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The Journal of Immunology, 1999, 162: 5805-5812.
Copyright © 1999 by The American Association of Immunologists

Costimulation Reverses the Defect in IL-2 But Not Effector Cytokine Production by T Cells with Impaired I{kappa}B{alpha} Degradation1

Thomas M. Aune*, Ana L. Mora{dagger}, Somee Kim{ddagger}, Mark Boothby2,{dagger} and Andrew H. Lichtman{ddagger}

Departments of * Medicine (Rheumatology) and {dagger} Microbiology/Immunology, Vanderbilt University Medical School, Nashville, TN 37232; and {ddagger} Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the transcriptional basis for states of unresponsiveness in primary T cells is unclear, tolerant B lymphocytes exhibit inhibition of both c-Jun N-terminal kinase induction and I{kappa}B{alpha} (inhibitor of NF-{kappa}B{alpha}) degradation, leading to lower levels of both nuclear AP-1 and NF-{kappa}B. Expression of an I{kappa}B{alpha} mutant resistant to signal-induced degradation in transgenic T cells caused markedly deficient effector cytokine (IL-4, IFN-{gamma}) production after primary TCR stimulation despite a detectable level of nuclear NF-{kappa}B. A TCR response element from the IFN-{gamma} promoter, despite lacking detectable NF-{kappa}B/Rel sites, was also unresponsive to TCR ligation. Nuclear induction of AP-1 proteins in response to T cell activation was diminished in transgenic T cells. Costimulation induced by anti-CD28 mAb increased IL-2 production, but failed to reverse the defects in effector cytokine production. Taken together, these data indicate that impaired NF-{kappa}B/Rel signaling in T cells interferes with the signal transduction pathways required for efficient induction of effector cytokine production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The spectrum of potential lymphocyte responses to Ag includes proliferation with cytokine production, unresponsive states with impaired proliferation and production of IL-2 or of effector cytokines such as IL-4 and IFN-{gamma}, and activation-induced apoptosis Refs. (1, 2, 3, 4, 5); reviewed in Refs. (6, 7, 8, 9, 10). Of note, Ag-specific T cells can proliferate or produce IL-2 yet exhibit a dramatic block of effector function in some in vivo models of T cell tolerance (11, 12, 13, 14), whereas in others a complete block of IL-2 production is observed (reviewed in 15). Despite the wealth of information regarding cell surface proteins required for a TCR-MHC interaction to provide growth signals Refs. (16) and (17); reviewed in Refs. (7), (18), and (19), it is unclear what alterations in transcription regulation are sufficient to render normal T lymphocytes unresponsive to TCR engagement.

In this regard, prior in vitro studies have indicated that three inducible transcription factor families are of particular importance in T cell activation: NF-ATs, the AP-1 family of basic leucine zipper proteins, and the NF-{kappa}B/Rel family reviewed in Refs. (20, 21, 22, 23). In vitro models of anergy induction using CD4+ T cell clones have focussed on the AP-1 family (24, 25, 26). This focus is consistent with the discovery that B lymphocytes rendered tolerant in vivo exhibit a failure to activate AP-1 (27). However, these B cells manifest a unique profile in which both I{kappa}B{alpha} degradation and AP-1 induction are inhibited, yet constitutive activation and nuclear translocation of NF-AT proteins remain intact, and low levels of RelA and c-Rel are present in the nuclei of tolerant cells (27). Similar alterations may be present in a model of superantigen-induced CD4+ cell tolerance (28). Such findings raise the question of whether functional unresponsiveness could result from inhibition of a single transcription factor family or instead requires the coordinate dysregulation of multiple signal transduction pathways.

To address this question we have created transgenic (Tg)3 mice whose T cells express I{kappa}B{alpha}({Delta}N) (29), an I{kappa}B{alpha} mutant that is refractory to signal-induced degradation (29, 30, 31). Such targeted inhibition impaired the development of a normal population in the CD8+ lineage with only a modest effect on the deployment of their CD4+ counterparts (29). This initial study did not investigate the effect of costimulation or the competence to express effector cytokines such as IL-4 or IFN-{gamma}, which may be regulated independently from IL-2. Although proliferation in response to mitogenic stimuli and IL-2 was inhibited by the mutant I{kappa}B{alpha} transgene, expression of the activation markers IL-2R{alpha} and CD69 by T lymphoblasts was normal. Because the cytosolic retention molecule I{kappa}Bß is degraded in response to TCR stimuli when cells are costimulated through CD28 (32), costimulatory signals could potentially lead to normal T cell activation despite inhibition of I{kappa}B{alpha} degradation. In this regard, previous results suggested that the inhibition of NF-{kappa}B/Rel signaling was not absolute, in that NF-{kappa}B/Rel proteins were still present at low levels in freshly isolated nuclei of Tg thymocytes (29). Moreover, CD28 may signal induction of T cell effector functions through NF-{kappa}B-independent mechanisms (33). Accordingly, we have investigated the nature of T cell responses when selective impairment of I{kappa}B{alpha} degradation was targeted to the T lineage. The result was a defect in the production of effector cytokines (IL-4, IFN-{gamma}) that could not be reversed by costimulation or IL-2 despite the spontaneous development of CD44high and CD62Llow cells in vivo and despite increased IL-2 production by T cell costimulation. In addition, induction of nuclear c-Jun, JunB, and NF-ATc following TCR stimulation was impaired. Taken together, these findings indicate that inhibiting the degradation of one I{kappa}B in vivo is sufficient to produce a state of lymphocyte unresponsiveness even though low levels of nuclear NF-{kappa}B are induced. The resulting cytokine production defect preferentially affects effector functions and differs from the phenotypes produced by the absence of individual NF-{kappa}B/Rel subunits (34, 35).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tg mice

I{kappa}B{alpha}({Delta}N)-Tg mice expressing a mutant I{kappa}B targeted to the T lineage using the lck proximal promoter and human CD2 locus control region have been described previously (27). Progeny derived from two separate founders were backcrossed to C57BL/6 mice for use in these studies. Dist.IFN-{gamma} luciferase mice bear a transgene encoding luciferase under the control of the distal (-98 to -72) IFN-{gamma} promoter AP-1-like element and a minimal IFN-{gamma} promoter TATA element; they were described in detail previously (36). Briefly, the 2.8-kb transgene contains a head-to-tail tetramer (5' to 3') of the distal conserved IFN-{gamma} AP-1-like element (-98 to -72 bp), the IFN-{gamma} minimal promoter (-39 to +64), and the luciferase reporter gene from the plasmid PRL-luc (37). The mice used in this study resulted from backcrossing onto a C57BL/6 genetic background. All mice were housed in microisolators under specific pathogen-free conditions.

Cell preparation and culture

Cells from the indicated sources were cultured, and B cell-depleted suspensions of spleen and lymph node cells were prepared for mobility shift analyses, as described previously (27). Two independent experimental methods were used to prepare and stimulate cytokine production, as indicated in the figure legends. Similar results were obtained with each method. In method 1, splenocytes (2.5 x 106/ml) were stimulated without fractionation, or CD4+ T cells (1 x 106/ml) were stimulated using APCs (1 x 106/ml) through culture with plate-bound Abs against CD3 (10 µg/ml of 145-2C11) in the presence or the absence of an activating hamster mAb against mouse CD28 (10 µg/ml; clone 37.51, PharMingen, San Diego, CA) as previously described (27, 36). CD4+ T cells (purity, ~90–95%) and syngeneic APCs were purified (subtraction of CD8+ T cells, NK cells, and class II-expressing cells from pooled splenocytes and lymph node cells, or negative selection with mAbs against CD4 and CD8, respectively) as described previously (36). In method 2, CD4+ T cells were purified from splenocytes by positive selection using magnetic beads derivatized with Abs against mouse CD4 and Detachabead (Dynal, Lake Success, NY). Unfractionated splenocytes and CD4+ T cells were cultured in RP/10F in flat-bottom 96-well microtiter plates (5 x 105 cells/0.2 ml) that had been coated with anti-CD3 (1 µg/ml in PBS). Where indicated, an activating Ab against mouse CD28 (PharMingen) was included in the culture (10 µg/ml).

Gel mobility shift and immunoblot analyses

Nuclear fractions were prepared from single cell suspensions by high salt extraction in the presence of protease inhibitors (31). Gel mobility shift assays of NF-{kappa}B/Rel proteins were performed using a double-stranded 32P-labeled oligonucleotide modified from {kappa}B enhancer sequences in the IL-2R{alpha} promoter ({kappa}B-pd; upper strand, 5'-CAACGGCAGGGGAATTCCCCTCTCCTT) (31). DNA binding reaction mixtures (20 µl) contained 4 µg of nuclear extract, 2 µg of double-stranded poly(dI-dC), and 10 µg of BSA buffered in 20 mM HEPES (pH 7.9), 5% glycerol, 1 mM EDTA, 1% Nonidet P-40, and 5 mM DTT. Similar reactions were performed using a labeled AP-1 oligonucleotide (Promega, Madison, WI). Nucleoprotein complexes were then resolved on native 5% polyacrylamide gels and visualized by autoradiography. Small scale nuclear extracts for immunoblot experiments were prepared from 5 x 106 cells as previously described (38, 39). Nuclear proteins were fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with specific primary Abs directed against c-Jun, JunB, and NF-ATc (all from Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were then washed, incubated with secondary horseradish peroxidase-conjugated second Abs, and developed using an enhanced chemiluminescent system according to the manufacturer’s instructions (Amersham, Arlington Heights, IL).

Analysis of cytokine and luciferase activities

ELISAs for the detection IL-4 and IFN-{gamma} were performed using Ab pairs (PharMingen, Sorrentino, CA) according to the manufacturer’s recommended procedures as described previously (36, 40). The lower limits of sensitivity of the ELISAs were 10 pg/ml (IL-4) and 1 U/ml (IFN-{gamma}), using as a reference standard mouse IFN-{gamma} with a sp. act. of 107 U/mg protein (PharMingen). As the upper limits of sensitivity were 2 ng/ml (IL-4) and 300 U/ml (IFN-{gamma}), additional quantitation was performed on serially diluted samples when indicated. To measure transcriptional activity directed by a minimal IFN-{gamma}-derived promoter, stimulated cells were harvested from duplicate cultures, washed twice in PBS, then suspended (30 min at 25°C) in 50 µl of lysis buffer (Luciferase Assay Kit, Promega, Madison, WI). The supernatant fluid was harvested, and duplicate 20-µl aliquots were assayed for luciferase activity after a 15-s incubation with 100 µl of luciferase reagent (Promega) in a luminometer (Turner TD20/20; Promega). The results are expressed as the mean relative light units per 106 cells produced by the replicates minus the blank value (background measurement using luciferase reagent alone).

Enumeration of apoptotic cells

To generate samples for quantitative TUNEL assays, single-cell suspensions were cultured for 40 h in the presence of plate-bound anti-CD3 mAb (10 µg/ml) and anti-CD28 mAb (10 µg/ml) as indicated, harvested, and stained with PE-conjugated mAb against CD4 or CD8. Cells were fixed with paraformaldehyde, permeabilized with 70% ethanol at -20°C, washed in PBS supplemented with 1% BSA, then resuspended in reaction mixtures containing terminal deoxynucleotidyltransferase (Life Technologies, Gaithersburg, MD) and biotin-16-dCTP (Boehringer Mannheim, Indianapolis, IN). After incubation at 37°C for 30 min, cells were washed in PBS/1% BSA, stained with FITC-conjugated avidin, and analyzed by flow cytometry. Control reactions lacking terminal deoxynucleotidyltransferase were performed to quantify nonspecific staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production of effector cytokines by CD4+ cells after TCR cross-linking requires NF-{kappa}B/Rel signaling

Normal activation of CD4+ T cells leads to increased IL-2 production, which may be regulated independently from effector proteins such as IL-4 and IFN-{gamma}. The NF-{kappa}B/Rel protein family binds to functionally important IL-2 promoter sequences (32, 36; reviewed in Ref. 21), and induction of this family of transcriptional activators is essential for the production of IL-2 in response to TCR cross-linking (29, 41, 42). However, the induction of proteins such as IL-2R{alpha}/CD25 and CD69 by TCR stimulation of I{kappa}B{alpha}({Delta}N)-Tg T cells was inhibited minimally (29). This selective inhibition raised the question of whether key effector cytokine genes such as IFN-{gamma} and IL-4, whose promoters may be regulated by NF-{kappa}B binding (43, 44), would be activated normally despite inhibition of NF-{kappa}B/Rel signaling. To investigate the effect of this targeted inhibitor on the ability of primary T cells to produce effector cytokines, we stimulated cells with immobilized Abs against the TCR (Fig. 1Go). As shown in Fig. 1GoA, an unfractionated population of splenocytes from I{kappa}B{alpha}({Delta}N)-Tg mice was dramatically impaired in its generation of both IFN-{gamma} and IL-4. This result was recapitulated when purified CD4+ T cells were used instead of unfractionated splenocytes (Fig. 1GoB). Thus, these responses of wild-type cells induced in vitro by anti-CD3 cannot be attributed to cytokine production by NK (IFN-{gamma}) or NK1.1+ T (IL-4) cells (45). Moreover, the defect of Tg T cells compared with controls is unlikely to be due only to enhanced apoptosis or decreased proliferation (see below). These findings indicate that the population of CD4+ T lymphocytes that developed with a defect in signal-induced degradation of I{kappa}B{alpha} was unable to activate the expression of key effector molecules.



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  		FIGURE 1. Targeted inhibition of the NF-{kappa}B/Rel signaling pathway blocks production of effector cytokines. Splenocytes (A) or CD4+ T cells (B) were prepared from I{kappa}B{alpha}({Delta}N)-Tg mice or NTg littermates (method 1, Materials and Methods; using subtraction of NK1.1+, I-A+, and B220+ cells). After a 48-h stimulation with immobilized anti-CD3, the levels of IFN-{gamma} and IL-4 in the culture supernatants were determined by ELISA. The indicated results represent the mean (±SEM) from three separate experiments, each using two Tg (filled bars) and two NTg littermates (open bars). Similar results were obtained in three additional experiments using an independent method of T cell preparation and stimulation (method 2, Materials and Methods).

 
CD28-dependent enhancement of IL-2, but not effector cytokine, production

The above data suggested that efficient production of effector cytokines by CD4+ T cells may depend on the NF-{kappa}B/Rel signaling pathway. However, maximum rates of I{kappa}B{alpha} degradation require coordinate signaling through the TCR and CD28 (32, 46). Of note, a significant pool of NF-{kappa}B/Rel dimers is retained in the lymphocyte cytoplasm by alternative inhibitory proteins such as I{kappa}Bß and I{kappa}B{epsilon}, rather than by I{kappa}B{alpha} (32, 47). In contrast to I{kappa}B{alpha}, I{kappa}Bß is not degraded in response to TCR signaling alone, requiring the combination of CD28 and a TCR signal (32), while hypophosphorylated I{kappa}Bß can protect NF-{kappa}B from I{kappa}B{alpha} and shepherd NF-{kappa}B into the nucleus (48, 49). Moreover, CD28 can signal through NF-{kappa}B-independent pathways (33). Indeed, stimulatory anti-CD28 Abs proved able to partially reverse the defect in IL-2 production by I{kappa}B{alpha}({Delta}N) T cells (Fig. 2GoA). In light of these findings, we investigated whether ligation of CD28 would either restore normal production of effector cytokines by I{kappa}B{alpha}({Delta}N) T cells or enhance their nuclear induction of NF-{kappa}B/Rel proteins. Unfractionated splenocytes and CD4+ T cells from individual Tg or control mice were stimulated with immobilized anti-TCR Abs, alone or in combination with activating Abs against CD28. We found that T cells from the Tg mice were deficient in their production of effector cytokines even when activated by the combination of costimulatory and TCR signaling (Fig. 2Go). Similar results were obtained in experiments with splenocytes (Fig. 2GoB) and purified CD4+ T cells (Fig. 2GoC).



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  		FIGURE 2. Costimulation through CD28 increases IL-2 production, but does not reverse the defects in cytokine production caused by I{kappa}B{alpha}({Delta}N). A, CD4+ T cells prepared from I{kappa}B{alpha}({Delta}N)-Tg mice or NTg littermates were stimulated for 48 h with immobilized anti-CD3 or anti-CD3 and anti-CD28 as indicated. Levels of IL-2 in culture supernatants were determined by ELISA. The indicated results represent the mean (±SEM) from two separate experiments. Splenocytes (B) or CD4+ T cells (C) were prepared from I{kappa}B{alpha}({Delta}N)-Tg mice or NTg littermates. Resultant cells were stimulated 48 h with immobilized anti-CD3 or anti-CD3 aand anti-CD28 as indicated. Levels of IFN-{gamma} and IL-4 in culture supernatants were determined by ELISA. The indicated results represent the mean (±SEM) from two separate experiments, each using two Tg mice and two NTg littermates. Similar results were obtained after 24-h stimulation and in two additional experiments using an independent method of T cell preparation and stimulation (method 2, Materials and Methods).

 
The prevalence of apoptotic CD4+ cells among splenocytes from I{kappa}B{alpha}({Delta}N)-Tg mice was only modestly higher than that among cells from littermate controls after stimulation with anti-CD3 or a combination of anti-CD3 and anti-CD28 (Fig. 3GoA). Moreover, the number of Tg T cells recovered after 2 days was greater than half that in non-Tg (NTg) controls, while the decrease in cytokine production was far greater (a 10- to 20-fold difference). It was also possible that only a small minority of cells from I{kappa}B{alpha}({Delta}N) mice respond to TCR stimulation. However, 40% of Tg cells acquired blast morphology after combined stimulation through the TCR and CD28 compared with 57% of wild-type control cells (Fig. 3GoB). It is thought that the bulk of effector cytokine production immediately after polyclonal stimulation under these conditions reflects activity of a pre-existing population of resting CD44high (or CD62Llow or CD45RBlow) CD4+ cells (50). As such, these results could have arisen if the activation defect of I{kappa}B{alpha}({Delta}N) Tg T cells blocked formation of such cells in vivo. However, the observed steady state frequencies of CD44high and L-selectinlow CD4+ T cells in spleen (data not shown) and lymph nodes (Fig. 3GoC) of young Tg mice were no less than those in their NTg littermates. Thus, the observed decrease in cytokine production is not simply due to a diminished population of responder cells stemming from enhanced apoptosis. We conclude that despite increased IL-2 production, costimulation is unable to reverse the inhibitory effect of the I{kappa}B{alpha}({Delta}N) protein on the effector cytokine production capacity of T cells.



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  		FIGURE 3. Costimulation through CD28 does not reverse the enhanced apoptosis caused by targeted expression of I{kappa}B{alpha}({Delta}N). A, Splenocytes from either NTg (open bars) or Tg (filled bars) mice were cultured in the presence of immobilized anti-CD3 Abs (10 µg/ml) with or without the addition of anti-CD28 (10 µg/ml) as indicated. After 40 h in culture, cells were divided equally, stained with PE-labeled Abs against CD4 or CD8, and subjected to TUNEL analysis. The data represent the mean percentage (±SEM) of TUNEL-positive cells in the CD4+ T cell subset. The mean was calculated using data from eight Tg mice and an equal number of NTg controls (four independent experiments with 6- to 8-wk-old mice). B, Forward light scattering of cells activated as described in A was determined for cells in the CD4+ gate. C, The normal steady state population of memory phenotype cells in I{kappa}B{alpha}({Delta}N) mice was determined by flow cytometry. Spleen and lymph node cells from 8-wk-old Tg mice and their NTg littermates were stained for CD4 and the indicated cell surface markers. The results represent the mean (±SEM) prevalence of cells with high level expression of the indicated marker. Previous work has shown that virtually no splenocytes or lymph node T cells in I{kappa}B{alpha}({Delta}N) mice are CD25+ or CD69+ (27), indicating that the majority of these CD44high cells are CD25-CD69-.

 
Inhibition of NF-{kappa}B/Rel signaling by I{kappa}B{alpha}({Delta}N) is refractory to costimulation

In light of the failure of costimulation to reverse the functional unresponsiveness of Tg T cells, we investigated whether these observations could be correlated with the biochemical status of NF-{kappa}B/Rel induction. T cells were stimulated with immobilized anti-TCR Abs, alone or in combination with activating Abs against CD28. Nuclear extracts from these T cell preparations were analyzed by gel mobility shift analysis to determine the relative levels of nuclear NF-{kappa}B/Rel proteins. These experiments revealed that costimulation with anti-CD28 was insufficient to reverse the observed inhibition of NF-{kappa}B/Rel induction by the I{kappa}B{alpha} mutant. Importantly, the results indicate that a low level of NF-{kappa}B/Rel proteins was able to bypass this inhibitory effect (Fig. 4Go), leading to detectable levels of NF-{kappa}B complexes reminiscent of the low levels of nuclear c-Rel and RelA in nuclei of tolerant B cells (27). These mobility shift experiments also demonstrated a shift in the ratio of faster migrating complexes (previously shown to represent p50/NF-{kappa}B1 without c-Rel or RelA) relative to the slower mobility NF-{kappa}B complexes (previously shown to represent p50/NF-{kappa}B1 with c-Rel or RelA). As such, these observations imply that even incomplete inhibition of the NF-{kappa}B/Rel pathway can lead to a defect in effector function.



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  		FIGURE 4. Costimulation through CD28 does not reverse the NF-{kappa}B/Rel-specific defect in nuclear protein induction. Pooled spleen and lymph node cell suspensions from NTg or Tg mice were B cell depleted by nylon wool column chromatography, then cultured for 16 h in the presence of immobilized anti-CD3 Abs (10 µg/ml) with or without the addition of anti-CD28 (10 µg/ml) as indicated. Nuclear extracts from equal numbers of the resultant cells were then used in gel mobility assays (1 µg protein/lane) with a probe specific for NF-{kappa}B/Rel proteins (31). Previous studies have established that the complexes include p50, c-Rel, and RelA (upper band, arrow) and p50 without c-Rel or RelA (lower band, open circle), respectively (27).

 
Failure of IL-2 to reverse the unresponsive state

Costimulation only partially reversed the defect in IL-2 production by T cells from I{kappa}B{alpha}({Delta}N) mice, and IL-2 signaling may influence the development of unresponsiveness or the activation of effector cytokine production (51). Therefore, we directly investigated whether exogenous IL-2 would reverse the defect in activation of effector cytokine genes in I{kappa}B{alpha}({Delta}N) T cells. The results (Fig. 5Go) show that exogenous IL-2 was unable to restore effector cytokine production by Tg T cells to the level generated by comparably treated cells from NTg littermates. We conclude that selective inhibition of I{kappa}B{alpha} degradation (27) in T cells can induce a functional defect that is not simply a consequence of deficient IL-2 production.



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  		FIGURE 5. Failure of IL-2 to reverse the defect in cytokine production by CD4+ T cells expressing I{kappa}B{alpha}({Delta}N). CD4+ T cells were stimulated (48 h) with immobilized anti-CD3 in the presence or the absence of exogenous rIL-2 (5 ng/ml). Culture supernatants were analyzed for IFN-{gamma} and IL-4 levels by ELISA. The results represent the mean values (±SEM) from two separate experiments using a total of five Tg mice and an equal number of NTg littermates.

 
Inhibition of a promoter lacking an NF-{kappa}B site

The above findings indicate that inhibition of I{kappa}B{alpha} degradation attenuated the activation of certain target cytokine genes by a mechanism refractory to exogenous IL-2. Since the IFN-{gamma} gene contains a potentially functional NF-{kappa}B site (44), it was possible that the observed decrease in IFN-{gamma} production was due to a requirement for a crucial NF-{kappa}B-dependent regulatory sequence. Thus, the observed decrease in effector cytokine production might represent either a general inhibition of T cell activation by I{kappa}B{alpha} or another direct transcriptional effect of NF-{kappa}B/Rel proteins. To help distinguish between these possibilities, we crossed I{kappa}B{alpha}({Delta}N) and dist.IFN-{gamma} reporter gene Tg mice (36). T cells from these latter mice transcribe a luciferase gene that is under the control of a minimal IFN-{gamma} promoter and an AP-1-like element that lacks an NF-{kappa}B site. This imperfect TRE element binds AP-1, but not NF-{kappa}B/Rel transcription factors (52). The activity of this minimal reporter gene mimics much of the specific regulation of the IFN-{gamma} gene despite the absence of an NF-{kappa}B binding site (36). Activation of the luciferase gene by the dist.IFN-{gamma} promoter was abrogated in I{kappa}B{alpha}({Delta}N)-expressing T cells compared with that in cells from littermates expressing only wild-type I{kappa}B{alpha} (Fig. 6GoA).



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  		FIGURE 6. Disruption of the NF-{kappa}B signaling pathway prevents activation of an AP-1-like element derived from the IFN-{gamma} promoter. A, Inhibition of reporter gene activity in normal T cells. I{kappa}B{alpha}({Delta}N) Tg mice were intercrossed with dist.IFN-{gamma}-luc reporter Tg mice. CD4+ T cells were purified from 5-wk-old Tg pups bearing the chimeric reporter gene dist.IFN-{gamma}, then stimulated with immobilized anti-CD3 Abs (see Materials and Methods). After 48 h, T cells were harvested and analyzed for levels of luciferase activity. The data represent the mean (±SEM) results derived using dist.IFN-{gamma}-Tg T cells from mice with (filled bar) or without (open bar) the I{kappa}B{alpha}({Delta}N) transgene in two separate experiments (five I{kappa}B{alpha}({Delta}N)-Tg mice and an equal number of control NTg littermates). B, Diminished AP-1 binding activity in the nuclei of cells from I{kappa}B{alpha}({Delta}N) Tg mice. Gel mobility shift assays were performed using equal amounts of protein (1 µg) derived from nylon wool-enriched T cells cultured in the presence or the absence of activating stimuli as indicated and an AP-1-specific oligonucleotide (53). Cells were obtained from pooled spleen and lymph nodes of I{kappa}B{alpha}({Delta}N) Tg mice and littermate controls as indicated. C, Inhibition of the nuclear induction of AP-1-related transcription factors. CD4+ T cells were purified from the spleens of I{kappa}B{alpha}({Delta}N) Tg mice and their non-Tg littermates, and then cultured in the presence of anti-CD28 and immobilized anti-CD3 as indicated. Proteins in the nuclear extracts of these cells were resolved by SDS-PAGE and subjected to immunoblotting using Abs against c-Jun, JunB, or NF-ATc as indicated.

 
Since the distal TCR-RE from the IFN-{gamma} promoter binds AP-1, but not NF-{kappa}B transcription factors, these data raise the question of whether the defect in induction of this promoter element may result from diminished induction of AP-1 transcription factors. To investigate this possibility, gel mobility shift assays of AP-1 binding activity were performed using nuclear extracts derived from unstimulated and activated cells (Fig. 6GoB). These experiments indicated that AP-1 activity in I{kappa}B{alpha}({Delta}N) T cells was diminished compared with that in wild-type controls. To extend this observation, additional nuclear extracts prepared from resting and activated primary wild-type and Tg T cells were analyzed for levels of c-Jun, JunB, and NF-ATc by Western blotting. We found that induction of nuclear c-Jun, JunB, and NF-ATc protein was inhibited in T cells with impaired NF-{kappa}B signaling (Fig. 6GoC). Although CD28 costimulation increased levels of nuclear JunB and NF-ATc in wild-type T cells, it did not restore levels of these transcription factors in Tg T cell nuclei to normal. Thus, the defect in effector cytokine production induced initially by inhibition of NF-{kappa}B signaling can be correlated with diminished induction of critical transcription factors that regulate IFN-{gamma} gene transcription. These findings suggest that the observed defect of IL-4 and IFN-{gamma} production by these lymphocytes is not simply a consequence of deficient IL-2 production and includes a propagation of inhibitory effects on T cell activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The response of lymphocytes to engagement of their Ag-specific receptor and the development of effector function represent key checkpoints in the regulation of immune responses. A crucial objective is to define which specific perturbations in signal transduction or transcription are sufficient to cause specific states of functional unresponsiveness. We present evidence that inhibition of I{kappa}B{alpha} degradation, through expression of a trans-dominant inhibitor, I{kappa}B{alpha}({Delta}N), is sufficient to generate an unresponsiveness that is manifested as impaired effector cytokine production after primary stimulation of normal CD4+ T cells. Importantly, this unresponsive state was achieved despite the induction of low levels of nuclear NF-{kappa}B and was not reversed by either costimulation through CD28 or provision of exogenous IL-2. An impaired IFN-{gamma} response was also manifested at the transcriptional level. Inhibition of NF-{kappa}B/Rel signaling blocked induction of a TCR response element from the IFN-{gamma} promoter despite the absence of an NF-{kappa}B site and also decreased the nuclear induction of AP-1 binding activity as well as that of c-Jun, JunB, and NF-ATc. In contrast, the low level of NF-{kappa}B/Rel proteins that escaped the inhibitory effect of I{kappa}B{alpha}({Delta}N) appeared sufficient to permit significant IL-2 production by T cells upon costimulation. This latter observation suggests that activation of signaling pathways other than NF-{kappa}B/Rel (33) makes quantitatively important contributions to CD28 costimulation of IL-2 production by primary T cells.

These experiments focussed on primary ex vivo stimulation of cells to reflect analyses of lymphocytes that have been exposed in vivo to conditions that induce unresponsiveness (12, 14, 27, 28). It is thought that the bulk of the IFN-{gamma} and IL-4 responses under these conditions derives from pre-existing CD44high CD4+ T cells (50; reviewed in 54). The observation that similar numbers of CD44high, CD62Llow, and CD45RBlow (data not shown) CD4+ cells were present in our wild-type and I{kappa}B{alpha}({Delta}N) samples suggests that the acquisition of these phenotypic characteristics does not depend on NF-{kappa}B/Rel signaling. Accordingly, our data cannot distinguish between an impairment of signaling during stimulation in vivo and the interesting possibility that the signaling defect prevented development or survival of a population of T cells in vivo that has the capacity to produce IFN-{gamma} within 24–48 h after in vitro stimulation. Of note, c-Jun is induced equally in naive and effector T cells following antigenic stimulation (55), indicating that CD4+ T cells of each functional type induce this transcription factor. In contrast, c-Jun induction in cells from I{kappa}B{alpha}({Delta}N) mice was impaired, suggesting that some inhibition of signaling during the in vitro stimulation was exerted. It is unlikely that preferential apoptosis of CD44high cells could account for the observed defect in cytokine production (Fig. 3Go). It also was conceivable that the defect in effector cytokine production reflects some block in proximal activation, an event that could be bypassed using PMA/ionomycin. However, we have found that although the inhibition of NF-{kappa}B/Rel signaling had little effect on the prevalence of early apoptotic cells compared with controls, IL-4 and IFN-{gamma} production by Tg cells remained profoundly inhibited (unpublished observations). In summary, our data indicate that an initial impairment of NF-{kappa}B signaling leads to a defect in effector cytokine production through inhibition of signaling at a differentiation step in vivo, during reactivation in vitro, or both.

The ability of a targeted perturbation in one signaling pathway to create such a spectrum of defects is a striking outcome. There has been little previous evidence that partial inhibition of one transcriptional pathway can cause lymphocyte activation defects refractory to costimulation and IL-2. Other correlates of T lymphocyte unresponsiveness induced in anergy models in tissue culture systems have included diminished or aberrant activation of signal-transducing kinases such as ZAP-70, the Ras/Raf/mitogen-activated protein kinase kinase (MAPKK) cascade, c-Jun N-terminal kinase (JNK), and extracellular signal-related kinase (ERK) (25, 26, 56, 57). While informative mutations in humans indicate that ZAP-70 kinase activity is required for CD4+ T cell activation, the precise roles of other elements in these pathways remain uncertain, because defects in the Ras/Raf/MAPKK pathway block T cell development (58, 59, 60, 61). These impairments in signal transduction lead to defective activation of the AP-1 family of basic leucine zipper transcription factors in anergized CD4+ T cell clones (24, 25, 26). However, it is unclear whether impaired activation of AP-1-like proteins is sufficient to render primary CD4+ T cells unresponsive and unable to perform effector functions. Gene-targeting experiments have created mice whose T cells lack the c-Fos and c-Jun transcription factors, but such mice exhibit apparently normal T cell activation, as do mice whose T cells express a dominant negative MAP/ERK kinase kinase (MEKK) (52, 62, 63). Thus, it remains to be determined whether a primary defect in AP-1 activation will inhibit the effector functions of mature T cells.

Although previous reports have focussed on the AP-1 family as a critical step in the transcriptional regulation of clonal anergy induction in vitro, I{kappa}B{alpha} degradation and NF-{kappa}B/Rel signaling are abnormal in lymphocytes rendered tolerant in vivo (27, 28). Moreover, the Ras/Raf/MEKK pathways may regulate I{kappa}B{alpha} degradation through MEKK1, and NF-{kappa}B induction may, in turn, influence transcriptional activation through both NF-{kappa}B and AP-1 sites (64, 65, 66). Alternatively, IL-2 responsiveness may be crucial for the acquisition of T cell competence to produce effector cytokines (51). In this regard it is interesting to note that although T cells from I{kappa}B{alpha}({Delta}N) mice can be induced to express normal levels of cell surface expression IL-2R{alpha}, -ß, and -{gamma}c subunits, the IL-2-dependent induction of STAT proteins is significantly decreased.4 Taken together, these findings suggest that degradation of the different I{kappa}Bs may be a key regulatory target in determining the characteristics of a T cell response to TCR engagement. In principle, independent regulation of the cytosolic retention of different trans-activating subunits of NF-{kappa}B may lead to different patterns of unresponsiveness in T cells. Specifically, when only c-Rel was eliminated from the NF-{kappa}B/Rel signaling pathway, the primary defect was the absence of IL-2 production, and almost all other defects were normalized if exogenous IL-2 was present (34, 42). When only the RelA component of signaling was impaired, IL-2 production was normal, and cells exhibited no increase in TCR-induced apoptosis, but T cells exhibited a proliferative defect refractory to the endogenously produced IL-2 (35). Finally, T cells with impaired I{kappa}B{alpha} degradation affecting both the RelA and c-Rel trans-activators exhibited defective production of effector cytokines (IL-4, IFN-{gamma}) and enhanced TCR-induced apoptosis despite costimulation or the provision of exogenous IL-2. Taken together, these observations can be reconciled if differential regulation of c-Rel and RelA lead to different patterns of CD4+ T cell unresponsiveness. However, it is not clear what was the prevalence of CD44high CD4+ cells in c-Rel or RelA-deficient samples, and the patterns of cytokine production could not be compared with those of I{kappa}B{alpha}({Delta}N) cells in the same experiments. Although inhibition of I{kappa}B{alpha} degradation leading to coordinate, but incomplete, cytosolic retention of RelA and c-Rel has been demonstrated in vivo (27), it also is not clear whether selective defects in the nuclear induction of specific members of the NF-{kappa}B/Rel family occur. Notwithstanding these issues, the present findings demonstrate that the inhibition of I{kappa}B{alpha} degradation, resulting in an incomplete blockade of NF-{kappa}B translocation to the nucleus, could suffice to inhibit an effector function of CD4+ T cells.


   Acknowledgments
 
We thank W. Armistead for technical assistance; J. Youn, D. Ballard, and G. Miller for helpful discussions; A. Abbas, J. Chen, G. Miller, T. Parks, and J. W. Thomas for critiques of the manuscript; and Vanderbilt University Cancer Center (CA 68485) and D.R.T.C. (DK20593) for tissue culture, DNA, and flow cytometry core functions.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants K01-AR02027 and PO1-DK20593 (to T.A.), R01-AI36997 and GM42550 (to M.B.), and PO1-HL36028 (to A.H.L.); the Arthritis Foundation (to T.A.); and Vanderbilt University Cancer Center Grant CA68485. M.B. is a Scholar of the Leukemia Society of America. Back

2 Address correspondence and reprint requests to Dr. Mark Boothby, Department of Microbiology and Immunology, AA-4214 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232-2363. E-mail address: Back

3 Abbreviations used in this paper: Tg, transgenic; NTg, nontransgenic; I{kappa}B{alpha}, inhibitor of NF-{kappa}B{alpha}; MEKK, MAP/ERK kinase kinase. Back

4 A. L. Mora, J. Youn, A. D. Keegan, and M. Boothby. NF-{kappa}B participation in the lymphokine-dependent proliferation of T lymphoid cells. Submitted for publication. Back

Received for publication September 28, 1998. Accepted for publication February 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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