify where the block in itk–/– NKT cell development occurs, we analyzed surface marker and cytokine transcript expression by WT and itk–/–tetramer+ thymocytes (Fig. 2). During maturation in the thymus, immature NKT precursors progress from NK1.1–CD44low (stage 1) to NK1.1–CD44high (stage 2) and finally to NK1.1+CD44high (stage 3) (1). Other markers, such as CD69 and CD122, are also up-regulated during the transition from stage 2 to stage 3. Analysis of thymocytes from Itk-deficient mice revealed that itk–/– NKT cells up-regulate CD44, but are impaired in up-regulation of CD122 and also, in agreement with previous studies, CD69 and NK1.1 (37) (Fig. 2A). These results are consistent with earlier studies on the developmental phenotype of Itk-deficient NKT cells (37), and together the studies suggest that in the absence of Itk, NKT cells up-regulate CD44, but are impaired in progression from stage 2 to stage 3, marked by defective up-regulation of CD69, CD122, and NK1.1.


Figure 2
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  		FIGURE 2. Absence of Itk results in a partial block in NKT cell maturation. A, Expression of CD44, CD69, CD122, and NK1.1 on WT and itk–/– NKT cells from thymus and spleen of 6- to 8-wk-old mice. Histograms were gated on PBS-57/CD1d tetramer+TCRbeta+ NKT cells and analyzed for expression of the indicated surface molecules. In each plot, WT cells are represented by the shaded histograms, and itk–/– cells are represented by the open histograms. The numbers indicate the percentages of WT and itk–/– cells within the histogram gate. B, Thymic NKT (TCRbeta+tetramer+) cells from 3-wk-old WT and itk–/– 4get mice were analyzed for CD44 (top) or CD122 (bottom) vs IL-4/GFP expression. Numbers represent the percentage of cells within the gate. Data are representative of at least three independent experiments, three mice per group.

 
In contrast to conventional CD4+ T cells, NKT cells have been shown to express cytokine mRNA during development. Bicistronic IL-4/enhanced GFP (eGFP) reporter (4get) mice have been used previously to track the induction of IL-4 transcription in conventional CD4+ T cells (39) and also NKT cells (4). During NKT cell maturation, IL-4 transcripts are up-regulated early, peaking in CD44low cells and decreasing upon transition to CD44high cells (4, 40). It is not clear whether the signals for NKT cell development are the same as those signals that induce cytokine transcripts. Given Itk’s role in conventional Th2 cells, we asked whether the absence of Itk affected expression of IL-4 mRNA in NKT cells. Analysis of thymocytes from 3-wk-old 4get mice showed that itk–/– NKT cells express eGFP and CD44 similarly to WT cells (Fig. 2B). However, consistent with a defect in maturation, the loss of GFP expression associated with thymic maturation to CD122+ was attenuated in the absence of Itk. Our results with IL-4 reporter expression, along with surface marker analysis, suggest that NKT cell precursors can progress through the early stages of NKT cell maturation, as itk–/– NKT cells express IL-4 and up-regulate CD44, but inefficiently transition to the CD44highCD122+ stage (stage 3) of maturation. The reduced frequency of immature eGFP+CD44low cells and elevated frequency of eGFP+CD44high thymocytes (Fig. 2B) are consistent with this notion.

Itk is not required for the induction and maintenance of constitutive cytokine mRNAs

NKT cells in the periphery of WT mice constitutively express elevated levels of IL-4, IFN-{gamma}, IL-5, and IL-13 mRNA (3, 4), a characteristic thought to allow for the rapid secretion of these cytokines. Our single-cell analysis of 4get lymphocytes showed that up-regulation of IL-4 occurs independently of Itk expression in thymic NKT cells (Fig. 2B). Despite thymic developmental defects, IL-4/eGFP+ NKT cells were present in peripheral organs of itk–/– 4get mice (Fig. 3, A and B). However, the number of eGFP+ NKT cells in the spleen was reduced and correlated with a decrease in the ratio of GFP+ to GFP– NKT cells between WT and itk–/– mice (Fig. 3, A and B). At the population level, constitutive mRNA expression for a variety of cytokines, including both IL-4 and IFN-{gamma}, was elevated in tetramer+ NKT cells of both WT and itk–/– splenocytes compared with conventional CD4+ T cells (Fig. 3, B and C). Thus, constitutive expression of many effector cytokine transcripts in NKT cells is Itk independent. Interestingly, constitutive T-bet expression was also unperturbed in the absence of Itk (Fig. 3D). Indeed, levels of T-bet were consistently slightly elevated in splenic TCRbeta+tetramer+ Itk-deficient NKT cells compared with WT NKT cells, despite differences in the frequency of mature NKT cells between the two groups (Fig. 2) (37). The failure to see a defect in T-bet expression in the absence of Itk is somewhat surprising given that T-bet has been reported to be required for terminal maturation of NK and NKT cells (41, 42). The maturation defects seen in the absence of Itk cannot therefore be explained by a defect in the expression of T-bet and may suggest that the stable induction of T-bet expression precedes the development of stage 3 "mature" NK1.1+ NKT cells (1).


Figure 3
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  		FIGURE 3. Constitutive expression of cytokine mRNAs by WT and itk–/– NKT cells. A, Representative histograms of eGFP expression by WT and itk–/– 4get NKT cells. Histograms are gated on TCRbeta+tetramer+ NKT cells from the spleens or livers of 6- to 8-wk-old WT and itk–/– 4get mice. Numbers represent the percentage of cells in the indicated histogram gate. Data are representative of at least two independent experiments, three mice per group. B, Number of TCRbeta+tetramer+ cells expressing eGFP. C, Real-time PCR analysis of IL-4, IL-5, IL-13, IFN-{gamma}, and D, T-bet transcript levels. Transcript levels in sorted splenic NKT cells (TCRbeta+tetramer+) from WT ({blacksquare}) and itk–/– (Figure 3) mice. Values are calculated relative to sorted naive CD4+ T cells (CD44lowCD62LhighTCR+) and calibrated to the endogenous control, CD3{delta}. Data are representative of three independent experiments.

 
Itk is required for NKT cell production of both IL-4 and IFN-{gamma} in vitro and in vivo

Previously, we described the ability of conventional itk–/–CD4+ T cells to initiate IL-4 transcription during Th2 differentiation (29). Under Th2-polarizing conditions, itk–/–CD4+ cells up-regulated IL-4 mRNA and other Th2 cytokines with the same kinetics and to the same magnitude as WT cells. However, upon a secondary stimulation, Th2-primed itk–/– cells failed to enhance IL-4 transcription, which correlated with a severe defect in IL-4 protein production. Given that Itk is required for conventional CD4+ T cells to exert, but not gain, Th2 effector function, we asked whether itk–/– NKT cells were similarly functionally impaired. With the reduced frequency of IL-4/eGFP-expressing NKT cells in the periphery of Itk-deficient mice, IL-4-committed eGFP+ NKT cells were sorted from WT and itk–/– 4get splenocytes for direct comparison of cytokine production and stimulated with either {alpha}GalCer and APCs or with plate-bound anti-TCRbeta (Fig. 4A). Both WT and itk–/– NKT cells were stimulated with {alpha}GalCer-loaded WT APCs to directly test functional defects intrinsic to Itk deficiency in the NKT cells themselves. Despite similar levels of eGFP expressed by both WT and itk–/–eGFP+ populations (eGFP mean fluorescence intensity of CD4+ T cells: WT, 93.8 ± 20.8; itk–/–, 95.5 ± 23.3), IL-4 protein was detected only from WT NKT cells; Itk-deficient NKT cells were impaired in IL-4 production following both Ab and {alpha}GalCer/APC stimulation (Fig. 4A). Thus, similar to conventional T cells, itk–/– NKT cells fail to produce IL-4 upon TCR stimulation, despite constitutive expression of IL-4 mRNA.


Figure 4
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  		FIGURE 4. Impaired production of both IL-4 and IFN-{gamma} by itk–/– NKT cells. A, Sorted eGFP+TCRbeta+tetramer+ NKT cells from WT ({blacksquare}) and itk–/– 4get (Figure 4) spleens were stimulated with plate-bound anti-TCRbeta or 100 ng/ml {alpha}GalCer plus WT APCs and the frequency of IL-4 secretors measured by ELISPOT. B, Sorted TCRbeta+tetramer+ NKT cells were stimulated with 100 ng/ml {alpha}GalCer and WT APCs for 24 h, and the frequency of cytokine-producing cells was measured by ELISPOT (APC cultures in the absence of NKT cells = <5 IFN-{gamma} spots and <14 IL-4 spots). Conventional CD4+ T cells from WT ({blacksquare}) and itk–/– (Figure 4) mice were primed under Th1- or Th2-polarizing conditions for 5 days and restimulated with plate-bound anti-TCRbeta mAb for 24 h for cytokine-producing cells by ELISPOT. C, Sorted TCRbeta+tetramer+ NKT cells from WT (black lines) and itk–/– (gray lines) mice were stimulated for 24 h with varying does of {alpha}GalCer and WT APCs or plate-bound anti-TCRbeta mAb. Graphs represent ELISPOT analysis for IL-4- and IFN-{gamma}-secreting cells per 104 NKT cells. D, IL-4 and IFN-{gamma} mRNA fold induction in NKT cells upon stimulation. Sorted CD3{epsilon}+tetramer+ NKT cells were stimulated with {alpha}GalCer and TCR C{alpha}–/– APCs for 12 h and harvested for RNA extraction. Real-time PCR analysis of IL-4 and IFN-{gamma} transcript levels in WT ({blacksquare}) and itk–/– (Figure 4) NKT cells is shown. Values are fold induction over the levels detected in unstimulated CD3{epsilon}+tetramer+ NKT cells and normalized to CD3{delta}. E, T-bet and GATA-3 transcript levels in unstimulated CD3{epsilon}+tetramer+ (–{alpha}GalCer) NKT cells or NKT cells stimulated for 24 h in the presence of 100 ng/ml {alpha}GalCer and APCs (+{alpha}GalCer). Values are calculated relative to sorted naive (CD44lowCD62Lhigh) CD4+ T cells and calibrated to the endogenous control, CD3{delta}. Data representative of at least three separate experiments. F, Sorted NK1.1+TCRbeta+tetramer+ NKT cells were stimulated for 24 h with 100 ng/ml {alpha}GalCer and WT APCs for analysis of IL-4 and IFN-{gamma} production by ELISPOT.

 
Although Itk is required for Th2 responses, Th1 responses develop in the absence of Itk both in vitro and in vivo (26, 27, 28). Indeed, Th1-primed itk–/–CD4+ cells produced similar levels of IFN-{gamma} as WT Th1 cells (Fig. 4B). Unexpectedly, in addition to a defect in IL-4, Itk-deficient NKT cells also failed to produce IFN-{gamma} upon TCR stimulation (Fig. 4B). These results suggest that NKT cells, unlike conventional CD4+ Th cells, are highly dependent on Itk for production of both IL-4 and IFN-{gamma}. In the absence of Itk, the magnitude of TCR signaling is attenuated and could contribute to functional defects (19, 27, 28). However, enhancing signal strength by increasing the stimulating dose also failed to bypass the Itk-dependent deficiency in IL-4 and IFN-{gamma} production (Fig. 4C), suggesting a qualitative rather than quantitative role for Itk in TCR signals for cytokine production.

Our previous studies had highlighted a key requirement for Itk signals in transcriptional enhancement of IL-4 on restimulation of Th2-primed CD4+ effector cells that in turn was critical for optimal cytokine production (29). Resting NKT cells express cytokine transcripts at a similar level to resting differentiated Th1 and Th2 cells (4), but it is unclear how much these existing transcripts contribute to cytokine protein production on Ag stimulation. On {alpha}GalCer stimulation, NKT cells also exhibit marked cytokine transcriptional up-regulation (100- to 1000-fold; Fig. 4D) similar to that seen for Th1 and Th2 effectors (4). Such transcriptional enhancement was attenuated in the absence of Itk (Fig. 4D), with up-regulation of IFN-{gamma} mRNA being most severely affected by the lack of Itk. The pronounced defect in IFN-{gamma} mRNA enhancement on Ag stimulation correlated with a failure to sustain T-bet expression (Fig. 4E). Interestingly, we found that down-regulation of T-bet occurred coordinate with an increase in GATA-3 mRNA (Fig. 4E). Peripheral NKT cells are thought to express both IL-4 and IFN-{gamma} at the single-cell level (4) but the regulation of cytokine production (relative levels of IL-4 vs IFN-{gamma}) by individual cells is not well understood. It is possible that within individual cells GATA-3 plays a negative regulatory role in IFN-{gamma} production, altering the relative levels of IL-4 and IFN-{gamma} production by NKT cells. The altered T-bet/GATA-3 balance in the absence of Itk may account for the more severe defect in IFN-{gamma} protein production compared to IL-4 production (Fig. 4C). Defects in IL-4 production despite GATA-3 expression in itk–/– NKT cells may reflect Itk’s role in regulating the calcium flux and the relative importance of NFAT2, over GATA-3, in IL-4 production by NKT cells (43). The WT and itk–/– NKT cell populations differed in their frequency of mature NK1.1+ NKT cells (Fig. 2A), with itk–/–tetramer+ cells containing one-half as many mature NKT cells, raising the possibility that developmental differences accounted for the changes in cytokine responses observed here. However, on analysis of sorted NK1.1+tetramer+ cells, itk–/– mature NKT cells also showed a reduced frequency of both IL-4- and IFN-{gamma}-producing cells (Fig. 4F and developed further in Fig. 6).


Figure 6
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  		FIGURE 6. Provision of ionomycin restores IL-4 and IFN-{gamma} production by mature NK1.1+itk–/– NKT cells. A, Splenic NKT (CD3{epsilon}+tetramer+) cells were sorted into NK1.1– and NK1.1+ fractions and stimulated for 18 h with plate-bound anti-TCRbeta mAb in the presence or absence of 1 µg/ml ionomycin for IL-4 and IFN-{gamma} ELISPOT analysis. Graphs represent the number of WT ({blacksquare}) and itk–/– (Figure 6) cytokine-secreting cells per 5 x 103 NKT cells. Representative of three separate experiments (*, p ≤ 0.05 Student’s t test; n.s. (not significant), p ≥ 0.05) B, WT and itk–/– mice were challenged with {alpha}GalCer or vehicle alone in vivo, as in Fig. 5. Splenocytes were incubated for 4 h in the presence or absence of 1 µg/ml ionomycin before the cytokine secretion assay. For IFN-{gamma} (top) and IL-4 (bottom) secretion analysis, splenocytes were labeled with the bispecific "catch" reagent, followed by incubation at 37°C for 45 min. Cells were washed and stained with PE-conjugated IL-4 and IFN-{gamma} Abs and anti-NK1.1 mAb. Dot plots are gated on CD3{epsilon}+tetramer+ NKT cells. Numbers in quadrants are the percentage of total tetramer+ NKT cells. Data representative of three separate experiments.

 
NKT cells are capable of secreting cytokines rapidly in vivo upon Ag stimulation. To determine whether our in vitro defects in cytokine production by itk–/– NKT cells were observed on in vivo activation, WT and itk–/– mice were injected with 2 µg of {alpha}GalCer or vehicle alone. After 2 h, splenocytes were harvested and incubated with brefeldin A for 4 h before intracellular cytokine staining (Fig. 5). Consistent with our in vitro data, NKT cells from itk–/– mice were impaired in production of both IL-4 and IFN-{gamma} protein upon stimulation with {alpha}GalCer in vivo. Similar defects were observed following in vivo activation with i.v. anti-CD3 mAb (data not shown and Ref. 44). Thus, despite evidence of developmental activation of both the IL-4 and IFN-{gamma} loci (represented by the elevated levels of cytokine mRNAs before Ag stimulation, "constitutive" cytokine mRNAs, Fig. 3), itk–/– NKT cells are severely functionally impaired in their ability to produce effector cytokines in vivo.


Figure 5
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  		FIGURE 5. Impaired IL-4 and IFN-{gamma} production by itk–/– NKT cells following in vivo challenge with {alpha}GalCer. WT and itk–/– mice were injected i.v. with 2 µg of {alpha}GalCer or vehicle alone. After 2 h, splenocytes were harvested and incubated for 4 h at 37°C in the presence of brefeldin A. Cells were then surface stained, fixed, and permeabilized for intracellular cytokine staining. Filled histograms represent CD3{epsilon}+tetramer+ cells from vehicle-treated mice and open histograms represent CD3{epsilon}+tetramer+ cells from {alpha}GalCer-treated mice. Numbers in the table are the percentages of cytokine-expressing NKT cells. Data representative of three independent experiments.

 
Distinct roles for Itk in NKT cell development and effector function?

NKT cells undergo a series of functional changes during thymic development with cells transitioning from a NK1.1-IL-4highIFN-{gamma}– phenotype to a NK1.1+IL-4lowIFN-{gamma}+ (37, 40, 45). Once in the periphery, further functional changes result in a peripheral NKT cell pool where the majority of mature NK1.1+ NKT cells express both IL-4 and IFN-{gamma} (3, 4). As suggested previously, given the clear role for Itk in NKT cell development, it was possible that the defects in peripheral NKT cell function were a consequence of defects in development. Therefore, we compared cytokine production from phenotypically immature (NK1.1–) and mature (NK1.1+) tetramer+ NKT cells following anti-TCRbeta stimulation in vitro and {alpha}GalCer stimulation in vivo (Fig. 6). In vitro, tetramer+ splenocytes were sorted into NK1.1– and NK1.1+ fractions and stimulated with WT APCs and anti-TCRbeta (Fig. 6A). In vivo, mice were challenged with {alpha}GalCer as in Fig. 5, and cytokine production was assessed directly ex vivo using the cytokine capture assay in combination with cell surface phenotyping for NK1.1 expression (Fig. 6B). Cytokine production, both IL-4 and IFN-{gamma}, remained severely compromised when mature itk–/– splenic NK1.1+ NKT cells were compared with WT NK1.1+ NKT cells, suggesting that Itk plays a role in the execution of NKT cell function independent from its role in NKT cell development.

IL-4 production can be restored in Itk-deficient Th2 effector cells by the provision of TCR signals with ionomycin (29). This result suggested that conventional CD4+ T cells differentiated into Th2 cells with similar cytokine potential as WT cells but lack the appropriate activation signals to exert effector function. To define the cytokine-producing potential of Itk-deficient NKT cells, we asked whether ionomycin could similarly rescue cytokine production. Indeed, supplementing {alpha}GalCer signals with ionomycin rescued both IL-4 and IFN-{gamma} production by NK1.1+itk–/– NKT cells activated in vitro or in vivo (Fig. 6, A and B). Similar restoration of IFN-{gamma} production was noted on PMA/ionomycin stimulation of itk–/–NK1.1+ NKT cells (37). Thus, phenotypically mature itk–/– NKT cells appear functionally mature when given the appropriate activation signals. These results imply that Itk and the calcium flux are dispensable for the gain of cytokine function but are critical for the production of effector cytokines by NKT cells.

In contrast, analysis of the NK1.1– immature NKT cells revealed an additional effect on the cytokine potential of splenic NKT cells that appears to arise from the Itk-dependent developmental block. The splenic itk–/–NK1.1– NKT cells produced IL-4 but not IFN-{gamma} on anti-TCRbeta stimulation in vitro in contrast to WT NK1.1– NKT cells that produced both IL-4 and IFN-{gamma} on anti-TCRbeta/ionomycin stimulation (Fig. 6A). The difference between Itk-deficient and WT immature NKT cells is consistent with a developmental block in the absence of Itk from stage 2 to 3 (Fig. 2); a stage where IFN-{gamma} potential is usually first established. Interestingly, we consistently observed an increased frequency of IFN-{gamma} producers in splenic WT NK1.1– cells compared to WT NK1.1+ cells when stimulated with anti-TCRbeta and ionomycin. This is in contrast to published studies on thymocyte subpopulations where the NK1.1+ fraction was enriched for IFN-{gamma} producers (37, 39, 43). This may simply reflect differences in the methods of cytokine detection; published studies measured the total amount of cytokine produced, whereas our study detects the frequency of IFN-{gamma} producers. The studies may be compatible if there are quantitative differences between NK1.1– and NK1.1+ NKT cells in the amount of IFN-{gamma} produced per cell. Alternatively, our results from peripheral tetramer+NK1.1– NKT cells may suggest additional functional maturation within the NK1.1– subset (without acquisition of NK1.1 itself) or possibly the presence within the NK1.1– fraction of contaminating mature NK1.1+ NKT cells that have lost expression of NK1.1 on activation in the periphery.

Once again, similar effects were seen following in vivo {alpha}GalCer challenge, NK1.1+itk–/– NKT cells produced both IL-4 and IFN-{gamma} on ex vivo provision of ionomycin. In contrast, the NK1.1– fraction produced IL-4 (of the NK1.1–tetramer+ fraction: 27% of WT cells were IL-4+ and 37% of itk–/– cells were IL-4+) but IFN-{gamma} production was severely attenuated (of the NK1.1–tetramer+ fraction: 65% of WT cells were IFN-{gamma}+ but only 8.8% of itk–/– cells were IFN-{gamma}+ (Fig. 6B). The ionomycin-dependent restoration of IL-4 production in both immature and mature NKT cell subsets suggests that Itk plays a direct role in IL-4 effector function, independent of defects in development. In contrast, the IFN-{gamma} defect in itk–/– NKT cells likely arises due to both the reduced frequency of mature (IFN-{gamma}-competent) NKT cells in the periphery and defects in the liberation of effector function in mature NKT cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The ability to rapidly secrete cytokines provides NKT cells with the potential to influence immune responses. Therefore, much interest is focused on the signals required for NKT cell development and function. Although conventional T cells and NKT cells both express T cell {alpha}beta+ receptors, a number of recent studies suggest that the signals required for NKT cell development and function may be distinct from those for conventional T cells. In this study, we confirm and extend analysis on the role of Itk in NKT cell maturation from thymic stage 2 (CD44highNK1.1–) to stage 3 (CD44highNK1.1+). Surprisingly, despite a partial block in maturation, mature NK1.1+ NKT cells were present in the periphery and constitutively contain mRNAs for several cytokines, including IL-4 and IFN-{gamma}. Strikingly, such itk–/– NKT cells that appear poised for cytokine synthesis failed to produce effector cytokines on stimulation with {alpha}GalCer. These results suggest that Itk-dependent signals, although not required for activation of cytokine loci in NKT cells, are required for optimal IL-4 and IFN-{gamma} production upon TCR stimulation in vitro and in vivo. That the functional potential of mature Itk-deficient NK1.1+ NKT cells could be revealed by provision of ionomycin supports a role for Itk in release of NKT cell function independent of Itk effects on NKT cell development.

The severe defect in IL-4 and IFN-{gamma} protein production in the absence of Itk correlates with an impairment in Ag-driven enhancement of IL-4 and IFN-{gamma} transcription. Although NKT cells constitutively express cytokine mRNAs, activated WT NKT cells further enhanced cytokine transcription 100- to 1000-fold and this response was attenuated in the absence of Itk. In conventional CD4+ T cells at least, such transcriptional enhancement is critical for effective IL-4 production by Th2 cells (29). For IL-4 therefore, NKT cells and conventional Th2 cells share a common requirement for Itk to exert effector function. Interestingly, our studies also highlight a marked dependency on Itk for IFN-{gamma} production in NKT cells. Recent studies also suggest that CD8+ T cells may be dependent on Itk for robust IFN-{gamma} production (46). Thus, Itk is not solely a Th2-dependent kinase. These observations support the idea that Itk regulates the availability of signaling components downstream of PLC{gamma} activation, such as NFAT and AP-1, which are commonly used by many cytokine genes. Interestingly, the pattern of cytokine genes affected by Itk deficiency appears to be cell type specific and likely reflects differences in activation thresholds between immune cells.

Notably, NKT cell production of IFN-{gamma} appears more dependent on a number of signaling molecules (PKC{theta}, Itk) that are dispensable for Th1 cell production of the same cytokine (Fig. 4B and Refs. 26 , 36 , and 47). Signals during Th1 differentiation may reset activation thresholds in a lineage-specific manner. Indeed, Th1 effectors acquire changes in the TCR-induced calcium flux that results in a more rapid and higher magnitude calcium response compared with naive and Th2 effectors (48). The removal of Itk, a key regulator of PLC{gamma} activity, leads to a reduced but still measurable calcium flux in Th1 cells (potentially providing sufficient NFATc/ERK activation for IFN-{gamma} transcription) while itk–/– Th2 calcium responses are ablated (B. B. Au-Yeung and D. J. Fowell, unpublished observations). Therefore, akin to the loss of Itk in Th2 cells, the loss of Itk in NKT cells may reduce the calcium flux below a critical threshold for both IL-4 and IFN-{gamma} transcription. The rescue of both IL-4 and IFN-{gamma} protein production in NKT cells by provision of ionomycin in combination with Itk-deficient TCR signals is consistent with such a notion.

These results suggest that execution of NKT cell function may be more tightly regulated than inferred from their ability to rapidly synthesize cytokines upon TCR ligation. Interestingly, a recent study highlighted another potential checkpoint in NKT cell effector function at the level of cytokine secretion. Unlike the defect presented here in the absence of Itk where cytokine mRNA is intact but cytokine synthesis is impaired (as measured by intracellular cytokine staining, Fig. 5), GM-CSF deficiency led to normal cytokine synthesis in mature NKT cells but an inability to secrete these proteins (38). Once again, the defect was restricted to NKT cells, as cytokine secretion from GM-CSF-deficient NK cells and CD4 and CD8 T cells was unimpaired. Itk-deficient NKT cell thymocytes and splenocytes express WT levels of GM-CSF (B. B. Au-Yeung and D. J. Fowell, unpublished observations), indicating that the two functional defects are distinct. Combined, these studies highlight the extraordinary lineage specificity in signal requirements for production and secretion of a given cytokine in different immune effector cell types.

Itk joins an expanding group of signaling components whose requirements for development and function differ between conventional T cells and NKT cells. For example, in mice lacking SAP, NF-{kappa}B1, or PKC{theta}, conventional T cell numbers were not affected but NKT cell development was impaired (31, 34, 36, 47, 49, 50, 51). At the functional level, SAP-deficient and PKC{theta}-deficient conventional CD4+ cells show selective defects in Th differentiation, capable of Th1 differentiation and IFN-{gamma} secretion but impaired Th2 responses in vitro and in vivo (47, 49), similar to the phenotype observed in itk–/– mice. Strikingly, NKT cells deficient in PKC{theta}, SAP, and now Itk exhibit a more severe functional defect with impaired production of both IL-4 and IFN-{gamma} (31, 34, 36, 50). However, the severe reduction in NKT cell number in the SAP-deficient mice may make accurate measurements of functional capacity problematic. The similar defects in cytokine production by NKT cells lacking PKC{theta}, Itk, SAP suggest that these molecules contribute to common signaling pathways that are critical for NKT cell effector function. Previous studies have linked signaling lymphocytic activating molecule family receptor stimulation with SAP, Fyn, and PKC{theta} recruitment, leading to activation of the NF-{kappa}B pathway (51, 52). How Itk could contribute directly to this pathway is unclear. The absence of Itk phosphorylation in T cells lacking Lck suggests that Fyn does not have a major role in phosphorylation of Itk (53). Currently, there is no evidence to suggest that Itk interacts with SAP. However, association between Itk and PKC{theta}, possibly resulting in enhanced PLC{gamma} activation, has been observed both in T cell lines and in mast cells (54, 55). This interaction may also occur in NKT cells, promoting transduction of signals important for enhanced cytokine transcription.

Given the potential immunomodulatory effects of NKT cell-derived cytokines, the striking defect in cytokine production (both IL-4 and IFN-{gamma}) by Itk-deficient NKT cells in vivo could significantly impact the magnitude and quality of the developing acquired immune response. It will be interesting to revisit in vivo models previously used to study immune responses in the absence of Itk to determine the relative contributions of distinct Itk-deficient immune cell types to the overall immune response. Our previous work on the role of Itk in Th2 development provided evidence for a CD4-intrinsic defect in Th2 cytokine production through the use of CD4+ T cell adoptive transfers (26). However, these studies did not rule out a contribution to the magnitude of the type 2 response by other immune cell types following infection of Itk-deficient mice. For example, mice lacking Itk have reduced airway hyperreactivity and lower levels of IL-4 and IL-13 in the bronchoalveolar lavage fluid in a model of allergic asthma (56, 57). This phenotype was attributed to reduced T cell infiltration to the lungs and impaired Th2 cytokine production. However, recent evidence suggests that NKT cells are sufficient to induce airway hyperreactivity, a hallmark of asthma, even in the absence of conventional CD4+ T cells (58). Given that Itk is critical for cytokine production by NKT cells and Th2 cells, it would be interesting to re-evaluate how these cell types contribute to attenuated airway hyperreactivity and type 2 cytokine production in itk–/– mice. The original description of immune defects in the absence of Itk demonstrated increased susceptibility of itk–/– mice to Toxoplasma gondii infection despite similar frequencies of IFN-{gamma}-producing immune cells on in vitro restimulation with soluble tachyzoite Ag 30 days after infection (59). The generation of antimicrobial effectors but subsequent death from T. gondii infection may also be explained by a defect in NKT cell function given a number of reports suggest a protective role for NKT cells in limiting T. gondii-induced immune pathology (60, 61).

The Tec kinases appear attractive therapeutic candidates for the modulation of TCR signal strength (62, 63, 64). However, the complexity of the signal requirements for individual cytokine genes in distinct immune cell types may make it difficult to predict the outcome of Tec kinase modulation in vivo: attenuation of IL-4 only or IL-4 and IFN-{gamma}. A common and encouraging theme to come of recent studies on Itk is that the kinase appears be critical, not for the gain of function but, for the execution of effector function in both CD4+ T cells and NKT cells, making it an appealing candidate for the termination of existing cytokine-mediated immunopathology.


   Acknowledgments
 
We thank Shoshana Katzman for assistance with experiments, the members of the Fowell laboratory for helpful comments, and Jim Miller for critical review of this manuscript.


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


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

1 This work was supported by National Institutes of Health Grant AI50201 (to D.J.F.), the Howard Hughes Medical Institute Biomedical Research Program for Medical Schools (to D.J.F.), and Training Grant T32-DE07165 (to B.A.). Back

2 Address correspondence and reprint requests to Dr. Deborah J. Fowell, Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, University of Rochester, 601 Elmwood Ave, Box 609, Rochester, NY 14642. E-mail address: deborah_fowell{at}urmc.rochester.edu Back

3 Abbreviations used in this paper: {alpha}Galcer, {alpha}-galactosyl ceramide; PLC{gamma}, phospholipase C{gamma}; SAP, signaling lymphocytic activation molecule-associated protein; PKC{theta}, protein kinase C{theta}; WT, wild type; eGFP, enhanced GFP. Back

Received for publication December 20, 2006. Accepted for publication April 22, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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