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The Journal of Immunology, 2006, 176: 3895-3899.
Copyright © 2006 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Itk-Dependent Signals Required for CD4+ T Cells to Exert, but Not Gain, Th2 Effector Function1

Byron B. Au-Yeung, Shoshana D. Katzman and Deborah J. Fowell2

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
The TCR signals for the release of CD4 effector function are poorly understood. Itk plays an essential role in Th2, but not Th1, responses. However, when Itk is required during Th2 development is unclear. We followed the fate of Itk-deficient T cells during Th2 development in vitro and in vivo using an IL-4/GFP reporter. Surprisingly, a similar frequency of itk–/– CD4+ cells differentiated and committed to the Th2 lineage as wild-type cells. However, Itk-deficient Th2 cells failed to exert effector function upon TCR triggering. Loss of function was marked by defective transcriptional enhancement of Th2 cytokines and GATA3. IL-4 production in itk–/– Th2s could be rescued by the expression of kinase-active Itk. Thus, Itk is necessary for the release, but not gain, of Th2 function. We suggest that the liberation of effector function is tightly controlled through qualitative changes in TCR signals, facilitating postdifferentiation regulation of cytokine responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Naive CD4+ T cell differentiation into Th1 and Th2 effector cells is regulated by coordinating signals from the TCR, cytokine receptors, and costimulatory/adhesion molecules (1). The role of cytokine receptor signaling for the induction of lineage-specific transcription factors (Tbet for Th1 and GATA3 for Th2) is well established. However, how TCR signals differentially regulate the development of CD4 effector subsets remains poorly understood. The development of specialized Th1 and Th2 effectors involves initial differential gene expression, chromatin remodeling, and subsequent transcriptional enhancement of cytokine genes (2). Cytokine and TCR signals are required for initial differentiation, but the ability to exert effector function appears to be TCR dependent and cytokine signal independent.

A number of signaling components have been implicated in the development of Th2, but not Th1, responses: requirements for Lck, Itk, SLAM/SAP, PKC{theta}, and the transcription factors NFATc1, JunB, and NF{kappa}B p50 have been independently reported (3). However, the interrelationship of these signaling molecules in Th2 development remains to be determined. A role for Itk in Th2 development has been established in Itk-deficient mice following pathogen challenge (4, 5) and allergic asthma (6); however, its mechanism of action has not been established. Itk regulation of the PLC{gamma}/calcium pathway and NFATc1 (4, 7), cytoskeletal reorganization at the immunological synapse (8, 9), and the early down-regulation of Tbet (10) are all potential Th2 control points.

To better understand Itk at a mechanistic level we set out to define when Itk is required: at initial differentiation, survival/expansion or release of effector function of Th2-primed cells. We used a IL-4/enhanced GFP (eGFP) reporter mouse (4get) to follow the fate of Itk-deficient CD4+ T cells during Th2 development in vitro and in vivo (11). Surprisingly, the frequency of naive itk–/– CD4+ T cells that initiated IL-4-gene expression and committed to the Th2 lineage was identical with that of wild-type (WT)3 CD4+ T cells. Rather, Itk was required for Th2-primed cells to exert their effector function in a calcium-dependent fashion. These studies highlight differential signal requirements for the gain and release of Th2 effector function.


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

Itk–/– BALB/c mice (C.129S2(B6J)-Itktm1Litt) (12) were crossed to BALB 4get mice (C.Cg-IL4tm1Lky) (11) or DO11.10 TCR transgenics on a TCR-C{alpha}–/– (C.Cg-Tcratm1Mjo TgDo11.1010Dlo) background. Mice were maintained in the pathogen-free animal care facility at the University of Rochester Medical Center (Rochester, NY).

Cell purification

CD4+ cells were enriched from the spleen and lymph node by Ab/complement-mediated lysis (4) and sorted (FACSAria; BD Biosciences) for naive CD4+ cells, >98.7% CD62LhighCD44low. Memory cells were enriched by depletion of CD62Lhigh cells (Miltenyi Biotec). T-depleted splenocytes (APC) were isolated from WT mice by complement lysis (4) and irradiated (2000 rads). Naive DO11.10+ T cells were isolated by FACS sorting as for nontransgenic T cells.

T cell priming

A total of 106 naive CD4+ T cells per well in 2 ml of complete RPMI 1640 medium with 10% heat-inactivated FCS was stimulated with plate-bound mAb H57.597 (0.5 µg/ml) and 37N51.1 (2 µg/ml) (37°C). Th1 priming: 10 U/ml human IL-2 (rhIL-2; National Institutes of Health Research and Reference Reagent Program, Frederick, MD), 10 ng/ml rIL-12 (Peprotech), and 40 µg/ml anti-IL-4 mAb (11B11). Th2 priming: 10 U/ml rhIL-2, 50 ng/ml murine rIL-4, and 50 µg/ml anti-IFN {gamma} mAb (XMG 1.2). After 5–6 days, cells were washed and restimulated at 1 x 105 cells in 200 µl in H57-coated plates. Where designated, rhIL-2, rIL-4, anti-IL-4R{alpha} mAb (M1, 50 µg/ml), anti-CD28 (2 µg/ml), ionomycin (1 µg/ml; Calbiochem), PMA (50 ng/ml), or cyclosporin A (2 µg/ml; Sigma-Aldrich) were added. Naive DO11.10 TCR-C{alpha}–/– CD4+ cells were stimulated with 1 µM OVA (323–339) peptide (pOVA) and APC.

Real-time RT-PCR

RNA was extracted (TRIzol; Invitrogen Life Technologies) and reverse-transcribed (RT for PCR kit; BD Clontech). Real-time PCR used Assays-on-Demand TaqMan primer/probe sets with an ABI prism 7900 sequence detection system (Applied Biosystems). Target levels were normalized to CD3{delta}.

Cytokine measurements

Cytokines from 48-h culture supernatants were measured by ELISA. The Cytokine Capture Assay (Miltenyi Biotec) was performed essentially as described (13). A total of 1 x 106 CD4+ T cells were stimulated with plate-bound anti-TCRbeta or PMA/ionomycin, harvested, and labeled with the bifunctional Ab "catch" reagent for 5 min on ice and warmed to 37°C with H57 or PMA/ionomycin for 45 min for cytokine secretion. Cytokine was detected by FACS using a second anti-cytokine mAb. Gates were drawn on cells labeled without the catch reagent ("no catch"). Intracellular cytokine staining was performed using a BD Pharmingen kit. Brefeldin A was added to cultures 4 h before harvest.

Leishmania major infections

Mice were infected intradermally in one ear with 2 x 105 L. major promastigotes (4) in 10 µl of PBS and PBS in the contralateral ear. Four weeks postinfection, cells were isolated from the ear tissue with 1 mg/ml collagenase/dispase (Roche) for 30 min at 37°C. L. major-specific cytokine production was determined by ELISPOT (4).

T cell transfections

WT Itk or kinase-dead (KD, K390R) Itk, GATA3, and constitutively nuclear NFATc1 (14) cDNA were cloned into a bicistronic internal ribosomal entry site-GFP expression vector. On day 4 of Th2 priming, itk–/– CD4+ cells were electroporated with 4 µg of plasmid DNA using the Amaxa Mouse T cell nucleofector kit (Amaxa Biosystems). Transfected cells were incubated at 37°C overnight before restimulation and cytokine secretion analysis.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
Th2 differentiation independent of Itk signals

CD44lowCD62Lhigh naive CD4+ T cells from IL-4/eGFP reporter mice (4get) were primed under Th2 conditions, and the emergence of eGFP+ cells was analyzed by flow cytometry (Fig. 1A). Surprisingly, the kinetics, frequency, and magnitude of early eGFP expression were identical in Th2-primed WT 4get and itk–/– 4get cells. Real-time PCR analysis confirmed the FACS data showing normal Th differentiation under strong polarizing conditions in the absence of Itk (10). Under Th2 priming, GATA3 and IL-4 were induced and Tbet was repressed, whereas Th1 priming induced Tbet and IFN-{gamma} expression and GATA3 repression in itk–/– cells (Fig. 1B). IL-5 and IL-13 also were induced in itk–/– cells similarly to WT (Fig. 2B). WT and itk–/– cells also looked similar with respect to the low frequency of eGFP+ cells observed after neutral priming (1.00% and 0.93% eGFP+, respectively). Despite induction of both Th1 and Th2 programs in itk–/– cells, only Th1 cells were capable of secreting effector cytokines on restimulation (Fig. 1C) (4). In contrast, although at very low frequency, both WT and itk–/– cells secreted IL-4 during the early Th2-priming period (Fig. 1D). We found no evidence for preferential death of itk–/– eGFP+ cells after either 5-day priming or following restimulation (24 h) (Fig. 1E) using 7-aminoactinomycin D exclusion as a marker of cell viability. Indeed, fewer activated itk–/– cells than WT cells were 7AAD positive on restimulation, consistent with itk–/– defects in activation-induced cell death (15).


Figure 1
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  		FIGURE 1. ITK-independent Th1 and Th2 differentiation. A, Naive CD4+ T cells from 4get mice were stimulated under Th2 priming conditions and analyzed by FACS for early eGFP expression. B, Naive CD4+ T cells were stimulated under Th2 or Th1 priming conditions and mRNA purified for quantitative RT-PCR. Data are expressed as fold change over unstimulated naive CD4+ T cells. C, Cells in B were restimulated with anti-TCRbeta and cytokine secretion measured by ELISA. D, Naive cells were Th2-primed for 72 h and cytokine secretion measured by cytokine capture (Miltenyi). E, Cells as in A were primed for 5 days, and eGFP+ cells were assayed for viability by FACS before (0 h) and after restimulation with anti-TCRbeta (24 h). Numbers in quadrants are the percentage of CD4+ T cells expressing the given marker. Results represent one of four comparable experiments.

 

Figure 2
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  		FIGURE 2. Defective transcriptional enhancement of IL-4 in absence of ITK. A, WT and itk–/– 4get Th2 effectors were analyzed by FACS for eGFP expression 24 h after restimulation. Numbers above each gate are the percentage of CD4+ T cells. B, Naive CD4+ cells were Th2 primed and mRNA purified during primary (1°) and secondary (2°) stimulation for quantitative RT-PCR: cytokine data expressed as fold change relative to resting WT Th2 cells, transcription factor data relative to naive CD4+ cells. Results represent one of three comparable experiments.

 
Itk-deficient signals fail to support transcriptional enhancement of Th2 cytokine genes

Secondary stimulation of WT Th2-primed cells is accompanied by a 100- to 1000-fold transcriptional enhancement of Th2 cytokine gene expression over primary stimulation (16) (Fig. 2B). Single-cell analysis of WT 4get cells showed that a significant population of Th2-primed cells became at least 10-fold brighter for eGFP (eGFPhigh) on restimulation (Fig. 2A), suggesting mRNA differences were, in part, due to enhancement of transcription in individual cells. However, the absence of Itk led to a striking block in transcriptional enhancement of Th2 cytokines on restimulation as measured by a failure to up-regulate eGFP expression by FACS (Fig. 2A) and transcripts by RT-PCR (Fig. 2B). In addition, itk–/– Th2-primed cells were unable to sustain GATA3 expression on restimulation in contrast to other transcription factors such as NFATc1 (Fig. 2B). This does not appear to be due to aberrant Tbet expression (10) as Tbet mRNA expression was down-regulated in Th2-primed effectors (Fig. 2B).

Transcriptional enhancement and the release of Th2 effector function

To correlate transcripts with IL-4 protein production, we used cells from 4get mice and the cytokine capture assay. On restimulation, we found a strong positive correlation between enhanced transcripts, eGFPhighcells, and the magnitude of IL-4 production in WT Th2 cells (Fig. 3A). Although the frequency of eGFP expressing itk–/– Th2-primed cells remained constant on restimulation (~65%), consistent with enhanced mRNA transcripts over naive cells (Fig. 2B), the failure to further enhance transcription led to a profound attenuation of IL-4 protein production (Fig. 3A). The absence of intracellular staining for IL-4 confirmed a defect in protein production and not secretion (Fig. 3B). Importantly, bypassing the TCR proximal signaling defects using PMA/ionomycin revealed the IL-4 potential of the Th2-primed itk–/– eGFP+ T cells: WT and itk–/– Th2-primed cells were identical in their ability to secrete IL-4, and both had lost the capacity to secrete IFN-{gamma} (Fig. 3C) consistent with Th2 differentiation.


Figure 3
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  		FIGURE 3. ITK-dependent release of Th2 effector function. Th2 effectors were restimulated with anti-TCRbeta mAb and assayed for IL-4 secretion by cytokine capture (A) or intracellular cytokine staining (B). C, Th2 effectors restimulated with PMA/ionomycin and analyzed for eGFP expression and IL-4 secretion (left panels, 6 h) or for IL-4 and IFN-{gamma} secretion (right panels, 24 h). Numbers in dot plots are the % of CD4+ T cells. D, DO11.10 CD4+ T cells were stimulated with 1 µM pOVA/APC under Th1 or Th2 priming conditions and then recultured with pOVA/APC for a further 5 days under additional Th1 or Th2 conditions. Cells were stimulated a third time with pOVA/APC and cytokines measured by ELISA. E, Th2 effectors were restimulated with anti-TCRbeta and additional cytokines or Abs for 24 h and IL-4 secretion from eGFP+ cells measured by cytokine capture. F, Th2 effectors were restimulated with anti-TCRbeta, TCRbeta and ionomycin (plus Iono 2°) or TCRbeta and cyclosporin A (plus CsA 2°), or with PMA/ionomycin for 6 h and IL-4 secretion from eGFP+ cells measured as in A. Some cells received ionomycin at the start of the primary culture, but not on restimulation (plus Iono 1°). Results represent one of four comparable experiments. G, itk–/– Th2 cells were transfected with empty vector, WT-Itk, kinase-inactive KD-Itk (K390R), GATA3, or constitutively nuclear NFATc1 and restimulated for IL-4 secretion in transfected cells at 24 h.

 
To directly address Th2 lineage commitment, WT and itk–/– DO11.10 TCR Tg+ T cells were primed under Th1 or Th2 conditions with pOVA/APC for 5 days, harvested, and restimulated for a further 5 days in the original or opposing polarizing conditions. Cells were stimulated for a third round in the absence of exogenous cytokine to determine their cytokine program (Fig. 3D). Itk-deficient CD4+ T cells failed to develop into IL-4 secreting cells even after multiple rounds of Th2 priming (Fig. 3D, middle panel) despite similar expansion as WT cells over three rounds of stimulation. Nevertheless, both Th2-primed WT and itk–/– cells were committed to the Th2 lineage as secondary priming under Th1 conditions failed to generate IFN-{gamma} producing cells (Fig. 3D, right panel). Again, PMA/ionomycin restimulation of pOVA/APC Th2-primed itk–/– cells revealed their Th2 potential. Thus, in the absence of Itk, CD4+ cells differentiate into cells competent and committed to the Th2 lineage but secondary Itk-deficient TCR signals fail to support enhancement of cytokine mRNA levels critical for the production of Th2 cytokines.

Itk is required for optimal IL-2 and IL-4 responses (4, 10, 12, 17) and may be a component of CD28 costimulation (18, 19). Supplementation of secondary cultures with these missing components was unable to restore IL-4 secretion in the itk–/– effectors (Fig. 3E). In addition, coculture of itk–/– cells with Th2-primed WT cells also failed to restore Th2 effector function, suggesting the defect is not due to a missing Itk-dependent factor made by WT Th2s. As described (20), IL-4 transcriptional enhancement and IL-4 production in WT cells was highly dependent on calcium mobilization and was blocked by the addition of CsA (Fig. 3F). Indeed, ionomycin-supplemented TCR signals on restimulation (plus Iono 2°), but not at the time of initial priming only (plus Iono 1°), restored IL-4 secretion in itk–/– Th2 effectors (Fig. 3F). Our studies implicate the calcium flux as a critical regulator of Th2 effector function. Indeed, given that itk–/– cells have defects in calcium signaling both on initial priming and on restimulation, our data suggest that initial induction of IL-4 transcription is less sensitive to changes in the magnitude of the calcium flux than secondary transcriptional enhancement. Interestingly, the calcium flux of Th2 cells is poor, compared with that of Th1 cells, both in magnitude and duration (21). Thus, Th2 responses are likely to be highly susceptible to small changes in signals that regulate such a modest calcium response, as highlighted here in the absence of Itk.

To formally demonstrate Itk’s requirement for Th2 effector function, we introduced the Itk gene into Th2-primed itk–/– effectors. We compared WT-Itk with kinase-inactive (KD-Itk) given potential kinase-independent functions for Itk (22). Genes were introduced at the end of the Th2 priming conditions (4). WT-Itk (Fig. 3G) restored the frequency of IL-4-secretors to that seen for WT Th2 cells (Fig. 3E), whereas the kinase-inactive Itk was less effective (Fig. 3G). Thus, Itk contributions to Th2 responses are necessary at the effector stage only. Moreover, full IL-4 activation appears to be Itk-kinase dependent.

Enhancement of IL-4 transcription in Th2 cells appears to be controlled by an inducible 3' enhancer element in the IL-4 gene: DNase 1 hypersensitivity site VA (16, 20). The VA region is essential for IL-4 production in Th2 cells and may be a determinant of probabilistic IL-4 production in Th2 cells (16, 23). Both GATA3 and NFAT bind to this site and are regulated by Itk, (NFATc1 (4) and GATA3; Fig. 2B) in Th2-primed effectors. Thus, Itk may play a role in the induction of this enhancer element and/or the transcription factors required for its activity. However, simple add-back of GATA3 or a constitutively nuclear NFATc1 to Th2-primed itk–/– cells failed to rescue IL-4 effector function (Fig. 3G). A recent report of Itk kinase activity in the phosphorylation of the transcription factor Tbet (24) suggests that Itk might act posttranslationally to enhance IL-4 gene expression.

Itk-dependent release, but not gain, of Th2 effector function in vivo

It is not known whether the impaired Th2 responses in in vivo models are due to defects in differentiation, Th2 survival, homing or cytokine release (4, 5, 6). Th2 function was analyzed directly ex vivo in CD44highCD62Llow effector/memory CD4+ T cells from 4get mice. WT and Itk-deficient effector/memory populations contained similar frequencies of CD4+ eGFP+ cells (Fig. 4A), suggesting that Itk-independent Th2 differentiation can occur to physiological stimuli. Upon in vitro restimulation, eGFP expression was dramatically enhanced and marked by the release of IL-4 protein in WT cells but not in Itk-deficient effector/memory cells (Fig. 4, A and B). As with in vitro-primed cells, PMA/ionomycin released their Th2 effector potential (data not shown). Similar defects in IL-4 were observed in memory cells from non-4get itk–/– mice.


Figure 4
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  		FIGURE 4. Defects in release, but not gain, of Th2 effector function ex vivo. A, Ex vivo-purified CD4+ CD44highCD62Llow effector/memory cells analyzed for eGFP expression before or after anti-TCRbeta stimulation. Numbers below the boxes are the percentage of CD44highCD62Llow CD4s, and numbers in parentheses are the percentage of total CD4s. B, Effector/memory cells were stimulated (anti-TCRbeta) for 24 h and analyzed for IL-4 secretion. Quadrant numbers are the percentage of CD4+ T cells. C, eGFP expression of CD4+ cells isolated from the ear tissue of 4-wk L. major-infected (open overlay) or PBS-injected (filled overlay) mice. Numbers are the percentage of CD4+ cells in each gate. D, L. major-specific ELISPOT from the L. major-infected ear. Results represent one of three experiments.

 
We have previously described the requirement for Itk in Th2 responses to L. major infection (4). We used the 4get mice to determine whether a similar defect in release, but not gain, of Th2 effector function could explain the absence of Th2 activity in infected BALB/c itk–/– mice. Indeed, we were surprised to see a similar frequency of CD4+ eGFP+ cells at the infection site in both WT and itk–/– mice (Fig. 4C), suggesting Itk-independent generation and localization of a IL-4-competent T cell pool. However, the itk–/– eGFP+ population at the tissue site had a reduced frequency of eGFPhigh CD4+ T cells directly ex vivo (Fig. 4C) corresponding to defects in IL-4 production, but not IFN-{gamma}, on restimulation to L. major Ags (Fig. 4D). Therefore, a similar defect in the ability to release Th2 effector function in the absence of Itk was observed ex vivo for both polyclonal and Ag-specific recall responses.

The requirement for Itk in the Th2 effector T cell response, but not in Th2 differentiation, reveals that TCR signals for initiation and liberation of effector function are distinct. Indeed, the recent use of a dual reporter system for analysis of IL-4 transcription and IL-4 secretion supports the notion that IL-4 transcriptional competency and IL-4 production can be separated (25). These signal differences may provide a means for secondary control of effector function subsequent to primary differentiation. Given effector cells are likely to exert their effector function at an infected tissue site and not the priming lymph node, we propose that these distinct signals might also be spatially separated.


   Acknowledgments
 
We thank members of the Fowell laboratory for helpful discussion and technical assistance, and Jim Miller for insightful comments on the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results and 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 Support Program for Medical Schools (to D.J.F.), and Training Grants T32-DE07165 (to B.B.A.-Y.) and T32-A107285 (to S.D.K.). Back

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

3 Abbreviation used in this paper: WT, wild type; KD, kinase dead; eGFP, enhanced GFP. Back

Received for publication November 14, 2005. Accepted for publication February 1, 2006.


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

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M. E. C. Lutsiak, Y. Tagaya, A. J. Adams, J. Schlom, and H. Sabzevari
Tumor-Induced Impairment of TCR Signaling Results in Compromised Functionality of Tumor-Infiltrating Regulatory T Cells
J. Immunol., May 1, 2008; 180(9): 5871 - 5881.
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N. Sahu, C. Mueller, A. Fischer, and A. August
Differential Sensitivity to Itk Kinase Signals for T Helper 2 Cytokine Production and Chemokine-Mediated Migration
J. Immunol., March 15, 2008; 180(6): 3833 - 3838.
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X. Zhao, B. Zheng, Y. Huang, D. Yang, S. Katzman, C. Chang, D. Fowell, and W.-p. Zeng
Interaction between GATA-3 and the Transcriptional Coregulator Pias1 Is Important for the Regulation of Th2 Immune Responses
J. Immunol., December 15, 2007; 179(12): 8297 - 8304.
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V. Sancho-Shimizu, R. Khan, S. Mostowy, L. Lariviere, R. Wilkinson, N. Riendeau, M. Behr, and D. Malo
Molecular Genetic Analysis of Two Loci (Ity2 and Ity3) Involved in the Host Response to Infection With Salmonella Typhimurium Using Congenic Mice and Expression Profiling
Genetics, October 1, 2007; 177(2): 1125 - 1139.
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B. B. Au-Yeung and D. J. Fowell
A Key Role for Itk in Both IFN{gamma} and IL-4 Production by NKT Cells
J. Immunol., July 1, 2007; 179(1): 111 - 119.
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Q. Qi, N. Sahu, and A. August
Tec Kinase Itk Forms Membrane Clusters Specifically in the Vicinity of Recruiting Receptors
J. Biol. Chem., December 15, 2006; 281(50): 38529 - 38534.
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