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The Journal of Immunology, 2006, 177: 5041-5050.
Copyright © 2006 by The American Association of Immunologists, Inc.

Evidence for a Role for Notch Signaling in the Cytokine-Dependent Survival of Activated T Cells1

Geetha Bheeshmachar2,*, Divya Purushotaman2,*, Hadassah Sade*, Vigneshkumar Gunasekharan{dagger}, Annapoorni Rangarajan{dagger} and Apurva Sarin3,*

* National Centre for Biological Sciences, Bangalore, Karnataka, India; and {dagger} Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, Karnataka, India


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peripheral T cell homeostasis results from a balance between factors promoting survival and those that trigger deletion of Ag-reactive cells. The cytokine IL-2 promotes T cell survival whereas reactive oxygen species (ROS) sensitize T cells to apoptosis. Two pathways of activated T cell apoptosis–one triggered by Fas ligand and the other by cytokine deprivation–depend on ROS, with the latter also regulated by members of the Bcl-2 family. Notch family proteins regulate several cell-fate decisions in metazoans. Ectopic expression of the Notch1 intracellular domain (NICD) in T cells inhibits Fas-induced apoptosis. The underlying mechanism is not known and the role, if any, of Notch in regulating apoptosis triggered by cytokine deprivation or neglect has not been examined. In this study, we use a Notch1/Fc chimera; a blocking Ab to Notch1 and chemical inhibitors of {gamma}-secretase to investigate the role of Notch signaling in activated T cells of murine origin. We show that perturbing Notch signaling in activated CD4+/CD8+ T cells maintained in IL-2 results in the accumulation of ROS, reduced Akt/protein kinase B activity, and expression of the antiapoptotic protein Bcl-xL, culminating in apoptosis. A broad-spectrum redox scavenger inhibits apoptosis but T cells expressing mutant Fas ligand are sensitive to apoptosis. Activated T cells isolated on the basis of Notch expression (Notch+) are enriched for Bcl-xL expression and demonstrate reduced susceptibility to apoptosis triggered by neglect or oxidative stress. Furthermore, enforced expression of NICD protects activated T cells from apoptosis triggered by cytokine deprivation. Taken together, these data implicate Notch1 signaling in the cytokine-dependent survival of activated T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Notch signaling plays a critical role in several cell-fate decisions during the development and life span of multicellular organisms (1, 2, 3). The Notch pathway is mediated by isoforms of the Notch receptor and ligands, which include Jagged and Delta in vertebrates. Signaling is initiated by ligands that liberate the Notch1 intracellular (NIC)4 domain (NICD) from the membrane, which then translocates to the nucleus (1). In the nucleus, NICD binds the C-promoter-binding factor-1/recombinant signal-binding protein-J-{kappa} complex converting it from a repressor to an activator of transcription (4, 5, 6). We have recently described an antiapoptotic role for the Notch pathway that depends on the tyrosine kinase p56lck and the serine-threonine kinase Akt/protein kinase B (PKB) in T cell lines (7). Both p56lck and Akt/PKB-dependent signaling have been shown to regulate the persistence/survival of activated T cells (8, 9).

A complex network of signaling cascades and molecules is required for the regulated survival of T cells at all developmental stages (10, 11). T cells activated by cognate Ag undergo several rounds of cell division and differentiate into functional subsets that perform specialized functions. Following clearance of Ag from the organism, the bulk of Ag-specific activated T cells is deleted. Two key pathways regulate activated T cell apoptosis. The stimulation of T cells in response to self-Ags is negatively regulated via a death pathway activated by receptors of the TNFR family. This is referred to as activation-induced cell death (AICD) and in CD4+ T cells is brought about by Fas-mediated activation of the caspase family of proteases (12). Unlike AICD, which removes autoreactive cells, the deletion of T cells activated in response to infection is believed to be triggered by a reduction of sustaining cytokines from the microenvironment. This process is referred to as activated T cell neglect-induced death (A-NID) and reactive oxygen species (ROS) are the principal mediators of this death pathway (13, 14). Members of the Bcl-2 family proteins, which comprise both pro- and antiapoptotic proteins, regulate this event (15, 16).

The cytokine IL-2 functions not only as a growth factor, promoting T cell proliferation and survival, but also primes T cells activated by self-Ags for AICD (17). Differential signaling of receptor components as well as the presence of concomitant stimuli regulate these opposing outcomes. Ectopic expression of NICD blocks AICD in T cell lines (7, 18) presumably by blocking signals downstream of FasR ligation (7). Although persistent signaling via Notch inhibits reactivation responses of T cells (19), in the early stages of T cell activation, Notch promotes IL-2 responsiveness and proliferation by regulating cytokine receptor expression (20, 21). Because IL-2 can drive both the survival and death of Ag-stimulated T cells, here we ask if Notch biases IL-2 signaling toward either one of these outcomes in activated T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Isolation of T cell subsets and T cell activation

C57BL/6 and C57BL/6gld/gld were maintained at the Small Animal Facility at National Centre for Biological Sciences (NCBS). All experiments involving animals were performed with the prior approval of the Institutional Animal Ethics Committees at NCBS. CD3+, CD4+, or CD8+ T cells were isolated from mouse spleens using MagCellect kits (R&D Systems). Activated T cell blasts (TCB) were generated from naive T cells isolated from spleens of 6- to 8-wk-old C57BL/6 mice. T cells were stimulated with beads coated with Abs to CD3-CD28 (Dynal) and in some experiments with plate-bound anti-CD3 (1 µg/ml, clone 2C11; R&D Systems) and soluble anti-CD28 (5 µg/ml; BD Biosciences). After 48 h, TCB were washed and used in assays or continued in culture with the cytokine IL-2 (R&D Systems). For peptide-stimulated T cells, splenocytes from 8-wk-old p14 TCR-transgenic mice were cultured with 200 ng/ml peptide for 12 h. Cells were washed to remove unbound peptide and cultured in Click’s medium containing 0.5 IU/ml IL-2.

Chemicals and reagents

Hoechst 33342, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP), the {gamma}-secretase inhibitors L-685,458 (GSI-X), WPE-III-31C (GSI-XVII), and DAPT (GSI-IX) were obtained from Calbiochem (EMD Biosciences). Dihydroethidium (DHE) was obtained from Molecular Probes (Invitrogen Life Technologies) and the recombinant rat Notch1/Fc chimera and recombinant mouse Fas/Fc chimera were obtained from R&D Systems.

Analysis of protein expression by flow cytometry

A total of 0.5 x 106 cells were fixed with 1% paraformaldehyde for 30 min, washed twice in permeabilization buffer (PBS with 0.3% saponin, 1% FCS, and 0.1% sodium azide), and stained with the appropriate isotype control or Ab of interest. Abs were diluted in permeabilization buffer and all incubations were of 40-min duration on ice. Cells were resuspended in saponin-free buffer for analysis by flow cytometry (CellQuest software; BD Biosciences). For the analysis of cell surface expression of Notch1, Ab (clone A6) was freshly conjugated to Zenon Alexa Fluor 488 and nonpermeabilized cells stained as per the manufacturer’s instructions (Invitrogen Life Technologies-Molecular Probes). Notch expression was analyzed by flow cytometry.

Isolation of Notch+ T cells

A total of 1–2 x 107 T cells stimulated for 24 h with anti-CD3/CD28 were washed and resuspended in 1 ml of MagCellect Binding Buffer (R&D Systems) containing 6 µg of anti-Notch1 Ab (clone A6). Cells were incubated for 20 min at 4°C, washed to remove unbound Ab and resuspended in 1 ml of MagCellect buffer containing an appropriate dilution of goat-anti-mouse Ig-Ferrifluid (R&D Systems) and incubated at 4°C according to the manufacturer’s instructions. Unbound cells isolated after three to four rounds of selection are the Notch-low (NotchL) subset and cells bound to beads after two rounds of selection used as the Notch+ subset. All subsets were normally divided into two groups for analysis; these include: cells continued in culture for functional assays and a second group used to generate lysates for Western blot analysis.

Manipulations of Notch processing in activated T cells

Stock solutions of GSI-X, GSI-XVII, and GSI-IX in DMSO were obtained commercially (Calbiochem). Activated T cells were cultured in cytokine in the presence of the GSI or the equivalent volume of DMSO. Clone A6 was added in soluble form and purified mouse Ig was used as the control. In experiments with the Notch/Fc, the reagent was coated on wells following the manufacturer’s instructions. The Fas/Fc-chimera from the same company was used as the control for Notch/Fc.

Retroviral infections

Retroviruses pseudotyped with vesicular stomatitis virus G envelope were produced by transient transfection of 293T cells with the pMIG retroviral vector carrying the gene of interest, and packaging plasmids encoding pCMV-VSV-G and pUMVC3-gag-pol (Aldevron) using Fugene 6 (Roche Molecular Biochemicals). Media was changed at 24 h and viral supernatant was collected 48 h posttransfection. pMIG-NIC-GFP comprises aa 1759–2556 of the human NICD subcloned into the pMIG vector backbone and has been used in an earlier study (7).

For retroviral infections, spleen cells were cultured at 2 x 106/ml with 1 µg/ml anti-CD3 (clone 2C11; R&D Systems). Eighteen to 24 h later, cells were spin-infected with pMIG-GFP or pMIG-NIC-GFP retrovirus suspension supplemented with 4 µg/ml protamine sulfate (Sigma-Aldrich) and HEPES (Invitrogen Life Technologies) for 90 min at 2500 rpm at 30°C. Following this step, 70% of the retroviral supernatant was replaced with fresh medium supplemented with 0.5 µg/ml CD3. One day later, the culture supernatant was replaced with medium supplemented with IL-2 and the cells either continued in cytokine or were used for experiments after a 24-h exposure to IL-2.

Apoptosis assays

Apoptotic nuclear morphology was assessed using Hoechst 33342. Culturing TCB in the absence of any exogenous cytokine for 18–24 h triggers A-NID. In experiments using hydrogen peroxide, day 2 or 3 TCB were cultured in IL-2-containing medium with 25–100 µM hydrogen peroxide for 18 h. Susceptibility to dexamethasone was tested in TCB generated by stimulating T cells for 24 or 48 h with CD3/CD28. In cells expressing GFP, preparations were counterstained with Hoechst 33342 as described and only GFP-positive cells were scored for nuclear morphology.

Abs, immunoprecipitations, and Western blot analysis

Abs were from the following sources: Akt, NICD (M20 or C-20), Hes-1 (sc-25392), Bcl-xL and p38MAPK (Santa Cruz Biotechnology); Akt-phospho serine 473 and phosphorylated substrates of Akt (Cell Signaling Technology); actin, Bax (clone 2C8), Notch-1 (clone A6), and tubulin (NeoMarkers); Jagged1, p56Lck, BIM, PE-conjugated Notch1-ICD (clone mNIA), and FITC-conjugated Bcl-2 as well as appropriate isotype controls for the latter two reagents (BD Biosciences); Notch1 cytoplasmic domain (rabbit polyclonal) and PI3K (Upstate Biotechnology); Notch-1 C-terminal domain clone C17.9C6 (DSHB); CD25, CD127, CD69, and isotype controls all conjugated to PE and CD28 (eBiosciences); anti-Hes-1 (Chemicon International).

For immunoprecipitations, 5–8 x 106 cells were lysed using buffer from Pierce Biotechnology supplemented with protease and phosphatase inhibitors. Cell lysates were incubated with relevant Abs (1 µg of Ab/106 cells) for 1 h. Immune complexes were precipitated for 2 h at 4°C using Sepharose-G plus beads (Pierce Biotechnology). The beads bound to immunoprecipitate were washed, before analysis of the immunoprecipitated proteins. In the Western blot analysis, Abs from Upstate Biotechnology and BD Biosciences were used at concentrations recommended by the manufacturers; clones A6 (NeoMarkers), C-20 and M20 (Santa Cruz Biotechnology) were used at 2 µg/ml, cleaved Notch1 (Cell Signaling Technology) at 1/300, and membranes were incubated for 12–18 h with the Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Notch expression is regulated by TCR-dependent signals

Activation of naive T cells triggers the induction and processing of Notch1 as indicated by the appearance of proteins recognized by Abs to the Notch C terminus (NCT)/NICD in Western blot analysis. These include reactivity to M-20, C-20, C17.9C6 (Fig. 1A), as well as to an Ab recognizing a specific cleavage product of Notch1 (NCT-Val 1744) (Fig. 1A). Although naive T cells demonstrate poor reactivity to the aforementioned Abs, they express full-length Notch1 (Fig. 1B). A comparable induction of the NICD also occurs in CD3+ T cells activated in vivo following i.v. or i.p. challenge with allogenic spleen cells (Fig. 1C). As reported by other groups, the Notch1 target protein Hes1 is induced when naive T cells are stimulated to generate activated TCB (Fig. 1D). No changes are apparent in the expression of the ligand Jagged1, which is detected in both naive and activated T cells (Fig. 1E).


Figure 1
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  		FIGURE 1. T cell activation triggers Notch1 expression. A, Detection of NICD by Abs to the NICD in freshly isolated naive (N) T cells (lane 1) or the same preparation activated with plate-bound Ab to CD3 and CD28 for 48 h (lane 2) to generate TCB. The arrow in A indicates the correct band. B, Expression of the 230-kDa full-length receptor in naive T cells and TCB C, Levels of NICD in CD3+ T cells isolated from mice injected i.v. or i.p. with allogenic spleen cells 48 h before harvest and analysis. CD3+ T cells from saline injected mice are similar to naive (N) T cells in patterns of Notch1 expression and data from a saline injected (i.v.) group is presented. D, Levels of Hes-1 protein in naive and TCB detected using rabbit polyclonal Abs from two sources, Chemicon International (upper panel) and Santa Cruz Biotechnology (lower panel). The arrow indicates the specific band. E, Expression of the ligand Jagged1 in naive and activated T cells. LC indicates the loading control.

 
Interfering with Notch in activated T cells triggers ROS-dependent apoptosis

Cytokines such as IL-2 promote the survival of activated T cells. To assess Notch function in T cell survival, reagents that block Notch processing were assessed for the effect on activated T cells. Notch processing is initiated by ligand binding to the Notch ectodomain, which triggers two successive cleavages that release the intracellular domain from its association with the ectodomain (22). The first cleavage is mediated by an extracellular metalloprotease, which in turn facilitates the cleavage and release of the intracellular domain by {gamma}-secretases. Two {gamma}-secretase inhibitors, GSI-X (L685,458) or GSI-XVII (WPE-III-31C), and a recombinant rat Notch1-Fc chimera and an Ab to the extracellular domain (clone A6) (19, 23) that would intercept ligand-receptor interactions, were tested. Differences in efficacy notwithstanding, the GSIs and rNotch/Fc triggered an increase in cellular ROS (measured by increased fluorescence of the oxidized form of the dye DHE) as compared with cells treated with IL-2 (Fig. 2A). Consistent with the increase in DHE oxidation, all reagents also compromised the viability of activated CD4+ or CD8+ T cells (Fig. 2, B––D). In both subsets, treatment with the GSI or the Ab resulted in a loss of processed Notch1 (Fig. 2E), which preceded the induction of apoptotic damage. This was accompanied by a minimal change if any in expression of total Notch1 (Fig. 2E). We also report a concomitant loss of expression of the Notch target Hes1 in activated T cells following treatment with the GSI (Fig. 2F). We tested whether blocking ROS induction ameliorates apoptosis triggered by the disruption of the Notch pathway. MnTBAP, a broad-spectrum antioxidant with catalase and superoxide dismutase activity which blocks mature T cell apoptosis (13, 14, 24), inhibited GSI-X induced apoptosis or A-NID (Fig. 2G). The loss of processed Notch1 in cells in the condition of A-NID was prevented in cells treated with MnTBAP (Fig. 2H). There was only a small increase noted in cellular levels of Notch protein (Fig. 2H). Similarly, Hes1 is not completely lost in cells treated with MnTBAP (Fig. 2H). Thus, the molecular features of apoptotic damage triggered by reagents that disrupt Notch signaling in activated T cells phenocopies those of T cells undergoing A-NID.


Figure 2
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  		FIGURE 2. ROS accumulation and the apoptotic response in activated T cells following disruption of Notch processing. A, Activated T cells/TCB were cultured in IL-2 as such (IL-2) or with the addition of GSI-X (15 µM) or GSI-XVII (10 µM) or recombinant rat Notch/Fc chimera (5 µg). After 6 h, cells were stained with DHE as described in Materials and Methods and analyzed by flow cytometry. B and C, CD3+, CD4+, and CD8+ TCB were cultured without (Figure 2) or with 10 µM GSI-XVII ({blacksquare}) or 15 µM of GSI-X ({blacksquare}) and apoptotic nuclear damage assessed after 12 h. {square} in C is a 10 µM concentration of GSI-X. D, Apoptotic nuclear damage assessed 18 h after activated CD4+ or CD8+ T cells were cultured in IL-2 in the presence of either plate-bound 5 µg/ml of the Notch/Fc chimera or 10 µg/ml of an Ab to the Notch1 extracellular domain (clone A6). TCB were also cultured with an isotype control Ab to clone A6 (Figure 2 in the A6 group) and on wells coated with Fas/Fc (Figure 2 in the Fc chimera group) at the same concentration. E, NICD levels as assessed by Western blot analysis of cell lysates of activated T cells of CD4 or CD8 origin cultured with IL-2 in the presence or absence of GSI-X (15 µM) or GSI-XVII (10 µM) for 8 h. The arrow indicates the specific band detected by clone M20. The flow cytometry histograms show the expression of NICD using clone mNIA (filled histogram) and the isotype control (open histogram) in activated CD4+ T cells cultured with IL-2 in the presence of an MsIg isotype control or A6 for 8 h. Shown below is the Western blot analysis of NICD expression in CD3+ T cells cultured with MsIg or A6. F, Cellular levels of Hes1 in CD3+ TCB treated with GSI-X (15 µM) or GSI-XVII (10 µM). G, Apoptotic nuclear damage (18–24 h) triggered by GSI-X in TCB in the absence or presence of indicated concentrations of MnTBAP (circles). Also shown is the inhibition of A-NID (squares) by MnTBAP in the same experiment. H, TCB cultured in the absence of IL-2 but in the presence or absence of 250 µM MnTBAP for 12 h were analyzed by Western blot analysis for the indicated proteins. Statistical significance (*, p < 0.005 and **, p < 0.05) of the differences between control and treated groups was assessed by the Student t test.

 
Apoptosis triggered by inhibiting Notch activity is not Fas dependent

ROS are key intermediates in both AICD and A-NID, performing distinct functions in both death pathways. Because ROS are also required for the induction of Fas ligand in AICD (25), we tested whether apoptosis triggered in cells treated with the GSIs culminates in Fas-mediated apoptosis. Two approaches were taken to address this question. First, a murine Fas-Fc chimera that blocks Fas ligand-Fas mediated apoptosis in TCB (data not shown) was tested and was observed to have a marginal effect on GSI-X-induced apoptotic damage (Fig. 3A). Another inhibitor GSI-IX was also tested in these experiments and gave similar results (Fig. 3A). Second, activated T cells were generated from gld/gld mice, which have a point mutation in Fas ligand and the effect of the GSIs tested on activated T cells derived from these mice. Both GSI-X and GSI-XVII triggered an accumulation of ROS as measured by DHE fluorescence (Fig. 3B), and a substantial loss in T cell viability (Fig. 3C). These effects were comparable to those observed in wild-type mice (Fig. 2). These data indicate that Fas receptor signaling plays a minimal role, if at all, in the apoptotic pathway triggered by the disruption of Notch in activated T cells.


Figure 3
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  		FIGURE 3. Apoptosis triggered by inhibiting Notch activity is Fas independent. A, Activated T cells were cultured with IL-2 ({square}) or IL-2+GSI-X (15 µM) or IL-2+GSI-IX (10 µM) in the presence (Figure 3) or absence ({blacksquare}) of 10 µg of soluble murine Fas/Fc chimera. Apoptotic nuclear damage was assessed after 18–24 h. Inset, Levels of Hes1 in cells treated with GSI-IX for 12 h. B, Activated Tgld/gld cells were cultured in IL-2 alone or with IL-2 and GSI-X (10 µM) or GSI-XVII (10 µM). After 5 h, cells were stained with DHE as described in Materials and Methods and analyzed by flow cytometry. C, Activated Tgld/gld cells were cultured in IL-2 alone or with indicated concentrations of GSI-X (squares) or GSI-XVII (circle). Apoptotic nuclear damage was assessed after 18 h.

 
Disrupting Notch processing reduces cellular levels of Bcl-xL

In an earlier study, we have shown that ectopically expressed NICD increases cellular levels of Bcl-xL in T cell lines (7). Induction of Bcl-xL also results from signals transmitted via IL-2 in T cells. We tested whether inhibiting Notch processing disrupts Bcl-xL expression in activated CD4+ and CD8+ T cells. Treatment with GSI-X or GSI-XVII resulted in a loss of Bcl-xL protein (Fig. 4A). Levels of the antiapoptotic protein Bcl-2 are also reduced but to a smaller extent (Fig. 4A). Similarly, a loss of Bcl-xL was also observed in cells treated with clone A6 (Fig. 4B).


Figure 4
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  		FIGURE 4. Disrupting Notch modifies cellular levels of anti apoptotic proteins and Akt/PKB activity. A, Western blot analysis of cell lysates derived from CD4+ or CD8+ TCB cultured for 12 h with IL-2 or with IL-2 and 15 µM GSI-X or IL-2 and 10 µM GSI-XVII for the expression of Bcl-xL and Bcl-2. B, TCB were cultured with IL-2, IL-2 + mouse Ig (MsIg), or IL-2 + clone A6 for 12 h and the expression of Bcl-xL assessed as in A. C, Whole cell lysates (WCL) made from T cells 2 days after activation with CD3-CD28 (TCB) were immunoprecipitated with an Ab to CD28, the immunoprecipitate resolved on SDS gels and assessed for Notch1 by Western blot analysis, using an Ab to the NCT. Membranes were reprobed for p56lck and PI3K in the immunoprecipitate. D, As in C except that the lysates were immunoprecipitated using clone C-20 (NICD) and assessed for the expression of CD28, p56lck, actin (specificity control) and in separate assays, C17.9C6 by Western blot analysis. E, Activated CD3+ T cells or CD8+ T cells were cultured with IL-2 in the presence or absence of GSI-X (15 µM) and assessed for phosphorylation of Akt Ser473 by Western blot analysis. F, Activated CD3+ T cells cultured as in E, were assessed for phosphorylated substrates (RXRXXS/T) of Akt/PKB. The data are representative of two to three separate experiments.

 
Notch triggers a PI3K and Akt/PKB-dependent survival pathway in T cell lines (7) and in immature T cells (26). IL-2 activates Akt/PKB, which regulates the expression and function of several proteins implicated in T cell survival (27). We have shown that Notch1 is present in a complex with PI3K and the Src-family nonreceptor tyrosine kinase p56lck in activated T cells and T cell lines (7). We now show that the NICD-PI3K-p56lck complex in activated T cells is immunoprecipitated by the T cell costimulatory molecule CD28, which is reported to contribute to the activation of PI3K in T cells (28) (Fig. 4C). Further, CD28 is present in a complex with C-20 (Fig. 4D). Notch1 immunoprecipitated by clone C-20 is recognized by C17.9C6 (Fig. 4D). Activation of Akt/PKB requires phosphorylation of serine 473 and GSI-X-treated CD3+ and CD8+ cells present a significant reduction in the phosphorylation of the serine 473 residue in Akt/PKB indicating reduced kinase activity (Fig. 4E). This was also confirmed by a loss of phosphorylated substrates of Akt/PKB in GSI-X treated cells, compared with cells cultured in IL-2 alone (Fig. 4F).

IL-2 and IL-7 receptor expression following disruption of Notch

The induction of apoptosis and modulations described in the preceding sections were observed in the continued presence of IL-2, prompting an assessment of the IL-2R {alpha}-chain (CD25) expression following interference with Notch signaling. Perhaps owing to differences in the activation state of the T cells and the continued presence of exogenous cytokine, the reduction of CD25 expression observed in activated T cells is not as complete as reported in T cells during primary encounter with Ag (20). However, surface expression of CD25 was consistently reduced in activated T cells cultured in IL-2 and concomitantly treated with either GSI-X or GSI-XVII (Fig. 5, A and B).


Figure 5
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  		FIGURE 5. Notch regulation of cytokine receptor expression in activated T cells. A, CD25 expression (filled histogram) on activated T cells cultured in IL-2 or in the presence of GSI-X (15 µM) or GSI-XVII (10 µM) for 6 h. The open histogram represents the isotype control. The data are representative of three separate experiments. B, MFI of CD25 expression from three separate experiments as described in A. The MFI for CD25 in each case has been corrected for the MFI of the isotype control. C, Apoptotic nuclear damage in activated T cells cultured for 18 h with IL-7 (10 ng/ml) in the presence of GSI-X (15 µM) or GSI-XVII (10 µM). D, CD127 expression (filled histogram) on activated T cells cultured in IL-7 or in the presence of GSI-X or GSI-XVII at the same concentrations as in C for 6 h. The open histogram represents the isotype control. The data are representative of two separate experiments. E, MFI of CD127 expression from two separate experiments as described in A.

 
Although small, the changes in CD25 expression could still account for the observed attenuation of IL-2-mediated survival by the GSI. Therefore, we tested the effect of the GSI in the context of activated T cell survival mediated by the cytokine IL-7. IL-2 and IL-7 share a common {gamma} ({gamma}c) chain in their receptor complexes (29) and A-NID is also inhibited by IL-7. The GSIs attenuate the IL-7-mediated survival of activated T cells (Fig. 5C), but cells continue to express substantial amounts of CD127, the {alpha}-chain of the IL-7R (Fig. 5, D and E). Thus, although GSI down-modulate CD25 expression even after relatively short exposures, the possibility that Notch signaling converges on other pathways downstream of receptor signaling is likely in these conditions.

Notch expression is modulated following TCR signaling

The experiments thus far indicate that Notch signaling impinges on several aspects of IL-2-mediated signaling and survival in peripheral T cells. However, IL-2 does not up-regulate Notch1 expression in the absence of concomitant signaling via the TCR. Thus, the levels of NICD were comparable in freshly isolated CD3+ T cells (Fig. 6A, day 0) and cells cultured for 24 (data not shown) and 48 h in IL-2 (Fig. 6A). NICD is indeed up-regulated if T cells of either the CD4+ or CD8+ subset are activated using CD3 and CD28 (Fig. 6. B and C). Cell surface Notch1 is undetectable on naive T cells (data not shown) but the expression is increased following stimulation with CD3-CD28 and is comparable in CD4+ and CD8+ T cells across multiple experiments (Fig. 6D). The expression of Notch on T cells following their activation has not been characterized and since activated T cells are destined to undergo apoptosis, we hypothesized that the pathway will not be sustained in activated T cells for extended durations. In agreement with this, flow cytometric analysis of cell surface Notch on activated T cells revealed heterogeneity emerging in the pattern of Notch expression following the discontinuation of Ag receptor stimulation, with only a subset of activated cells expressing higher levels of the receptor following culture in IL-2 (Fig. 6D).


Figure 6
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  		FIGURE 6. Distribution of Notch in activated T cells in culture. A, Flow cytometric analysis of freshly isolated T cells (left panel) and cells cultured for 48 h with 20 U/ml IL-2 (right panel), permeabilized, and stained with a PE-conjugated Ab (clone mNIA) to the NICD (black histogram). B and C, NICD expression (black histogram) in CD3+ T cells (B) or CD4+ or CD8+ T cells (C) cultured for 48 h with CD3-CD28. In A and B, gray histograms and in C the open histograms represent staining with an isotype-matched Ab. D, Cell surface expression of Notch-1 (clone A6) in CD4+ or CD8+ T cells stimulated with CD3-CD28 for 48 h. The filled histogram represents staining with A6. Data shown are representative of two to three experiments. E, Cell surface expression of Notch-1 (clone A6) in naive T cells stimulated with CD3-CD28 for 48 h, followed by culture with IL-2 for an additional 2 days. Upper panel, staining with an isotype-matched control. NECD indicates Notch extracellular domain. F, Splenocytes from p14-TCR-transgenic mice were stimulated (200 ng/ml peptide) in vitro for 12 h and continued in culture without peptide. Cells were stained for Thy 1.2 and gated cells analyzed for cell surface Notch1 by flow cytometry. Panels on the left are isotype controls. The results in all panels represent three to four independent experiments.

 
To confirm the modulation of Notch expression observed in polyclonally stimulated T cells, we extended the analysis to peptide-stimulated CD8+ transgenic T cells from P14-transgenic mice, which recognize the lymphocytic choriomeningitis virus Db-gp33 epitope. Closely paralleling the pattern of polyclonally stimulated T cells (data not shown), cell surface Notch1 is induced in CD8+ T cells stimulated by peptide using established protocols (14). Following stimulation with antigenic peptide, there is gradual increase in the number of cells positive for cell surface Notch1 (Fig. 6E). Subsequent to this increase, Notch+ expression is down-regulated by 72 h and is detected only on a comparatively small subset (Fig. 6E). In summary, Notch1 expression is modulated following Ag receptor signaling and is not retained on the surface of all activated T cells.

Notch+ cells are resistant to apoptotic stressors

Receptivity to Notch signaling is determined by the expression of the receptor on the cell surface. Because T cells express Notch ligands, the Notch+ subset offers a system to study Notch signaling in a more physiological context wherein conventional activation of Notch is possible. Therefore, we isolated and analyzed the Notch+ subset for survival capabilities. Using an Ab to extracellular Notch (clone A6), T cells expressing cell surface Notch (Notch+) were isolated 24–36 h poststimulation. The expression of cell surface Notch1 in Notch-low (NotchL) cells is clearly lower than unfractionated (UF) or Notch+ cells (Fig. 7A). However, both subsets express comparable levels of activation Ags such as CD25, CD69, and ScaI (data not shown). In comparison to NotchL cells, the Notch+ subset demonstrates improved survival in an assay of A-NID (Fig. 7B), and apoptosis induced by oxidative stress i.e., exogenously added hydrogen peroxide (Fig. 7C). Apoptosis triggered by a range of concentrations of the synthetic corticosteroid dexamethasone is also substantially lower in the Notch+ subset (Fig. 7D). Notch+ cells are not generally resistant to cell death, as susceptibility to apoptosis triggered by the genotoxic drug etoposide or staurosporine is unchanged (data not shown). To assess reactivation responses of cells isolated by this protocol, the following analysis was undertaken. Following a 7-day rest phase in low dose IL-2, restimulation via the TCR resulted in an induction (relative to the unstimulated cells) of CD25 and CD69 in the Notch+ and the UF/NotchL subsets as compared with cells cultured in IL-2 alone (Fig. 7E). Furthermore, consistent with their improved survival and expression of Notch1, NICD is detected in Notch+ cells even when cultured for 48 h in the absence of cytokine (Fig. 7F). Expectedly, NotchL cells present a dramatic loss of this species when cultured in the absence of IL-2 (Fig. 7F). Analysis of Bcl-2 family proteins showed that expression of Bcl-2 is not substantially different between subsets (Fig. 7G). However, Notch+ cells express substantially high levels of Bcl-xL (Fig. 7H). Unexpectedly, the expression of the proapoptotic protein BIM is reduced in this subset. The expression of Bax is comparable in both groups (Fig. 7H). Similarly, Notch+-activated subsets of p14 T cells are also enriched for the expression of Bcl-xL (Fig. 7I).


Figure 7
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  		FIGURE 7. Characterization of Notch+ T cells. A, UF, NotchL, and Notch+ activated T cells were stained (filled histogram) for cell surface expression of Notch-1 (clone A6). B, Apoptotic damage in NotchL and Notch+ activated T cells cultured for 18 h in the presence (Figure 7) or absence ({blacksquare}) of IL-2. C, Apoptotic damage in NotchL and Notch+ activated T cells cultured for 18 h with 50 µM H2O2 ({blacksquare}). D, Apoptotic damage in NotchL and Notch+ activated T cells cultured for 10 h with dexamethasone. E, NotchL and Notch+ cells were cultured for 5 days in low levels of IL-2. Cells were continued as such or restimulated for 8 h with beads coated with Abs to CD3-CD28 and stained for CD25 and CD69. The histograms (open: isotype control; filled: CD69; open gray: CD25) represent expression profiles of stimulated cells. The MFI for cells cultured in IL-2 alone or IL-2 and CD3-CD28 are shown in the graph. A representative experiment of two separate analysis is shown. F, Cell lysates of NotchL or Notch+ cells cultured for 48 h without IL-2 and levels of NICD (C17.9C6) assessed by Western blot analysis. G, NotchL and Notch+ T cells were permeabilized and stained for Bcl-2 expression by flow cytometry. The open histogram indicates the isotype control. H, Cell lysates derived from NotchL and Notch+ activated T cells were analyzed for the levels of indicated proteins by Western blot analysis. I, Cell lysates of NotchL and Notch+ cells isolated 24 h after peptide stimulation of p14-transgenic T cells analyzed for the expression of Bcl-2 and Bcl-xL. The Student t test was used as the test of significance (*, p < 0.005).

 
Overexpression of activated Notch1 in T cells

The experiments thus far are consistent with a protective role for Notch in A-NID in T cells. To test this more directly and analyze the specificity of the reagents used, we constitutively expressed activated human NIC using a retroviral expression system in activated T cells. Activated T cells infected with pMIG-NIC-IRES-GFP or pMIG-IRES-GFP were tested for susceptibility to apoptosis induced by the GSI. Apoptotic damage triggered by GSI-X (at 20 and 10 µM) and GSI-XVII (at 15 and 10 µM) was significantly lower (p < 0.005) in cells expressing NIC as compared with cells infected with GFP alone (Fig. 8A). Subsequently, T cells infected with pMIG-NIC-IRES-GFP or pMIG-IRES-GFP were assessed for the induction of apoptotic nuclear damage in conditions of A-NID. Induction of neglect-induced death was lower in activated T cells expressing the NICD cultured in the absence of IL-2 as compared with cells transfected with GFP (Fig. 8B). When the difference in induction of apoptotic damage relative to the IL-2-treated group is considered, cells expressing NIC are significantly more protected (p < 0.005) from apoptosis triggered by IL-2 withdrawal than cells expressing GFP alone.


Figure 8
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  		FIGURE 8. Ectopic expression of NIC protects activated T cells from GSI-induced apoptosis and A-NID. A, Activated T cells expressing pMIG-GFP or pMIG-NIC-GFP were cultured with IL-2 in the presence of the indicated concentrations of GSI-X and GSI-XVII. After 18 h, apoptotic nuclear damage was assessed as described in Materials and Methods. B, Activated T cells expressing pMIG-GFP or pMIG-NIC-GFP were cultured in the presence or absence of IL-2. After 18 h, apoptotic nuclear damage was assessed in the different groups as described in Materials and Methods. The data presented are derived from five independent infections. Differences between various groups have been analyzed using the Student t test (*, p < 0.05; **, p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Cytokine deprivation-induced apoptosis is characterized by irreversible changes in cellular metabolism triggered by reduced nutrient uptake and a decreased rate of cellular glycolysis. This process is negatively regulated by Bcl-2 family proteins, which promote mitochondrial integrity and by protein kinases that regulate the uptake and assimilation of nutrients (30, 31). In this study, we describe a role for the Notch pathway in the cytokine-dependent survival of activated T cells. A Notch1/Fc chimera or an Ab to Notch1 that interferes with Notch-ligand interactions and chemical inhibitors of {gamma}-secretase that disrupt Notch processing, implicate Notch signaling in T cell survival. The antiapoptotic function suggested by these experiments was substantiated by the ectopic expression of an active form of Notch1 that protected activated T cells from A-NID. Furthermore, the consequences of Notch signaling in activated T cells are manifested in elevated levels of the antiapoptotic protein Bcl-xL and enhanced Akt/PKB activity.

Cell-cell interactions regulating proliferation, survival, fate specification, and differentiation are critical at many stages in development and the Notch pathway is implicated in these events in flies, worms, and mammals (1, 2, 3). Notch1 regulates commitment to the T cell lineage (32) and the survival of pre-T cells following beta-selection (26). Mature T cells express Notch receptors and their ligands, which renders them susceptible to regulation via this pathway. Thus, the role of the Notch pathway in mature T cell function is being investigated by several groups. Both inhibitory and activating functions have been attributed to Notch in the context of peripheral T cell proliferation (reviewed in Ref. 33). Notch signaling is implicated in the acquisition of effector function by mature T cells (34, 35, 36, 37, 38) and interactions between Notch and other transcription factors have been demonstrated to regulate the TH1 or TH2 polarization of mature T cells (34, 37). In this context, it is noteworthy that the conditional deletion of Notch1 in mature T cells indicated that there is redundancy in Notch-receptor signaling during T cell differentiation (39). Furthermore, in peripheral T cells, Notch receptor engagement concomitant with Ag-receptor stimulation is tolerogenic to T cells (19, 36). Consequently, the expectation from these studies that the pathway is stringently regulated in activated T cells, is validated by the observed down-regulation of cell surface Notch1 that occurs in T cells following their activation (Fig. 6). Another member of the family, Notch-3, is implicated in the generation of regulatory T cells (40). More recently, regulatory T cells have been reported to exert immunosuppressive effects by the activation of the Notch pathway in target cells (41). Thus, the Notch pathway influences diverse functions in the T cell lineage.

In the immune response to infection, Ag-receptor engagement triggers multiple rounds of cell division, resulting in a dramatic increase in effector T cells. Ag clearance is accompanied by the deletion of a majority of activated cells and the emergence of an Ag-specific, memory pool capable of extended survival in the absence of Ag (42). The requirement for memory T cells to transit through an activated effector phase before differentiation is unresolved (43, 44) and may not be equivalent for CD4 and CD8+ T cell subsets. Nonetheless, surviving the contraction phase of the immune response is necessary for the generation of memory and the Notch pathway may be critical for the transition through this survival checkpoint. Emerging data indicates that ROS-mediated postactivation apoptosis is a key event defining this checkpoint that cells must overcome before differentiation into memory (13, 14, 45). Thus, inhibitors of ROS block activated T cell death (13) and cause an increase in circulating pools of memory cells in vivo (14). In our experiments, both the GSIs and the Notch/Fc chimera trigger accumulation of ROS and consequent loss of viability, suggesting that Notch can negatively regulate cellular ROS, a key intermediate in the deletion of Ag-reactive T cells. An earlier study has reported that cells treated with MnTBAP accumulate Notch transcripts (13). We find that the inhibition of ROS by a broad-spectrum antioxidant stabilizes the expression of NICD although we do not observe a substantial difference in the expression of Notch protein in these cells. Although regulation of Notch expression by ROS may have a transcriptional component, the accumulation of ROS following disruption of Notch signaling may not require new gene expression. These data suggest interactions between cellular ROS and the Notch pathway and possible feedback circuits regulating outcomes, although the mechanism(s) are yet to be elucidated. In earlier studies, mutations in Notch have been linked to mitochondrial dysfunction in humans and Notch has also been reported to regulate expression of mitochondrial enzymes in Drosophila (46, 47). Furthermore, a more recent study has shown that hypoxia modulates NIC turnover and Notch activity (48). Notch processing is closely allied to TCR stimulation and is not observed in naive T cells cultured with IL-2 (Fig. 6) or IL-7 (data not shown). Thus, the Notch pathway may offer a means of regulating effector T cell survival without compromising the deletion of autoreactive or inappropriately activated T cells.

Enhanced cellular levels of the antiapoptotic protein Bcl-xL also marks the subset that stabilizes cell surface Notch expression. Conversely, perturbation of Notch signaling by the GSI in activated T cells results in a substantial reduction in cellular levels of Bcl-xL, which cannot be overcome by IL-2. Because IL-2 sustains Bcl-xL expression and promotes T cell survival, it may be argued that the observed effects of Notch are a reflection of its regulation of IL-2R{alpha} expression. Although a small but consistent reduction in levels of IL-2R expression is indeed observed, the GSIs do not modulate the expression of the IL-7R{alpha} although they regulate both IL-2- and IL-7-mediated survival. Without excluding the possibility that the regulation of IL-2R expression is a component of Notch-dependent survival, we expect that Notch signaling in activated T cells likely intersects with multiple signaling cascades in T cells.

We extend earlier data to show that in activated T cells the Notch1-PI3K-p56lck complex includes CD28, which is a key molecule for T cell survival and the development of effectors (28) and confirm that the Notch pathway converges on the activation of Akt/PKB (Fig. 4). Although we report on Akt/PKB signaling, it should be noted that the possibility of Notch interacting with other signaling pathways that regulate cell survival (30, 49, 50) is yet to be examined. In agreement with our earlier study and the data presented here, Zúñiga-Pflücker and colleague (26) have reported a positive interaction between Notch and the Akt/PKB pathway. The activation of Akt/PKB favors the argument that Notch signaling can modulate nutrient uptake to maintain bioenergetic homeostasis (29, 30). Indeed, Notch-ligand interactions have been shown to maintain glucose transport and metabolism in double-negative thymocytes (26). Whether these outcomes reflect a transcriptional function of Notch or interactions with membrane-localized receptor complexes (27, 28, 29) is, as yet, unclear.

One mechanism by which this or similar interactions may be effected is via associations with receptors for cytokines. In activated T cells, disruption of Notch signaling compromises both IL-2- and IL-7-mediated survival. The receptors for these cytokines, along with some others, contain a {gamma}c chain in their receptor complexes, which is implicated in the activation of JAK-STAT, PI3K, and Ras (MAPK) pathways (29). Despite considerable overlap in signaling pathways, cytokines trigger distinct outcomes because of the differential regulation of receptor expression and because the {gamma}c functions in association with other (IL-7R{alpha}, IL-2R{alpha}, or IL-2Rbeta) receptor chains (29, 49). In the absence of clear evidence of a physical association with cytokine receptor components, the possibility that Notch signaling independently converges on common intermediates cannot be excluded. A recent report of Notch localization in the immunological synapse in CD4+ T cells (51) is consistent with Notch functioning as a component of the program that regulates activation and survival of effector T cells. Thus, we propose that in addition to the functions described for Notch in promoting Ag-dependent T cell proliferation and differentiation, Notch augments immune function by modulating cytokine-dependent survival of Ag-stimulated T cells.


   Acknowledgments
 
We acknowledge Veronica Rodrigues (Tata Institute of Fundamental Research, Mumbai, India), Satyajit Mayor (NCBS), Vineeta Bal, and Satyajit Rath for comments and discussion. We are very grateful to Satyajit Rath, Vineeta Bal, and Anna George (National Institute of Immunology, New Delhi, India) for generous access to reagents. We acknowledge the NCBS Flow Cytometry Facility for all fluorescence-based analysis. Clone C17.9C6 developed by Spyroes Artavanis-Tsakonas was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by a Senior Research Fellowship in Biomedical Sciences from the Wellcome Trust, U.K. (to A.S.). P.D. was funded by a Junior Research Fellowship from the Council of Scientific and Industrial Research, India. Back

2 B.G. and P.D. contributed equally. Back

3 Address correspondence and reprint requests to Dr. Apurva Sarin, National Centre for Biological Sciences, Bellary Road, Bangalore 560065, Karnataka, India. E-mail address: sarina{at}ncbs.res.in Back

4 Abbreviations used in this paper: NIC, Notch1 intracellular; NICD, NIC domain; PKB, protein kinase B; AICD, activation-induced cell death; A-NID, activated T cell neglect-induced death; ROS, reactive oxygen species; TCB, T cell blast; MnTBAP, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride; DHE, dihydroethidium; NotchL, Notch-low; GSI, {gamma}-secretase inhibitor; NCT, Notch C terminus; {gamma}c, common {gamma}-chain. Back

Received for publication December 9, 2005. Accepted for publication July 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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