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The Journal of Immunology, 2002, 169: 1151-1158.
Copyright © 2002 by The American Association of Immunologists

Essential Role of NF-{kappa}B-Inducing Kinase in T Cell Activation Through the TCR/CD3 Pathway1

Mitsuru Matsumoto2,*, Takuji Yamada{dagger}, Steven K. Yoshinaga{ddagger}, Tom Boone{ddagger}, Tom Horan{ddagger}, Shigeru Fujita{dagger}, Yi Li* and Tasuku Mitani*

* Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, Tokushima, Japan; {dagger} First Department of Internal Medicine, School of Medicine, Ehime University, Ehime, Japan; and {ddagger} Amgen, Thousand Oaks, CA 91320


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NF-{kappa}B-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-{beta} receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-{kappa}B activity in both mature and immature T cells; the impaired NF-{kappa}B activity in mature T cells was also associated with the failure of maintenance of activated NF-{kappa}B. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-{kappa}B activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell receptor ligation by Ags coupled with MHC molecules triggers the activation of multiple protein kinases downstream of TCR/CD3, which then phosphorylate various cellular substrates. This is followed by the deployment of several signaling cascades, resulting in the efficient transcription of many genes required for T cell function (1). Stimulation of T cells through the TCR/CD3 complex alone, however, is not sufficient to activate optimal cytokine production and T cell proliferation, and additional costimulatory signals provided by the APC are required. These costimulatory molecules include CD80 (B7.1), CD86 (B7.2), and other newly discovered B7 superfamily members (2). Because T cell activation plays a pivotal role in the initiation of Ag-specific immune responses required for host defense, the mechanisms of T cell activation have been the subject of intense investigation.

The diverse signaling pathways downstream of TCR/CD3 converge on several key transcription factors, including NF-AT, AP-1, and NF-{kappa}B. NF-{kappa}B plays a wide variety of roles in the regulation of innate immunity, stress responses, inflammation, and the inhibition of apoptosis (3, 4). In most cell types, NF-{kappa}B is present as a heterodimer comprising 50 (p50)- and 65-kDa (p65) subunits, and is sequestered in the cytoplasm by a member of the I-{kappa}B family of inhibitory proteins. NF-{kappa}B activation requires the degradation of I-{kappa}B proteins, which is tightly regulated by cytokines and other external stimuli (5, 6, 7). Although the signaling pathways downstream of the TNFR leading to NF-{kappa}B activation have been well studied, the exact roles of NF-{kappa}B activation in T cell function as well as the signaling pathway leading to NF-{kappa}B activation downstream of TCR/CD3 still remain elusive.

NF-{kappa}B-inducing kinase (NIK)3 is structurally related to mitogen-activated protein kinase kinase kinase (8) and has been shown to phosphorylate both I-{kappa}B kinase-{alpha} (IKK{alpha}) and IKK{beta}, which may sequentially activate the downstream I-{kappa}B proteins necessary for NF-{kappa}B activation (3, 4, 5, 6, 7). The alymphoplasia (aly) mouse is a natural strain with a mutated NIK, which provides a novel and unique model for studying the abnormal development of lymphoid organs as well as immunodeficiency (9, 10); these mice completely lack lymph nodes and Peyer’s patches, and exhibit disturbed spleen architecture (for example, the development of germinal centers and follicular dendritic cell clusters is abnormal) (9, 10, 11, 12). Aly mice also exhibit disturbed thymic structure; the segregation of thymic cortex and medulla is unclear (9, 10). We have demonstrated that abnormal lymphoid organogenesis in aly mice is caused by impaired signaling for lymphotoxin-{beta} receptor (LT{beta}R) (13), a receptor essential for lymphoid organogenesis (14, 15). With the use of NIK mutant (aly mice) and IKK{alpha}-deficient mice, we have further demonstrated that NIK-IKK{alpha} constitutes an essential pathway for the induction of NF-{kappa}B through LT{beta}R, whereas this pathway is dispensable in TNFR-I signaling (16). The indispensable role of NIK in LT{beta}R signaling, but not in TNFR-I signaling, has also been demonstrated by the use of NIK-deficient mice generated by gene targeting (17). These studies have clearly shown that diverse pathways converging on NF-{kappa}B activation exist downstream of a variety of cell surface receptors.

Aly mice manifest various signs of immunodeficiency, including impaired Ab responses and defective allogeneic skin graft rejection (9, 10, 11, 12), suggesting that the aly mutation affects not only lymphoid organogenesis, but also immune regulation. In fact, IL-2 production from TCR/CD3-stimulated T cells is impaired in aly mice (12). Although these studies suggested that NIK plays an important role in mediating the signals downstream of TCR/CD3, it remains largely unknown how NIK mutation contributes to impaired T cell function.

In the present study, with the use of in vivo mouse models, rather than with enforced gene expression systems and/or leukemic cell lines, we have demonstrated that NIK mutation affects NF-{kappa}B activity downstream of TCR/CD3 in a distinct, but cooperative fashion with protein kinase C (PKC).


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

Aly/+ mice and aly/aly mice (9) were purchased from CLEA Japan (Osaka, Japan). The mice were maintained under pathogen-free conditions, and handled in accordance with the Guidelines for Animal Experimentation of Tokushima University, School of Medicine. The experiments were initiated at 8–12 wk of age.

Functional analysis of T cells

T cells were purified, as described previously, with slight modification (12). Briefly, spleen cell suspensions were prepared by teasing the tissues apart between two frosted microscope slides in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME, hereafter referred to as R10. The spleen cell suspensions were depleted of RBC by osmotic lysis, and mononuclear cells from the spleen were first purified with Histopaque-1119 (Sigma-Aldrich, St. Louis, MO). T cells were then purified with MACS CD90 (Thy-1.2) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. The resulting preparations contained ~90% CD3-positive cells from both aly/+ and aly/aly mice, and no contaminating B cells were detectable. The purified T cells (1 x 105 cells/well) were stimulated with immobilized anti-CD3 mAb (clone 145-2C11; BD PharMingen, San Diego, CA) and anti-CD28 mAb (clone 37.51; Serotec, Oxford, U.K.). To cross-link TCR/CD3 complexes with different strength, we have used various concentrations of anti-CD3 mAb diluted with PBS for the preparation of anti-CD3 mAb-coated plates. For the last 8 h of the 48-h culture period (spleen T cells) or 72-h culture period (thymocytes), the cells were pulsed with 0.5 µCi [3H]thymidine per well, and 3H incorporation was determined, as described previously (12). For blockade of PKC, bisindolylmaleimide I (ICN Pharmaceuticals, Costa Mesa, CA) was added to the culture of purified T cells. IL-2 production by the culture supernatants was determined with an ELISA kit (Amersham, Little Chalfont, U.K.) after 48 h (spleen T cells) or 72 h (thymocytes) of culture.

EMSA

Purified T cells (106) from spleen or thymus were stimulated with immobilized anti-CD3 mAb. The cells were washed once with PBS, and were resuspended in 400 µl ice-cold lysis buffer containing 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM DTT, 0.5% Nonidet P-40 (Sigma-Aldrich), and a mixture of protease inhibitors. The cells were vortex mixed vigorously, and the mixture was centrifuged for 1 min at 5,000 x g. The nuclear pellets were resuspended in 30 µl ice-cold lysis buffer containing 20 mM HEPES, 1 mM EDTA, 1 mM DTT, 0.4 M NaCl, 20% glycerol, and a mixture of protease inhibitors, and incubated for 20 min on ice. After centrifugation for 2 min at 12,000 x g, the supernatant was collected and 2–4 µg of each extract was subjected to the reaction. The following pairs of oligonucleotides were used: NF-{kappa}B, 5'-CTCGAGCCTCTCGGAAAGTCCCCTCTGTTG-3' and 5'-AGCTTCAACAGAGGGGACTTTCCGAGAGGC-3'; NF-AT, 5'-GATCGAAGAGGAAAATTTGTTTCATACA-3' and 5'-GATCGTGTATGAAACAAATTTTCCTCTT-3'; AP-1, 5'-AGCTTAAAGCATGAGTCAGACA-3' and 5'-TCAGGTGTCTGACTCATGCTTTA-3'. The binding buffer was 10 mM HEPES-NaOH, 50 mM KCl, 1 mM EDTA, 5% glycerol, 5 mM DTT, and 250 µg/ml poly(dI:dC). A total of 2 x 104 cpm labeled probe was used in each reaction, and bandshifts were resolved on 4% polyacrylamide gels in 0.5x Tris-borate/EDTA running buffer. The specificity of the signals for NF-{kappa}B was demonstrated by the supershift assay with rabbit antipeptide Abs directed against p50 (catalog sc-114) and p65 (catalog sc-109) from Santa Cruz Biotechnology (Santa Cruz, CA), as well as by the cold inhibition assay. The densities of the bandshifts were analyzed by scanning densitometry.

Assessment of costimulatory pathway in T cells

The purified T cells (1 x 105 cells/well) were cultured with recombinant costimulatory molecules in the presence or absence of TCR/CD3 ligation by immobilized anti-CD3 mAb, and the proliferative responses were determined by 3H incorporation, as described above. The following recombinant costimulatory molecules were used: B7RP-1 human IgG1 Fc fusion protein (B7RP-1-Fc), B7.2 human IgG1 Fc fusion protein (B7.2-Fc) (18), and homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed on T cells-flag fusion protein (LIGHT-flag) (19).

Flow cytometric analysis

For the assessment of inducible costimulatory (ICOS) expression, T cells were stimulated with immobilized anti-CD3 mAb for 18 h. Cells were washed twice with PBS and incubated with B7RP-1-Fc and then with PE-conjugated anti-human IgG (Calbiochem, La Jolla, CA). The cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) with CellQuest software (13).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Impaired T cell activation in NIK mutant mice

We have previously demonstrated that the aly-NIK mutation affects hemopoietic cell function, including that of T cells; IL-2 production by aly/aly T cells after in vitro stimulation with immobilized anti-CD3 mAb or in allogeneic MLR was dramatically reduced compared with that from aly/+ T cells (12). In contrast, proliferative T cell responses assessed by [3H]thymidine incorporation were moderately retained in aly mice; reduction of 3H incorporation by aly/aly T cells was 30–40%, at most (12). Because we used rather high concentrations of mAb (10 µg/ml) for the preparation of anti-CD3 mAb-coated plates in these experiments, we thought that this discrepancy might be a result of the experimental setting. We therefore prepared anti-CD3 mAb-coated plates for T cell stimulation with various concentrations of mAb. Cross-linking of TCR/CD3 complexes with high concentrations of anti-CD3 mAb (i.e., 10 and 20 µg/ml) elicited good proliferative T cell responses from aly mice, although the dose-response curve did not show a plateau even at the highest concentration of mAb used (Fig. 1Go, top). As the concentration of mAb decreased, the impairment of proliferative T cell responses from aly mice became more evident; less than 25% of [3H]thymidine incorporation was observed using anti-CD3 mAb-coated plates at the concentration of 5.0 µg/ml. Thus, NIK mutation affects T cell responsiveness induced by TCR/CD3 ligation in a dose-response fashion. IL-2 production from aly/aly T cells was severely impaired throughout the range of different concentrations of anti-CD3 mAb tested (Fig. 1Go, bottom). However, costimulation through CD28 significantly restored IL-2 production from aly/aly T cells in a dose-response manner of TCR/CD3 ligation (12) (our unpublished data).



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  		FIGURE 1. NIK mutation affects T cell responsiveness induced by TCR/CD3 ligation in a dose-response fashion. Purified splenic T cells (1 x 105 cells/well) from aly/+ mice (solid line) and aly mice (dotted line) were stimulated with anti-CD3 mAb-coated plates prepared with various concentrations of mAb ranging between 1 and 20 µg/ml. Cells were pulsed for the last 8 h of the 48-h culture period with [3H]thymidine, and 3H incorporation was measured using a beta counter (top). The results are expressed as the mean ± SEM for triplicate wells. The concentration of IL-2 in the culture supernatants was measured with an ELISA kit after the 48-h culture period (bottom). The results are expressed as the mean for triplicate wells. One representative experiment from a total of five repeats is shown.

 
Attenuated NF-{kappa}B activity accounts for impaired T cell activation through the TCR/CD3 pathway in aly mice

We and others have recently demonstrated that NIK is essential for NF-{kappa}B transactivation through LT{beta}R, but not through TNFR-I (16, 17). We have reasoned that the impaired function of T cells from aly mice described above might be caused by defective induction of NF-{kappa}B downstream of TCR/CD3. We therefore prepared nuclear extracts from T cells stimulated with differential concentrations of anti-CD3 mAb for 8 h, and examined NF-{kappa}B activation with EMSA using a NF-{kappa}B-binding oligonucleotide probe (Fig. 2GoA, top). In aly/+ T cells, increasing the concentration of anti-CD3 mAb induced proportional NF-{kappa}B activation compared with the basal level of NF-{kappa}B activity from unstimulated T cells. In contrast, 1.0 µg/ml anti-CD3 mAb-coated plates, which induced obvious NF-{kappa}B activation from aly/+ T cells, induced only minimal NF-{kappa}B activation in aly/aly T cells; densitometric analysis showed that the amount of p50/p65 complex from aly/aly T cells was only one-third of that from aly/+ T cells (Fig. 2GoB, top). With the use of 5.0 µg/ml anti-CD3 mAb-coated plates, NF-{kappa}B activation in aly/aly T cells became evident, but still weaker compared with that from aly/+ T cells. However, 10 µg/ml anti-CD3 mAb-coated plates induced similar magnitudes of NF-{kappa}B activation in aly/+ and aly/aly T cells. The specificity of the EMSA was confirmed by supershifting with anti-p50 and anti-p65 Abs as well as by the cold inhibition assay. Thus, the impaired T cell function induced by TCR/CD3 ligation was in parallel with the attenuated NF-{kappa}B activation downstream of TCR/CD3 in aly mice. In contrast, activation of NF-AT (Fig. 2Go, middle) and AP-1 (Fig. 2Go, bottom) induced by TCR/CD3 ligation was indistinguishable between aly/+ and aly mice throughout the whole range of anti-CD3 mAb concentrations tested.



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  		FIGURE 2. Attenuated NF-{kappa}B activity induced by TCR/CD3 stimulation of splenic T cells from NIK mutant mice. Nuclear extracts were prepared from purified splenic T cells (106) after stimulation with various concentrations of immobilized anti-CD3 mAb for 8 h. NF-{kappa}B activation was assessed with EMSA using NF-{kappa}B (A, top)-, NF-AT (A, middle)-, and AP-1 (A, bottom)-binding oligonucleotide probes. The specificity of the EMSA was confirmed by supershifting with anti-p50 and anti-p65 Abs for NF-{kappa}B as well as by the cold inhibition assay (A, top). The densities of the bandshifts from aly/+ mice (solid line) and aly mice (dotted line) were analyzed by scanning densitometry (B). The percentages of densities of the bandshifts (p50/p65 heterodimer) from aly mice compared with those from aly/+ mice are shown for NF-{kappa}B activity (B, top). One representative experiment from a total of three repeats is shown.

 
Kinetic analysis after anti-CD3 stimulation revealed that attenuated NF-{kappa}B activity in mature T cells from aly mice was associated with the failure of maintenance of activated NF-{kappa}B. With low concentration of anti-CD3 mAb-coated plates (i.e., 1 µg/ml, as demonstrated in Fig. 3Go), initial NF-{kappa}B activation (i.e., 2 h after stimulation) occurred to similar levels in both aly/+ and aly/aly T cells. NF-{kappa}B DNA-binding activity from aly/+ T cells continued to increase until 4 h after stimulation, and then decreased gradually; it still remained higher than that of the basal level 16 h after stimulation. In contrast, activated NF-{kappa}B from aly/aly T cells, of which peak was lower than that from aly/+ T cells, returned to the basal level immediately thereafter. Failure of the maintenance of activated NF-{kappa}B was also observed when the peak of NF-{kappa}B activity was similar between aly/+ and aly/aly T cells with higher concentrations of anti-CD3 mAb stimulation (i.e., 2 µg/ml, data not shown). These results suggest that NIK also affects the magnitude of NF-{kappa}B activity by regulating the maintenance of activated NF-{kappa}B downstream of TCR/CD3 in mature T cells.



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  		FIGURE 3. Impaired maintenance of NF-{kappa}B activity in NIK mutant T cells after anti-CD3 stimulation. Nuclear extracts were prepared from purified splenic T cells (106) at various time points after stimulation with immobilized anti-CD3 mAb (1 µg/ml). The densities of the bandshifts from aly/+ mice (solid line) and aly mice (dotted line) were analyzed by scanning densitometry (B). One representative experiment from a total of six repeats is shown.

 
Indispensable role of NIK in NF-{kappa}B activation in immature T cells

PKC-{theta} is a novel Ca2+-independent PKC isoform that plays an important role in T cell activation (20, 21, 22, 23, 24). Recent studies with gene-targeted mice have demonstrated that PKC-{theta} is essential for TCR-mediated NF-{kappa}B activation in mature T cells, but not in immature T cells, suggesting that the requirement for PKC-{theta} in T cell activation is dependent on maturation stage (22). We have examined whether NIK plays an important role in NF-{kappa}B activation in immature T cells as well as in mature T cells. NF-{kappa}B DNA-binding activity in thymocytes from aly mice was decreased compared with that from aly/+ mice upon stimulation with anti-CD3 ligation even with the highest concentration of anti-CD3 mAb used (i.e., 10 µg/ml anti-CD3 mAb, as demonstrated in Fig. 4Go). The attenuated NF-{kappa}B activity in thymocytes from aly mice was observed throughout the time course examined (Fig. 5Go). Thus, in contrast to PKC-{theta}, NIK is required for NF-{kappa}B activity not only in mature T cells, but also in immature T cells. Consistent with the impaired NF-{kappa}B activity, thymocytes from aly mice showed impaired proliferative T cell responses and IL-2 production upon stimulation with anti-CD3 mAb (Fig. 6Go).



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  		FIGURE 4. Attenuated NF-{kappa}B activation induced by TCR/CD3 stimulation of thymocytes from NIK mutant mice. Nuclear extracts were prepared from thymocytes (106) after stimulation with various concentrations of immobilized anti-CD3 mAb for 8 h. The densities of the bandshifts (p50/p65 heterodimer) from aly/+ mice (solid line) and aly mice (dotted line) were analyzed by scanning densitometry (B). One representative experiment from two repeats is shown.

 


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  		FIGURE 5. Kinetic analysis of NF-{kappa}B activation in thymocytes from NIK mutant mice. Nuclear extracts were prepared from thymocytes (106) at various time points after stimulation with immobilized anti-CD3 mAb (10 µg/ml). One representative experiment from a total of seven repeats is shown.

 


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  		FIGURE 6. NIK mutation affects thymocyte responsiveness induced by TCR/CD3 ligation. Purified thymocytes (1 x 105 cells/well) from aly/+ mice (filled bars) and aly mice (open bars) were stimulated with anti-CD3 mAb-coated plates prepared with different concentrations of mAb indicated. Cells were pulsed for the last 8 h of the 72-h culture period with [3H]thymidine (top). The results are expressed as the mean ± SEM for triplicate wells. In the same set of experiment, the concentration of IL-2 in the culture supernatants was measured with an ELISA kit after the 72-h culture period (bottom). One representative experiment from a total of five repeats is shown.

 
Retained responsiveness to T cell costimulation in NIK mutant mice

In order for T cells to become fully activated, they need to be provided with costimulatory signals such as CD28 from APC. We have previously demonstrated that impaired IL-2 production from anti-CD3-activated aly/aly T cells could be restored by coligation with CD28; IL-2 production by aly/aly T cells was significantly increased after CD28 coligation, although still reduced ~50% compared with that by aly/+ T cells (12). Consistent with this finding, CD28 costimulation by rB7.2 coupled with suboptimal TCR/CD3 ligation resulted in augmented proliferative T cell responses from aly mice, similar to that in aly/+ mice (Fig. 7GoA, middle). Proliferative T cell responses by the costimulation with CD28 in aly mice were increased by increasing concentration of anti-CD3 mAb used, as observed when the T cells were stimulated by TCR/CD3 ligation alone. Consistent with these findings, NF-{kappa}B DNA-binding activity from aly/aly T cells was increased after CD28 coligation, although still reduced compared with that from aly/+ T cells (Fig. 8Go). These results favor the notions that CD28 induces costimulation by acting as a general amplifier of early TCR signals (20, 25, 26) and that NIK does not appear to be uniquely coupled to the CD28 costimulatory pathway.



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  		FIGURE 7. Retained responsiveness to T cell costimulation in NIK mutant mice. Purified T cells (1 x 105 cells/well) were cultured with recombinant B7.2 (A, middle) or B7RP-1 (A, bottom) in the presence or absence of TCR/CD3 ligation by immobilized anti-CD3 mAb. The proliferative responses were determined as described in Fig. 1Go. Filled and open bars represent aly/+ and aly/aly T cells, respectively. Purified T cells were stimulated with various concentrations of immobilized anti-CD3 mAb for 18 h, and then ICOS expression was assessed by flow cytometry using B7RP-1 human IgG1 Fc fusion protein (B). Filled profiles represent ICOS expression from unstimulated T cells.

 


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  		FIGURE 8. Retained responsiveness to T cell costimulation in NIK mutant mice assessed by NF-{kappa}B activation. Nuclear extracts were prepared from purified splenic T cells (106) stimulated with immobilized anti-CD3 mAb or anti-CD3 mAb plus anti-CD28 mAb for 8 h. One representative experiment from two repeats is shown.

 
The role of NIK in the newly identified costimulatory pathway, ICOS, was investigated. Because it has been demonstrated that ICOS expression on T cells is activation induced (18, 27), we first examined ICOS expression by flow cytometry of both aly/+ and aly/aly T cells after TCR/CD3 stimulation. On aly/+ T cells, ICOS could be detected with B7RP-1-fusion protein when T cells were stimulated with a rather low concentration of anti-CD3 mAb (i.e., 2.5 µg/ml) (Fig. 7GoB, top). In contrast, we could not detect ICOS expression on aly/aly T cells with the same condition; detection of ICOS on aly/aly T cells required higher concentrations of anti-CD3 mAb (i.e., 10.0 µg/ml) (Fig. 7GoB, bottom). This impaired expression of ICOS was reflected in part by the fact that costimulation by ICOS coupled with TCR/CD3 ligation was less effective in aly mice, especially when the concentration of anti-CD3 mAb was low (Fig. 7GoA, bottom); compare the difference of proliferative T cell responses between aly/+ and aly mice when T cells were stimulated either with rB7.2 or rB7RP-1 in the range from 0.2 to 0.5 µg/ml of anti-CD3 mAb. Thus, although costimulation with ICOS did augment proliferative T cell responses from aly mice, the costimulatory effect of ICOS was weaker compared with that of CD28 ligation, possibly because of the TCR/CD3-dependent inducible expression of ICOS.

NIK and PKC in mature T cells regulate NF-{kappa}B activation downstream of TCR/CD3 in a cooperative fashion

Both NIK and PKC-{theta} play important roles in NF-{kappa}B activity through TCR/CD3 signaling in mature T cells, whereas NIK, but not PKC-{theta}, is indispensable for full NF-{kappa}B activation in immature T cells (as demonstrated by the present study and Ref. 22). Despite the differential requirement for NIK and PKC-{theta} for NF-{kappa}B activity in distinct T cell maturation stages, cross-talk between two pathways is possible, at least in mature T cells. To test this possibility, the effect of blocking PKC pathway with bisindolylmaleimide, a PKC inhibitor, was assessed in aly/aly T cells. Increasing the dose of PKC inhibitor caused proportional decreases in proliferative responses from aly/+ T cells after anti-CD3 stimulation (Fig. 9Go, top); addition of 0.6 and 1.2 µM PKC inhibitor resulted in 20 and 40% reduction of the proliferative responses from aly/+ T cells, respectively (Fig. 9Go, bottom). The effect of blocking PKC was more dramatic in aly/aly T cells; 80 and 95% of the proliferative responses were lost from aly/aly T cells in the presence of 0.6 and 1.2 µM PKC inhibitor, respectively. This suggests that NIK and PKC control T cell activation in a cooperative fashion in the mature stage.



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  		FIGURE 9. NIK and PKC in mature T cells regulate NF-{kappa}B activity downstream of TCR/CD3 in a cooperative fashion. Purified splenic T cells (1 x 105 cells/well) were stimulated with anti-CD3 mAb (10 µg/ml)-coated plates in the presence or absence of different concentrations of bisindolylmaleimide, a PKC inhibitor (top). Proliferative responses were determined as described in Fig. 1Go. Relative reduction of [3H]thymidine incorporation was calculated by defining the proliferative responses without PKC inhibitor as 100% for both aly/+ and aly/aly T cells (bottom). Filled and open bars represent aly/+ and aly/aly T cells, respectively. One representative experiment from a total of three repeats is shown.

 
Blockade of PKC together with aly-NIK mutation severely disturbed T cell function, as described above. The relationship between NIK and PKC in NF-{kappa}B activation was further investigated. The 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester, stimulates PKC directly and induces NF-{kappa}B activation in T cells (22). If NIK regulates NF-{kappa}B activation downstream of PKC (including PKC-{theta}), NF-{kappa}B activation induced by TPA should also be impaired in aly/aly T cells, in a similar way as after anti-CD3 stimulation. In contrast to anti-CD3 stimulation, different concentrations of TPA induced similar levels of NF-{kappa}B DNA-binding activity in splenic T cells as well as in thymocytes from both aly/+ and aly mice (data not shown), suggesting that NIK does not regulate NF-{kappa}B activation just downstream of PKC. Taken all together, these results demonstrate critical roles for NIK in controlling the T cell function by modulating NF-{kappa}B activity downstream of TCR/CD3 in a distinct, but cooperative fashion with PKC (most likely PKC-{theta}; see Discussion).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that NIK plays an essential role in T cell function by regulating NF-{kappa}B DNA-binding activity downstream of TCR/CD3. NIK was originally identified as a kinase required for NF-{kappa}B activation induced by a wide variety of ligand binding (8). It is now clear, however, that the requirement for NIK for NF-{kappa}B activation is strictly signal dependent; NF-{kappa}B activation induced by TNF takes place without NIK, whereas NIK is essential for NF-{kappa}B activation downstream of LT{beta}R (16, 17). A signal-dependent requirement for IKK{alpha} has been also demonstrated; IKK{alpha} is dispensable for TNF-induced NF-{kappa}B activation (28, 29), whereas IKK{alpha} plays an essential role in LT{beta}R-mediated NF-{kappa}B activation (16). Furthermore, we have demonstrated that the association of aly-type NIK with IKK{alpha} was disrupted by the mutation (16). Thus, NIK-IKK{alpha} constitutes an essential pathway in LT{beta}R signaling, but not in TNFR-I signaling. The requirement for NIK downstream of TCR/CD3 suggests that IKK{alpha} may also be involved in NF-{kappa}B activation in this pathway. Consistent with this hypothesis, it has been demonstrated that NIK induces IKK{alpha} phosphorylation in Jurkat T cells (21).

In contrast to NIK, PKC-{theta} has been suggested to activate NF-{kappa}B through selective induction of IKK{beta} enzymatic activity after CD3/CD28 costimulation; PKC-{theta} induces phosphorylation of IKK{beta}, but not IKK{alpha} (21). Because NF-{kappa}B activation, as well as CD69 expression, induced by direct PKC stimulation with TPA was indistinguishable between aly/+ and aly/aly T cells (our unpublished observation), it is reasonable to speculate that the PKC-{theta}-induced IKK{beta} activation process itself remains unaffected in aly/aly T cells. However, given that anti-CD3 stimulation induces activation of both IKK{alpha} and IKK{beta}, and that activation of IKKs is unidirectional (i.e., through IKK{alpha} to IKK{beta}) (30, 31), NIK-IKK{alpha} should constitute an essential step that consequently controls IKK{beta} activity as well. It is also possible that the PKC-{theta}-driven NF-{kappa}B activation pathway regulates the NIK-driven NF-{kappa}B activation pathway before the convergence on IKK activation, which in turn controls the total enzymatic activity of IKKs. Without normal NIK in T cells, dose response of TCR/CD3 ligation required for the induction of IKK activities is affected.

There is another line of finding suggesting that NIK and PKC-{theta} are involved in distinct NF-{kappa}B activation pathways downstream of TCR/CD3; PKC-{theta} is indispensable in mature T cells, but not in immature T cells (22). In contrast, we have demonstrated in the present study that NIK plays an important role in NF-{kappa}B activity not only in mature T cells, but also in immature T cells. We have also demonstrated that proliferation of mature T cells from aly mice was abrogated more severely compared with that from aly/+ mice after anti-CD3 stimulation in the presence of PKC inhibitor (Fig. 9Go), suggesting that NIK and PKC control T cell activation in a cooperative fashion. Although we cannot determine which PKC isoform(s) is involved in this process, PKC-{theta} is a good candidate for this action; transfection studies with Jurkat T cells have demonstrated that PKC-{theta} was the only isoform that could induce significant NF-{kappa}B activation among PKC isoforms tested, including PKC-{theta}, -{epsilon}, -{delta} (novel PKCs), PKC-{alpha} (conventional PKC), and PKC-{zeta} (atypical PKC) (20, 21). Furthermore, PKC-{theta}, but not other PKC isoforms, translocates to the site of cell contact between Ag-specific T cells and APCs (32). Together with the indispensable role of PKC-{theta} in NF-{kappa}B activation in mature T cells (22), we speculate that PKC-{theta} most likely accounts for a cooperation with NIK in TCR-mediated NF-{kappa}B activation. Thus, full activation of NF-{kappa}B requires both NIK and PKC-{theta} activities, at least in mature T cells.

Impairment of NF-{kappa}B activity in immature T cells was more dramatic compared with that from mature T cells in aly mice; attenuated NF-{kappa}B activity in thymocytes from aly mice was observed over a broad range of anti-CD3 mAb concentration throughout the time course examined. Although not so obvious as PKC-{theta} (22), differential requirement of NIK for NF-{kappa}B activity in mature and immature T cells might be possible.

We have demonstrated that impaired NF-{kappa}B activity in aly/aly T cells was associated with a lack of sustained NF-{kappa}B activation (Fig. 3Go). In fact, CD69 expression on aly/aly T cells after anti-CD3 stimulation disappeared earlier than that on aly/+ T cells, although initial CD69 expression was similar between aly/+ and aly mice (our unpublished observation). These results suggest critical roles for NIK in the turnover of NF-{kappa}B components. Consistent with this hypothesis, it has been demonstrated that NIK regulates the processes involved in the generation of mature NF-{kappa}B components (p52) from their precursor proteins (p100) (12, 33). Thus, NIK may regulate T cell function by controlling the maintenance of NF-{kappa}B activity after TCR/CD3 ligation by Ags in vivo.

In addition to NIK and PKC-{theta}, Bcl-10 and Bcl-3 have recently been demonstrated as modulators of NF-{kappa}B activation in T cells. T cells from mice deficient for Bcl-10, a caspase recruitment domain-containing protein identified from the breakpoint in mucosa-associated lymphoid tissue lymphomas, failed to activate NF-{kappa}B and did not proliferate after stimulation with anti-CD3 plus anti-CD28 (34). On the basis of the lack of NF-{kappa}B activation and proliferative responses in Bcl-10-deficient T cells after phorbol ester stimulation, unlike in aly mice, it was proposed that Bcl-10 acts at the level of, or downstream from, PKC-{theta} (34). With the use of microarray technology, Bcl-3 was identified as a gene whose expression was significantly increased upon T cell activation in combination with adjuvant treatment (35). Increased expression of NF-{kappa}B prosurvival target genes by Bcl-3 was induced by growth factors such as IL-4, IL-7, and IL-9, rather than by TCR/CD3 ligation. Bcl-3 might modulate the activity of NF-{kappa}B by functioning as a transcriptional coactivator of p52 homodimers. Alternatively, Bcl-3 could remove repressive p50 homodimers from DNA, allowing them to be replaced by NF-{kappa}B heterodimers with potent transcription-activating activity (36). These results suggest the existence of a group of genes that together control NF-{kappa}B activity in T cells by distinct mechanisms.

The molecular mechanisms by which NIK becomes activated after anti-CD3 stimulation remain unknown. Although the protooncogene Tpl2/Cot had been hypothesized to be a component that activates NIK downstream of the CD3/CD28 pathway (37), the phenotypes of Tpl2/Cot-deficient mice do not support this hypothesis; in contrast to aly/aly T cells, Tpl2/Cot-deficient splenic T cells showed no obvious defect in cytokine production, including IL-2, as well as in proliferative responses after stimulation with anti-CD3 plus anti-CD28 (38). In light of the fact that NIK does not determine the presence or absence of NF-{kappa}B activation (as is the case with the LT{beta}R pathway), but rather modulates the activity of NF-{kappa}B transcription factors in the TCR/CD3 signaling pathway, we speculate that putative molecule(s) upstream of NIK plays similar regulatory roles in T cell activation. It is also possible that the molecule(s) that activates NIK downstream of TCR/CD3 is distinct from the molecule(s) that acts downstream of LT{beta}R (39).

We have demonstrated that aly/aly T cells could respond significantly to the costimulatory signals tested (i.e., CD28 and ICOS), although the total effect induced by TCR/CD3 stimulation coupled with costimulation was still compromised. We also tested the impact of NIK mutation on LIGHT-induced T cell activation through herpesvirus entry mediator, another costimulatory system for T cell function (19, 40). As observed for CD28 and ICOS costimulation, LIGHT induced significant costimulation in anti-CD3-activated aly/aly T cells, although the total proliferative responses were still reduced compared with those from aly/+ mice (our unpublished observation). These results suggest that NIK is not uniquely coupled to the costimulatory pathways, including CD28, ICOS, and herpesvirus entry mediator. Instead, NIK regulates the first signal, the TCR/CD3 pathway, by the mechanisms discussed above, so that the final effect of costimulation is determined at the first signaling step. This first signal-aided T cell regulation by NIK, however, does not control all aspects of T cell function; for example, we did not see any difference in CD40 ligand expression level between aly/+ and aly/aly T cells induced by anti-CD3 stimulation over a broad range of mAb concentration (our unpublished observation). Thus, both NIK-dependent and NIK-independent TCR/CD3-induced activation processes will determine overall T cell function.

Impaired NF-{kappa}B activation was associated with more profound effect on IL-2 production than that on the proliferative responses from mature T cells in aly mice. This phenomenon may also reflect the existence of both NIK-dependent and NIK-independent TCR/CD3-induced activation processes. Although there are similar reports in which the defective TCR-mediated signals in gene-targeted mice caused differential effect on T cell function as observed in the present study, the mechanisms of this remain undetermined. In this respect, the different effects of IL-2 on T cell function may merit attention. IL-2 is an important growth and survival factor for T cells, but also sensitizes these cells to Fas-mediated activation-induced cell death. The molecular basis of these different effects of IL-2 has revealed that T cell proliferation and promoted Fas ligand expression are preferentially mediated by Stat5 activation downstream of IL-2R {beta}-chain, whereas T cell survival was dependent on a receptor region that activated Akt and the expression of Bcl-2 (41). Likewise, it would be important to determine which signaling pathways and related molecules, including NIK and PKC-{theta}, are involved in which specific T cell function after TCR/CD3 ligation in a complex T cell activation process.

Typically, thymocytes differentiate in direct physical contact with thymic stromal cells, and this interaction affects T cell maturation and shapes T cell function in the periphery (42). Thymic structure is disorganized in aly mice; the boundary of cortex and medulla is unclear (9, 10), and ER-TR5-positive cells (medullary epithelial cells) are sparse (43). We also found that epithelial cells that bind the lectin UEA-1 (UEA-1+ medullary epithelial cells) (44) were absent from the thymus of aly mice (our unpublished observation). The signaling pathways that control thymic structure in a NIK-dependent fashion are unknown at present. In this respect, it is interesting to note that mice with a mutation disrupting the RelB gene also manifest disorganized thymic structure devoid of UEA-1+ medullary epithelial cells (45). Thus, the integration of detailed phenotypic analyses of these mutant mice with current knowledge of Rel gene family members and/or modulators of NF-{kappa}B activation such as NIK may illuminate many aspects of thymic organogenesis as well as the T cell maturation and function.


   Acknowledgments
 
We thank M. Morioka and S. Sun for technical assistance.


   Footnotes
 
1 This work was supported in part by Special Coordination Funds of the Science and Technology Agency of the Japanese Government; by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports, Japan; by the Yamanouchi Foundation for Research on Metabolic Disorders; and by the Suzuken Memorial Foundation. Back

2 Address correspondence and reprint requests to Dr. Mitsuru Matsumoto, Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. E-mail address: mitsuru{at}ier.tokushima-u.ac.jp Back

3 Abbreviations used in this paper: NIK, NF-{kappa}B-inducing kinase; ICOS, inducible costimulatory; IKK, I-{kappa}B kinase; LT{beta}R, lymphotoxin-{beta} receptor; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate. Back

Received for publication September 19, 2001. Accepted for publication May 16, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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