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The Journal of Immunology, 2002, 168: 3847-3854.
Copyright © 2002 by The American Association of Immunologists

Molecular Mechanisms Underlying Differential Contribution of CD28 Versus Non-CD28 Costimulatory Molecules to IL-2 Promoter Activation1

Xu-Yu Zhou*, Yumi Yashiro-Ohtani*, Masakiyo Nakahira*, Woong Ryeon Park*, Ryo Abe{dagger}, Toshiyuki Hamaoka*, Mayumi Naramura{ddagger}, Hua Gu{ddagger} and Hiromi Fujiwara2,*

* Department of Oncology, Osaka University Graduate School of Medicine, Osaka, Japan; {dagger} Research Institute for Biological Sciences, Science University of Tokyo, Chiba, Japan; and {ddagger} Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell costimulation via CD28 and other (non-CD28) costimulatory molecules induces comparable levels of [3H]TdR incorporation, but fundamentally differs in the contribution to IL-2 production. In this study, we investigated the molecular basis underlying the difference between CD28 and non-CD28 costimulation for IL-2 gene expression. Resting T cells from a mutant mouse strain generated by replacing the IL-2 gene with a cDNA encoding green fluorescent protein were stimulated with a low dose of anti-CD3 plus anti-CD28 or anti-non-CD28 (CD5 or CD9) mAbs. CD28 and non-CD28 costimulation capable of inducing potent [3H]TdR uptake resulted in high and marginal levels of green fluorescent protein expression, respectively, indicating their differential IL-2 promoter activation. CD28 costimulation exhibited a time-dependent increase in the binding of transcription factors to the NF-AT and NF-{kappa}B binding sites and the CD28-responsive element of the IL-2 promoter, whereas non-CD28 costimulation did not. Particularly, a striking difference was observed for the binding of NF-{kappa}B to CD28-responsive element and the NF-{kappa}B binding site. Decreased NF-{kappa}B activation in non-CD28 costimulation resulted from the failure to translocate a critical NF-{kappa}B member, c-Rel, to the nuclear compartment due to the lack of I{kappa}B{beta} inactivation. These observations suggest that unlike CD28 costimulation, non-CD28 costimulation fails to sustain IL-2 promoter activation and that such a failure is ascribed largely to the defect in the activation of c-Rel/NF-{kappa}B.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The activation of resting T cells requires two distinct signals (1, 2, 3, 4). The first signal stems from recognition by the TCR of processed Ag peptides plus MHC molecules on APCs. This signal leads to an effective T cell response when a second costimulatory signal is provided by the APC. Intensive research has characterized the T cell molecule CD28 as the principal receptor that generates costimulatory signals in T cells (2, 3, 4) based on the observations that CD28 engagement by either anti-CD28 mAb or ligands along with TCR stimulation results in full T cell activation (5, 6, 7).

In addition to CD28, multiple molecules on the T cell have been shown to costimulate resting T cells, including CD5 (8), CD2 (9), CD44 (10), and CD9 (Ref. 11 and reviewed in Ref. 12). In fact, in the presence of suboptimal doses of anti-CD3, each of the mAbs against these molecules costimulated resting T cells as potently as anti-CD28 mAb (11, 13). In most studies costimulation has been evaluated as the capacity of each molecule to enhance [3H]TdR incorporation of resting T cells stimulated with suboptimal doses of anti-CD3. In addition, both CD28 and costimulatory molecules other than CD28 (designated non-CD28 costimulatory molecules) were recently shown to function for enhanced association of TCR and lipid rafts (14). These results provided a mechanistic explanation that CD28 and non-CD28 similarly induce an initial phase of T cell activation. However, we also found a fundamental difference in the capacity to induce proliferation of costimulated T cells between CD28 and non-CD28 (13). Despite comparable levels of [3H]TdR incorporation, the proliferative expansion of CD28- and non-CD28-costimulated T cells differed greatly, and the difference in cellular proliferation was due to the fundamentally distinct levels of IL-2 production (13). The role for CD28 costimulation in the promotion of IL-2 gene expression has been well established. A number of elements in the IL-2 gene promoter have been shown to be involved in the regulation of IL-2 gene transcriptional activity (15, 16). These include the binding site of NF-AT (17), the NF-{kappa}B binding site (18), and the CD28-responsive element (CD28RE)3 (19).

The present study was undertaken to determine whether there exists a difference in the capacity of CD28 and non-CD28 costimulation to induce the binding of transcription factors to their respective binding sites in the IL-2 promoter. CD28 costimulation may also increase IL-2 production by prolonging the IL-2 mRNA half-life (20, 21, 22). Therefore, we first compared the capacity of the two categories of costimulation to induce IL-2 promoter activity in the system without an influence on mRNA stabilization. CD28 and non-CD28 (CD5 or CD9) costimulation of resting T cells bearing the IL-2 gene replaced with a cDNA encoding green fluorescent protein (GFP) resulted in high and marginal levels of GFP expression, respectively. The results also demonstrated that there exists a great difference between CD28 and non-CD28 costimulation in the induction of NF-{kappa}B that interacts with the NF-{kappa}B binding site as well as the CD28RE. Among the NF-{kappa}B family, enhanced translocation of c-Rel to the nuclear compartment was induced by CD28, but not by non-CD28, costimulation. This was associated with the failure of non-CD28 costimulation to induce I{kappa}B{beta} inactivation. These results suggest that the non-CD28 costimulation fails to induce sufficient levels of IL-2 promoter activity due to the decreased activation of c-Rel/NF-{kappa}B that would result from the lack of signaling to I{kappa}B{beta} inactivation.


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

Female BALB/c mice were obtained from Charles River Laboratories (Yokohama, Japan). A mutant mouse strain was previously generated by replacing the IL-2 gene with a cDNA encoding GFP (23). This strain mouse with the knocked-in GFP cDNA was designated IL-2-GFPki. IL-2-GFPki mice with the BALB/c background were used.

Reagents

Anti-CD3 (145-2C11) (24), anti-CD28 (Pv-1) (25), anti-CD5 (53-7-313) (26), anti-CD9 (9D3) (11), and anti-I-Ad/b (34-5-3S) (27) mAbs were purified from culture supernatants or ascitic fluids of the relevant hybridoma cells. The following Abs against NF-{kappa}B members were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): rabbit anti-c-Rel (sc-71), rabbit anti-p50 (sc-114x and sc-1190x), rabbit anti-RelB (sc-226x), rabbit anti-I{kappa}B{beta} (sc-945), goat anti-c-Rel (sc-71x), and goat anti-p65 (sc-372x). Rabbit anti-I{kappa}B{alpha} Ab (13996E) was obtained from BD PharMingen (San Diego, CA). Normal rabbit Ig and normal goat Ig were also obtained from Santa Cruz Biotechnology. Cycloheximide (CHX) was purchased from Wako Pure Chemicals (Osaka, Japan).

Preparation of a purified T cell population

Lymph node cells were depleted of B cells and Ia+ APCs by immunomagnetic negative selection as previously described (28). Briefly, Ia+ APC in a lymph node cell population were allowed to react with anti-I-Ad/b mAb. Lymph node cells containing the labeled cells and surface Ig+ cells (B cells) were incubated with magnetic particles conjugated to goat anti-mouse IgG (Polysciences, Warrington, PA). Surface Ig- and Ia- cells (B cell- and APC-depleted population) were obtained by removing cell-bound magnetic particles with a rare earth magnet (Polysciences). The purity of the resulting population was checked by flow cytometry with anti-CD3. Purified T cells were consistently >98% CD3 positive.

T cell cultures for stimulation with mAbs

mAbs were diluted to indicated concentrations in PBS and immobilized to individual wells of 96-well flat-bottom microculture plates (Corning 25860; Corning Glass Works, Corning, NY) in a final volume of 0.1 ml or in 24-well culture plates (Corning 25820) in a volume of 1 ml. After 3 h solutions were discarded, and plates were washed with PBS twice. Purified T cells were cultured in 0.2 ml of RPMI 1640 medium supplemented with 10% FCS and 2-ME at 1.0 or 2.0 x 105 cells/well of mAb-immobilized 96-well microculture plates in a humidified atmosphere at 5% CO2 at 37°C for a various number of days. The cultures were harvested after a 6-h pulse with 20 KBq/well [3H]TdR. Results were calculated from the uptake of [3H]TdR and were expressed as the mean cpm ± SE of triplicate culture. For assays other than [3H]TdR incorporation, purified T cells (2 x 106 cells/well) were cultured in mAb-immobilized 24-well culture plates in a volume of 2 ml. Cells were harvested after various times in culture and either subjected to flow cytometry or used to prepare nuclear factor.

Measurement of IL-2 concentration

The IL-2 concentration was measured by ELISA. Mouse rIL-2 was provided by Shionogi Research Laboratories (Osaka, Japan). The mouse IL-2 ELISA system was prepared using two types of anti-mouse IL-2 mAbs (JES6-1A12 and biotinylated JES6-5H4, purchased from BD PharMingen).

Flow cytometry

Flow cytometric analysis for the detection of intracellular GFP expression was performed with a FACSCalibur (BD Biosciences, San Jose, CA). GFP fluorescence was detected with the FL1 detector (530 ± 30 nm). In some experiments, activated T cells were stained with PE-conjugated anti-CD4 or anti-CD8 mAb (BD Biosciences).

Preparation of nuclear extracts

Nuclear extracts were prepared as follows. After washing with PBS, cells were resuspended in cell lysis buffer (20 mM HEPES-NaOH (pH 7.9), 20 mM NaF, 1 mM Na3VO4, 1 mM EDTA, and 0.1 mM EGTA) supplemented with 0.2% Nonidet P-40, 1 mM DTT, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.1 mM Pefabloc (Roche, Mannheim, Germany). The nuclei were pelleted and then extracted with vigorous agitation at 4°C in the above buffer without Nonidet P-40, but containing 0.42 M NaCl, 20% glycerol, and protease inhibitors as described above.

EMSA

The binding reaction was performed in a total volume of 20 µl in the following buffer: 10 mM HEPES-NaOH (pH 7.9), 1 mM EDTA, 30 mM NaCl, 0.1% Nonidet P-40, 1 mM DTT, 1 mg/ml BSA, and 5% glycerol. Each reaction, also containing 0.7 µg poly(dI-dC) and 32P end-labeled probe, was initiated by the addition of ~9 µg nuclear extract and allowed to incubate at room temperature for 30 min before electrophoretic analysis on a 5% polyacrylamide gel in 0.25x Tris-borate-EDTA buffer. The following oligonucleotide probes corresponding to the NF-AT/AP-1 (5'-CCAAAGAGGAAAATTTGTTTCATACAGAAGGCGT-3'), NF-IL-2A (5'-GACTCTTTGAAAATATGTGTAATATGTAAAACATCGTGAC-3'), and NF-{kappa}B (5'-AACCCGACCAAGAGGGATTTCACCTAAATCCAT-3') binding sites as well as the CD28RE (5'-TGGGGGTTTAAAGAAATTCCAGAGAGTCATCAG-3') were purchased from Pharmacia (Tokyo, Japan) and annealed by standard protocol in our laboratory.

Immunoblotting

Stimulated cells were lysed in buffer containing 0.2% Nonidet P-40. Lysates were resolved on 10% SDS-polyacrylamide gels and transferred to Immobilon (Millipore, Bedford, MA). For immunoblotting, membranes were blocked in PBS containing 5% nonfat dry milk, and 0.05% Tween 20, and sequentially incubated with anti-c-Rel Ab and HRP-conjugated donkey anti-rabbit IgG F(ab')2 (Amersham, Little Chalfont, U.K.). Detection was performed using ECL (Amersham).

Oligo DNA precipitation

Biotinylated oligonucleotide corresponding to the NF-{kappa}B binding sequence present in the IL-2 promoter and streptavidin-coupled agarose beads were purchased from Pharmacia and Sigma-Aldrich (St. Louis, MO), respectively, and mixed at 4°C for 1 h to prepare agarose beads coupled to oligonucleotide corresponding to the NF-{kappa}B binding sequence. The procedure of oligo DNA precipitation was essentially the same as previously described (29). Nuclear extracts were incubated with agarose beads coupled to oligonucleotide for the NF-{kappa}B binding site. The binding reaction was performed for 60 min at room temperature in a binding buffer containing 30 mM NaCl, HEPES-NaOH (pH 7.9), 1 mM EDTA, 1 mM DTT, 5% glycerol, 1 mg/ml BSA, and 35 µg/ml poly(dI-dC). The agarose beads were washed four times with binding buffer. The bound proteins were released with SDS loading buffer, separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and visualized with the relevant Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD28 and non-CD28 costimulations induce comparable levels of an initial phase of T cell activation, as evaluated by [3H]TdR incorporation, but not by IL-2 production

Our earlier studies demonstrated that CD28 and non-CD28 costimulations induce comparable levels of [3H]TdR incorporation of anti-CD3-stimulated T cells, but exhibit strikingly different levels of IL-2 production (13, 30). We confirmed these results (Fig. 1Go). Purified BALB/c T cells were cultured in wells coated with mAb (10 µg/ml) against CD5 or CD9 as representatives of non-CD28 molecules together with a suboptimal dose (1 µg/ml) of anti-CD3. All mAbs increased [3H]TdR incorporation of anti-CD3-triggered T cells on days 1 and 2 (Fig. 1GoA). The magnitude of costimulation by non-CD28 molecules (CD5 and CD9) was comparable to that by CD28. Fig. 1GoB shows that IL-2 production fundamentally differs between CD28 and non-CD28 costimulation. The level of IL-2 production by non-CD28 costimulation was not zero, but was strikingly low (5–10 U/ml) for the initial 48 h.



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  		FIGURE 1. [3H]TdR incorporation and IL-2 production by resting T cells costimulated by CD28 and non-CD28 molecules (CD5 or CD9). A, Purified lymph node T cells (1 x 105/well) were cultured in 96-well microculture plates coated with 10 µg/ml anti-CD5, -CD9, or -CD28 mAb together with 1 µg/ml anti-CD3 mAb for 1–3 days. [3H]TdR uptake at each time point is expressed by the mean ± SE of triplicate cultures. B, Purified T cells were cultured in 24-well culture plates coated with anti-CD3 (1 µg/ml) alone or with anti-CD3 plus the indicated mAbs (10 µg/ml). Supernatants were harvested at various time points and tested for IL-2 concentrations. The results are expressed as the mean ± SE of triplicate cultures. The results are representative of five (A) or three (B) similar experiments.

 
A fundamental difference in IL-2 promoter activation between CD28 and non-CD28 costimulation

Decreased IL-2 production may result from reduced IL-2 promoter activation and/or IL-2 mRNA stability. We investigated whether differential IL-2 production between CD28 and non-CD28 costimulation is ascribed at least partly to the difference in IL-2 promoter activation. An experimental system was first defined that can evaluate the capacity to induce IL-2 promoter activation. Purified T cells from IL-2-GFP knockin (IL-2-GFPki) mice were stimulated with immobilized anti-CD3 alone or together with coimmobilized anti-CD28 (Fig. 2Go). IL-2 promoter activation was evaluated by detecting intracellular GFP expression. As shown in Fig. 2Go, upper panels, stimulation with anti-CD3 alone induced marginal levels of GFP expression, whereas CD28 costimulation induced time-dependent IL-2 promoter activation. The results also show that such activation occurs exclusively in CD4+ T cells (Fig. 2Go, lower panels). We next compared the capacity to induce IL-2 promoter activity between CD28 and non-CD28 costimulation (Fig. 3Go). CD28 costimulation again induced IL-2 promoter activation. In contrast, CD5 and CD9 costimulation elicited markedly decreased levels of GFP expression, although the levels were slightly higher than those stimulated with anti-CD3 alone. These results demonstrate that CD28 and non-CD28 costimulation lead to strikingly distinct levels of IL-2 promoter activation.



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  		FIGURE 2. Time course of IL-2 promoter activation in CD28-costimulated CD4+ and CD8+ T cells. Purified T cells from IL-2-GFPki mice and control BALB/c mice were stimulated in 24-well culture plates coated with anti-CD3 alone or along with anti-CD28 for the indicated number of hours. Harvested cells were unstained or stained with PE-conjugated anti-CD4 or PE-conjugated anti-CD8 mAb. GFP expression in these activated T cells was detected with the FL1 detector. The third and fourth rows are the histograms of GFP fluorescence gated on a CD4 or CD8 population. Thin lines are control T cells; IL-2-GFPki T cells are shown in heavy lines. The results are representative of two similar experiments.

 


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  		FIGURE 3. Differential IL-2 promoter activation by CD28 vs non-CD28 costimulation. Purified IL-2-GFPki and control T cells were stimulated with immobilized anti-CD3 (1 µg/ml) plus coimmobilized anti-CD28, anti-CD5, or anti-CD9 (10 µg/ml) for the indicated number of hours. The results are representative of three similar experiments.

 
Differential effects of CD28 and non-CD28 costimulation on the induction of various IL-2 transcription factors

Stimulation of T cells with anti-CD3 plus anti-CD28 induces a number of transcription factors that bind to the IL-2 promoter. These include the NF-AT/AP-1 complex (17), NF-IL-2A (31), NF-{kappa}B (18), and the CD28RC (19). By EMSA we first confirmed that these are also induced in the present stimulation conditions, and that the binding of each factor to the relevant oligonucleotide probe is inhibited in the presence of an excess of unlabeled oligonucleotide (data not shown). Then we examined whether there exists any difference in the induction of each factor between CD28 and non-CD28 costimulations. At an initial phase (8 h) of stimulation, CD5 and CD9 costimulations induced rather higher levels of NF-AT/AP-1 and NF-IL-2A compared with those produced by CD28 costimulation (Fig. 4Go) and similar levels of NF-{kappa}B and CD28RC as those by CD28 costimulation (Fig. 4Go). In contrast to a decrease in these transcription factors following non-CD28 costimulation, CD28 costimulation exhibited a time-dependent increase in all transcription factors. When these factors were compared throughout the entire time course (8–24 h of stimulation), there existed a great difference in the induction of NF-{kappa}B and CD28RC.



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  		FIGURE 4. Comparison of CD28 and non-CD28 costimulations in the induction of various IL-2 transcription factors. Purified BALB/c T cells were stimulated with immobilized anti-CD3 alone or together with coimmobilized anti-CD5, anti-CD9, or anti-CD28 for the indicated number of hours. Nuclear extracts were examined for binding to oligonucleotides corresponding to various IL-2 promoter binding sites. The results are representative of two similar experiments.

 
Analysis of the protein compositions of the NF-{kappa}B complexes binding to the NF-{kappa}B binding site and the CD28RE

NF-{kappa}B is the transcription factor that binds to the NF-{kappa}B site itself as well as to the CD28RE and is composed of dimeric complexes of various NF-{kappa}B members. To analyze the protein compositions of the NF-{kappa}B complexes binding to the NF-{kappa}B site and the CD28RE, nuclear extracts from CD28-costimulated T cells were treated with Abs that specifically recognize NF-{kappa}B members and were subjected to EMSA using the oligonucleotide probes corresponding to the NF-{kappa}B binding sequence and CD28RE. Fig. 5Go shows that the NF-{kappa}B complex (upper panel) induced by CD28 costimulation was strongly supershifted by anti-p50 Ab and was partially blocked by anti-c-Rel Ab, but was only weakly influenced by anti-p65 Ab. Anti-Rel-B Ab failed to affect the mobility. Similar patterns of supershifts using these Abs were observed in the EMSA with CD28RE oligonucleotide (Fig. 5Go, lower panel). Thus, the NF-{kappa}B complexes binding to the NF-{kappa}B-binding site and CD28RE are composed of p50, p65, and c-Rel.



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  		FIGURE 5. Supershift analysis of NF-{kappa}B and CD28RC induced by CD28 costimulation. Purified T cells were stimulated with anti-CD3 alone or together with anti-CD28 mAb. Nuclear extracts of CD28-costimulated T cells were incubated with the indicated supershift Abs or control (Co.) rabbit or goat Ig for 20 min on ice before EMSA for the NF-{kappa}B binding site (upper) and the CD28RE (lower). The results are representative of three similar experiments.

 
The NF-{kappa}B complex consists of the upper and lower bands (Figs. 4Go and 5Go), which have been described to be a p50 homodimer and heterodimers of p50 and other components, respectively (32, 33). We compared the supershift of each component in these two bands between CD28- and non-CD28-costimulated T cells (Fig. 6Go). There was no great difference in the lower thick band of the NF-{kappa}B complex, and this band in both types of T cells was completely supershifted by anti-p50 Ab, but not by Abs against other components. The upper band, which was also eliminated by anti-p50 Ab, was partially or slightly supershifted by anti-c-Rel or anti-p65 Ab (Fig. 6Go, lower panels), but not by anti-Rel B Ab (data not shown). Consistent with previous reports (32, 33), these results indicate that the upper band contains two types of complexes: c-Rel plus p50, and p65 plus p50. It should be noted that CD28 costimulation induced higher levels of the upper band containing heterodimers than non-CD28 costimulation (Figs. 4Go and 5Go), and that the upper band induced by CD28 costimulation was more strongly reduced by anti-c-Rel Ab than by anti-p65 Ab (Figs. 5Go and 6Go).



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  		FIGURE 6. The protein compositions of the NF-{kappa}B complexes induced by CD5, CD9, and CD28 costimulations. Nuclear extracts of non-CD28- and CD28-costimulated T cells were incubated with the indicated supershift Abs before EMSA for the NF-{kappa}B-binding site. The results are representative of two similar experiments.

 
c-Rel is a critical member in the NF-{kappa}B family induced by CD28 costimulation

The results presented in Fig. 6Go suggest that CD28 costimulation induces greater amounts of the c-Rel-p50 complex than non-CD28 costimulation. However, this was not clear from the observation of the supershift. To confirm it, we examined the amount of each NF-{kappa}B component present in nuclear factors by immunoblotting. Purified T cells were stimulated with anti-CD3 alone or together with anti-CD5, anti-CD9, or anti-CD28. Proteins from cytosolic and nuclear fractions of these activated cells were subjected to SDS-PAGE and immunoblotted with anti-p50, anti-p65, and anti-c-Rel Abs (Fig. 7Go). After TCR stimulation, p50/p105 (p50 precursor) and c-Rel were induced in the cytosolic fraction regardless of whether T cells were uncostimulated or CD28- or non-CD28-costimulated, although the amounts of these proteins induced by CD28 costimulation were slightly larger than those induced by non-CD28 costimulation (Fig. 7GoA, left). However, the translocation of p50 and c-Rel into the nuclear fraction greatly differed between CD28 and non-CD28 costimulation groups; both p50 and c-Rel translocations occurred in a strikingly enhanced manner selectively in CD28-costimulated cells (Fig. 7GoA, middle). This was the case during the entire time course up to 24 h after stimulation (Fig. 7GoB). Like p50/c-Rel, the induction of p65 protein was also enhanced, but unlike p50/c-Rel, intranuclear translocation was marginally induced in both CD28 and non-CD28 costimulations (Fig. 7GoA, middle).



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  		FIGURE 7. c-Rel is a critical NF-{kappa}B member that is induced by CD28 costimulation and interacts with the NF-{kappa}B binding sequence. Purified T cells were stimulated with anti-CD3 alone or together with anti-CD5, anti-CD9, or anti-CD28. A, Left and middle, The cytosolic and nuclear fractions from 24-h stimulated T cells were separated by SDS-PAGE and immunoblotted with the relevant Abs. A, Right, nuclear extracts were incubated with agarose beads coupled to NF-{kappa}B oligonucleotide for 45 min. The bound proteins were separated by SDS-PAGE and immunoblotted with the relevant Abs. B, Purified T cells were stimulated with the indicated Abs for various numbers of hours. Nuclear extracts were subjected to SDS-PAGE, followed by immunoblotting with anti-p50 and anti-c-Rel Ab. The results are representative of four (A) and three (B) similar experiments.

 
Further, we examined the binding activity of NF-{kappa}B members translocated into the nuclear fraction by oligo DNA precipitation (Fig. 7GoA, right). Considerable amounts of p50 from uncostimulated and CD28- or non-CD28-costimulated T cells interacted with agarose beads coupled to the NF-{kappa}B binding oligonucleotide sequence, although the amount of bound p50 was appreciably larger in the CD28 costimulation than in other groups. Unlike p50, the binding of c-Rel was enhanced only in nuclear extracts from CD28-costimulated T cells. These results indicate that binding of the c-Rel/p50 heterodimer to the NF-{kappa}B binding sequence is enhanced by CD28 costimulation, but not by non-CD28 costimulation, although the p50 homodimer is induced by both types of costimulation.

Decreased levels of I{kappa}B{beta} degradation following non-CD28 costimulation

The NF-{kappa}B complexes are sequestered in the cytoplasm at latent precursors by physical association with inhibitory proteins such as I{kappa}B{alpha} (34, 35) and I{kappa}B{beta} (36). Each NF-{kappa}B member is allowed to translocate into the nuclear compartment upon the degradation of these inhibitory proteins. Earlier studies have shown that the degradation of inhibitory proteins, particularly of I{kappa}B{beta}, in Jurkat T cells occurs largely depending on the CD28 costimulatory signal (37). The results presented in Fig. 8GoA (lower panels) show that the amounts of I{kappa}B{alpha} and I{kappa}B{beta} are small in freshly prepared resting T cells, but increase rapidly after TCR triggering (8 h after stimulation) by replenishment through de novo synthesis regardless of whether the cells are not costimulated or are costimulated with anti-CD5, anti-CD9, or anti-CD28. The amount of I{kappa}B{beta} did not increase, but, rather, slightly decreased from 8 to 24 h after CD28 costimulation. In contrast, non-CD28 costimulation increased the amount of I{kappa}B{beta} from 8 to 24 h. Thus, there was a significant difference in the amount of I{kappa}B{beta} between CD28- and non-CD28-costimulated T cells, which correlated with differential translocation of c-Rel protein (Fig. 8GoA, upper panels). Unlike I{kappa}B{beta}, the level of cytoplasmic I{kappa}B{alpha} did not differ between CD28 and non-CD28 costimulation.



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  		FIGURE 8. Differential effects of CD28 and non-CD28 costimulation on the degradation of I{kappa}B{beta}. A, The nuclear and cytosolic fractions were prepared from the activated T cells and unstimulated T cells. The nuclear and cytosolic fractions were examined for c-Rel and I{kappa}B{beta}/I{kappa}B{alpha}, respectively. The results are representative of four similar experiments. B, CHX (100 µg/ml) was added to cultures of T cells that had been stimulated with anti-CD3/anti-CD28 or anti-CD3/anti-CD5 for 20 h. Cells were harvested 1, 2, and 4 h later. Cytoplasmic lysates were subjected to Western blotting with anti-I{kappa}B{beta}. Autographs were quantitated by densitometer. Data are expressed as a ratio to the I{kappa}B{beta} level before CHX treatment. Data in two independent experiments of four similar experiments are shown.

 
To more exactly determine the rate of I{kappa}B{beta} degradation, we examined the I{kappa}B{beta} levels in CD28- and non-CD28 (CD5)-costimulated T cells at various hours after treatment with the protein synthesis inhibitor CHX. After T cells were stimulated with anti-CD3 plus mAb against CD28 or CD5 as a representative of non-CD28 molecules for 20 h, CHX was added, and the cells were subsequently cultured in the presence of CHX for an additional 1–4 h. Cytosolic lysates were analyzed by Western blotting, and autographs were quantitated by densitometer. Fig. 8GoB shows that a time-dependent breakdown of I{kappa}B{beta} occurred in CD28-costimulated T cells, whereas the level of I{kappa}B{beta} did not change greatly in CD5-costimulated T cells during the entire time period (4 h). Taken together, the results indicate that low levels of c-Rel translocation in non-CD28 costimulation are associated with the reduced degradation of I{kappa}B{beta} compared with that in CD28 costimulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows that CD28 and non-CD28 costimulations differ in the capacity to sustain IL-2 production by TCR-triggered resting T cells and that this difference is based on the differential IL-2 promoter activation. Compared with CD28 costimulation, non-CD28 costimulation induced apparently reduced levels of transcription factors that interact with the IL-2 promoter region. In addition to the induction of NF-AT/AP-1, there was a great difference in the induction of NF-{kappa}B capable of binding the NF-{kappa}B sequence as well as the CD28RE. By focusing on this transcription factor, we found that the activation of NF-{kappa}B c-Rel/p50 heterodimer was markedly decreased in non-CD28 costimulation. More importantly, both c-Rel and p50 proteins were similarly induced through de novo synthesis in the cytoplasm of CD28 and non-CD28-costimulated T cells. However, non-CD28 costimulation failed to activate the newly synthesized c-Rel/p50 for the translocation into the nuclear compartment. Thus, these results provide a mechanistic explanation of how non-CD28 costimulation fails to sustain IL-2 expression.

Our previous studies have shown that there exists a fundamental difference in the capacity to induce proliferation of TCR-triggered resting T cells between CD28 and other costimulatory molecules (13, 30). Anti-CD28 mAb and mAbs against CD5, CD9, CD2. CD44, or CD11a all induced activation of resting T cells in the absence of APC when coimmobilized with a submitogenic dose of anti-CD3 mAb. [3H]TdR incorporation determined 2 days after costimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 costimulation, costimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL-2 production; CD28 costimulation induced progressively increasing IL-2 production, whereas non-CD28 costimulation failed to sustain IL-2 production (13, 30).

IL-2 gene expression is controlled by its 5'-flanking sequences that contain critical regulatory regions corresponding to binding sites of various transcription factors (15, 16). CD28 costimulation has been demonstrated to enhance the generation of most important transcription factors (17, 18, 19). In addition to its well-recognized effects on transcriptional initiation, CD28 costimulation enhances cytoplasmic IL-2 mRNA stability (20, 21, 22). Like many cytokine mRNAs, the IL-2 mRNA contains several copies of an AU-rich sequence (AUUUA) element with its 3' untranslated region (38). Deletion of these sequences allows the prolonging of mRNA expression and an increase in cytokine production (39, 40). It has been widely assumed that this sequence element is also associated with the regulation of IL-2 mRNA stability. A more recent study demonstrated that the coding region of the IL-2 mRNA contains CD28-responsive sequence elements that also contribute to enhancing mRNA stability (41). The present experiments using T cells from IL-2-GFPki mice (23) permitted us to investigate the difference in IL-2 gene expression between CD28 and non-CD28 costimulation by focusing on IL-2 promoter activation. Our results illustrated a fundamental defect of non-CD28 costimulation in IL-2 promoter activation.

A number of transcription factors have been shown to interact with the IL-2 gene promoter, including NF-AT/AP-1 (17), NF-{kappa}B (18), and CD28RC (19). NF-AT/AP-1 was first identified as a key regulator of IL-2 gene transcription (42). However, it is becoming increasingly evident that NF-{kappa}B plays more important roles in T cell activation by CD28 costimulation. In this view, induction of NF-{kappa}B requires a costimulatory signal derived from engagement of the CD28 receptor (43, 44). NF-{kappa}B regulates not only the NF-{kappa}B enhancer element (45), but also the CD28RE of the IL-2 promoter (19). The CD28RE interacts with several members of the NF-{kappa}B family and NF-AT (42, 43, 46) and is essential for integrating the CD28 costimulatory signal. In the present study CD28 costimulation up-regulated the induction of both NF-AT/AP-1 and NF-{kappa}B in a time-dependent manner. In contrast, non-CD28 costimulation induced higher levels of NF-AT/AP-1 and NF-IL-2A at an early time point after stimulation than CD28 costimulation, but failed to exhibit a time-dependent increase in all transcription factors examined. Particularly, there was a fundamental defect in the induction of NF-{kappa}B that interacts with the NF-{kappa}B enhancer element and the CD28RE.

Members of the NF-{kappa}B family consist of several distinct subunits, including c-Rel, RelB, p52, and ubiquitous proteins in the family, Rel-A (p65) and p50 (47, 48, 49). Some components such as c-Rel are inducibly synthesized in response to NF-{kappa}B-activating stimuli. NF-{kappa}B is comprised of dimeric complexes of these subunits and is sequestered in the cytosol as a complex associated with inhibitory family molecules I{kappa}B. Distinct dimeric complexes are activated in response to various stimuli capable of phosphorylating/degrading I{kappa}B and are translocated into the nuclear compartment. These include signals from TCR/CD28 (43, 44, 46, 50) and from TNF receptor (48, 49, 51). Particularly, the inability of T cells from c-Rel-deficient mice to produce IL-2 supported a central role for c-Rel in regulation of the IL-2 gene (52). Our present results demonstrated that CD28 costimulation can induce translocation of c-Rel/p50, which is consistent with the previous studies (52). In contrast, non-CD28 costimulation failed to activate the NF-{kappa}B complex, including a c-Rel component. This explains the failure of non-CD28 costimulation to sustain IL-2 gene expression.

Furthermore, our results provided an additional mechanistic explanation for the failure of non-CD28 costimulation to activate c-Rel. As described previously (47, 48, 49), c-Rel was induced following TCR triggering. There was no substantial difference in the amount of synthesized c-Rel protein between CD28 and non-CD28 costimulations. However, the translocation of c-Rel into the nuclear compartment greatly differed. Although I{kappa}B degradation is required for the translocation of NF-{kappa}B members such as c-Rel, the amounts of I{kappa}B proteins increase along with c-Rel induction through de novo synthesis after TCR triggering. In this context our results showed that CD28 costimulation did not induce an increase in I{kappa}B{beta} from 8 to 24 h after TCR triggering, whereas non-CD28 costimulation increased the amount of this inhibitory protein between these two time points. More importantly, experiments using CHX demonstrated that unlike CD28 costimulation, non-CD28 costimulation induced only marginal levels of I{kappa}B{beta} degradation. Thus, our results suggest that non-CD28 costimulation generates a signal for the inducible synthesis of c-Rel protein, but fails to trigger the signaling that leads to NF-{kappa}B/c-Rel activation.

Costimulation via various molecules other than CD28, including CD5 and CD9, induces enhanced [3H]TdR incorporation comparable to those of CD28 costimulation (Refs. 13, 30 ; this study). This is assumed to be based on the fact that non-CD28 costimulation results in a very low, but detectable, level of IL-2 production as well as higher levels of IL-2R induction than CD28 costimulation (30). Moreover, our previous studies (13, 30) demonstrated that apoptosis occurs in non-CD28-costimulated T cells as early as 48 h after stimulation. This apoptosis was also found to be associated with the failure of non-CD28 costimulation to induce the anti-apoptosis protein Bcl-xL (30), that is induced by CD28 costimulation (53). In this context it should be noted that the Bcl-xL promoter contains the NF-{kappa}B binding site (54, 55) and that the NF-{kappa}B cascade is important in Bcl-xL expression and for the anti-apoptotic effect of CD28 (56).

In addition to NF-{kappa}B, CD28 costimulation induced higher levels of NF-AT/AP-1 at a later time point (24 h after stimulation) than non-CD28 costimulation. However, non-CD28 costimulation could also elicit this transcription factor, and at an early time point (8 h after stimulation) the levels were higher in non-CD28 than in CD28 costimulation. Further studies will be required to investigate whether the protein compositions of NF-AT/AP-1 induced in CD28 and non-CD28 costimulations are similar and why/how the time course of NF-AT/AP-1 induction is different between these two categories of costimulation.

Our results illustrate that in contrast to CD28 costimulation, non-CD28 costimulation fails to translocate a critical NF-{kappa}B member, c-Rel, and to prepare an activated form of c-Rel/p50 heterodimer. As a result, non-CD28-costimulated T cells produce a modest amount of IL-2 that is sufficient for an initial phase of T cell activation as evaluated by [3H]TdR uptake, but not for the induction of cellular expansion. The lack of NF-{kappa}B activation is also considered to underlie non-CD28 costimulation-induced apoptosis that occurs due to the shortage of IL-2 (13, 30). Further studies to investigate the failure of non-CD28 costimulation to induce the NF-{kappa}B activation cascade could, in turn, contribute to a better understanding of the molecular mechanisms by which CD28 costimulation induces full T cell activation.


   Acknowledgments
 
We thank M. Yasuda for secretarial assistance.


   Footnotes
 
1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. Back

2 Address correspondence and reprint requests to Dr. Hiromi Fujiwara, Department of Oncology (C6), Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail address: hf{at}ongene.med.osaka-u.ac.jp Back

3 Abbreviations used in this paper: CD28RE, CD28-responsive element; CD28RC, CD28RE-reactive complex; CHX, cycloheximide; GFP, green fluorescent protein. Back

Received for publication December 7, 2001. Accepted for publication February 13, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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