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The Journal of Immunology, 2006, 176: 2299-2306.
Copyright © 2006 by The American Association of Immunologists

Thymocyte Negative Selection Is Mediated by Protein Kinase C- and Ca2+-Dependent Transcriptional Induction of Bim1

Kirsten Canté-Barrett*, Elena M. Gallo{dagger}, Monte M. Winslow{dagger} and Gerald R. Crabtree2,*,{dagger}

* Departments of Developmental Biology and Pathology, Howard Hughes Medical Institute, and {dagger} Program in Immunology, Stanford University, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The processes of positive and negative selection in the thymus both determine the population of T cells that will enter the peripheral immune system and eliminate self-reactive T cells by apoptosis. Substantial evidence indicates that TCR signal intensity mediates this cell fate choice: low-intensity signals lead to survival and differentiation, whereas high-intensity signals generated by self-Ag lead to cell death. The molecular mechanism by which these graded signals are converted to discrete outcomes is not understood. Positive selection requires the Ca2+-dependent phosphatase calcineurin, whereas negative selection requires the proapoptotic Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim). In this study, we investigated the regulation of Bim expression and the role of Ca2+ in mediating negative selection. Our results show that transcription is necessary for both negative selection and Bim induction. Surprisingly, we also found that Ca2+ is necessary for Bim induction. Induction of bim transcription appears to involve protein kinase C, but not calcineurin, JNK, p38 MAPK, or MEK. These results localize the decision point in positive vs negative selection to a step downstream of Ca2+ signaling and suggest that negative selection signals induce Ca2+-dependent bim transcription through PKC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Developmental cell fate decisions are often made on the basis of signal intensity, yet relatively little is known about how these graded signals are converted to nonoverlapping cell fates. For example, this analog-to-digital conversion is seen in sonic hedgehog and bone morphogenetic protein signaling, where genetic studies indicate that all components of these signaling pathways are required for all fates and that the mechanisms converting graded signals to discreet outcomes involves cross-regulation between transcription factors downstream of the pathway (1, 2). In contrast, our current understanding of T lymphocyte development is that the pathways for positive and negative selection appear to diverge, with a Bim-dependent pathway leading to negative selection and calcineurin- and ERK-dependent pathways leading to positive selection. Although other proteins are critical for negative selection, the present data indicate that Bim is a final common mediator of cell death in these pathways and is both necessary and sufficient for cell death (3, 4, 5, 6).

Substantial evidence now supports the notion that self-Ags induce a strong TCR signal, leading to death of self-reactive thymocytes, whereas weaker signals produced by the binding of self-MHC lead to cell survival, differentiation, and entry into the peripheral immune system (reviewed in Ref.7). Thus, the currently accepted view is that the affinity and avidity between MHC/peptide and TCR determines the ultimate fate of the developing T lymphocyte.

One approach to understanding how signals of different intensity can produce radically different fates would be to identify the proteins critical for both positive and negative selection vs those needed for one cell fate but not the other. Signal transduction through the TCR involves a number of proteins common to both the positive and negative selection pathways, including the tyrosine kinases lck and Zap70, Tec kinases, the adaptor proteins Src homology 2 domain-containing leukocyte protein of 76 kDa and linker for activation of T cells, and the guanine nucleotide exchange factor Vav (8, 9, 10, 11, 12, 13). Bouillet et al. (3) showed that Bcl-2-interacting mediator of cell death (Bim)3 is required for negative selection of immature CD4/CD8 double-positive (DP) thymocytes, but does not play a role in positive selection. In contrast, thymocytes lacking calcineurin b1 have a complete block in positive selection but exhibit normal negative selection (14). Moreover, thymocytes lacking ERK or serum response factor accessory protein 1 function have a reduction in positive selection (15, 16, 17, 18, 19, 20). Thus, the current view is that low-intensity signals lead to positive selection via calcineurin and ERK, whereas high-intensity signals trigger the dominant-negative selection pathway via the proapoptotic proteins Bim and Bcl-2 antagonist/killer/Bcl-2-associated X protein (3, 21).

During negative selection, DP thymocytes undergo apoptosis. The Bcl-2 family of proteins consists of both prosurvival and proapoptotic proteins and these proteins are crucial in regulating apoptosis by controlling mitochondrial damage and the subsequent activation of caspases. One subset of Bcl-2 family members contains only the third Bcl-2 homology (BH) domain and all of these BH3-only proteins discovered to date have a proapoptotic effect. Different apoptotic stimuli trigger apoptosis through activation of different BH3-only proteins (reviewed in Ref.22). The proapoptotic BH3-only protein Bim is essential for apoptosis after T and B cell Ag-receptor cross-linking (23, 24), as well as for apoptosis after cytokine withdrawal in several cell types (25, 26). Importantly, as little as a 2-fold increase in Bim appears to be sufficient for cell death, while bim heterozygosity reduces cell death and negative selection (4, 5, 6). Thus, Bim appears to be a highly dosage-sensitive and genetically dominant regulator of cell death, most likely acting downstream of other important regulators.

In this study, we explored the mechanism by which Bim is induced during negative selection upon strong TCR activation. In contrast to previous studies indicating the involvement of posttranslational modifications (27, 28), we show that transcription of bim is necessary for negative selection of DP thymocytes. Consistent with the requirement of Ca2+ in negative selection (29, 30), we show that Ca2+ also plays a critical role in Bim induction. This provides evidence that the decision for positive or negative selection is made at or downstream of Ca2+. Finally, we present evidence that the Ca2+-activated kinase protein kinase C (PKC) plays a role in regulating Bim expression and mediating negative selection. Based on these data, we present a model for cell-fate determination of DP thymocytes, in which Ca2+ plays a central role in both pathways and leads to induction of bim transcription in negative selection.


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

C57BL/6 mice between 6 and 10 wk of age were used in all experiments, unless otherwise noted. Mice were maintained in the animal facility of Stanford University in accordance with federal and institutional guidelines. The conditional calcineurin b1 (Cnb1) mice have been described (14). OT-I transgenic mice (31) on the TAP0 background were a gift from K. A. Hogquist (University of Minnesota, Minneapolis, MN).

Cells and reagents

Thymocytes were obtained by straining the whole thymus through a 70-µm nylon cell strainer. In all experiments, cells were incubated at 37°C/5% CO2 in RPMI 1640 containing 10% FBS supplemented with penicillin, streptomycin, L-glutamine, sodium pyruvate, nonessential amino acids, 2-ME, and HEPES (pH 7.4). For peptide stimulation, EIINFEKL and SIINFEKL peptides (Anaspec) were incubated at 2 µM with CH27-H2Kb cells (CH27 cell line transfected with H-2Kb (32), provided by M. M. Davis, Stanford University, Stanford, CA) for 2 h. Cells were then washed and fixed with 0.1% gluteraldehyde. The OT-I/TAP0 thymocytes were added in a 2:1 ratio to the fixed APCs. In vivo stimulation was done by i.p. injecting 50 µg of anti-CD3{epsilon} Ab (145-2C11; BD Biosciences), 24 and 44 h before thymocytes were harvested. For in vitro Ab stimulation, tissue culture plates were coated with 10 µg/ml anti-CD3{epsilon} (145-2C11; BD Biosciences) and 50 µg/ml anti-CD28 (37.51; BD Biosciences) in PBS overnight at 4°C. The Ab solution was removed before the addition of thymocytes. Incubations with PMA (25 or 50 ng/ml), ionomycin (0.5 µM), and thapsigargin (5–50 nM; Calbiochem) were done for 3 h. Calcium chelators EGTA (4 mM) and BAPTA-AM (100 µM; Molecular Probes) and 5,6-dichlorobenzimidazole riboside (DRB), Gö 6976 (up to 1 µM), Gö 6850 (1 µM), Gö 6983 (1 µM), JNK inhibitor I (20 µM), p38 MAPK inhibitor III (7.6 µM), and the MEK inhibitor U0126 (1.4 µM) (all from Calbiochem) were added to the cells in prewarmed medium. EGTA and the inhibitors were present for 20 min before and throughout the experiment, unless otherwise noted. DRB was removed at indicated time points by replacing the medium on the cells. BAPTA-AM was washed out after the preincubation time of 20 min.

Flow cytometry

Abs used for flow cytometry analysis were anti-CD4 (H129.19; BD Biosciences) and anti-CD8a (53-6.7; BD Biosciences). All flow cytometry data are presented as all-points histograms of CD4/CD8 DP thymocytes. Annexin V (BD Biosciences) and CMX-Rosamine (Molecular Probes) were used to quantify dying and viable cells, respectively. CMX-Rosamine stains mitochondria of live cells and thus CMX-positive cells indicate the viable population, measured as a percentage of the viability at 0 h. Staining was done according to standard procedures following the manufacturer’s recommendations. For intracellular Bim staining, cells were first stained for CD4 and CD8, then fixed at room temperature with 4% formaldehyde in PBS. All subsequent steps were performed on ice and in 0.3% (permeabilization step) or 0.1% saponin (wash and incubation steps), 5% FBS, and 10 mM HEPES (pH 7.4) in PBS. Cells were permeabilized for 30 min followed by an incubation of 1 h with anti-Bim (1:200; StressGen Biotechnologies) and a secondary incubation of 30 min with (1:100) PE-conjugated anti-rabbit IgG (The Jackson Laboratory).

Western blotting

Total cell lysates from 1 to 5 x 106 thymocytes were prepared in radioimmunoprecipitation assay buffer, typically after 3 or 4 h of stimulation and/or treatment in culture. For the experiment shown in Fig. 1D, CD4/CD8 DP thymocytes were sorted before lysates were prepared. For the experiment shown in Fig. 4A, thymocytes were enriched using CD8 MACS beads (Miltenyi Biotec) before lysates were prepared. Lysates were resolved on 4–12% gradient Bis-Tris NuPage gels (Invitrogen Life Technologies) and transferred to polyvinylidene difluoride. The Abs used were: rabbit anti-Bim (1:1000; StressGen Biotechnologies), rabbit anti-Egr1 (1:200; Santa Cruz Biotechnology), rabbit anti-phospho-ERK1/2 (1:1000; Cell Signaling Technology), goat anti-calcineurin b1 (1:1000; Santa Cruz Biotechnology), rabbit anti-actin (1:2500; Sigma-Aldrich), and mouse anti-heat shock protein (Hsp) 90 (1:2500; BD Biosciences). Signal was detected with ECL followed by exposure to autoradiograph film. For all Western blots, a longer exposure was necessary to visualize BimS. In Fig. 3, a separate exposure for BimL, as well as for BimS, is shown.


Figure 1
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  		FIGURE 1. Both negative selection and Bim induction require transcription. A, Flow cytometric analysis of CD4/CD8 DP thymocytes with or without 3 h of stimulation by plate-bound anti-CD3/anti-CD28 or 100 nM dexamethasone, using annexin V staining as a marker for apoptosis. Stimulation-induced apoptosis was completely blocked by 125 µM DRB. B, Time course of viable DP thymocytes, measured by flow cytometry using the mitochondrial dye CMX-Rosamine and expressed as a percentage. Data were normalized to the 0-h time point, which was set as 100% viability. Solid symbols, unstimulated; open symbols, stimulation with plate-bound anti-CD3/anti-CD28. Black symbols, without DRB; gray symbols, with DRB present during the experiment as indicated by the solid gray bar. This experiment was done in triplicate, and data are presented as mean ± SD. C, From the same experiment as in A, total thymocytes were lysed for Western blot analysis. In this and subsequent blots, actin was probed as a loading control, unless otherwise indicated. D, Western blot analysis of DP thymocyte lysates of mice that were i.p. injected 24 and 44 h earlier with anti-CD3. Hsp90 shows equal loading.

 

Figure 4
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  		FIGURE 4. Calcineurin is not required for Bim induction and a negative-, but not positive-, selecting ligand induces Bim. A, Western blot analysis of CD8-enriched Cnb1f/f thymocytes. Cnb1f/f; lck-cre– (expressing functional calcineurin) and Cnb1f/f; lck-cre+ (calcineurin-deficient) thymocytes have comparable Bim induction after anti-CD3/anti-CD28 stimulation for 4 h. B, Flow cytometric analysis of DP thymocytes from the same experiment as in A. C, Western blot analysis of OT-I/TAP0 thymocytes after incubating 4 h with APCs presenting no peptide (no selection), SIINFEKL (negative selection), or EIINFEKL (positive selection). Bim is induced only when SIINFEKL is presented. Egr-1 induction is shown as a positive control for both peptides.

 

Figure 3
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  		FIGURE 3. Calcium is necessary for Bim induction in DP thymocytes. A, Western blot analysis of unstimulated and stimulated thymocytes treated with or without the Ca2+ chelators EGTA and BAPTA-AM. B, Flow cytometric analysis of DP thymocytes treated with calcium chelators as in A. Cells were fixed after staining for CD4/CD8, then stained for intracellular Bim. C, Flow cytometric analysis of DP thymocytes stained for intracellular Bim. Left panel, Cells were either unstimulated or stimulated for 3 h with plate-bound Abs. Right panel, Unstimulated cells were treated with various concentrations of thapsigargin as indicated for 3 h to increase cytosolic [Ca2+]. In this and subsequent figures, cells were stained for intracellular Bim, and Bim staining was measured in DP thymocytes.

 
RNase protection assay

Total RNA from nonstimulated and stimulated thymocytes was purified with an RNA isolation kit (Roche). One microgram of total RNA per sample was hybridized with bim and L32 probes, using the RPA III kit (Ambion). A 246-nt bim probe, which spans exons 5 and 6, and a 94-nt L32 probe against the large ribosomal subunit were synthesized using [{alpha}-32P]UTP (Amersham) in a transcription reaction using an RNA labeling kit (Roche). After the assay, samples were run on a 5.5% acrylamide/bisacrylamide (19:1)/7 M urea gel. After drying, the gel was exposed overnight to a PhosphorImager screen (Molecular Dynamics). Quantitative analysis of band intensities was performed with the ImageQuant software and the bim/L32 mRNA ratio was normalized to the 0-h time point. The protected bim fragment of 210 nt (exons 5 and 6) is common to the three main bim splice variants bimEL, bimL, and bimS, and was used to calculate the bim/L32 ratio. The other bim fragment (111 nt) represents bim splice variants that contain exon 5, but lack exon 6 (33, 34).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Both negative selection and Bim induction require transcription

Since Bim is both necessary and sufficient for negative selection of CD4/CD8 DP thymocytes (3), we tested whether transcription is required for negative selection, using the rapid and reversible transcription inhibitor DRB. To mimic negative selection, DP thymocytes were stimulated with plate-bound anti-CD3/anti-CD28, and DRB was applied to block transcription; apoptosis was measured by annexin V staining 3 h after stimulation (Fig. 1A). Stimulation of DP thymocytes increased apoptosis (10.4% compared with 4.1% for unstimulated cells), and this increase was completely blocked by treatment with DRB (4.5%). As a positive control, glucocorticoid-mediated apoptosis was induced with dexamethasone (35); dexamethasone increased apoptosis (19.5%), and this increase was completely blocked by DRB (4.5%).

We also tested the ability of DRB to maintain cell viability following Ab stimulation, using the mitochondrial dye CMX-Rosamine (Fig. 1B). Consistent with increased percentages of annexin V-positive cells, 3 h after stimulation DP thymocyte viability was reduced (Fig. 1B, open circles). DRB (gray lines) protected thymocytes from apoptosis, and this effect was reversed after DRB removal (right panel in Fig. 1B), providing further evidence that transcription is necessary for negative selection.

Having shown that transcription is required for negative selection in DP thymocytes, we examined whether Bim itself is induced upon stimulation. Samples from the experiment in Fig. 1A were used for Western blot analysis (Fig. 1C). Both plate-bound Ab stimulation and dexamethasone treatment caused increased levels of BimL and BimS, two Bim isoforms with the strongest apoptotic activity (4). Consistent with the in vitro induction of Bim, injecting anti-CD3 Ab as an in vivo model of negative selection also caused increased Bim levels (Ref.3 and Fig. 1D). In contrast with what was reported recently (36), we found that Bim levels in DP thymocytes increased 24 and 44 h after i.p. injection of anti-CD3 Ab (Fig. 1D). As with apoptosis and cell viability, DRB completely blocked Bim induction following both Ab stimulation and dexamethasone treatment in vitro, suggesting that transcription of bim is necessary for apoptosis, and therefore negative selection. It is interesting to note that DRB also maintained the viability of unstimulated thymocytes (Fig. 1B) and reduced Bim levels compared with unstimulated cells in the absence of DRB (Fig. 1C), presumably because thymocytes in a suboptimal in vitro environment undergo apoptosis through transcriptional induction of Bim.

bim mRNA levels increase following plate-bound Ab stimulation

For other cell types, posttranslation modifications or protein-protein interactions are believed to regulate Bim activity (27, 37). However, our results with DRB suggest that, in thymocytes, stimulation increases bim transcription. Therefore, increased bim message should be detectable following Ab stimulation. We used an RNase protection assay to measure bim message levels 1, 3, and 6 h following stimulation (Fig. 2). At all time points, stimulated thymocytes had higher levels of bim mRNA compared with unstimulated cells. As an internal control, L32 mRNA was also measured, and used to normalize bim message levels (Fig. 2B). At 1 h, the bim mRNA level in stimulated thymocytes was ~2.5-fold that of unstimulated thymocytes, consistent with the increased Bim protein we observed at 3 h (Fig. 1C). A similar increase in bim message was seen when measured by real-time PCR (our unpublished data). We also observed increased bim mRNA in unstimulated thymocytes after 1 h in culture (Fig. 2), consistent with increased cell death in the absence of stimulation (Fig. 1, A and B).


Figure 2
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  		FIGURE 2. bim mRNA levels increase following plate-bound anti-CD3/anti-CD28 stimulation. A, RNase protection assay of bim mRNA using a 246-nt probe which protects two fragments (210 and 111 nt; see Materials and Methods). The upper bim band (210 nt) represents the protected fragment common to the three main bim splice variants bimEL, bimL, and bimS. The lower bim band (111 nt) represents other bim splice variants. A 94-nt probe which protects a 78-nt fragment of the large ribosomal subunit L32 was used as a loading control. Negative and positive controls included yeast RNA treated with or without RNase after the hybridization step, respectively. –, unstimulated; +, anti-CD3/anti-CD28 stimulation for the times indicated. B, The ratio of 210-nt protected bim bands to their respective protected L32 bands in A was determined, and normalized to the 0-h time point.

 
Our results with DRB suggest that the increased bim mRNA in stimulated thymocytes is likely the result of increased bim transcription, and not simply increased stability of bim mRNA. This was confirmed by inhibiting transcription in unstimulated thymocytes and measuring bim mRNA at time points ranging from 15 min to 3 h. We found that under these conditions, bim mRNA levels were unchanged (our unpublished data). From this we conclude that increased bim mRNA (and thus protein) in stimulated thymocytes is not the result of bim mRNA stabilization, but represents an increase in bim transcription. Although the increases in bim mRNA and protein levels are modest (ranging from 2- to 3-fold), it is likely sufficient to account for cell death since previous studies have shown that cells are highly sensitive to Bim levels and that a 2-fold change can dramatically affect cell death (4, 5).

Calcium is necessary for Bim induction in DP thymocytes

In developing thymocytes, Ca2+ influx and calcineurin activation are necessary for positive selection, whereas calcineurin is not required for negative selection (14). To determine the role of Ca2+ in negative selection, we tested the effect of Ca2+ chelators on Bim induction following Ab stimulation. Bim levels were measured by both Western blot analysis and intracellular Bim staining followed by flow cytometry. The chelators EGTA (4 mM) and BAPTA-AM (100 µM) were used to capture extra- and intracellular Ca2+, respectively. Treatment with either EGTA or BAPTA-AM alone partially blocked Bim induction, while cotreatment with EGTA and BAPTA-AM completely blocked Bim induction (Fig. 3, A and B). Moreover, chelating Ca2+ reduced Bim levels in unstimulated cells after 3 h in culture. These results implicate both intracellular and extracellular Ca2+ in Bim expression. Consistent with this notion, real-time PCR analysis revealed decreased bim mRNA levels in stimulated thymocytes treated with EGTA and BAPTA-AM for 1 h (our unpublished data). We therefore conclude that, in addition to playing a critical role in positive selection, Ca2+ is also necessary for inducing bim in thymocytes during negative selection.

We next asked whether increased intracellular Ca2+ is sufficient to induce Bim, using the endoplasmic Ca2+-ATPase inhibitor thapsigargin to induce increased cytosolic [Ca2+]. Even at 50 nM, well above its IC50, thapsigargin failed to increase Bim levels after 3 h (Fig. 3C). Thus, simply raising cytosolic [Ca2+] does not appear to be sufficient to induce Bim.

Calcineurin is not required for Bim induction

As mentioned above, calcineurin does not play a role in negative selection. Thus, calcineurin should not be necessary for Bim induction following Ab stimulation of DP thymocytes. To test this, we used a conditional knockout mouse in which the calcineurin b1 subunit is specifically deleted in the thymus upon lck-cre expression (14). Control (Cnb1f/f; lck-cre–) and Cnb1 deficient (Cnb1f/f; lck-cre+) thymocytes showed a similar increase in Bim level 4 h after plate-bound Ab stimulation (Fig. 4, A and B). Unstimulated Cnb1-deficient thymocytes had slightly increased Bim levels compared with unstimulated control thymocytes, consistent with the moderate decrease in viability of these thymocytes in culture (14). Consistent with the fact that calcineurin does not play a role in negative selection, these results show that calcineurin is not required for Bim induction.

Bim is induced by a negative-, but not positive-, selecting ligand in OT-1 thymocytes

If transcription of bim is critical in the differentiation between positive and negative selection then we would predict that positively selecting stimuli would not induce Bim, whereas negatively selecting stimuli would. To test this, we used transgenic mice carrying the OT-I TCR (31) on a TAP0 background. Thymocytes in these mice express the OT-1 TCR but are not positively or negatively selected in vivo due to TAP deficiency. Thus, isolated thymocytes can be driven to either positive or negative selection by presentation of peptides on class I MHC molecules: presentation of the agonist peptide SIINFEKL induces negative selection of these thymocytes, while a synthetic variant with the sequence EIINFEKL induces a weak signal and mimics positive selection (31). When cocultured for 4 h with APCs expressing H-2Kb MHC (32), OT-I/TAP0 thymocytes presented with SIINFEKL had increased Bim levels compared with no peptide, while presentation with EIINFEKL did not induce Bim (Fig. 4C). As expected for positive selection, EIINFEKL presentation led to Egr-1 induction (Fig. 4C) and ERK phosphorylation (38, 39, 40), as well as CD69 induction (our unpublished data). From these data, we conclude that Bim induction is specific for the negative selection pathway of thymocyte development.

PKC inhibitors block anti-CD3/anti-CD28-dependent Bim induction and apoptosis

Because our thapsigargin experiments suggested that increased cytosolic [Ca2+] is not sufficient to induce Bim, some other factor(s) might be necessary for inducing Bim and negative selection. The phorbol ester PMA is commonly used in combination with the Ca2+ ionophore ionomycin to activate T cells (41). As with T cells, Egr-1 and pERK1/2 were induced in thymocytes after treatment with PMA and ionomycin (Fig. 5A). We therefore hypothesized that cotreatment with PMA and ionomycin would mimic negative selection and induce Bim in thymocytes. PMA treatment alone caused moderate Bim induction, while ionomycin only slightly induced Bim (Fig. 5). However, treatment with both PMA and ionomycin increased Bim to a comparable or even greater level than stimulation with anti-CD3/anti-CD28 (Fig. 5B), suggesting that PMA and ionomycin treatment is sufficient to induce negative selection.


Figure 5
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  		FIGURE 5. PMA and ionomycin induce Bim in thymocytes. A, Western blot analysis of total thymocytes treated with PMA (25 ng/ml) and/or ionomycin (0.5 µM). Cotreatment with PMA and ionomycin strongly induces Bim. Egr-1 and pERK1/2 are shown as positive controls, and Hsp90 is shown as a loading control. B, Flow cytometric analysis of DP thymocytes stimulated with plate-bound Abs or treated with PMA (50 ng/ml) and/or ionomycin (0.5 µM).

 
PMA is a diacylglycerol analog commonly used to activate PKC, and thus the ability of PMA to induce Bim in thymocytes implicates PKC in the negative selection pathway. Therefore, we tested the effect of three selective PKC inhibitors (42, 43, 44) on Bim induction and apoptosis during plate-bound Ab stimulation. The inhibitor Gö 6976 blocked Bim induction in a dose-dependent manner, with 1 µM sufficient for a complete block (Fig. 6, A and B). Similar results were obtained with 1 µM Gö 6850 and Gö 6983. In contrast, Egr-1 induction was not fully blocked by these inhibitors, consistent with its selective role in positive selection. To examine the role of other kinases in Bim induction, we tested inhibitors of JNK, p38 MAPK, and MEK, and found that blocking each of these kinases had no effect on Bim induction following Ab stimulation (Fig. 6B). Consistent with the block in Bim induction, all three PKC inhibitors also blocked thymocyte apoptosis after plate-bound Ab stimulation, whereas JNK, p38 MAPK, and MEK inhibitors had no effect on cell death (Fig. 6C and data not shown).


Figure 6
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  		FIGURE 6. PKC inhibitors block anti-CD3/anti-CD28-induced Bim. A, Western blot analysis of thymocytes shows that Bim induction by plate-bound anti-CD3/anti-CD28 is blocked in the presence of increasing amounts of the PKC inhibitor Gö 6976. In contrast, Egr-1 induction is not fully blocked by Gö 6976. B, Top row, Flow cytometric analysis of DP thymocytes from the same experiment as in A. In different experiments, additional kinase inhibitors were tested for their effect on Bim induction. Middle row, PKC inhibitors Gö 6976, Gö 6850, and Gö 6983 (each at 1 µM) all blocked Bim induction. Bottom row, JNK inhibitor I (20 µM), p38 MAPK inhibitor III (7.6 µM), and the MEK inhibitor U0126 (1.4 µM) did not inhibit Bim induction. C, Flow cytometric analysis of unstimulated (top row) and plate-bound Ab stimulated (middle row) DP thymocytes. PKC inhibitors Gö 6976, Gö 6850, and Gö 6983 (each at 1 µM) all blocked plate-bound Ab-induced apoptosis, as measured by annexin V staining. The MEK inhibitor U0126 (1.4 µM) had no effect on apoptosis.

 
It was shown previously that activation of the ERK1/2 pathway phosphorylates and degrades BimEL (45, 46). Indeed, we found that BimEL was phosphorylated upon stimulation (see Figs. 4, A and C, 5A, and 6A). This phosphorylation was blocked by the MEK inhibitor U0126 (our unpublished data), while Bim induction was unaffected by this inhibitor (Fig. 6B). This suggests that while phosphorylation of BimEL through the ERK1/2 pathway does occur in thymocytes upon stimulation, it does not affect Bim levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The currently accepted view of positive and negative selection is that weak TCR signals lead to positive selection and cell survival, while strong signals lead to negative selection by apoptosis. In the present study, we investigated the mechanism of negative selection, focusing on the regulation of the proapoptotic protein Bim. Our data are consistent with a model in which strong TCR activation triggers a Ca2+-dependent pathway involving PKC activation and subsequent transcriptional induction of Bim (Fig. 7). Surprisingly, we found that cells undergoing negative selection have only a modest increase in Bim levels, ~2- to 3-fold. However, this small increase in Bim protein appears to be sufficient to induce cell death, suggesting that thymocytes are extremely sensitive to apoptotic signals. Two previous observations support this conclusion. First, mice haploid for bim have reduced negative selection (6), and secondly, overexpression studies have shown that only a small increase in Bim protein levels induces cell death (4, 5). Therefore, we conclude that, in DP thymocytes, transcriptional induction of bim is the primary mechanism leading to increased levels of Bim.


Figure 7
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  		FIGURE 7. Model for signal discrimination in negative vs positive selection, with Ca2+ as a key player in both pathways. The two pathways diverge downstream of proximal signaling molecules required for both positive and negative selection. A high-intensity signal through the TCR leads to negative selection of thymocytes through Ca2+-dependent PKC activation and subsequent bim transcription. This pathway is dominant over the calcineurin and ERK pathways for positive selection. PKC activation and subsequent Bim induction may require higher Ca2+ levels than are necessary for calcineurin activation. Alternatively, negative selection could induce PKC activation through a conformational change at the TCR (faint dashed arrow). Our model allows for both possibilities. A putative transcription factor X is believed to drive bim transcription upon negative selection. In addition to Bim, misshapen/Nc{kappa}-interacting kinase-related kinase and growth factor receptor-bound protein 2 have been implicated in negative selection (63 64 ).

 
Previous studies have reported protein-protein interactions or posttranslational modifications as a means of controlling Bim levels. Association of Bim with the dynein motor complex through binding the dynein L chain (LC8) has been suggested (27); however, we found no evidence of such an interaction in thymocytes (our unpublished data). Additionally, several studies have called attention to phosphorylation of BimEL by ERK1/2 after survival signals, which in turn results in ubiquitination and proteosome degradation, thereby decreasing Bim protein levels and protecting the cell from apoptosis (28, 37, 45, 46). Even though this mechanism keeps Bim levels low in other cell types such as neurons, we have not observed proteolytic regulation in thymocytes (our unpublished data). Thus, withdrawal of survival signals leads to apoptosis by both transcriptional regulation of Bim, as well as Bim accumulation due to reduced proteolysis. In thymocytes, however, negative selection appears to use a different pathway induced by Ag-receptor cross-linking and requires bim transcription. Our data are in agreement with microarray analysis in NOD mice, which showed defective bim mRNA induction correlating with impaired negative selection in these mice (47, 48).

What factor(s) might regulate bim transcription in thymocytes? One candidate is the forkhead transcription factor FOXO3A, which responds to IL-3 or neurotrophin withdrawal (25, 26). However, our preliminary studies with the Bim promoter, which contains one FOXO3A-binding site and drives expression in HEK-293T cells (49), suggest that it does not respond to negative selection stimuli in thymocytes (our unpublished data). Therefore, in thymocytes, additional transcription factor(s) or regulatory elements (which we call factor X in Fig. 7) may be necessary to induce bim transcription.

In addition to requiring transcription, we found that both Bim induction and thymocyte apoptosis require PKC activity, as demonstrated by three different PKC inhibitors. All three inhibitors (Gö 6976, Gö 6850, and Gö 6983) block at least PKC{alpha} and betaI, conventional isoforms that are activated by diacylglycerol and Ca2+. Previous work has indicated that Gö 6850 and Gö 6976 (partially) inhibit thymocyte death induced by anti-CD3 and anti-CD28 Abs (50). Furthermore, experiments with PKC knockout mice have ruled out PKCbeta and PKC{epsilon} in T cell development and signaling (51, 52), leading us to propose that PKC{alpha} is the most likely isoform involved in Bim induction. However, we cannot exclude the possibility that other PKC isoforms, such as PKC{theta} (50, 53, 54), are involved in negative selection.

Consistent with the requirement for a Ca2+-dependent PKC isoform, we found that Bim induction is Ca2+-dependent. Moreover, the Ca2+-dependent phosphatase calcineurin, which is necessary for positive selection, is required neither for negative selection (14) nor Bim induction. Thus, Ca2+ is common to both pathways and may serve as the final decision point sorting signals to distinct cell fates.

Weak TCR signals activate calcineurin, leading to positive selection; strong signals additionally induce Ca2+-dependent PKC activation, Bim, and cell death, which is dominant over positive selection. The PKC response in negative selection may be the result of enhanced Ca2+ influx in terms of duration and/or frequency reported to correlate with negatively selecting stimuli (55, 56, 57). If the levels of Ca2+ required to activate PKC were higher than for calcineurin, this could be a mechanism for discriminating the stronger negatively selecting stimuli from weaker positively selecting signals. Alternatively, conformational changes in the TCR or associated CD3 chains induced by negatively selecting peptides (58) might lead to PKC activation by an unidentified mechanism; this is supported by the finding that conformational changes in specific CD3 chains following interaction with Nck might discriminate positive vs negative selection (59). However, the CD3{epsilon}-Nck interaction is not required for negative selection (60) and negatively selecting peptides or strong agonists do not appear to induce specific conformational changes in the TCR (61). In addition, the conformational changes induced by weak or strong agonists are indistinguishable and not indicative of signal strength (62). Thus, we favor a model in which the decision point in thymocyte selection is the differential requirement for Ca2+-induced PKC activation vs calcineurin and ERK activation.


   Acknowledgments
 
We thank Mark Davis and Kristin Hogquist for generously providing reagents and Curtis Barrett for critically reviewing the manuscript.


   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 the Howard Hughes Medical Institute (HHMI), National Institutes of Health Grant No. 1-RO1-AI-60037-01, the Boehringer Ingelheim Fonds (to K.C.-B.), Stanford Graduate Fellowships (to E.M.G. and M.M.W.), and an HHMI Predoctoral Fellowship (M.M.W.). Back

2 Address correspondence and reprint requests to Dr. Gerald R. Crabtree, Stanford University, Beckman Center Room B211, 279 Campus Drive, Stanford, CA 94305. E-mail address: crabtree{at}stanford.edu Back

3 Abbreviations used in this paper: Bim, Bcl-2-interacting mediator of cell death; DP, double positive; BH, Bcl-2 homology; PKC, protein kinase C; DRB, 5,6-dichlorobenzimidazole riboside; Hsp, heat shock protein. Back

Received for publication September 12, 2005. Accepted for publication November 23, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
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 References
 

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