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The Journal of Immunology, 2001, 166: 2218-2227.
Copyright © 2001 by The American Association of Immunologists

NF-{kappa}B/Rel Participation in the Lymphokine-Dependent Proliferation of T Lymphoid Cells1

Ana L. Mora*, Jeehee Youn2,*, Achsah D. Keegan{dagger} and Mark Boothby3,*

* Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232; and {dagger} Immunology Department, Holland Labs, American Red Cross, Bethesda, MD 20855


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proliferative responses of lymphoid cells to IL-2 and IL-4 depend on activation of the cells, but the mechanism(s) by which activation enhances cellular competence to respond to cytokines is not fully understood. The NF-{kappa}B/Rel family represents one signal transduction pathway induced during such activation. We show in this study that inhibition of NF-{kappa}B through the expression of an I{kappa}B{alpha} (inhibitory protein that dissociates from NF-{kappa}B) mutant refractory to signal-induced degradation (I{kappa}B{alpha}({Delta}N)) interfered with the acquisition of competence to proliferate in response to IL-4 as well as IL-2. Thymocytes and T cells from I{kappa}B{alpha}({Delta}N) transgenic mice expressed normal levels of IL-2R subunits. However, transgenic cells exhibited a dramatic defect in Stat5A activation treatment with IL-2, and a similar defect was observed for IL-4-induced Stat5. In contrast, T lymphoid cells with inhibition of NF-{kappa}B showed normal insulin receptor substrate-2 phosphorylation and only a modest decrease in Stat6 activation and insulin receptor substrate-1 phosphorylation after IL-4 stimulation. These results indicate that the NF-{kappa}B/Rel/I{kappa}B{alpha} system can regulate cytokine receptor capacitation through effects on the induction of downstream signaling by the Stat transcription factor family.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytokines regulate functions in lymphoid cells that include proliferation, survival, and differentiation (1, 2). Certain responses of lymphocytes to cytokines require a cellular state of competence that is acquired only after T cell activation. However, the molecular mechanisms that lead to this enhanced responsiveness are not fully understood. Cells may not respond to IL-2, a growth factor for activated T cells, unless first activated so that the IL-2R{alpha}-chain is expressed (3, 4, 5). After expression of the full high-affinity IL-2R triggered by engagement of the TCR, IL-2 can drive responding cells through the cell cycle, inducing T cell proliferation in an autocrine fashion (4, 5). Similarly, activation-induced increases in IL-4R{alpha}-chains may be required to achieve sufficient IL-4 signaling or participate in growth regulation (6, 7, 8, 9, 10, 11). In contrast to these examples of receptor induction as a basis for competence, it is possible that the competence of a cytokine receptor to transduce signals may be regulated. For instance, the expression of IL-2R may not be sufficient for maximal proliferation in response to IL-2 (5, 12, 13, 14, 15), and there may be circumstances under which T cells expressing IL-4R may fail to activate certain signaling events normally (16, 17, 18).

One signaling pathway that is induced during T cell activation and regulates cytokine-inducible gene expression in normal T cells involves the NF-{kappa}B/Rel transcription factor family (19, 20). Regulation of NF-{kappa}B/Rel proteins is tightly controlled by inhibitory proteins, which include inhibitory protein that dissociates from NF-{kappa}B (I{kappa}B{alpha}).4 The noncovalent association of NF-{kappa}B/Rel dimers with these inhibitory molecules prevents nuclear translocation of the NF-{kappa}B proteins in lymphocytes. During normal T cell activation, I{kappa}B{alpha} is subject to sequence-specific phosphorylation and ubiquitination leading to I{kappa}B{alpha} degradation and the nuclear import of NF-{kappa}B (19, 20). We have developed transgenic (Tg) mice whose T lineage expresses I{kappa}B{alpha}({Delta}N), an I{kappa}B{alpha} mutant that is refractory to signal-induced degradation (21, 22). The resultant inhibition of the NF-{kappa}B/Rel signaling pathway was associated with a T cell proliferative defect refractory to the addition of exogenous IL-2 (21). Consistent with this phenotype, T cells lacking the individual subunit RelA (p65) exhibited an impaired proliferative response to various mitogens despite normal production of IL-2 and expression of IL-2R{alpha} (23). Taken together, these data suggested NF-{kappa}B/Rel involvement in regulation of the competence to generate proliferative responses following the engagement of the IL-2R. The requirement for induction of competence applies to cytokines in addition to IL-2. For instance, IL-4 is a cytokine that can promote the survival, proliferation, and differentiation of T lymphocytes (6, 7, 24, 25). To investigate whether the involvement of NF-{kappa}B/Rel proteins in lymphokine-responsive proliferation of T lineage cells extends to a different T cell growth factor, we have measured the responses to IL-4 of cells derived from I{kappa}B{alpha}({Delta}N) Tg mice and investigated mechanisms for altered IL-2- and IL-4-dependent proliferation to determine whether there are any lymphokine-specific differences underlying the hyporesponsiveness to these cytokines.


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

I{kappa}B{alpha}({Delta}N) Tg mice, in which expression of a trans-dominant inhibitor of NF-{kappa}B/Rel transcription factors is targeted specifically to the T lineage using the proximal lck promoter and cointegration of a CD2 locus control region, have been described previously (21). In selected experiments, these mice were crossed with mice in which the lck proximal promoter alone leads to T cell-specific expression of a transgene encoding a chimeric cytokine receptor in which the mouse IL-2R{beta} extracellular and transmembrane domains are fused to a mouse IL-4R{alpha} cytoplasmic tail (26). All mice were maintained in specific pathogen-free conditions using microisolator cages and were used at 6–8 wk of age in accordance with the federal and state government regulations after institutional approval.

Cell preparations

Single cell suspensions were prepared from thymus, spleen, and lymph nodes by crushing the organs in complete media (RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, and 0.1% penicillin-streptomycin), followed by hypotonic lysis of erythrocytes. Splenic and lymph node T lymphocytes were depleted of B cells by chromatography through nylon wool columns, as described previously (21). Briefly, single cell suspensions from pooled spleen and lymph nodes were loaded onto nylon wool columns preequlibrated with RPMI 1640 supplemented with 5% FBS at 37°C. After 45 min at 37°C, the nonadherent cells were eluted from the columns. The resultant population was <10% B220+ and 75–90% T cells, as determined by flow cytometry.

Proliferation assays

Thymocyte suspensions were counted and plated (5 x 104 cells per 100 µl of media) in microtiter wells. Triplicate samples were cultured for 48 h at 37°C in the presence of PMA (50 ng/ml), ionomycin (1 µg/ml), IL-2 (10 ng/ml), IL-4 (10 ng/ml), IL-12 (10 ng/ml), PMA and IL-4, PMA and IL-2, PMA and IL-12, or PMA and ionomycin, as indicated. Tritiated thymidine (1 µCi in 100 µl of media) was added to each well for the final 8 h before determination of radioisotope incorporation into DNA. T lymphocyte preparations from spleen and lymph node, depleted of B cells as described above, were plated in microtiter wells previously incubated overnight with PBS or anti-CD3 mAb (10 µg/ml, clone 2C11; PharMingen), as indicated. Triplicate samples were then cultured (48 h at 37°C) in the presence of IL-2, IL-4, an activating mAb against CD28 (10 µg/ml, clone 37.51; PharMingen, San Diego, CA), anti-CD28 and IL-2, or anti-CD28 and IL-4, or as for thymocytes, as indicated; DNA synthesis was then quantified as above.

Gel mobility shift analyses

Total cell extracts were prepared from single cell suspensions of thymocytes or T cells using high-salt extraction in the presence of protease inhibitors, as previously described (21, 22). These extracts were then used for gel mobility shift assays of NF-{kappa}B/Rel proteins (21). The probe used was a double-stranded 32P-labeled oligonucleotide modified from a {kappa}B enhancer sequence in the IL-2R{alpha} promoter ({kappa}B-pd) (5'-CAACGGCAGGGGAATTCCCCTCTCCTT-3'). DNA-binding reaction mixtures (20 µl) contained 5 µg of nuclear extract, 2 µg double-stranded poly(dI-dC), and 10 µg BSA buffered in 20 mM HEPES (pH 7.9), 5% glycerol, 1 mM EDTA, 1% Nonidet P-40, and 5 mM DTT. Nucleoprotein complexes were then resolved on a native 5% polyacrylamide gel and visualized by autoradiography.

For detection of Stat5- and Stat6-binding activities, thymocytes or B cell-depleted T lymphocytes were cultured overnight at 37°C in the presence of PMA and ionomycin, or plate-bound anti-CD3 (10 µg/ml) and anti-CD28 (2.5 µg/ml), respectively. These cells were then rinsed once with RPMI medium without serum, cultured 1 h at 37°C in serum-free media, treated for 30 min with IL-2, IL-4, or medium alone, and then lysed in 0.5% Nonidet P-40, 50 mM Tris-Cl (pH 8), 0.1 mM EDTA, 150 mM NaCl, 100 mM Na3VO4, 50 mM NaF, 1 mM DTT, 0.4 mM PMSF, 3 mg/ml aprotinin, 1 µg/ml leupeptin, and 10% glycerol. Lysates were cleared of insoluble material by centrifugation at 15,000 x g for 5 min. For the EMSA, cell extracts (5 µg of total protein) were incubated with 1 µg of poly(dI-dC) for 30 min with 32P-labeled double-stranded oligonucleotide probes containing Stat binding sites, then resolved by electrophoresis on nondenaturing 4.5% PAGE. The probes used contained a consensus binding site for Stat5 (upper strand, 5'-AGATTTCTAGGAATTCAATCC3') (Santa Cruz Biotechnology, Santa Cruz, CA) or a Stat6 binding site from the mouse germline epsilon Ig heavy chain promoter (5'-AACTTCCCAAGAACAGA-3') (27, 28).

Flow cytometric analysis

Single cell suspensions of thymocytes treated overnight with PMA (50 ng/ml), ionomycin (1 µg/ml), or T cells from spleen and lymph node treated with anti-CD3 (10 µg/ml) and anti-CD28 (2.5 µg/ml) were incubated with fluorochrome-conjugated or biotinylated Abs (PharMingen) against IL-2R{alpha}/CD25 (FITC), {gamma}-chain (biotin), IL-2R{beta}/CD122, or IL-4R{alpha} (biotin) at 4°C, as described (20). CD122 was detected by a three-step staining, including biotinylated goat anti-rat Ig and streptavidin-PE (PharMingen).

Immunoprecipitation and immunoblotting

Whole cell extracts obtained from thymocytes and T cells (pooled splenic and lymph node cells) were prepared as described above. These extracts were then subjected to immunoprecipitation with antisera against Stat5A, Stat5B (Zymed Laboratories, San Francisco, CA), insulin receptor substrate-1 (IRS-1; Santa Cruz Biotechnology), and IRS-2 (Upstate Biotechnology, Lake Placid, NY). The precipitates were washed in lysis buffer, solubilized in SDS sample buffer, resolved on 7.5% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. The membranes were probed in accordance with manufacturer’s protocols using mAbs against phosphotyrosine (RC-20; Transduction Laboratories, Lexington, KY; and 4G10; Upstate Biotechnology), Stat5A, Stat5B, IRS-1, or IRS-2, as indicated. After rinsing with PBS-0.1% Tween, bound Abs were detected using enhanced chemiluminescence and secondary Abs, where indicated (NEN, Dupont, Boston, MA).

Isolation and analysis of RNA

Suspensions of thymocytes and B cell-depleted T cells were plated (2.5 x 106 cells/ml) and cultured overnight at 37°C in the presence of PMA (50 ng/ml), ionomycin (1 µg/ml), IL-4 (10 ng/ml), PMA and IL-4, or PMA and ionomycin for thymocytes, or with IL-4 and anti-CD3 plus anti-CD28 for T cells. Total cellular RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Life Technologies, Bethesda, MD). After resolving RNAs by electrophoresis on formaldehyde-agarose gels, nucleic acids were transferred to Nylon membranes (Amersham, Arlington Heights, IL), fixed by UV cross-linking, then probed using cDNAs labeled by random hexamer priming and hybridization in 50% formamide, 0.2% SDS, 0.6 M NaCl, 4x Denhardt’s, 50 µg/ml sheared, denatured salmon sperm DNA, and 5% dextran sulfate at 42°C. The filters were washed in 2x SSC, 0.1% SDS at room temperature, then in 0.5x SSC, 0.1% SDS at 60°C. Equal loading of RNAs was verified by ethidium bromide staining and by hybridization with a rRNA oligonucleotide probe (29). Band intensities were quantified using a Fuji BAS 1000 phosphor imager.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of NF-{kappa}B signaling in IL-4-dependent proliferation of T lymphoid cells

We previously reported that thymocytes and T cells from mice expressing a constitutive repressor of NF-{kappa}B/Rel signaling (I{kappa}B{alpha}({Delta}N)) proliferated poorly after mitogenic stimuli, and that addition of exogenous IL-2 did not reverse this abnormality (21). These data were interpreted as indicating the presence of a defect refractory to IL-2, but it was unclear whether receptor expression was diminished or whether activation of specific signaling pathways downstream from the IL-2R was affected by the inhibition of NF-{kappa}B. As outlined above, it seemed possible that the findings were due to an inability of activating signals to create a state of competence. To determine whether NF-{kappa}B/Rel signaling is required for responses of T lymphoid cells to any other lymphokine, we measured proliferative responses to IL-4 using thymocytes and T cells derived from I{kappa}B{alpha}({Delta}N) Tg mice and their wild-type littermates (nontransgenic (NTg) mice). The proliferative response to IL-4 of PMA-treated thymocytes from I{kappa}B{alpha}({Delta}N) Tg mice was one-tenth that of control cells (Fig. 1GoA). A similar proliferative defect was observed using cells cultured in PMA and IL-2, compared with the 4-fold difference between NTg and Tg samples stimulated by PMA and ionomycin (21) (Fig. 1GoA legend). As expected, thymocytes did not proliferate appreciably when treated with PMA, IL-2, or IL-4 alone. These data indicate that PMA was required to induce thymocyte competence to proliferate in response to IL-2 or IL-4, and that NF-{kappa}B/Rel signaling was essential for induction of this competent state.



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  		FIGURE 1. I{kappa}B{alpha}({Delta}N) inhibition of the NF-{kappa}B/Rel pathway impairs the competence of T cells to respond to IL-4 growth signaling. A, Thymocytes from either NTg ({square}) or Tg ({blacksquare}) I{kappa}B{alpha}({Delta}N) mice were treated for 40 h with PMA (50 ng/ml), ionomycin (1 µg/ml), IL-2 (10 ng/ml), and IL-4 (10 ng/ml), as indicated. Cells were pulsed for an additional 8 h with tritiated thymidine and harvested for scintillation counting. The results are shown as the mean (±SEM) tritiated thymidine incorporation in three independent experiments with pooled thymocytes from six NTg and eight Tg mice in each experiment. Thymidine incorporation into these pooled thymocytes treated was 21,521 cpm (Tg) compared with 91,947 cpm (NTg) when treated with PMA and ionomycin. B, B cell-depleted T cells prepared from pooled lymph nodes and spleens of Tg and NTg mice were stimulated as indicated, using plate-bound anti-CD3 (10 µg/ml), agonistic Abs against CD28 (10 µg/ml), or PMA (50 ng/ml), each in the presence of IL-2 (10 ng/ml) or IL-4 (10 ng/ml). Tritiated thymidine incorporation was determined as in A. C, Gel mobility shift analysis of nuclear NF-{kappa}B/Rel proteins. Pooled thymocyte suspensions were prepared from six NTg and eight Tg mice and cultured overnight in the absence of stimulation, or in the presence of PMA or PMA plus ionomycin. Equal amounts of nuclear extracts from these cells were added to DNA-binding mixtures containing a 32P-labeled {kappa}B probe. DNA-protein complexes were resolved on polyacrylamide gels and visualized by autoradiography.

 
To determine whether a similar requirement applies to mature T cells, proliferative responses to IL-2 and IL-4 were measured using B cell-depleted T lymphocytes from secondary lymphoid organs. Lymphokine-dependent proliferation of I{kappa}B{alpha}({Delta}N) cells stimulated with PMA was again observed to be reduced by an order of magnitude (Fig. 1GoB). T cell activation by anti-CD3 depends on IL-2 production and responsiveness. I{kappa}B{alpha}({Delta}N) T cells exhibited a proliferative defect refractory to IL-4 as well as IL-2 after activation with anti-CD3 (Fig. 1GoB), although the magnitude of the deficit was less dramatic. Costimulatory signaling by CD28 has been described as critical for the enhancement of T cell proliferation (30). To determine whether the observed decrease in proliferation was due to inadequate costimulation in these cultures and if signaling through the CD28 receptor could reverse the impairment of cytokine-dependent proliferation, we added an activating Ab against CD28 to the anti-CD3 stimulus. However, the presence of agonist Abs against CD28 did not reverse the failure of cells to proliferate normally in IL-2 or IL-4 (Fig. 1GoB). We have previously shown that induction of NF-{kappa}B under biochemical conditions different from these (i.e., by anti-CD3/28 or by PMA plus ionomycin) is potently inhibited by the I{kappa}B{alpha}({Delta}N) transgene (21, 31), but had not assayed PMA alone. To determine the effect of I{kappa}B{alpha}({Delta}N) on the nuclear induction of NF-{kappa}B/Rel during PMA treatment, mobility shift assays were performed using nuclear extracts derived from NTg and Tg mice. These experiments demonstrated that PMA treatment alone was sufficient to induce high nuclear levels of NF-{kappa}B activity in cells from wild-type animals, but not I{kappa}B{alpha}({Delta}N) Tg mice (Fig. 1GoC). We conclude that T cell competence to proliferate in response to both growth signals, IL-2 and IL-4, depends on normal NF-{kappa}B signaling.

Selective decreases in IL-4-induced gene expression

It has been proposed that growth regulation and differentiation/regulation of gene expression represent mechanistically distinct effects of IL-2 and IL-4 signaling that are dependent on different portions of the cytokine receptors and different signaling pathways (32, 33). IL-4 does not activate NF-{kappa}B, but the finding that one downstream effect of cytokine receptor signaling, proliferation, was diminished in lymphoid cells whose NF-{kappa}B/Rel signaling is inhibited prompted us to investigate whether IL-4 induction of target gene transcription was normal. Few genes have been identified whose expression is IL-4 dependent in T cells, but IL-4 does increase IL-4R{alpha}-chain expression (8) and transcription of the mouse IL-2R{beta}-chain gene (34). Therefore, we performed Northern blot analyses to analyze the effect of I{kappa}B{alpha}({Delta}N) on this measure of IL-4 signaling. Levels of IL-4R{alpha} and IL-2R{beta} mRNA were substantially diminished (0.3 x control) in cells from I{kappa}B{alpha}({Delta}N) mice after IL-4 stimulation (Fig. 2Go). We have observed a decrease in c-myc mRNA in Tg T cells after activation by anti-CD3 and anti-CD28 (unpublished observations), perhaps reflecting the presence of {kappa}B-binding sites in the c-myc promoter (35). Since this proto-oncogene promoter is a target of IL-4-mediated signaling that may be independent of the IL-4-inducible transcription factor Stat6 in a nonlymphoid cell line (36), we investigated the levels of c-myc mRNA expression. As shown in Fig. 2Go, levels of c-myc mRNA expression observed before and after IL-4 stimulation of Tg T cells were little different from those of wild-type controls. Thus, the inhibitory effects of I{kappa}B{alpha}({Delta}N) on IL-4 signaling pathways include decreased expression of selected target genes, including a key signaling subunit of the IL-4R.



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  		FIGURE 2. Decreased expression of IL-4 target genes. Northern blot analysis was performed on RNAs derived from cultured lymphoid cells of Tg mice and littermate controls (WT). Thymocytes, or B cell-depleted T cell preparations, as indicated, were cultured in media with IL-4 (10 ng/ml), or without exogenous cytokine, as indicated. Samples of total cellular RNA (10 µg) were fractionated under denaturing conditions on formaldehyde-agarose gels, transferred to nylon membranes, and sequentially hybridized with radiolabeled IL-4R{alpha}, IL-2R{beta}, and c-myc cDNA probes, then with an rRNA probe. B, Quantification of the intensity of the T cell IL-4R{alpha} band was performed using a phosphor imager within its linear range. Values plotted represent arbitrary units of signal intensity.

 
Activation of IL-4 signal transduction pathways in I{kappa}B{alpha}({Delta}N) Tg mice

The IL-4R{alpha} cytoplasmic tail lacks intrinsic tyrosine or serine/threonine kinase activity, but upon ligand binding, resident Janus tyrosine kinases (Jak) become activated (37, 38). These kinases are thought to induce phosphorylation of downstream substrates, which include conserved tyrosine residues in the IL-4R{alpha} tail, IRS proteins IRS-1/2, and the IL-4-associated Stat6 (37, 38, 39). Recruitment of the IRS proteins to IL-4R{alpha} in particular has been implicated as an essential step in IL-4-induced proliferation of certain cell lines (39, 40, 41). Thus, engagement of the IL-4 (but not IL-2) receptor activates a signaling pathway in which recruitment of IRS-1 and -2 to a distinct tyrosine-phosphorylated residue in IL-4R{alpha} leads to increased tyrosine phosphorylation of the IRS proteins in cell lines, followed by recruitment of downstream signal transducers (37, 42). IRS proteins are indispensible for IL-4-induced mitogenesis in the hemopoietic cell line 32D, and thus a defect in IRS activation might lead to decreased IL-4-dependent proliferation of lymphoid cells (40, 41). To investigate whether IRS activation is altered in I{kappa}B{alpha}({Delta}N) Tg cells compared with normal primary cells, we performed immunoprecipitations of IRS-1 or IRS-2, followed by antiphosphotyrosine immunoblotting analyses (Fig. 3Go). When thymocytes were first activated, rinsed, and then stimulated, we discovered that the preactivation step induced tyrosine phosphorylation of IRS-2 independent of the addition of exogenous IL-4 (panel A). The amount of tyrosine phosphorylation in I{kappa}B{alpha}({Delta}N) Tg cells was normal. To investigate further whether the inhibition of NF-{kappa}B affected the ability of IL-4 to influence the IRS-2 pathway, responses to IL-4 were tested in cells that had not undergone preactivation (B). Under these conditions, the primary cells demonstrated IL-4-dependent increases in IRS-2 phosphorylation that could not be mimicked by IL-2 and were unaffected by I{kappa}B{alpha}({Delta}N). Short-term IL-4 treatment induced the tyrosine phosphorylation of IRS-1 in resting cells (not shown) and those that had been preactivated (C). In contrast to the results with IRS-2, the magnitude of IL-4-induced IRS-1 phosphorylation was moderately reduced in Tg cells compared with wild-type preparations. Inasmuch as control immunoblots revealed similar levels of IRS proteins in wild-type and Tg cells (Fig. 3Go), we conclude that the inhibition of NF-{kappa}B by I{kappa}B{alpha}({Delta}N) led to a diminution in the degree of tyrosine phosphorylation of IRS-1, but not IRS-2 in response to IL-4. Since IRS-2 appears be the predominant IRS protein in primary T lymphoid cells rather than IRS-1 (43), the degree of inhibition selective for IRS-1 seemed unlikely to explain fully the more substantial impairment of proliferation.



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  		FIGURE 3. Effect of I{kappa}B{alpha}({Delta}N) transgene on cytokine-induced IRS-1 and IRS-2 phosphorylation. A, Activated thymocytes from control and Tg mice were generated by overnight culture in the presence of PMA (50 ng/ml) and ionomycin (1 µg/ml). These preactivated cells were rinsed, replated in complete medium, and then stimulated with IL-4 (10 ng/ml for 30 min), as indicated. B, Thymocytes from wild type and Tg mice were cultured for 4 h in medium alone, and then stimulated for 30 min with IL-2 (10 ng/ml) or IL-4 (10 ng/ml), as indicated. C, Activated thymocytes were prepared as in A. Cell lysates were subjected to immunoprecipitation using Abs against IRS-2 (A and B) or IRS-1 (C). Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with Abs to phosphotyrosine (4G10). Filters were subsequently stripped and reprobed with Abs against IRS-2 or IRS-1, as indicated.

 
In addition to IRS recruitment and activation, members of the Stat transcription factor family are induced by IL-4 treatment of activated T cells (37, 38, 44). Following IL-4-induced phosphorylation, cytosolic Stat6 proteins dimerize, translocate to the nucleus, and bind to consensus sequences in the promoter regions of IL-4-regulated genes. Stat6 activation represents a key pathway for IL-4-induced gene induction and differentiation (10, 11, 45), but it has been suggested that Stat6 also may contribute to the regulation of T cell proliferation by IL-4 (46). To determine the effect of I{kappa}B{alpha}({Delta}N) on this IL-4 signaling pathway, we compared Stat6-binding activity in thymocytes and T cells from NTg and Tg mice. As shown in Fig. 4Go, induction of nuclear Stat6 was 2-fold higher in wild-type cells as compared with I{kappa}B{alpha}({Delta}N)-expressing cells. mRNA levels for IL-4R{alpha} were substantially decreased due to defective NF-{kappa}B/Rel signaling (Fig. 2Go), and this inhibitory effect was also observed at the cell surface (see below). Stat6 activation depends on the availability of IL-4R{alpha}-associated phosphotyrosine residues in the cytoplasm (32, 44), so we infer that there is no apparent decrease in the biochemical efficiency of Stat6 activation per IL-4R molecule when the net induction of this transcription factor in the nucleus was diminished to a modest extent.



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  		FIGURE 4. IL-4 induction of Stat6- and Stat5-binding activity in I{kappa}B{alpha}({Delta}N) T cells. A, B cell-depleted lymphoid cells (T cells) from spleen and lymph nodes of wild-type and Tg mice were activated 16 h with plate-bound anti-CD3 and anti-CD28, harvested, rinsed, and recultured 30 min in complete media, alone or supplemented with IL-4 (10 ng/ml). B, Thymocytes of wild-type and Tg mice were activated 16 h with PMA (50 ng/ml) and ionomycin (1 µg/ml), harvested, rinsed, and then stimulated with IL-4 (10 ng/ml for 30 min). Whole cell extracts from these cells were then assayed for Stat6 by mobility shift analyses using an N4-spacer (Stat6-specific) oligonucleotide probe spanning nucleotides -122 to -104 of the mouse Ig heavy chain germline epsilon promoter (28 ). The identity of the indicated complexes was confirmed by supershift analyses using antiserum specific for the C terminus of Stat6 (black dot). C, Impairment of Stat5 activation by IL-4. B cell-depleted lymphoid cells (T cells) from spleen and lymph nodes of wild-type and Tg mice were activated and stimulated with IL-4, as is described in A. Whole cell extracts were then assayed for Stat5 by mobility shift analyses using an oligonucleotide probe that contains a Stat5 consensus sequence. The identity of the indicated complexes was confirmed by supershift analyses using antiserum specific for Stat5 (A/B) (black dot). Similar results were obtained with thymocytes.

 
In addition to the activation of Stat6 by IL-4, more recent work has established that IL-4 induces the tyrosine phosphorylation and nuclear translocation of Stat5 isoforms A and B in activated T cells, followed by increased expression of Stat5-dependent reporters (47, 48). Importantly, several groups have implicated Stat5 as a key link between the IL-4R and a proliferative response to this cytokine (48, 49, 50). We therefore assayed Stat5 induction after IL-4 treatment of activated cells from wild-type and I{kappa}B{alpha}({Delta}N) mice (Fig. 4GoC). In contrast to the relatively modest effect on IRS proteins and Stat6, Stat5 induction was greatly diminished in cells from the Tg mice as compared with controls. We conclude that interference with the NF-{kappa}B/Rel pathway led to a preferential inhibition of Stat5 induction as compared with Stat6.

IL-4R expression

The IL-4R consists of an IL-4R{alpha}-chain, which heterodimerizes with a second subunit, the common {gamma}-chain ({gamma}c), which is shared with the receptors for IL-2 and other members of the hemopoietin receptor superfamily (37, 51, 52, 53). Importantly, loss-of-function mutants and Ab-blocking studies indicate that {gamma}c plays a critical role in IL-4-induced proliferation (52, 53), and the induction of Stat5 by IL-4 in activated T cells appears to be due to interactions with Jak3 that is noncovalently associated with {gamma}c rather than IL-4R{alpha} (47, 48). To investigate further how I{kappa}B{alpha}({Delta}N) leads to diminished responses to IL-4, we measured the cell surface expression of IL-4R {alpha} and {gamma}c subunits. We observed a substantial reduction in IL-4R{alpha} expression on Tg thymocytes and T cell lymphocytes compared with wild-type counterparts that had been rendered competent to proliferate (Fig. 5Go). In contrast, expression of the {gamma}c subunit was normal. These data indicate that NF-{kappa}B/Rel proteins serve a regulatory role in the expression of IL-4R{alpha}. Morever, decreased IL-4-induced proliferation correlated with a diminution in IL-4 signaling reflected most by Stat5 activation, and to a lesser extent by IRS-1 phosphorylation and Stat6 induction.



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  		FIGURE 5. Decreased expression of IL-4R{alpha} and normal levels of IL-2R in I{kappa}B{alpha}({Delta}N) mice. Activated thymocytes (PMA plus ionomycin) and T cells (anti-CD3 plus anti-CD28) from Tg mice and their NTg littermates were stained with mAbs specific for IL-2R{alpha} (CD25), IL-2R{beta} (CD122), {gamma}c, IL-4R{alpha}, CD3, and TCR{alpha}{beta}, and then analyzed by flow cytometry. The expression of the indicated surface molecules on wild-type and Tg mice was assessed by gating on TCR{alpha}{beta}+ thymocytes (A) or on CD3+ T cells (B) and then displaying the indicated histograms. Solid lines indicate specific staining, while dashed lines (—-) show the relevant control histogram. Of note, production of IL-2 by anti-CD3/28-activated I{kappa}B{alpha}({Delta}N) T cells is essentially normal (31 ) so that differences in receptor internalization are unlikely to contribute to the similarity of receptor expression on wild-type and Tg T cells. Minor variability leading to the apparent presence of IL-2R{beta}high or {gamma}chigh subpopulations is unlikely to account for differences in proliferation in light of the high frequency of T cells that undergo blast transformation under identical conditions (31 ) and 5-bromo-2'-deoxyuridine-labeling indices of 12–17%/h for wild-type CD4 and CD8 T cells (A. L. Mora and M. Boothby, unpublished observations).

 
In transfected B lymphoma cells, the activation of some signaling pathways by IL-4 was influenced by the number of receptors expressed (33). Therefore, we explored whether a decreased pool of available IL-4R{alpha} cytoplasmic tails in these primary cells is the mechanistic basis for the observed diminution in IL-4-induced proliferation. To do so, we sought to increase the pool of IL-4R{alpha} tails by an approach independent of prior activation. We had previously characterized Tg mice in which the T lineage specifically expresses a chimera of mouse IL-2R{beta} and the IL-4R{alpha} cytoplasmic tail (26). Both thymocytes and T cells from these mice demonstrated inducibility of Stat6 by IL-2 and other evidence that these cytoplasmic tails functioned in an IL-4-specific manner (26). Accordingly, we crossed chimeric cytokine receptor Tg mice with those expressing the inhibitor of NF-{kappa}B signaling and performed proliferation assays measuring the response to IL-2, IL-4, or a combination of these cytokines (Fig. 6Go). Strikingly, under no condition (IL-4 alone or IL-2 with IL-4) did the increased pool of IL-4R{alpha} cytoplasmic tails in double-Tg mice promote an enhancement of proliferation above that obtained with I{kappa}B{alpha}({Delta}N) single-Tg animals. These data suggest that a limiting pool of cytoplasmic tails is not the sole basis for a decreased response to IL-4.



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  		FIGURE 6. Failure of increased IL-4R{alpha} cytoplasmic tails to enhance proliferation of I{kappa}B{alpha}({Delta}N) cells. I{kappa}B{alpha}({Delta}N) Tg mice were crossed with a Tg mice encoding a chimeric IL-2/IL-4 receptor to the T lineage (chimeric (Chi) IL-2/4R) (26 ). B cell-depleted T cells were prepared from pooled lymph nodes and spleens from wild-type, Chi IL-2/4R, I{kappa}B{alpha}({Delta}N), and Chi IL-2/4R x I{kappa}B{alpha}({Delta}N) double-Tg littermate mice. Cell preparations were stimulated for 48 h, as indicated, using plate-bound anti-CD3 (10 µg/ml), agonistic Abs against CD28 (10 µg/ml), or PMA (50 ng/ml), each in the presence of IL-2 (10 ng/ml) or IL-4 (10 ng/ml). Cells were pulsed for an additional 8 h with tritiated thymidine and harvested for scintillation counting. The results are shown as the mean (±SEM) of the stimulation indexes in four independent experiments. Stimulation index was calculated as the mean cpm stimulated [3H]thymidine incorporation divided by the arithmetic mean cpm unstimulated [3H]thymidine incorporation.

 
Defective Stat5A activation in I{kappa}B{alpha}({Delta}N) T cells despite IL-2R expression

The functional IL-2R in mice is composed of three subunits: IL-2R{alpha}, IL-2R{beta}, and the {gamma}c (4, 5). In light of the finding that IL-4R expression on cells from I{kappa}B{alpha}({Delta}N) mice was substantially decreased, we measured the cell surface expression of IL-2R subunits. The signal-transducing components of the IL-2R, IL-2R{beta}, and {gamma}c were expressed at comparable levels in thymocytes and T cells from Tg and wild-type mice (Fig. 5Go). The IL-2R{alpha} subunit is critical for the binding of mouse IL-2 to its receptor (3, 4, 5) and for mouse IL-2R function (3, 12, 13), so that the proliferative defect of thymocytes could arise from a decrease in IL-2R{alpha} expression. Despite reports that NF-{kappa}B/Rel proteins are potent trans-activators of the IL-2R{alpha} promoter in transfected cells, IL-2R{alpha}/CD25 expression on I{kappa}B{alpha}({Delta}N) T cells was at most slightly lower than on wild-type cells, and we observed only a modest decrease in IL-2R{alpha} expression on mature thymocytes (Fig. 5Go). Since cell surface expression of the IL-2R subunits on T lymphoid cells appeared normal, the data indicate that decreased proliferation of I{kappa}B{alpha}({Delta}N) reflects a failure to capacitate signal transduction by the receptor subunits. Although a subject on which there is disagreement, the nuclear induction of Stat5 has been reported to be a key event in IL-2-induced proliferation (49, 54, 55, 56). Accordingly, our data on IL-4-induced Stat5 raised the possibility that inhibition of Stat5 induction might represent a defect shared by IL-2R and IL-4R signaling when NF-{kappa}B was inhibited. To test this possibility, we measured activation of Stat5 by IL-2.

Like IL-4, IL-2 induces phosphorylation of Stat proteins expressed from related genes (Stat5A and Stat5B) (38). To determine whether the influence of I{kappa}B{alpha}({Delta}N) on IL-2-induced proliferation could be correlated with phosphorylation of Stat5, we activated thymocytes and T cells from Tg and wild-type mice, followed by mobility shift assays using extracts of control and IL-2-treated cells. This activation step was designed to mimic the conditions of proliferation assays and promote induction of IL-2R{alpha}. It also leads to the production of IL-2 by wild-type thymocytes and T cells. This IL-2 production is blocked by the Tg inhibition of NF-{kappa}B in thymocytes (21). The binding activity of DNA-protein complexes that comigrate with Stat5 was reduced in thymocytes (Fig. 7GoA) and T cells (Fig. 7GoB) from I{kappa}B{alpha}({Delta}N) Tg mice. Endogenous production of IL-2 by wild-type but not Tg cells led to high basal levels of binding activity in the extracts from wild-type thymocytes, but not those from I{kappa}B{alpha}({Delta}N) Tg mice, and even the addition of exogenous IL-2 was unable to induce substantial binding activity. The presence of Stat5 in this complex was validated using an antiserum specific for both Stat5A and B, which eliminated the Stat5 complex and created a supershifted band of slower mobility (Fig. 7GoA). An independent Ab specific for Stat5A also supershifted the indicated complex, whereas a control serum did not (data not shown). Stat5 induction in T cells also was impaired (B). To understand better which form(s) of Stat5 contributed to the mobility shift activity, extracts were subjected to immunoprecipitation with antiserum against the individual isoforms of Stat5, Stat5A, and Stat5B, followed by immunoblotting with an antiphosphotyrosine Ab. As in A, endogenous IL-2 production by wild-type thymocytes led to a substantial level of Stat5A tyrosine phosphorylation that was not further increased by exogenous IL-2. In contrast, we observed far less phosphorylation of Stat5A in cells from I{kappa}B{alpha}({Delta}N) mice as compared with controls, and the addition of exogenous IL-2 did not induce phosphoStat5A (Fig. 7GoC). Although at lower signal intensity overall, the level of Stat5B phosphorylation in IL-2-treated cells from Tg animals was indistinguishable from wild-type samples (Fig. 7GoC). This finding suggested that IL-2 induction of Stat5B homodimers and perhaps Stat5AB heterodimers was quantitatively less affected than that of Stat5A homodimers. While the decreased Stat5A phosphorylation might have been attributed to a requirement for NF-{kappa}B/Rel signaling in the induction of Stat5A protein levels, wild-type and I{kappa}B{alpha}({Delta}N) mice expressed similar levels of total Stat5A and Stat5B protein (Fig. 7GoC). We conclude that the presence of I{kappa}B{alpha}({Delta}N) in Tg T lineage cells is associated with impaired phosphorylation and activation of Stat5A despite expression of all subunits of the IL-2R. Taken together with observations linking Stat5 to T cell proliferation (49, 54, 55, 56) through association with IL-2 but not Ag receptors (49), these findings suggest that the observed impairment in IL-2-inducible proliferation by I{kappa}B{alpha}({Delta}N) T cells is associated with defective Stat5A activation.



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  		FIGURE 7. Defective induction of Stat5A in activated, IL-2-treated T lymphoid cells. A, Thymocytes from wild-type and Tg mice were activated overnight with PMA plus ionomycin and anti-CD28, harvested, rinsed, and recultured 30 min with media, alone or supplemented with IL-2 (10 ng/ml). B, B cell-depleted lymphoid cells (T cells) from spleen and lymph nodes of wild-type and Tg mice were activated 16 h with plate-bound anti-CD3 and anti-CD28, harvested, rinsed, and recultured 30 min in complete media, alone or supplemented with IL-2 (10 ng/ml). Whole cell extracts from these treated cells were then assayed for Stat5 by mobility shift analyses using an oligonucleotide probe that contains a Stat5 consensus sequence. The background of Stat5-binding activity in cells not treated with IL-2 is due to IL-2 release from the activated cells; this band was not present when using extracts of resting cells. The identity of the indicated complexes was confirmed in thymocyte preparations by supershift analyses using antiserum specific for Stat5 (A/B). C, Activated thymocytes (16-h culture in PMA and ionomycin) were recultured 30 min in complete media, alone or in the presence of mouse IL-2 (10 ng/ml). Whole cell extracts from these stimulated cells were subjected to immunoprecipitation using an anti-Stat5A or Stat5B antiserum. Immune precipitates were resolved by SDS-PAGE, then subjected to immunoblotting using an anti-phosphotyrosine Ab (RC-20). Following this procedure, these filters were then divided, stripped, separately reprobed using the anti-Stat5A or Stat5B antiserum, as indicated, repositioned, and exposed to x-ray film. The position of the Stat5A and Stat5B band is marked with an asterisk and a diamond, respectively.

 
The evidence that the I{kappa}B/NF-{kappa}B/Rel system can regulate receptor capacitation raises the question as to whether such a role is limited to cytokines whose receptor uses Jak1 and Jak3 to induce Stat5, or instead may be more general. To explore this issue, we measured the ability of IL-12 to enhance the proliferation of T lymphoid cells. PMA-primed thymocytes could not be costimulated by IL-12 (data not shown). As shown in Fig. 8Go, IL-12-enhanced proliferation of PMA-primed T cells was inhibited by I{kappa}B{alpha}({Delta}N), although there was no enhancement of the response to other proliferative stimuli. Thus, while the mechanism of this effect remains to be determined, the action of this cytokine through different Janus kinases (Jak2 and Tyk2) and Stat proteins (Stat3 and Stat4) suggests the potential for additional NF-{kappa}B-dependent mechanisms of receptor capacitation.



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  		FIGURE 8. Decreased proliferation of PMA-treated cells costimulated with IL-12. B cell-depleted T cells were prepared from pooled lymph nodes and spleens from wild-type and Tg mice. Cell preparations were stimulated for 48 h, as indicated, using plate-bound anti-CD3 (10 µg/ml), agonistic Abs against CD28 (10 µg/ml), or PMA (50 ng/ml), each in the presence or absence of IL-12 (10 ng/ml). Cells were pulsed for an additional 8 h with tritiated thymidine and harvested for scintillation counting. The results are shown as the mean (±SEM) tritiated thymidine incorporation in three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The activation of lymphocytes after engagement of their Ag-specific receptor is a critical step in the regulation of immune responses. Lymphocyte activation leads to the production of cytokines such as IL-2 and IL-4. Importantly, this activation step also induces the competence to respond to the proliferative signals provided by these lymphokines (5, 12, 13, 14, 15, 16). However, the set of molecular mechanisms involved in this enhancement of cytokine responsiveness of normal lymphoid cells is not clear. Signal transduction pathways activated after engagement of the TCR include the NF-{kappa}B/Rel transcription factor family (19, 20), but neither IL-2 nor IL-4 induces NF-{kappa}B/Rel transcription factors. In the present study, 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 impair the competence of T cells to respond to the proliferative stimulus of IL-4 as well as IL-2. Moreover, the ability of each cytokine to induce Stat5 was diminished. This finding raises the possibility that NF-{kappa}B/Rel proteins may play a general role in the cytokine responsiveness of lymphoid cells by a Stat5-dependent mechanism.

Two general mechanisms for enhancing the competence of cells to respond to lymphokines can be envisaged. In the first, the composition and number of receptors are regulated in response to stimuli. Examples of this mechanism include induction of the IL-2R{alpha}-chain, whose association with IL-2R{beta} and {gamma}c dramatically influences ligand affinity, and regulated changes in the number of IL-2R{beta}-chains (4, 5). Capacitation, in which the functional effects of receptors are potentiated after cellular activation or differentiation (5, 15, 16, 17, 18, 57), provides an alternative mechanism of regulation. Receptor capacitation offers a powerful means for integration of disparate stimuli. For instance, in some systems, IL-4 may not provide a sufficient signal for lymphocyte proliferation unless costimulatory or Ag receptors have also bound to a ligand, while in others the response of T cells to TCR, IFN-{gamma}, or IL-12 signaling may lead to inhibition of Stat6 activation by IL-4R (15, 16, 17, 18, 58).

The data presented in this work support a role for NF-{kappa}B/Rel signaling in the regulation of lymphoid proliferation and Stat5 induction by IL-4. Such mechanisms may also apply to the regulation of apoptosis in T cells. IL-4 protects lymphoid cells against apoptosis in several contexts: programmed cell death of resting and activated T cells (59, 60, 61), and Fas-induced apoptosis of activated B cells (62). We have observed that while the addition of IL-4 to cultures decreased the apoptotic susceptibility of anti-CD3-treated wild-type T cells, this cytokine did not protect I{kappa}B{alpha}({Delta}N) T cells from apoptosis induced by TCR cross-linking (A. Mora, unpublished observations). In this regard, it is interesting to note that IL-4-induced Stat6 was entirely dispensable for the IL-4-mediated protection against apoptosis (61). Since Stat5 activity may be critical for mediating IL-2-induced survival signals (63), it is possible that the inhibition of IL-4-induced Stat5 is one mechanism by which I{kappa}B{alpha}({Delta}N) blocks survival signaling.

In contrast to the expression of IL-2R subunits after activation of I{kappa}B{alpha}({Delta}N) T lymphoid cells, hyporesponsiveness to IL-4 was associated with a significant decrease in IL-4R expression (Figs. 2Go and 5Go). The decrease in basal IL-4R{alpha} mRNA levels (Fig. 2Go) may reflect an important role of NF-{kappa}B in the activity of this promoter. Moreover, the ability of Stat6 to participate in the activation of IL-4-dependent promoters can require cooperation with adjacent cis-acting elements (64, 65). NF-{kappa}B/Rel proteins can cooperate with Stat6 in the activation of the IL-4-dependent germline epsilon promoter from the Ig heavy chain locus. Thus, the relatively modest decrease in IL-4-induced Stat6-binding activity may be amplified functionally in I{kappa}B{alpha}({Delta}N)-expressing T lymphoid cells because the nuclear import of the inducible trans-activators c-Rel and RelA is inhibited (21, 22). This decrease in receptor number was associated with a diminished capacity to activate downstream signaling pathways, particularly that leading to Stat5 induction. However, we consider it unlikely that the decreased expression of IL-4R{alpha} fully accounts for the impaired IL-4-dependent proliferation of I{kappa}B{alpha}({Delta}N) T lymphoid cells since a constitutively expressed Tg receptor competent to generate IL-4-specific signals (26) was unable to ameliorate the proliferative defect stemming from inhibition of NF-{kappa}B.

While NF-{kappa}B/Rel proteins clearly participate in an important signal transduction pathway after T cell activation, most studies on their potential role in cytokine signaling have focused on culture-adapted cell lines. In these settings, nuclear translocation of NF-{kappa}B has been associated with transcriptional activation of the IL-2R{alpha} gene (66, 67). How these findings with cell lines apply to normal T cells is unclear, since complete inactivation of either c-Rel or RelA by gene targeting had no apparent effect on IL-2R{alpha} (CD25) gene expression (23, 68). These gene inactivation studies could be explained on the basis that either trans-activating subunit is sufficient to achieve normal transcriptional induction of IL-2R{alpha} in cell lines. However, I{kappa}B{alpha}({Delta}N) interferes with the nuclear induction of both c-Rel and RelA (21, 22), yet activated T cells and thymocytes can express normal levels of IL-2R{alpha}, {beta}, and {gamma}c. Despite normal expression of the IL-2R subunits, direct evidence of a capacitation mechanism is provided by the observation that overall Stat5 activation was dramatically decreased, with a particularly substantial effect on Stat5A (Fig. 7Go). The observed tyrosine phosphorylation of Stat5B in I{kappa}B{alpha}({Delta}N) T cells further suggests that these cells express functional IL-2R. The mechanism by which NF-{kappa}B participates in this capacitation mechanism remains to be determined. It is thought that both Jak1 and Jak3 are essential for activation of Stat5, whereas only Jak1 appears essential for Stat6 induction (69, 70, 71, 72, 73, 74). Moreover, Jak3 deficiency leads to a homeostatic defect characterized by a preferential decrease in the CD8+ lineage relative to CD4+ cells (75, 76, 77), which is reminiscent of the phenotype in I{kappa}B{alpha}({Delta}N) mice. Thus, an attractive hypothesis is that Jak3 is inhibited as a consequence of I{kappa}B{alpha}({Delta}N) expression in T lymphoid cells. Although it remains to be determined whether the inhibition of Stat5A induction accounts for the entire proliferative defect of I{kappa}B{alpha}({Delta}N) T cells, the magnitude of the observed decreases in Stat5 expression suggests a parallel to studies of IL-2-dependent T cell proliferation in Stat5A-deficient mice (54, 55, 56, 78). Anti-CD3-stimulated splenocytes from Stat5A-deficient mice exhibited normal IL-2R{alpha} expression and decreased proliferation not unlike that observed in cultures of I{kappa}B{alpha}({Delta}N) T cells supplemented with IL-2 (Fig. 1Go). A failure to sustain increased CD25 expression was observed only when cells were rinsed and cultured for 2 days in IL-2 and the absence of a TCR stimulus (78). Thus, the present observations using T cells subjected to inhibition of NF-{kappa}B/Rel signaling are consistent with the phenotype of Stat5A-null T cells when compared under similar condition and with other studies suggesting differential roles for these proteins (79, 80). Moreover, the present data are consistent with the conclusion of previous studies that Stat5 is critical for IL-2- as well as IL-4-induced proliferation in T cells (48, 49, 50, 54, 55, 56), thereby suggesting that one major mechanism by which the I{kappa}B/NF-{kappa}B/Rel system regulates cytokine receptor capacitation is mediated through changes in the ability to induce nuclear Stat5.


   Acknowledgments
 
We thank W. Armistead, S. Stanley, B. Enerson, and S. McCarthy for expert technical assistance; J. Zamorano, M. Rojas, and S. Goenka for helpful discussions and critical review of the manuscript; Immunex (Seattle, WA) and F. W. Alt for cDNAs; J. Price and D. McFarland for expert flow cytometry through the Vanderbilt Cancer Center and Howard Hughes Medical Institute FACS cores; and the Vanderbilt Ingram Cancer Center and Diabetes Research and Training Center for Tissue Culture, DNA, Molecular Biology and Flow Cytometry core functions.


   Footnotes
 
1 This work was supported by The Vanderbilt Cancer and Diabetes Research and Training Centers (National Institutes of Health Grants CA68485 and P60 DK20593) through core functions (FACS, oligonucleotide synthesis) and a Pilot Project (DK20593). M.B. was a Scholar of the Leukemia Society of America, and funding for this work was provided by the National Institutes of Health (AI-36997 and GM-42550 (A.L.M., J.Y., and M.B.) and AI-38985 (A.D.K.)). Back

2 Current address: Department of Anatomy/Cell Biology, College of Medicine, Hanyang University, Seoul, Korea. Back

3 Address correspondence and reprint requests to Dr. Mark Boothby, Department of Microbiology and Immunology, Vanderbilt University Medical School,AA-4214 M. C. N., Nashville, TN 37232-2363. Back

4 Abbreviations used in this paper: I{kappa}B, inhibitory protein that dissociates from NF-{kappa}B; IRS, insulin receptor substrate; Jak, Janus kinase; NTg, nontransgenic; Tg, transgenic; {gamma}c, common {gamma}-chain; Chi, chimeric. Back

Received for publication July 11, 2000. Accepted for publication November 17, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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