Protein kinase B (PKBα/Akt1) a PI3K-dependent serine-threonine kinase, promotes T cell viability in response to many stimuli and regulates homeostasis and autoimmune disease in vivo. To dissect the mechanisms by which PKB inhibits apoptosis, we have examined the pathways downstream of PKB that promote survival after cytokine withdrawal vs Fas-mediated death. Our studies show that PKB-mediated survival after cytokine withdrawal is independent of protein synthesis and the induction of NF-κB. In contrast, PKB requires de novo gene transcription by NF-κB to block apoptosis triggered by the Fas death receptor. Using gene-deficient and transgenic mouse models, we establish that NF-κB1, and not c-Rel, is the critical signaling molecule downstream of the PI3K-PTEN-PKB signaling axis that regulates lymphocyte homeostasis.
Proper execution of apoptotic cell death is an essential component of lymphocyte development, the maintenance of naive T cell numbers, and the generation of T cell memory. The inability to initiate specific apoptotic programs can have severe consequences for the immune system. Mice deficient for proapoptotic members of the Bcl-2 family, such as Bim, Bax, and Bak, accumulate peripheral lymphocytes and develop autoimmune disease (1, 2). Important regulators of lymphocyte homeostasis in both mice and humans are the receptor-ligand pair of the TNF, Fas and Fas ligand (FasL)2 (3, 4). Mutations in Fas, FasL, or other downstream signaling components predispose individuals to the development of autoimmunity (5, 6, 7, 8). Given the significance of normal apoptotic signaling in the maintenance of lymphocyte homeostasis, it is important to identify the signaling networks that influence apoptosis in lymphocytes and Fas-mediated death.
Lipid second messengers generated downstream of the TCR and the costimulatory molecule CD28 play an important role in regulating T cell viability (9). One of the central targets of lipid-based signal transduction in T cells is the serine/threonine kinase protein kinase B (PKB/Akt). PKB is activated in response to phosphatidylinositol-3,4,5-trisphosphate, a lipid second messenger generated by PI3K and dephosphorylated by the lipid phosphatase and tensin homologue deleted on chromosome 10 (PTEN) (10, 11). Activation of PKB, through either enhanced PI3K signaling or impaired PTEN activity, promotes viability in response to multiple apoptotic stimuli in several cell types. Various models also demonstrate that enhanced PKB-mediated viability in T cells triggers lymphoid hyperplasia and autoimmunity. T cells from mice with deregulated PKB activity, through transgenic expression of active PI3K (12), haploinsufficiency or conditional deletion of PTEN (13, 14), or overexpression of active PKB (15, 16), develop a lpr-like lymphoproliferative disorder marked by an accumulation of activated T and B cells and evidence of autoimmune disease. T cells from these various mutant mice display impaired sensitivity to apoptosis induced by Fas, suggesting that, like Fas signaling-deficient lpr or gld mouse strains, mice with hyperactive PKB activity develop lymphoid hyperplasia as a consequence of impaired Fas signaling. To date, the molecular mechanisms linking PKB signaling to Fas-driven aberrations in lymphoid homeostasis have remained unclear.
Understanding how PKB controls lymphocyte homeostasis depends on dissecting the mechanism by which PKB antagonizes Fas-mediated apoptosis in T cells. PKB has been shown to prevent apoptosis either through direct phosphorylation of proapoptotic molecules or by modulating the activity of transcription factors (11). One potentially relevant indirect target for PKB-mediated antiapoptotic signaling in T cells is NF-κB. Members of the NF-κB family of transcription factors play key roles in the immune system and help govern such diverse processes as lymphoid organ development, proliferation, and the regulation of apoptosis (17). In quiescent T cells, NF-κB is rendered inactive through sequestration in the cytoplasm by inhibitory IκB proteins. TCR and CD28 stimulation induces the rapid phosphorylation of IκB by the IκB kinase (IKK) complex, leading to ubiquitin-dependent degradation of IκB and subsequent nuclear translocation of NF-κB to the nucleus. PI3K/PKB signaling enhances TCR- and CD28-mediated NF-κB activation (18, 19). Given the controversial, yet potential, link that NF-κB activity may protect T cells from Fas-mediated apoptosis (20, 21, 22, 23, 24, 25), we hypothesized that PKB may antagonize Fas-mediated apoptosis, and ultimately promote lymphocyte homeostasis, through a NF-κB-dependent process.
Using transgenic and gene-deficient mice, we have examined the contribution of NF-κB signaling to the antiapoptotic effects of PKB. We have found that PKB-NF-κB signaling plays an essential role in the prevention of Fas-mediated death, but is largely dispensable for PKB-mediated protection from cytokine deprivation. Transgenic expression of a dominant negative form of IκB (IκBΔN) or mutation of nf-κb1/p50 renders PKB-transgenic T cells susceptible to Fas-mediated cell death, but not to death induced by cytokine withdrawal. Although PKB inhibits Fas-induced death and cleavage of procaspase-8, defective NF-κB signaling results in normal processing of procaspase-8. The physiological consequences of PKB-NF-κB signaling were also shown through studies examining the dynamics of lymphoid homeostasis. Aging PKB-transgenic mice normally develop lymphoid hyperplasia, but this process is blocked by inhibiting NF-κB. These results suggest that NF-κB controls distinct survival pathways downstream of PKB in T cells and couples PKB signaling to the regulation of lymphocyte homeostasis in vivo.
Materials and Methods
Human CD2 (hCD2)-gag-PKB transgenic (B6/PKB; backcrossed eight generations to C57BL/6J) (18), IκBΔN transgenic (26), NF-κB1-deficient (27), and c-Rel-deficient (28) mice have been previously described. C57BL/6J (B6) mice were obtained from The Jackson Laboratory. All mice were maintained in a specific pathogen-free environment at the Ontario Cancer Institute according to institutional guidelines.
Reagents and Abs
Recombinant hCD8-mouse FasL fusion protein was supplied by M. Bray (Advanced Medical Discovery Institute, Toronto, Ontario, Canada). Purified anti-CD3 (2C11) and anti-CD28 (37.51) were purchased from BD Pharmingen. The following mAbs were used for flow cytometry (FITC-, PE-, or biotin-conjugated): anti-CD4, anti-CD8, anti-CD3, anti-B220, anti-CD69, anti-CD25, anti-CD23, anti-CD44, anti-CD19, and anti-Fas (BD Pharmingen). Biotinylated Abs were detected with streptavidin-CyChrome (BD Pharmingen).
Cell preparation and flow cytometry
Single-cell suspensions of lymphoid organs were generated by gently pressing organs through sterile wire mesh. Cells were resuspended in IMDM; supplemented with 10% heat-inactivated FCS (Invitrogen Life Technologies), 50 μM 2-ME, 2 mM glutamine, and 0.1% penicillin/streptomycin and counted. For flow cytometric analysis, 0.2–1 × 106 cells were stained with combinations of mAbs at 4°C in PBS containing 2% FCS and 2% NaN3. All flow cytometric analyses were performed on a FACScan instrument (BD Biosciences). Samples were gated based upon forward and side scatter parameters (10,000 events/sample) and analyzed using CellQuest software (BD Biosciences).
Detection of nuclear NF-κB complexes
Nuclear extracts were harvested according to established protocols (29). In brief, 1–5 million purified T cells were left untreated or were stimulated with anti-CD3 and CD28 Abs or PMA and calcium ionophore for various time periods. Cells were washed twice with PBS and resuspended in 250 μl of buffer A (10 mM HEPES (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 1 mM PMSF). After incubation on ice for 5 min, Nonidet P-40 was added to a final concentration of 0.6%. After centrifugation, cytoplasmic proteins were removed, and the pelleted nuclei were resuspended in 25 μl of buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). After a 30-min agitation at 4°C, the samples were centrifuged, and supernatants containing nuclear proteins were collected. NF-κB binding complexes in nuclear lysates were quantified by ELISA (TransAM NF-κB ELISA kit) or by EMSA analysis using a NF-κB-specific oligonucleotide probe containing two tandemly positioned NF-κB binding sites (5′-ATC AGG GAC TTT CCG CTG GGG ACT TTC CG-3′ and 5′-CGG AAA GTC CCC AGC GGA AAG TCC CTG AT-3′).
Splenocytes from 10- to 12-wk-old mice were isolated and cultured with plate-bound anti-CD3 (2C11; 10 μg/ml) and anti-CD28 (37.51; 5 μg/ml; BD Pharmingen) for 24 h, followed by culture in medium containing murine rIL-2 (50 U/ml; PeproTech) for 4 days. Viable, activated lymphocytes were isolated using Lympholyte-M (Cedarlane Laboratories). T cell apoptosis was assessed as follows. For death by cytokine withdrawal, activated lymphocytes were washed three times in IMDM and cultured in the absence of exogenous cytokines. Fas-specific apoptosis was assessed by culturing activated lymphocytes in the presence of IL-2 (50 U/ml) and varying amounts of rhCD8-mouse FasL. Apoptosis was determined by flow cytometry using Annexin VFITC (BD Pharmingen) and 7-aminoactinomycin D (7AAD; Sigma-Aldrich).
Western blot analysis
Single-cell suspensions were lysed by incubation on ice in Gentle Soft Buffer (10 mM NaCl, 20 mM PIPES (pH 7.4), 0.5% Nonidet P-40, 5 mM EDTA, 5 μg/ml leupeptin, 1 mM benzamidine, 0.5 mM sodium fluoride, and 100 μM Na3VO4) for 20 min. Lysates were cleared by centrifugation, and supernatants were normalized for total protein (Bio-Rad). Proteins were resolved by 10–12% SDS-PAGE, electroblotted to a polyvinylidene difluoride membrane (Costar), blocked in 5% TBS/0.1% Tween 20, and probed with primary Abs. Anti-poly(ADP-ribose) polymerase (anti-PARP), anti-IκBα, and phospho-specific IκBα Abs were purchased from Cell Signaling Technology. Anti-glycogen synthase kinase (GSK) 3 was purchased from Upstate Biotechnology. Rabbit polyclonal anti-caspase-8 was a gift from Dr. R. Hakem (University of Toronto, Ontario, Canada). After incubation with HRP-conjugated goat anti-rabbit or goat anti-mouse (Santa Cruz Biotechnology), bound Igs were detected using ECL (Amersham Biosciences) according to the manufacturer’s directions.
Caspase activity assays
Fas-specific apoptosis was induced in activated T cells as described. Caspase activity was determined using capsase-3- or caspase-8-specific colorimetric protease assay kits according to the manufacturer’s instructions (Chemicon International). In brief, FasL-stimulated (10 μg/ml) or unstimulated T cells were lysed, and protein levels were normalized to 2 mg/ml. Samples were incubated in the presence of reaction buffer and colorimetric caspase substrate for 90 min at 37°C (capase-3 substrate, DEVD-pNA; caspase-8 substrate, IETD-pNA). OD values at 405 nm were determined for each sample and corrected for spontaneous release of pNA. The fold increase in caspase activity is expressed as the ratio of activity in FasL-treated samples relative to that in untreated controls. All time points were measured in triplicate, and results were expressed as the mean ± SD.
PKB requires de novo gene expression to block death receptor-induced apoptosis
Using a CD2-gag-PKB transgenic mouse model we have demonstrated a role for PKB signaling in the prevention of T cell apoptosis in culture, after radiation- and Fas-induced death (18, 30). Because PKB has been linked with IL-2R signaling (31, 32, 33), we also wanted to determine whether PKB played a role in survival after cytokine withdrawal. As shown in Fig. 1 A, activated T cells deprived of IL-2 rapidly undergo programmed cell death, and this process is antagonized by PKB activity. Therefore, two clear phenotypes observed with PKB transgenic T cells are the ability to maintain the viability of T cells upon the removal of growth-promoting cytokines or after the initiation of death receptor-induced apoptosis.
The mechanism by which active PKB prevents these different types of cell death remains unclear. Current models of PKB function suggest that antiapoptotic PKB signaling may act by direct phosphorylation of downstream effectors or by altering gene transcription via regulation of transcription factors (11). To investigate the requirement for de novo gene expression in PKB-mediated protection from cytokine deprivation or Fas-mediated apoptotic pathways, we examined the ability of PKB to prevent T cell death in the presence of the protein synthesis inhibitor cycloheximide. The addition of cycloheximide did not alter PKB-mediated survival in the absence of IL-2 (Fig. 1,B), suggesting that the growth factor-dependent survival mediated by PKB does not require the transcription of additional genes. These data are in contrast to the effect of cycloheximide on death receptor-induced apoptosis; cycloheximide renders PKB-transgenic T cells susceptible to Fas-mediated apoptosis and reduces their viability to levels similar to those in wild-type cells (Fig. 1 C). Treatment with cycloheximide did not affect PKB protein levels or the level of PKB phosphorylation in wild-type or PKB transgenic cells (data not shown), suggesting that cycloheximide does not affect PKB activation itself. However, it is possible that the overexpression of an active form of PKB constitutively alters other pathways in unstimulated cells. Nonetheless, these data suggest a bifurcation of PKB survival signaling in T cells; de novo transcription is dispensable for PKB-mediated protection from cytokine-dependent survival, but is absolutely essential for the prevention of death receptor-induced apoptosis.
PKB enhances and sustains NF-κB activity in T cells
The observation that de novo gene transcription is required to mediate the antiapoptotic effects of PKB against Fas led us to hypothesize that PKB activates a specific transcriptional program to counter death receptor signaling in lymphocytes. One of the key signaling events implicated in the prevention of T cell apoptosis is the activation of NF-κB (34). In our transgenic mouse model, NF-κB activity was highly induced in PKB transgenic T cells upon TCR and CD28 signaling at a single time point (18). To further examine the link between PKB and NF-κB, we used an ELISA-based assay to detect NF-κB DNA-binding complexes. In this assay, nuclear lysates are incubated with plate-bound NF-κB oligonucleotides, and NF-κB DNA-binding complexes are detected by sandwich ELISA using Abs against the RelA (p65) subunit of NF-κB. We found that levels of nuclear NF-κB DNA-binding complexes were elevated in PKB transgenic T cells compared with those in wild-type controls only after triggering of CD3 and CD28 (Fig. 2,A). Unstimulated PKB transgenic T cells did not show elevated levels of NF-κB activity. The specificity of this assay was confirmed using competing oligonucleotides containing canonical NF-κB binding sites. Interestingly, higher levels of NF-κB DNA-binding complexes were detected in the nucleus of PKB transgenic T cells relative to control T cells over time (Fig. 2 B). This finding demonstrates that PKB promotes both elevated and persistent binding of nuclear NF-κB complexes to DNA after T cell activation.
NF-κB regulates PKB-mediated survival to Fas-dependent, but not cytokine-dependent, pathways
It is possible that enhanced NF-κB activity may be important for blocking apoptosis in T cells. To address this question, we generated PKB-transgenic mice lacking the ability to activate NF-κB by breeding CD2-gag-PKB transgenic mice with mice expressing a constitutive repressor of NF-κB (denoted IκBΔN) (26). IκBΔN is a truncated a form of IκBα in which the N-terminal phosphorylation sites have been deleted, such that IκB cannot be phosphorylated and inactivated by the IKK complex (35). To determine whether the transgenic expression of IκBΔN could reduce the number of NF-κB DNA-binding complexes in the nucleus of PKB-transgenic T cells, T cells from nontransgenic (B6), PKB-transgenic (PKB), IκBΔN-transgenic (IκBΔN), or double-transgenic (PKB/IκBΔN) mice were activated with various mitogens, and nuclear NF-κB levels were measured. Translocation of NF-κB-binding complexes to the nucleus was significantly increased in PKB-transgenic T cells after mitogenic stimulation (Fig. 3,A). The expression of IκBΔN effectively reduced this response, because nuclear NF-κB-DNA complexes were absent in stimulated PKB/IκBΔN double-transgenic T cells (Fig. 3,A). IκBΔN expression also affected nuclear NF-κB activity in T cells before the induction of apoptosis (Fig. 3,B). Levels of nuclear NF-κB DNA-binding complexes remained elevated in PKB-transgenic T cells after the activation period (24-h treatment with anti-CD3 and anti-CD28 Abs, followed by 3 days in the presence of IL-2). In contrast, NF-κB activity in PKB-transgenic T cells expressing IκBΔN was not detectable (Fig. 3 B). These data suggest that PKB-mediated translocation of NF-κB complexes to the nucleus requires IκB degradation. Moreover, these results demonstrate that NF-κB activation downstream of the TCR and CD28 is ablated in PKB/IκBΔN double-transgenic T cells, giving us a model to examine the function of PKB in T cells in the absence of NF-κB activity.
To assess the ability of PKB-transgenic T cells deficient for NF-κB activity to initiate programmed cell death, we measured the resistance of PKB/IκBΔN double-transgenic T cells to apoptosis induced by cytokine withdrawal or FasL treatment. Twenty-four hours after the removal of IL-2, the majority of activated T cells from IκBΔN-transgenic animals displayed phenotypic signs of apoptosis (Fig. 3,C). This process was reversed through transgenic expression of PKB, indicating that in the absence of cytokines, PKB affects T cell viability through NF-κB-independent means. However, this paradigm did not apply to apoptosis triggered by the death receptor Fas. Consistent with previous reports, activated T cells from IκBΔN-transgenic mice were more susceptible to Fas-mediated apoptosis than wild-type or PKB-transgenic T cells (Fig. 3,D), indicating a protective role for NF-κB in Fas apoptosis in primary T cells (23, 26). However, T cells from PKB/IκBΔN double-transgenic mice were also sensitive to Fas-mediated apoptosis and died with kinetics similar to those of IκBΔN transgenic T cells (Fig. 3 D). This indicates that the protective effects of PKB against Fas lie upstream of IκB degradation and nuclear translocation of NF-κB. Collectively, these data support the model that NF-κB transcriptional activity is critical for PKB-mediated protection from Fas death signals.
To evaluate the role of NF-κB in PKB-mediated resistance to Fas-induced apoptosis in vivo, PKB-transgenic mice expressing the IκBΔN transgene were challenged with staphylococcal enterotoxin B (SEB), a superantigen that induces expansion and Fas/FasL-dependent deletion of Vβ8+ T cells (36, 37). Wild-type mice (B6), PKB-transgenic mice (PKB), or PKB/IκBΔN double-transgenic mice were injected with SEB, and the expansion and deletion of the Vβ8+CD4+ T cell population in these mice was tracked over time (Fig. 4). Deletion of Vβ8+CD4+ T cells was reduced in PKB-transgenic animals, as previously observed (18). Blocking nuclear NF-κB translocation in PKB/IκBΔN double-transgenic mice abrogated this protective effect. The percentage of Vβ8+CD4+ T cells in PKB-transgenic mice decreased ∼25% between day 3 postinfection (the peak of the response) and day 7, a decrease from 36.6 ± 2.4 to 27.8 ± 5.0%, respectively. In contrast, the number of Vβ8+CD4+ T cells decreased by almost 50% in PKB/IκBΔN mice over the same time period (34.6 ± 2.7 vs 18.4 ± 1.3%). These observations support our findings in vitro and suggest that NF-κB signaling is required for PKB to inhibit Fas/FasL-dependent T cell deletion in vivo.
PKB requires NF-κB1/p50 to inhibit Fas-mediated death
There are five members of the NF-κB family in mammals: Rel (c-Rel), RelA (p65), RelB, NF-κB1 (p50 and its precursor p105), and NF-κB2 (p52 and its precursor p100), which exist as homo- or heterodimers in vivo (38). Differential activity of NF-κB subunits can lead to different biological outcomes. Several lines of evidence indicate that the NF-κB1/p50 subunit is important for antiapoptotic function in T cells. TCR and CD28 stimulation results in strong binding of NF-κB1/p50 to NF-κB-specific DNA sequences in the nucleus (24). NF-κB1/p50, in partnership with either RelA/p65 or c-Rel, has been implicated in promoting T cell viability in response to death stimuli (23, 39). To assess the contribution of NF-κB1/p50 to the antiapoptotic effects of PKB in primary T cells, we examined the apoptotic threshold of activated PKB transgenic T cells harboring either hetero- or homozygous mutations of the nf-κb1 gene. Deletion of nf-κb1 had no effect on TCR-mediated phosphorylation and degradation of IκB regardless of the transgenic expression of gag-PKB (Fig. 5,A). However, loss of NF-κB1 significantly impaired TCR- and CD28-driven nuclear translocation of NF-κB complexes in PKB/nf-κb1−/− T cells (Fig. 5 B), indicating that NF-κB1/p50 is a critical component of NF-κB-dependent PKB signaling.
To examine the role of NF-κB in protecting T cells from apoptosis, T cells from nf-κb1+/−, PKB/nf-κb1+/−, nf-κb1−/−, and PKB/nf-κb1−/− mice were activated with Abs against CD3 and CD28 for 24 h, followed by culture with IL-2 for 3 days. The death of activated T cells was measured 2 days after removal of exogenous cytokines, and Fas-specific apoptosis of viable CD4+ T cells was assessed after treatment with soluble FasL (Fig. 5, C and D, respectively). As shown in Fig. 5,C, T cells lacking NF-κB1 displayed reduced viability upon the removal of cytokines. However, the viability of nf-κb1−/− T cells was rescued through the expression of active PKB, again indicating that PKB-mediated rescue from cytokine withdrawal is NF-κB independent. In contrast, Fas-specific apoptosis was strongly enhanced in nf-κb1−/− CD4+ T cells after exposure to FasL and could not be rescued by forced expression of PKB activity (Fig. 5,D). Because the nf-kb1−/− T cells were more susceptible to Fas-mediated death than nf-κb1+/− T cells, we examined whether c-Rel−/− T cells would also display a differential susceptibility. Fig. 5 E shows that the Fas-induced death of c-Rel−/− T cells was similar to that of T cells from C57BL/6 mice. Thus, as observed in our experiments with IκBΔN-transgenic mice, PKB-mediated NF-κB signaling regulates distinct survival pathways in T cells. Signaling mediated though NF-κB1/p50 is dispensable for PKB-mediated survival signaling in the absence of cytokines, but is absolutely required to antagonize Fas-mediated death in T cells.
PKB-NF-κB signaling regulates caspase activation by Fas
Transduction of the apoptotic signal downstream of Fas is mediated by a series of proapoptotic adapter molecules and caspases, including the adaptor molecule Fas-associated molecule with a death domain (FADD) and the initiator caspase, caspase-8 (3). FADD recruits procaspase-8 to the Fas death-inducing signaling complex (DISC), where it becomes activated by proteolysis and activates other death-inducing molecules, such as procaspase-3 and Bid. We have previously shown that the expression of gag-PKB prevents the activation of procaspase-8 by inhibiting its recruitment to DISC (30). In light of our data indicating that PKB requires NF-κB activation to mediate its protective effects against Fas-mediated apoptosis, we speculated that PKB-dependent activation of NF-κB acts to counter caspase-8 activation. To test this hypothesis, we examined the level of caspase activity in PKB-transgenic T cells with impaired activation of NF-κB. T cells unable to activate NF-κB (IκBΔN and PKB/IκBΔN) displayed enhanced caspase activity compared with wild-type controls or PKB-transgenic T cells after treatment with FasL (Fig. 6,A). Interestingly, PKB-transgenic T cells, which normally display a 2- to 3-fold reduction in caspase-8 activity compared with wild-type cells, are completely dependent upon NF-κB activation to mediate this effect. These data were supported by Western blot experiments analyzing the level of caspase-8 activation in nf-κb1−/− T cells. The processing of procaspase-8 was significantly enhanced in both nf-κb1−/− and PKB/nf-κb1−/− T cells compared with both nf-κb1+/− and PKB/nf-κb1+/− T cells (Fig. 6,B, top panel). The degradation of PARP, a caspase-3 target, was also enhanced in T cells lacking NF-κB1/p50 regardless of gag-PKB expression (Fig. 6,B, middle panel). Procaspase-8 processing was also elevated in both IκBΔN single-transgenic and PKB/IκBΔN double-transgenic T cells compared with controls (data not shown). Elevated PKB activity in PKB-transgenic T cells had little effect on the levels of FLIP or FADD upon T cell activation (Fig. 6,C) despite resistance of these cells to Fas-mediated apoptosis (Fig. 6 C). Collectively, these data suggest that PKB activity inhibits Fas-mediated caspase-8 activation and only does so in NF-κB-competent cells. Procaspase-8 cleavage and activity are enhanced with the loss of NF-κB1/p50 signaling or proper IκB degradation, indicating that the link between PKB and NF-κB is required to antagonize caspase-8 activation.
PKB-NF-κB signaling controls lymphocyte homeostasis
The PI3K/PTEN/PKB pathway has been implicated as a key regulator of lymphoid homeostasis and autoimmunity (12, 13, 14, 15, 16). However, because PKB modulates T lymphocyte viability through multiple pathways, we have been unable to determine whether PKB-mediated inhibition of the Fas death pathway directly contributes to lymphoid hyperplasia in PKB-transgenic mice. The data described in Figs. 3–5 suggest that PKB-mediated activation of NF-κB is selectively required to prevent Fas-mediated apoptosis, but not growth factor withdrawal. Because Fas-mediated apoptosis can be successfully triggered in NF-κB-null PKB-transgenic T cells, we hypothesized that NF-κB may be the signaling intermediary linking PKB to the disruption of lymphocyte homeostasis. To address this question, we analyzed 5- to 7-mo-old PKB-transgenic mice expressing the inhibitory IκBΔN transgene for the development of lymphoid hyperplasia. As expected, we observed a significant expansion of lymphocytes in the spleen and lymph nodes of PKB-transgenic mice compared with wild-type controls; lymphoid hyperplasia was markedly absent in PKB-transgenic mice expressing the IκBΔN repressor transgene despite the fact that these T cells are resistant to cytokine withdrawal-induced apoptosis (Fig. 7,A). One prominent feature of the lymphoid disorder in PKB-transgenic mice is the enlargement of Peyer’s patches in the intestine. However, the number of lymphoid cells in the Peyer’s patches of PKB-transgenic mice was clearly reduced upon expression of IκBΔN (Fig. 7,A). The reduction of lymphocyte numbers in PKB/IκBΔN double-transgenic mice was primarily attributed to changes in the T cell population; total numbers of splenic CD4+ and CD8+ T cells in PKB-transgenic animals were reduced by the expression of IκBΔN (Fig. 7,B). This reduction is striking because the CD4+ T cell population normally increases 5-fold over that in wild-type controls in 5-mo-old PKB-transgenic mice. B cell expansion was also impaired in PKB/IκBΔN mice (Fig. 7,C), although both IκBΔN and PKB/IκBΔN mice displayed elevated B cell numbers over wild-type controls. Another feature of PKB-driven lymphoid hyperplasia is the accumulation of activated lymphocytes. Blocking NF-κB signaling by transgenic expression of IκBΔN prevented this aspect of the lymphoproliferative phenotype; the proportion of activated T and B cells in secondary lymphoid tissues from PKB/IκBΔN mice was comparable to that in wild-type animals (Fig. 7 D and data not shown). Similar results were observed in PKB-transgenic mice lacking NF-κB1 (data not shown). Thus, abolishing NF-κB signaling prevents the accumulation of activated lymphocytes that is promoted through deregulated PKB activity in T cells. Our results suggest that functional NF-κB signaling is required for the development of lymphoid hyperplasia in PKB-transgenic mice, and that NF-κB is a critical regulator of lymphoid homeostasis downstream of PKB.
PKB/Akt has been shown to promote survival in lymphocytes in multiple studies (16, 18, 39, 40, 41, 42). Naive T cells expressing active PKB have a selective survival advantage despite having received no previous stimulation (16, 18, 43). Using T cells from p50−/−c-Rel−/− double-knockout mice, Zheng et al. (16, 41) have shown that PKB promotes spontaneous T cell survival (cultured in the absence of anti-CD3 or IL-2) in a NF-κB-independent manner. PKB may also promote cell survival through the regulation of other survival pathways, such as the regulation of cellular metabolism.
To understand the molecular pathways that promote lymphocyte survival, we have bred PKB transgenic mice with IκBΔN-transgenic mice or NF-κB1/p50 gene-deficient mice. We have demonstrated that PKB can protect from death induced by cytokine withdrawal by a NF-κB-independent pathway. Conversely, our studies have also shown that the NF-κB pathway is important for inducing factors that inhibit Fas-mediated death. In the absence of NF-κB, the active PKB transgene can no longer inhibit caspase 8 activation and Fas-mediated death. In addition, unlike PKB-transgenic mice, lymphadenopathy was not observed in PKB/IκBΔN double-transgenic mice. Therefore, our results indicate that PKB controls distinct survival pathways in T cells, and that the PI3K-PKB-NF-κB1 pathway is critical in inhibiting Fas-mediated death.
Other studies have examined the association involving IL-2, PKB, and T cell survival. Experiments have shown that IL-2 can promote survival by activating PKB (31, 32, 33). Studies have also shown that survival induced by IL-2 does not require both p50 and c-Rel (39). Together with our studies, this suggests that IL-2 survival is mediated through PKB in a NF-κB-independent manner. Collectively, these studies support a model in which many molecular pathways sustain T cell viability via PKB and/or NF-κB.
Other studies have examined the link between costimulatory molecules and the role of PKB in survival. Studies by Song et al. (42) have shown that OX40 costimulation leads to enhanced PKB activity, and the downstream effector GSK-3 is important for T cell survival. Our gag-PKB-transgenic model reveals that the consequence of enhanced PKB signaling downstream of TCR/CD28 is enhanced nuclear localization of NF-κB over extended periods of time. This suggests that overexpression of PKB in activated T cells may promote long-term expression of NF-κB target genes, including those responsible for preventing Fas-mediated apoptosis. However, the expression of active PKB is not sufficient to activate nuclear translocation of NF-κB on its own, but potentiates NF-κB activity after TCR and CD28 triggering. This suggests that PKB must act with other players to connect TCR/CD28 signaling with NF-κB activation. Genetic analysis has revealed a signaling pathway mediated by Bcl10/CARMA-1/MALT1 upstream of IKK activity that is critical for TCR-mediated NF-κB activation (44, 45, 46, 47, 48). PKB may act to enhance signaling by these molecules or may act through alternate players, such as Cot (49) or PKCθ (50, 51), to affect IKK activity. PKB has been shown to enhance NF-κB signaling through the transactivation of RelA/p65 (21, 52, 53, 54), and IKKα is required for PI3K- or PKB-dependent phosphorylation of the p65 transactivation domain (55). Recent work has also suggested that PKB may influence Fas-induced apoptosis through the expression of c-FLIP (56, 57), a caspase-8 homologue that inhibits death receptor-induced apoptosis by forming heterodimers with FADD or procaspase-8 (58). However, this possibility is unlikely, because levels of c-FLIP are normal in activated gag-PKB T cells (Fig. 6 C) (30). The same is true with other effector proteins shown to be involved in regulating DISC formation downstream of the TNFR, such as cellular inhibitor of apoptosis (IAP)-1, cellular IAP-2 and X-linked IAP.
Previous studies by several laboratories have shown that the PI3K-PTEN-PKB/Akt pathway plays an important role in maintaining lymphocyte homeostasis. Transgenic mice or gene-deficient mice that enhance this signaling pathway develop lymphoid hyperplasia, multiorgan inflammation, and autoantibody production (12, 13, 14, 16). An increase in active PKB in vivo correlates with the impaired Fas-mediated death (13, 15) due to an inability to recruit procaspase-8 to the DISC (30). Although controversial data exist (24), previous work has generally supported the idea that Fas-mediated death can be inhibited by NF-κB (21, 23). In addition, previous studies have shown that gld and lpr mice expressing IκBΔN transgenes that impair NF-κB activation do not develop lymphoid hyperplasia (25, 59). However, in these studies it was not clear whether lymphocytes did not accumulate because of an impaired ability to proliferate due to the absence of NF-κB, or whether NF-κB played a role in regulating Fas death. In addition, the mechanism that linked NF-κB with Fas death was unknown. Our study provides clear evidence that NF-κB1 is a key mediator downstream of the PI3K-PKB pathway that directs the transcription of a gene(s) that prevents Fas-mediated death. In addition, this study demonstrates a unique function for NF-κB1 vs c-Rel.
Genetic susceptibility to autoimmunity has also been associated with perturbation of the NF-κB pathway. T cells from lupus patients have been shown to resist anergy and apoptosis by up-regulating cylooxygenase-2 expression (60). Interestingly, another recent report has linked Forkhead box O-3a (Foxo3a), a transcription factor inhibited by PKB phosphorylation (61), to the repression of NF-κB activity in T cells. Foxo3a-deficient T cells are hyperactive and display enhanced NF-κB activity despite normal levels of IκBα, and mice lacking Foxo3a function develop profound splenomegaly and lymphadenopathy (62). Therefore, the threshold of NF-κB activity in T cells, controlled in part by the activity of PKB or Foxo3a, appears to control the balance between normal lymphoid homeostasis and lymphoproliferation.
Importantly, many studies have linked PKB with lymphocyte survival and autoimmune disease. In this study we show that PKB can inhibit Fas death via a NF-κB1-dependent mechanism. There are clearly multiple receptor-ligand interactions that trigger multiple pathways that sustain T cell viability. It will be important to understand these molecular connections to manipulate T cell responses, tolerance, and autoimmunity.
We thank Christine Mirtsos for technical assistance, Dr. Mark Bray for recombinant FasL, and Rosa Pileggi for administrative help.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Abbreviations used in this paper: FasL, Fas ligand; 7AAD, 7-aminoactinomycin D; ALPS, autoimmune lymphoproliferative syndrome; DED, death effector domain; DISC, death-inducing signaling complex; FADD, Fas-associated molecule with a death domain; Foxo3a, Forkhead box O-3a; h, human; IKK, IκB kinase; PARP, poly(ADP-ribose) polymerase; PKB, protein kinase B; PKC, protein kinase C; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SEB, staphylococcal enterotoxin B; IAP, inhibitor of apoptosis.