Itk and Txk/Rlk are Tec family kinases expressed in T cells. Itk is expressed in both Th1 and Th2 cells. By contrast, Txk is preferentially expressed in Th1 cells. Although Itk is required for Th2 responses in vivo and Txk is suggested to regulate IFN-γ expression and Th1 responses, it remains unclear whether these kinases have distinct roles in Th cell differentiation/function. We demonstrate here that Txk-null CD4+ T cells are capable of producing both Th1 and Th2 cytokines similar to those produced by wild-type CD4+ T cells. To further examine whether Itk and Txk play distinct roles in Th cell differentiation and function, we examined Itk-null mice carrying a transgene that expresses Txk at levels similar to the expression of Itk in Th2 cells. Using two Th2 model systems, allergic asthma and schistosome egg-induced lung granulomas, we found that the Txk transgene rescued Th2 cytokine production and all Th2 symptoms without notable enhancement of IFN-γ expression. These results suggest that Txk is not a specific regulator of Th1 responses. Importantly, they suggest that Itk and Txk exert their effects on Th cell differentiation/function at the level of expression.
The kinases Txk/Rlk (hereafter referred to as Txk) and Itk are distantly related members of the Tec family of tyrosine kinases that are involved in signaling downstream from the TCR. Whereas Txk has a palmitoylation site instead of a pleckstrin homology (PH)5 domain that allows it to be constitutively associated with lipid raft membrane fractions, Itk requires the activation of PI3K for recruitment to the membrane via its PH domain (1, 2). Mutations affecting Itk in mice lead to altered T cell development and mature T cell function with reduced TCR-induced proliferation and impaired IL-2 production in vitro (Refs. 3, 4, 5, 6 and see Ref. 7 for review). One of the most dramatic phenotypes of Itk−/− mice is their defect in Th2 responses in vivo. Itk−/− mice are incapable of developing allergic asthma and have decreased responses to challenge with a number of Th2-inducing parasites, including the eggs of Schistosoma mansoni or the worm Nippostrongylus brasiliensis (8, 9, 10, 11). Indeed, in some cases Itk-deficient mice have been found to mount Th1 responses to Th2-inducing pathogens. In contrast, overexpression of Txk has been associated with increased expression of IFN-γ, a Th1 cytokine (12, 13, 14, 15). Txk has been found to bind directly to a sequence in the IFN-γ promoter, suggesting a direct role for Txk in driving IFN-γ transcription and Th1 responses (16). Together, these data suggested that Itk and Txk have distinct roles in Th2 and Th1 differentiation or function, respectively.
Nonetheless, the exact mechanism by which the Tec kinases influence Th cell differentiation remains controversial. Although some data suggest that Itk induces Th2 differentiation by suppressing the expression of T-bet (17), other reports propose that Itk but not Txk directly interacts with and tyrosine phosphorylates T-bet, promoting its interaction with GATA3, which suppresses the latter’s activity (18). It has also been proposed that Itk may modulate Th2 differentiation by virtue of its expression. Itk is expressed at higher levels than Txk in naive T cells and, although both Itk and Txk are expressed in Th1 cells, Txk is down-regulated in Th2 cells, leaving Itk as the major Tec kinase (17). Consistent with this idea, recent data argue that Itk is not required for Th2 differentiation per se but rather is required for effector function of differentiated Th2 cells (19). These data suggest it may not be the intrinsic function of these kinases but rather their patterns of expression that determine their roles in Th cell differentiation and function.
To evaluate these questions, we examined whether forced overexpression of Txk in T cells could rescue Th2 responses in Itk−/− mice using transgenic mice that drive expression of Txk at levels similar to those of Itk in Th2 cells. If these kinases have distinct Th1- and Th2-inducing properties, one would predict that overexpression of Txk would preferentially drive Th1 responses and that Th2 defects in Itk−/− mice may be exacerbated. However, if defects in Th2 responses in Itk−/− mice are secondary to the low levels of expression of Tec kinases in Th2 cells in the absence of Itk, expression of the Txk transgene may rescue these responses. Using two systems, a murine model of allergic asthma and challenge with the eggs of S. mansoni, a strong Th2-inducing parasite (8, 10), we demonstrate that Itk−/− mice expressing a Txk transgene leads to rescue of Th2 responses with no evidence of overexpression of Th1 cytokines. Our results thus strongly suggest that the effects of Itk and Txk on Th cell function may result from the differential patterns of expression of these kinases.
Materials and Methods
Wild-type (WT), Txk−/− (20), Itk−/− (6), and Tg(CD2-Txk) (13), were used between 6- and 12-wk of age and were backcrossed on the C57BL/6 background for at least five generations. Tg(CD2-Txk)Itk−/− mice were generated by breeding Itk−/− and Tg(CD2-Txk) mice. All experiments were approved by the Office of Research Protection’s Institutional Animal Care and Use Committee at Pennsylvania State University and by the National Institutes of Health.
Single cell preparations were made from thymi and spleens. RBC were lysed from spleens using ammonium chloride lysis solution. Cells were preincubated with anti-CD16 and then stained with combinations of anti-CD8α (clone 53-6.7 and FITC or PE), CD62L (MEL-14 and PE), CD4 (RM4-5 and PerCP-Cy5.5), CD44 (clone IM7 and allophycocyanin), and CD24 (M1/69, FITC) (eBioscience or BD Biosciences/Pharmingen).
Allergic asthma induction
Mice were sensitized with OVA (Sigma-Aldrich) complexed to aluminum hydroxide (10 μg of OVA/1 mg of alum; Pierce) i.p. in a total volume of 200 μl on days 0 and 5. Mice were later challenged intranasally with OVA from days 12 through 15 (at a concentration of 2 mg/ml, for a total of 40 μg of total exposure). Development of allergic asthma was measured by analyzing airway hyperresponsiveness (AHR) on day 16 using a custom-made mechanical ventilator as previously described (9, 10, 21). Mice were then sacrificed, bronchoalveolar lavage (BAL) fluid was obtained, and lungs were sectioned and stained using H&E or periodic acid-Schiff as detailed (9, 10).
Schistosome egg injection and analysis of response
Quantitative real-time PCR (qRT-PCR) analysis
After analysis of AHR, RNA was extracted from the lungs of mice using TRIzol reagent (Invitrogen). cDNA was generated with a kit from Amersham Biosciences. qRT-PCR was then performed for IL-4, IL-13, IFN-γ, CCL-7, and CCL-11, with GAPDH used as a housekeeping gene and the data expressed as 2−ΔΔCT (where CT is threshold cycle) (9). To quantify the expression level of Itk and Txk, Quantitative RT-PCR was performed on cDNA from naive CD4+ T cells or CD4+ T cells differentiated to Th2 cells. Signals were compared with standards generated from plasmids carrying the cDNA for Itk and Txk.
Analysis of calcium responses
CD4+ T cells were purified from spleens by negative selection using MACS mouse CD4+ T cell isolation kit microbeads (Miltenyi Biotec) with the addition anti-NK1.1(BD Biosciences/Pharmingen). Cells were labeled with 5 μg/ml fluo-3 and 5 μg/ml fura red (Molecular Probes/Invitrogen). Cells were kept at room temperature and then stimulated at 37°C with 15 μg/ml biotinylated anti-CD3 (clone 2C11) with the addition of 8 μg/ml streptavidin. Data were collected on a FACSCalibur flow cytometer for 10 min and analyzed using Flow Jo software (Tree Star).
Analysis of lymphocyte proliferation and cytokine secretion in response to Ag challenge
Purified splenocytes obtained from OVA-challenged mice were cultured with 10 and 100 μg/ml OVA (2 × 105 cells/well). After 72 h of culture, cells were pulsed with [3H]thymidine for 18 h and incorporated radioactivity was determined. Cytokine secretion was analyzed by stimulating splenocytes at 2 × 106/ml with 100 μg/ml OVA for 96 h following which supernatants were harvested. Cytokine concentration in supernatants and BAL were determined using a Luminex system (Bio-Rad) with plates from LINCOplex (Millipore). In mice exposed to S. mansoni eggs, cells from draining mediastinal lymph nodes and spleens were collected and restimulated with schistosome egg Ag (SEA) as described (8). Three days later, cells were stimulated with PMA and ionomycin in the presence of GolgiStop (BD Biosciences/Pharmingen), stained for CD4 and intracellular IL-4, IL-5, IL-10, and IFN-γ using specific Abs, and analyzed by flow cytometry.
In vitro differentiation to Th1 and Th2 cells
T cells were purified by T cell isolation columns (R&D Systems) and then sorted for CD4+CD62Lhigh and CD44low naive cells. Cells were stimulated with 1 μg/ml anti-CD3 plus anti-CD28 in the presence of mitomycin-treated, T-depleted splenocytes for 2–3 days and then restimulated with PMA and ionomycin in the presence of GolgiPlug, stained for intracellular levels of IFN-γ and IL-4, and analyzed by flow cytometry as above. Th0 conditions contained no extra cytokines; Th1 conditions included 40 ng/ml IL-12 and 10 μg/ml anti-IL-4; Th2 conditions included 40 ng/ml IL-4 and 10 μg/ml anti-IL-12 and anti-IFN-γ. Cytokines were obtained from Peprotech.
Statistical evaluation was conducted by using the Student’s t test with a probability value of p < 0.05 considered statistically significant.
Txk-null T cells are not defective in Th1 or Th2 cytokine secretion
Txk has been suggested to specifically regulate the production of IFN-γ in Th1 cells. To determine whether T cells lacking Txk have defects in this function, we analyzed T cells from Txk-null mice (20). We first confirmed that Txk-null mice had similar T cell subpopulations as those of WT mice (Fig. 1,A; Ref. 20). Consistent with these observations, Txk−/− CD4+ T cells showed normal TCR-induced Ca2+ mobilization (Fig. 1,B), supporting the conclusion that at this level of analysis T cells from Txk-null mice exhibit no significant abnormalities. We next analyzed naive CD4+ T cells that were differentiated under Th1 or Th2 conditions (Fig. 1 C). Txk-null T cells were able express both Th1 and Th2 cytokines similarly to WT T cells. Thus, although it has been proposed that Txk is a Th1-inducing kinase, these data support the conclusion that Txk is not required for the generation of IFN-γ in Th1 cells.
The CD2 promoter-driven Txk transgene is expressed in Th2 cells at similar levels as those of endogenous Itk
Although the above results suggest that Txk is not required for IFN-γ expression, Txk is expressed at lower levels than Itk (Fig. 1,D) and may have unique functions that might not be obvious due to its low level of expression. To evaluate whether Txk can compensate for Itk if expressed at higher levels, we used a transgenic mouse model in which Txk was overexpressed using the CD2 promoter (Tg(CD2-Txk)). When crossed onto an Itk-null background (referred to here as Tg(CD2- Txk)Itk−/−), expression of Txk was able to rescue the known calcium signaling defect downstream of TCR stimulation that occurs in T cells lacking Itk (Fig. 1 B), similar to what has been reported in the thymus (13). However, although expression of this Txk transgene has been shown to improve positive selection in Itk-deficient mice, Tg(CD2-Txk)Itk−/− mice still had low numbers of peripheral T cells, even lower than Itk-deficient mice (supplemental Table I).6
To further evaluate the Txk transgene, we used qRT-PCR to evaluate the relative expression levels of Txk and Itk mRNA in freshly isolated CD4+ T cells and in cells that were differentiated under Th2-inducing conditions. Although the expression of Txk in freshly isolated CD4+ T cells was higher (17-fold) than that of endogenous Txk in WT mice, it was only 5-fold higher than the level of expression of Itk mRNA (Fig. 1,D). As previously reported, endogenous Txk expression in WT Th2 cells was dramatically reduced in Th2 cells (17). However, in Th2 cells from Tg(CD2-Txk)Itk−/− mice, Txk expression remained high, similar to levels of Itk in WT Th2 cells (Fig. 1 D). Thus, in Th2 cells, Txk expression was similar to Itk expression on a copy number basis. We therefore used this transgene on the Itk−/− background to evaluate the ability of Txk to complement Itk function in Th2 cells.
Rescue of allergic airway inflammation and AHR in mice lacking Itk by expression of Txk transgene
To determine the effect of expression of Txk in Th2 cells, we analyzed the development of allergic asthma, a disease dependent on Th2 cells and cytokines. OVA-immunized and -challenged WT and Itk−/− mice, as well as mice expressing the Txk transgene on an Itk−/− background (Tg(CD2-Txk)Itk−/−), were evaluated for airway resistance in response to methacholine challenge as a measure of AHR. In these experiments, while WT mice developed significant airway resistance, Itk−/− mice responded poorly as previously reported (Fig. 2,A) (9). However, Tg(CD2-Txk)Itk−/− transgenic mice exhibited significant levels of airway resistance, similar to the WT mice (Fig. 2 A).
We next analyzed airway inflammation and mucous production, factors that can contribute to the development of allergic asthma in this model. Histological evaluation of lung sections revealed that Tg(CD2-Txk)Itk−/− mice exhibit massive leukocyte infiltration in the lung, which was similar to or higher than that observed in WT mice (Fig. 2,B). In contrast, Itk−/− mice showed reduced leukocyte infiltration, as previously reported (9, 10). Increased thickening of the epithelial cell lining of the bronchioles and mucous production by airway goblet cells was also observed in Tg(CD2-Txk)Itk−/−mice, similar to that seen in WT mice (Fig. 2 B).
Expression of Txk transgene in Itk-null mice enhances production of Th2 cytokines in response to allergic inflammation
Th2-specific cytokines such as IL-4, IL-5, and IL-13 are involved in inducing allergic airway inflammation (22, 23). To examine whether expression of Txk transgene rescued production of Th2 cytokines, we first analyzed cytokine production from splenic T cells of mice immunized and challenged with OVA. Stimulation with OVA in vitro induced proliferation of T cells from WT, Tg(CD2-Txk)Itk−/−, and Itk−/− OVA-challenged mice, although those from Itk−/− mice exhibited reduced proliferation in comparison to WT mice (Fig. 3,A) (10). Splenocytes from Tg(CD2-Txk)Itk−/− mice, however, had proliferative responses equivalent to those of WT mice, and the Txk transgene rescued IL-4, IL-5, and IL-13 secretion from these cells in vitro (Fig. 3,B). Because Txk is suggested to regulate the expression of Th1 cytokines such as IFN-γ, we also examined the expression of IFN-γ. Strikingly, Tg(CD2-Txk)Itk−/− mice did not secrete elevated levels of IFN-γ as would be expected if it specifically regulated IFN-γ (Fig. 3 B).
To further examine the expression level of Th2 cytokines in the lungs upon induction of allergic asthma, we measured the expression level of IL-4 and IL-13 mRNA in the lungs of OVA-immunized and -challenged WT, Itk−/−, and Tg(CD2-Txk)Itk−/− mice using qRT-PCR. As shown in Fig. 3,C, there was enhanced expression of IL-4 and IL-13 mRNA in the Tg(CD2-Txk)Itk−/− mice in comparison to WT and Itk−/− mice. By contrast, the mRNA levels of IFN-γ were similar in all three strains of mice (Fig. 3,C). These findings were further confirmed by the analysis of BAL fluid for the level of Th2 cytokines in these mice, indicating that expression of Txk rescued the Th2-mediated inflammation in the lungs of OVA-challenged mice (Fig. 3 D). Therefore the Txk transgene does not appear to lead to enhanced Th1 differentiation or IFN-γ production, nor does it block Th2 differentiation as would be observed if Txk were a Th1-inducing kinase (12, 13, 14, 15, 16). Higher levels of IL-13 mRNA, but not protein, were also observed in the Tg(CD2-Txk)Itk−/− mice in comparison to WT mice. These results could be due to IL-13 mRNA expression in other inflammatory cells, such as eosinophils and mast cells, recruited to the lungs in response to the disease. Nonetheless, we did not observe higher levels of the IL-13 protein, suggesting that this message may not be fully translated in these cells. Altogether, these data indicate that in the absence of Itk, Txk expression can lead to the generation of a Th2 response both systemically and in the lungs.
Rescue of CD4+ T cell recruitment in Itk-null mice expressing Txk transgene
Analysis of CD4+ T cell numbers in the lungs revealed that although WT mice could recruit these cells into the lung during airway inflammation, as previously reported, Itk-null mice could not (10, 24). However, Tg(CD2-Txk)Itk−/− mice had similar numbers of CD4+ T cells in the lungs compared with WT mice, indicating that expression of Txk was able to rescue migration and recruitment of T cells into the lung (Fig. 4). These results were not secondary to increased numbers of CD4+ T cells in Tg(CD2- Txk)Itk−/− mice, as these mice have even lower numbers of mature CD4+ T cells than Itk−/− mice (supplemental Table I). These data thus confirm that, in vivo, Txk can rescue specific functions of Itk that lead to the recruitment of leukocytes into the lungs during the development of allergic asthma.
The Txk transgene rescues Th2 responses to S. mansoni eggs
To examine induction of Th2-mediated responses using a different model, we analyzed responses to i.v. injection of S. mansoni eggs. Injection of S. mansoni eggs i.v. into mice results in the formation of eosinophilic granulomas in the lung in a Th2 cell-dependent manner. In Itk−/− mice, the size of these granulomas is reduced (Fig. 5,A) (8). However, consistent with our observations in the OVA-induced model, injection of S. mansoni eggs into Tg(CD2-Txk)Itk−/− mice resulted in granulomas of a size similar to that seen in WT mice (Fig. 5,A). In contrast, overexpression of Txk on a WT background also did not increase granuloma size (Fig. 5,A). To evaluate Th2 cytokine production in this model, we examined cytokine production by intracellular staining of cells from the spleen and draining mediastinal nodes of mice injected with S. mansoni eggs. Analyses of splenic and lymph node T cells restimulated in vitro with SEA confirmed that Itk-null mice had fewer cells producing the Th2 cytokines IL-4, IL-5, and IL-10. However, Itk−/− mice carrying the Txk transgene had similar numbers of cells making these cytokines as those seen in WT mice, confirming that Th2 differentiation was rescued. Again, Tg(CD2-Txk)Itk−/− mice did not show elevated numbers of IFN-γ-producing cells (Fig. 5, B and C). These mice also did not show increased percentages of cells expressing Th2 cytokines compared with WT cells, demonstrating that the cells were not merely over-responsive due to the expression of the Txk transgene. Similarly, overexpression of Txk on a WT background did not increase the level of Th1 or Th2 cytokines (Fig. 5, B and C), again arguing that expression of the Txk transgene did not merely result in increased activation of these cells. These data are in keeping with the finding that T cells from these mice do not exhibit higher levels of calcium influx in response to TCR stimulation (see Fig. 1). Thus, expression of the Txk transgene can rescue patterns of Th2 cytokine production in response to multiple Th2-inducing agents.
The Tec kinases Itk and Txk are both expressed in T cells and regulate their development, activation, and function (7). A role for Itk has been demonstrated for the production of Th2 cytokines, whereas roles for Txk are less clear (8, 9, 10, 11, 17, 19). Prior work suggests that Txk regulates IFN-γ production and thus Th1 development; however, a specific role of Txk in the regulation of Th1-specific cytokine production is still unresolved (12, 13, 14, 15, 16). Our data argue that Txk does not specifically drive expression of this Th1 cytokine. First, Txk-null T cells do not exhibit defects in producing IFN-γ, arguing that Txk is not essential for IFN-γ production. More importantly, in the models examined here, overexpression of Txk did not induce elevated levels of IFN-γ and prevent the development of Th2 cell-mediated disease. Instead, overexpression of Txk rescued production of Th2-type cytokines including IL-4, IL-5, and IL-13 in the lungs of Tg(CD2-Txk)Itk−/− mice in response to Th2-inducing agents. It should be noted that these observations do not rule out the possibility that Txk could indirectly regulate IFN-γ production via interaction with other factors that are specifically expressed in Th1 cells.
Txk is the most distantly related family member of the Tec family of tyrosine kinases. This protein has an N-terminal palmitoylation site instead of the PH domain found in Itk and other Tec kinases, allowing it to be anchored constitutively in the plasma membrane. Thus Txk, unlike Itk, does not require the activation of PI3K to be recruited to the plasma membrane (1, 2). This would suggest specific and unique functions for Txk. However, we show that when expressed at similar levels as Itk, Txk can functionally replace Itk for the induction of predominant Th2 responses in vivo by enhancing the expression of Th2-specific cytokines. Our data provide strong evidence that Txk can function to rescue Th2 responses in the absence of Itk, including AHR, airway inflammation, granuloma formation, T cell recruitment, and cytokine production in the lungs in vivo as well as Th2 cytokine production from T cells restimulated in vitro. Our data thus support the idea that these kinases have overlapping functions.
Although Txk is normally expressed at very low levels in Th2 cells, our observations with these transgenic mice nonetheless help provide a better understanding of the specific functions of Txk and Itk that would have been otherwise difficult to address. In addition, these observations provided greater insight about the relevance of selective expression of Txk and Itk in Th1 and Th2 cells, respectively. Our work also confirms previous studies by Berg, Luban, and colleagues that show that Txk expression is specifically down-regulated in Th2 cells (17, 25). In contrast, these groups observed similar levels of Itk expression in Th1 as well as Th2 cells. Thus, negative regulation of Txk expression may occur in a Th2-specific manner, perhaps via negative regulation by Th2-specific transcription factors. Alternatively, expression of Itk may be specifically maintained by Th2-specific transcription factors. In either case, this expression pattern could result in a critical dependence of Th2 cells on the function of Itk. Our work here lends support to this model, because expression of Txk using the T cell-specific CD2 promoter would allow for continuous expression of Txk, even in differentiating Th2 cells, and thus provide crucial Tec kinase signals needed for functional responses in the absence of Itk. Hence, our observations suggest that the selective expression of Txk and Itk in Th1 and Th2 cells, respectively, may provide the delicate balance of signals required for inducing or maintaining different types of T cell responses. It should be noted that although the CD2-Txk transgene has been reported to improve positive selection in Itk−/− thymocytes, we find that it does not rescue peripheral T cell numbers (perhaps because the low T cell numbers are secondary to earlier defects in Itk−/− thymocytes (26)). These observations suggest that the rescue of Th2 responses are not merely the result of increased CD4+ T cell numbers, although we cannot rule out other effects on development (3, 5, 27, 28)). However, the normal levels of TCR-induced Ca2+ influx in Tg(CD2-Txk) and Tg(CD2-Txk)Itk−/− peripheral T cells also argues that our results are not the result of nonspecific hyperactivation of cells expressing the Txk transgene.
In previous studies, we have found that although the absence of Itk leads to defective Th2 responses, absence of both Txk and Itk surprisingly leads to normal Th2 responses in Rlk−/−Itk−/− mice (8). Although these results may appear to contradict our current findings, it is possible that these findings result from the fact that CD4+ T cells lacking both Txk and Itk maintain high levels of GATA3 following stimulation compared with WT and Itk-null mice. These results suggested that a defect in GATA3 down-regulation may lead to a propensity to develop into Th2 cells in T cells from the Txk/Itk double knockout mice, perhaps because this was the only type of response that could occur. Our data presented here indicates that expression of Txk at similar levels as Itk can lead to normalized Th2 responses in Itk-deficient mice and that potential redundancy in function between Txk and Itk may explain the Th2-specific response observed in Tg(CD2-Txk)Itk−/− mice.
Our data are also in support of recent data from Fowell and colleagues, who demonstrated that Itk-deficient T cells can differentiate into Th2 cells; however, they cannot elaborate and secrete Th2 cytokines upon restimulation (19). The enforced expression of Txk in these Th2 cells allows for functional rescue of this event. Given previous results suggesting that rescue of calcium signaling can rescue Th2 cytokine production in Itk−/− CD4+ T cells (11, 19), it is likely that rescue of Ca2+ mobilization by expression of the Txk transgene contributes to our observations. Thus, Txk may be able to rescue Th2 responses by restoration of intracellular Ca2+ increases that are defective in Itk−/− T cells, perhaps by participating in the Slp76/GADS/LAT complex, which regulates phospholipase C (PLC)γ1 activation. Indeed, expression of the Txk transgene has also been shown to be able to rescue PLCγ1 tyrosine phosphorylation in Itk−/− double-positive thymocytes (13).
Other cell types such as mast cells and eosinophils have also been suggested to play an important role in the development of Th2-specific responses (29, 30, 31). These cells are capable of producing Th2 cytokines and chemokines that can induce initial immune responses and mediate further activation and migration of Th2 cells to the lungs, thereby exacerbating the immune responses (32). However, because the CD2 promoter cassette that drives Txk expression in these transgenic mice is primarily active in T cells (including NKT cells), these findings suggest that the defect in Itk-null mice in developing effective Th2 responses in our models is unlikely to be due to the lack of expression of Itk in other cell types such as mast cells (33).
In recent years there has been a growing interest in the Tec kinases Itk and Txk as potential therapeutic targets for Th2- and Th1-mediated diseases, respectively. Our data here support the model in which the Tec kinases Txk and Itk regulate Th cell-mediated responses via their differential expression in Th1 and Th2 cells, respectively, and not due to intrinsic functional differences as previously suggested (12, 13, 14, 15, 16). Our data also suggest that the defective Th2 response in the absence of Itk is due to reduced Tec kinase signals and that either Itk or Txk can fulfill this role if expressed at high enough levels. Overall, these findings provide novel insight into the role of Tec kinases Txk and Itk in the regulation of Th cell differentiation/function and disease.
We thank members of the Center for Molecular Immunology and Infectious Disease at Penn State for helpful comments. We also thank E. Kunze, N. Bem, S. Magargee, Dr. D. Grove, and M. Potter for technical assistance.
The authors have no financial conflict of interest.
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.
This work was supported by National Institutes of Health Grants AI051626 and AI065566, the American Health Association (to A.A.), and by the intramural program of the National Institutes of Health, National Human Genome Research Institute (to A.M.V., J.C., J.G.-R., and P.L.S.), the National Institute of Allergy and Infectious Disease (to D.J. and A.S.), and the National Institute of Child Health and Human Development (to C.S. and P.L.). N.S. thanks the American Academy of Allergy, Asthma and Immunology’s Strategic Training in Allergy Research (ST∗AR) Award for support.
Abbreviations used in this paper: PH, pleckstrin homology; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; qRT-PCR, quantitative real-time PCR; SEA, schistosome egg Ag; WT, wild type.
The online version of this article contains supplemental material.