Bim and Bcl-2 Mutually Affect the Expression of the Other in T Cells

Trine N. Jorgensen, Amy McKee, Michael Wang, Ella Kushnir, Janice White, Yosef Refaeli, John W. Kappler and Philippa Marrack
J Immunol September 15, 2007, 179 (6) 3417-3424; DOI: https://doi.org/10.4049/jimmunol.179.6.3417

Abstract

The life and death of T cells is controlled to a large extent by the relative amounts of Bcl-2-related proteins they contain. The antiapoptotic protein Bcl-2 and the proapoptotic protein Bim are particularly important in this process with the amount of Bcl-2 per cell dropping by about one-half when T cells prepare to die. In this study we show that Bcl-2 and Bim each control the expression of the other. Absence of Bim leads to a drop in the amount of intracellular Bcl-2 protein, while having no effect on the amounts of mRNA for Bcl-2. Conversely, high amounts of Bcl-2 per cell allow high amounts of Bim, although in this case the effect involves increases in Bim mRNA. These mutual effects occur even if Bcl-2 is induced acutely. Thus these two proteins control the expression of the other, at either the protein or mRNA level.

It is well known that members of the Bcl-2 family of proteins interact with each other to control the fate of the cell (1, 2, 3). In many types of cells, overexpression of antiapoptotic proteins, such as Bcl-2, inhibits cell death and, conversely, underexpression of these same proteins promotes death (4, 5, 6, 7, 8, 9, 10). Not unexpectedly, opposite effects result from over or underexpression of the proapoptotic members of the same family. Moreover, experiments have shown that Bcl-2-related proteins bind to each other, with different pairs present in different cells under different circumstances (11, 12).

Because the Bcl-2 family controls the life and death of many kinds of cells, including cancer cells, a lot of attention has been paid to the mechanisms that control transcription of their genes and much is now known about the processes. For example, bcl-2 transcription is induced by CREB and other factors (13, 14, 15, 16) and may be inhibited by factors binding to negative regulatory elements (17). The transcription factors that increase bcl-2 transcription are themselves in turn induced by extracellular signals acting through well-known intracellular pathways. In normal T cells, for example, bcl-2 is induced by members of the IL-2 family of cytokines acting via PI3K and AKT (18). Expression of the gene for the proapoptotic protein, Bim, has likewise been studied in detail. The bim transcription is controlled by transcription factors in the FOXO family (19), and the activity of these proteins, in turn, is inhibited by phosphorylation via PI3K and AKT and also perhaps by MAPK pathways (20, 21).

As is the case for many proteins, the activities of Bcl-2-related proteins are controlled not only by transcription of their genes but also by posttranslational modifications, their location in the cell, and their rates of synthesis and degradation. The activity of Bcl-2 is no exception, being affected by phosphorylation, nitrosylation, and degradation (22, 23, 24, 25). Likewise the effectiveness of Bim is controlled in several ways. In some cell types, Bim is sequestered away from target organelles such as the mitochondrion, and is only released to bind Bcl-2 on mitochondria and precipitate death under particular circumstances (26, 27). In T cells, however, Bim is always mitochondrially located bound to Bcl-2 (11).

Our laboratory has been studying the relative contributions of Bcl-2 and Bim to the death of Ag-activated T cells. In resting T cells much of the Bim is bound to Bcl-2 on mitochondria (11). The level and location of Bim does not change when activated T cells die. However, levels of Bcl-2 fall to ∼50% of normal just before activated T cells die (28). Inhibition of this fall in Bcl-2, by overexpression of the protein or by its induction via antioxidants such as MnTBAP, prevents activated T cells from dying (8, 29, 30). Thus it seems that this quite subtle change to the ratio between Bcl-2 and one of its binding partners, Bim, has dramatic effects on T cell life expectancy.

Because of these ideas we became interested in the relative amounts of Bcl-2 and Bim in T cells. A preliminary experiment led us to measure the amounts of Bcl-2 mRNA and protein in T cells that contained or lacked Bim. Absence of Bim had no statistically significant effect on the amounts of Bcl-2 mRNA. However, T cells lacking Bim halved their amounts of Bcl-2 protein, by comparison with wild-type cells. This effect was also observed with Bcl-2 expressed via a transgene; T cells expressing a Bcl-2 transgene had about one-half the amount of the transgenic Bcl-2 protein per cell if they lacked, rather than contained, Bim. The effect proved to be reciprocal, that is, overexpression, via a transgene, of Bcl-2 raised intracellular amounts of Bim and simultaneously reduced the amounts of endogenous Bcl-2 protein. These phenomena were not caused solely by long time effects, due to development in the absence of Bim or in the presence of high levels of Bcl-2. Thus Bcl-2 and Bim mutually control each other at the protein level.

Materials and Methods

Mice

Mice lacking Bim and backcrossed at least 10 times to C57BL/6 animals were the gifts of Drs. P. Bouillet and A. Strasser (The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia) (4). Mice expressing human Bcl-2 (hBcl-2)4 under the control of the μ-chain promoter, but only in T cells, were the gifts from Drs. J. Adams and S. Cory (Walter and Eliza Hall Institute of Medical Research Parkvilla, Australia) (8). These animals were bred to be homozygous for the human bcl-2 transgene and express or lack Bim at the Biological Resource Center (National Jewish Medical and Research Center, Denver, CO). C57BL/6J animals were purchased from The Jackson Laboratory. All animals were bred and maintained under the guidelines of the Institutional Animal Care and Use Committee as mandated by federal guidelines.

H2k mice expressing mouse mammary tumor virus (MMTV)1 and expressing or lacking Bim were bred from the intercross of H2k.MMTV1 mice (31) and Bim-deficient (Bim−/−) animals, selecting the F2 offspring for expression of H2k and the MMTV1 Vβ3-deleting superantigen, lack of H2b, and homozygous expressing of, or deletion of, Bim.

Isolation of mRNA and analysis of gene expression

Naive CD4 and CD8 T cells were prepared from the lymph nodes of Bim knockout and B6 mice, which did or did not in addition express the hBcl-2 transgene. The cells were stained with anti-CD4, anti-CD8, anti-CD44, and anti-B220. CD4+ and CD8+ naive T cells were isolated by gating out B220+ CD44high cells and then high sorting separately the cells bearing CD4 or CD8 using a MoFlo instrument (Cytomation). From B6 mice, 1.4 × 107 CD4 and 1.7 × 107 CD8 cells were isolated with purities of 97.1% and 97.8%, respectively. From Bim−/− mice, 2.6 × 107 CD4 and 2.7 × 107 CD8 cells were isolated with purities of 97.9% in both cases. RNA was isolated from the cells, converted to cDNA and cRNA as previously described, and analyzed on Affymetrix 403A and 403B Chips. Gene expression was analyzed using Affymetrix GeneChip Operating software.

Real-time PCR

RNA isolated from sorted CD4 or CD8 T cells was converted to cDNA using random hexamers and Superscript II reverse transcriptase. cDNA (100 ng) from each sample was subjected to real-time PCR using SYBR Green Master mix from Applied Biosystems and the following primers specific for: mouse Bcl-2 (mBcl-2) 5′-TGAGTACCTGAACCGGCATCT and 3′-GCATCCCAGCCTCCGTTAT), the hBcl-2 transgene 5′-CTCGGCCTCTGAGCTATTCCAG and 3′-CCCAGCGTGCGCCATATT), mouse Bim 5′-CGGATCGGAGACGAGTTCA and 3′-TTCCAGCCTCGCGGTAATCA), and actin 5′-TGGGAATGGGTCAGAAGGAC and 3′-GGTCTCAAACATGATCTGGG. PCRs were analyzed in real time using an ABI Prism 7700 sequence detector and software from Applied Biosystems.

Cell staining

T cells were analyzed for levels of Bcl-2 and Bim protein by staining of permeabilized cells with mAbs to the two proteins (32, 33). Briefly, B6 or Bim−/− T cells were stained with Abs to either CD4 or CD8, permeabilized with 0.03% saponin, and stained with hamster monoclonal anti-mBcl-2, 3F11, or with monoclonal anti-Bim, Ham151-149, prepared in our laboratory. Controls for Bim staining included cells deficient in Bim.

In experiments in which human and mBcl-2 were to be distinguished, BD Biosciences anti-human Bcl-2 (catalog no. 556535) and 3F11 anti-mouse Bcl-2 (34) were used to stain the two proteins, respectively. Controls confirmed the species specificity of these two Abs.

CD4 and CD8 T cells were stained for expression of various Vβ regions as we have previously described (31).

Western blotting

Spleen cells from B6 or Bim−/− mice were treated with ammonium chloride to lyse RBC. These cells were then combined with lymph node cells and T cells purified by passage over nylon wool. The cells were then lysed in 2% CHAPS or 1% Nonidet P-40 with protease inhibitors (Complete Mini; Roche Diagnostics) and 100 μM PMSF, and the lysates were run without reduction on SDS-PAGE 10–20% gradient Criterion gels and blotted onto nitrocellulose. The mBcl-2 was analyzed with hamster anti-mouse Bcl-2, 3F11 (34), Bim with BD Biosciences rabbit anti-Bim, Bax, with Santa Cruz Biotechnology rabbit anti-Bax (N-20) and actin with Santa Cruz Biotechnology goat anti-actin. Secondary Abs were HRP goat anti-hamster Ig or HRP donkey anti-rabbit Ig from The Jackson Laboratory or HRP anti-goat Ig from Santa Cruz Biotechnology. Western blots were developed with the ECL reagents.

Cell culture

Spleen cells from B6 or Bim−/− mice were lysed with ammonium chloride and then pooled with lymph node cells from the same animals. The cells were then cultured at 2 × 106/ml for various lengths of time in complete culture medium with 5 ng/ml IL-7. Before analysis, live cells were isolated by spinning on mouse Lympholyte gradients (Cedarlane Laboratories).

Viral transduction

The Vβ8+ T cells in C57BL/6 mice were activated in vivo by i.v. injection of 150 μg staphylococcal enterotoxin B (28). Two days later, spleen cells were isolated, RBC-lysed with ammonium chloride, and pooled with lymph node cells and T cells purified by passage over nylon wool. The cells were then transduced with retroviruses expressing Thy1.1 only (MIT) or Thy1.1 and Bcl-2 (MIT-Bcl-2) as we have previously described (35). Alternatively, cells were transduced with retroviruses expressing enhanced GFP only (MIG) or hBcl-2 and enhanced GFP (MIG-hBcl2) (36). The cells were cultured for 2 days, and live cells were purified on Lympholyte gradients and stained for Thy1.1, CD4, CD8, Bcl-2, and Bim expression as described. Results were analyzed on a FACScan instrument with CellQuest software.

Results

Levels of Bcl-2 mRNA are unaffected by the absence of Bim

Bim is one of the proteins that affects the life expectancy of normal T cells (4, 28, 33). To understand how Bim achieves this affect, we used Affymetrix arrays to compare gene expression in T cells from normal C57BL/6 (B6) and Bim−/− mice. Normal CD4 and CD8 T cells from the two kinds of mice were isolated by high-speed cell sorting. RNA was isolated from the purified cells and analyzed on Affymetrix gene chips by standard methods. We noticed that the amount of mRNA for some members of the Bcl-2 family were affected by the absence of Bim, and Puma and A1 mRNA amounts were raised by more than 2-fold both in the CD4 and CD8 T cell analyses (data not shown). There was also some effect on Bcl-2 mRNA amounts in the CD4 cell preparations (data not shown). Because Bim function is related to that of Bcl-2 (8, 29, 30), we were particularly interested in Bcl-2 mRNA result and decided to check it. We therefore sorted CD44low, CD4, or CD8 naive T cells from three different C57BL/6 mice, and three animals that had been backcrossed over 10 times onto the C57BL/6 background, but lacked Bim. mRNA was prepared from the individual mice and compared, using triplicate samples, for expression of Bcl-2 and actin using real-time PCR. As shown in Fig. 1⇓A, there was no significant difference in the expression of Bcl-2 mRNA in CD4 or CD8 resting T cells from the two types of mice, as shown by an average result from six mice in an experiment performed in triplicate twice. The results were similar if individual assays or individual mice were compared. Therefore we concluded that the array results that led us to do these experiments were inaccurate, and that in fact there is no statistically significant difference in expression of Bcl-2 mRNA in the resting T cells of mice that express or lack Bim.

FIGURE 1.
FIGURE 1.

The amount of Bcl-2 protein but not mRNA is reduced in T cells that lack Bim. A, CD4 and CD8 CD44low naive T cells were purified from the lymph nodes of B6 and Bim−/− mice by high-speed sorting. RNA was prepared from the cells and analyzed by real-time PCR for the amounts of mBcl-2 mRNA they contained, with actin as a control. The cycle number for 50% maximum expression of actin was subtracted from the number for Bcl-2. This number for B6 was then subtracted from that for Bim−/− cells and then converted to linear numbers. Data shown are the mean and SEM for two determinations of three independent mice of each type. B, T cells from B6 (Bim+/+) and Bim−/− mice were stained with anti-CD4 or anti-CD8, lysed, and stained intracellularly for mouse Bcl-2 and Bim. The cells were analyzed on a FACScan instrument. Results shown are typical of at least six independent experiments. C, Cells were analyzed as in B. Data shown are the averages of the mean fluorescent intensity and SE of three determinations and are typical of at least six independent experiments. Probabilities represent the likelihood that the samples shown are the same, calculated using two-tailed Student’s t test of the data.

The amount of Bcl-2 protein is reduced in cells lacking Bim

Changes in mRNA levels are not always reflected in the proteins for which they code. Therefore we used Western blot analysis to compare the amounts of Bcl-2 in T cells that contained or lacked Bim and found in one experiment that the amount of Bcl-2 protein per cell was reduced to about one-half the amount found in wild-type cells that lacked Bim (data not shown). However, Western blots are hard to quantitate, especially when the differences to be analyzed are on the order of 2-fold, so we decided to use the more accurate method of flow cytometry to measure Bcl-2 amounts per cell. Lymph node cells were therefore stained with anti-CD4 or anti-CD8, lysed with saponin, and stained for internal expression of Bcl-2. In confirmation of the previous data, Bim−/− CD4 and CD8 T cells had about one-half the amount of Bcl-2 per cell when compared with wild-type cells (Fig. 1⇑, B and C). Similar results were observed for bulk CD4−CD8− lymph node cells, most of which are B cells (data not shown), so this effect may not be restricted to T cells.

Lowered Bcl-2 levels in Bim−/− T cells are not caused by lack of negative selection in the thymus

The lowered levels of Bcl-2 protein in naive T cells from Bim−/− mice could have been caused in several ways. For example, Bim deficiency is known to affect negative selection in the thymus (4, 37, 38), so the phenomenon could have been due to cells that would normally have been negatively selected, but which had escaped due to their lack of Bim. These escapees might, for some unknown reason, express lower levels of Bcl-2. However, we do not believe that this escape is the explanation for two reasons. First, if the shift in amounts of Bcl-2 per cell were due to cells that had avoided negative selection in Bim−/− mice, then we would expect to observe two populations of Bim−/− cells in the anti-Bcl-2 staining experiments, one population representing normal T cells and the other, cells that had avoided negative selection. However, this expectation is not what was observed. The anti-Bcl-2 staining experiments show that Bcl-2 staining shifted for the entire CD4 and CD8 T cell population that lacked Bim vs wild-type cells (Fig. 1⇑B).

Secondly, in fact only a few T cells escape negative selection because of Bim deficiency. This is illustrated in Fig. 2⇓A, in which the effects of MMTV superantigen deletion of thymocytes (39) in B6 mice, which did or did not express Bim, were evaluated. C57BL/6 mice express MMTV8, 9, 17, and 30 (31, 40). The superantigens of these MMTVs react with mouse T cells bearing Vβ5x, Vβ7, Vβ11-Vβ13, and Vβ17, albeit only partially in B6, H2b, mice (31, 40). The results in Fig. 2⇓A show that the percentages of immature CD4+CD8+ thymocytes bearing Vβ5x, Vβ11, and Vβ12 were unaffected by lack of Bim and, in the case of mature CD4+ or CD8+ thymocytes, Bim deficiency affected only those cells expressing Vβ5x. Deletion of thymocytes bearing Vβ11 and Vβ12 was unaffected.

FIGURE 2.
FIGURE 2.

Clonal deletion of autoreactive thymocytes is only slightly inhibited by absence of Bim. A, Thymocytes were isolated from B6 (Bim+/+) and Bim−/− mice, cultured for 3 h to increase expression of their TCRs, stained with anti-CD4, anti-CD8, and Abs to various Vβ regions, and analyzed on a FACScan instrument. Results shown are the mean and SE of three independently analyzed animals of each type. B, Lymph node T cells from H2k mice expressing MMTV1 and expressing or lacking Bim were stained with anti-CD4, anti-CD8, and Abs to various Vβ regions and analyzed as in B. Also shown are the results of comparable measurements, previously published, for H2k mice lacking all MMTVs. Results shown are the mean and SE of at least three independently analyzed animals of each type. Probabilities represent the likelihood that the samples shown are the same, calculated using the two-tailed Student t test of the data.

One difficulty in interpreting this experiment is that we do not know to what extent the low levels of T cells expressing Vβ5x, Vβ11, and Vβ12 in B6 animals is due to deletion driven by MMTV superantigens vs lack of positive selection. However, this extent is known for mice expressing H2k because we have previously reported the percentages of T cells bearing different Vβ regions in H2k mice that lack all MMTV superantigens (31). Therefore we rechecked the effects of Bim deficiency in H2k mice that had been bred to express or lack Bim. To provide an additional superantigen, MMTV1, whose superantigen reacts with mouse T cells bearing Vβ3, was bred into both the Bim+ and Bim−/− mice. The results are shown in Fig. 2⇑B. For comparison, data from our previous work showing Vβ expression on T cells in H2k mice, which lacked all superantigens, are also shown. The results show that there was considerable deletion, via the MMTV superantigens, of T cells bearing Vβ3, Vβ5x, Vβ11, and Vβ12, in these animals, regardless of whether the T cells expressed Bim. Again T cells bearing Vβ5x were most affected by lack of Bim.

These results suggest that the number of T cells that escape thymic deletion because of lack of Bim are too few to account for the almost 50% average loss of Bcl-2 in Bim−/− T cells.

Exposure to IL-7 does not correct the Bcl-2 defect in Bim−/− T cells

In animals, resting T cells are kept alive by IL-4 and IL-7 (41, 42, 43). Bcl-2 mRNA is induced in T cells by exposure to these cytokines (44, 45). Even though the effects in these experiments do not seem to be due to changes in mRNA, it was possible that IL-4 and/or IL-7 might have some unsuspected effect on the stability of the Bcl-2 protein in cells that lacked Bim. It was also possible that the Bim−/− mice, which contain an abnormally large number of lymphocytes (4), might contain selectively lower levels of these constitutive cytokines. Therefore Bim−/− T cells might express less Bcl-2 because Bim−/− mice might have reduced levels of IL-4 or IL-7. If so, culture of Bim−/− cells in these cytokines should restore their expression of Bcl-2 to normal. To check this case, T cells from wild-type and Bim−/− animals were cultured in IL-7, a cytokine that induces Bcl-2 levels in naive T cells and keeps the cells alive without driving them into division. At various times, T cells were assayed by flow cytometry for the amounts of Bcl-2 they contained. Culture in IL-7 steadily increased Bcl-2 expression in both wild-type and Bim−/− T cells (Fig. 3⇓), but at no time did the amounts of Bcl-2 in the Bim−/− cells rise to those in wild-type cells cultured for the same length of time. Similar results were obtained by culture of the T cells in IL-4, a cytokine that acts similarly to IL-7 on naive T cells (data not shown).

FIGURE 3.
FIGURE 3.

Culture in IL-7 does not raise the amount of Bcl-2 in Bim−/− T cells compared with the amount in wild-type cells. Spleen and lymph node cells from B6 (Bim+/+) and Bim−/− mice were cultured in 5 ng/ml IL-7. At the indicated times, live cells were purified and analyzed for their content of Bcl-2 as described in Fig. 1⇑B. Results shown are the mean and SE of triplicate cultures and are representative of three independent experiments.

These results show that the Bcl-2 defect in Bim−/− cells is not caused by exposure to limited amounts of IL-7 and related cytokines, but rather is intrinsic to the T cells themselves.

Bim deficiency affects Bcl-2 at the protein level

The fact that the change in amounts of Bcl-2 per cell did not correlate with changes in mRNA suggested that the effects of Bim on amounts of Bcl-2 was at the level of the proteins. In support of this idea, there are, of course, numerous examples in which proteins affect each other directly. To test the idea by a different means than that already described, we measured the effects of Bim deficiency on hBcl-2 protein, expressed via a transgene (8), and bred into Bim+/+ or Bim−/− mice such that the transgene was homozygously present. hBcl-2 was measured by flow cytometry using a mAb that reacts with human but not mBcl-2. The analyses showed that only ∼70% of the T cells in the transgenic mice expressed the transgene. Therefore the effects of Bim deficiency were measured in terms of effects on both the percentage of cells, which expressed the transgene, and in terms of the amount of hBcl-2 per cell. The frequency of human Bcl-2-expressing cells was not affected by lack of Bim (data not shown). However, Bim−/− T cells contained about one-half the amount of hBcl-2 compared with wild-type cells (Fig. 4⇓A).

FIGURE 4.
FIGURE 4.

Expression of transgenic hBcl-2 protein, but not transgenic hBcl-2 mRNA, is reduced in Bim−/− T cells. A, Spleen and lymph node T cells from hBCL-2 homozygous transgenic mice that expressed (Bim+/+) or lacked (Bim−/−) Bim were analyzed for their content of hBcl-2 protein by staining with anti-CD4, anti-CD8, and a mAb specific for hBcl-2. Gates were placed on the cells that expressed hBcl-2, and the results shown are the mean and SE of hBcl-2 protein expression in the positive cells. Results are typical of at least three independent experiments. B, Spleen and lymph node CD4 and CD8 naive T cells were purified from B6 (Bim+/+) and Bim−/− mice by high-speed sorting, lysed, and analyzed by real-time PCR for their content of hBcl-2 mRNA. Results were normalized to levels of actin mRNA. Results shown are the mean and SE of data from one set of cells of each type of animal and are typical of at least three independent measurements. Probabilities represent the likelihood that the samples shown are the same, and are calculated using the two-tailed Student t test of the data.

The human bcl-2 transgene is driven by an SV40 long terminal repeat and the Eμ promoter (8, 46, 47) and therefore should not be subject to the transcriptional controls that apply to endogenous bcl-2. However, it has been reported that bcl-2 transcription is affected by transcription factors that bind to the Bcl-2 coding region itself (48). Therefore it was possible that the Bim deficiency acted on human bcl-2 transcription via factors binding to the SV40 long terminal repeat, Eμ promoter, or the hBcl-2 coding region. To check this possibility, CD4 and CD8 T cells were sorted from hBcl-2 transgenic Bim+/+ or Bim−/− mice. RNA was prepared from the cells and assayed for hBcl-2 and control actin mRNA via real-time PCR. To avoid confusion with endogenous mBcl-2, the hBcl-2 5′ primer used in this experiment was a sequence in the Eμ promoter. These assays showed that Bim+/+ and Bim−/− T cells contained statistically indistinguishable amounts of hBcl-2 mRNA (Fig. 4⇑B).

Taken together, these data support the idea that Bim affects the amounts of Bcl-2 protein present in the cell at the protein level.

Bcl-2 affects Bim expression

We wondered whether the effect of Bim on Bcl-2 was reciprocal, that is, whether a change in the amount of Bcl-2 per cell would affect Bim. To test this effect, we used flow cytometry to measure the amount of Bim protein in wild-type cells and cells expressing the human bcl-2 transgene. Expression of the human bcl-2 transgene approximately doubled the amount of Bim in T cells (Fig. 5⇓A). We have also tried to evaluate the effects of Bcl-2 deficiency on the amounts of Bim per cell. Unfortunately, we cannot produce Bcl-2−/− or Bim+/+ mice in Denver. However, as reported by others, we can produce mice lacking Bcl-2 and heterozygous for Bim. There was no significant difference in the amounts of Bim per cell between animals that are Bim+/−, Bcl-2+/+ and Bim+/−, Bcl-2−/− (data not shown). This result cannot be due to an unexpected requirement for Bim in order for T cells to exist because T cells, indeed the entire animal, survives well in the complete absence of Bim (4). Perhaps other proteins, such as Bcl-xL or Mcl-1 compensate for the lack of Bcl-2 in Bcl-2−/− cells, bind the Bim that would otherwise be engaged by Bcl-2, and restore equilibrium, at the normal level of Bim, to the cells.

FIGURE 5.
FIGURE 5.

Overexpression of Bcl-2 increases the amount of Bim per cell and reduces the amount of endogenous Bcl-2 per cell. A, Spleen and lymph node T cells from Bim+/+ mice that expressed homozygously or lacked an human bcl-2 transgene were stained with anti-CD4 or anti-CD8, lysed, and stained for expression of Bim protein. Results shown are the mean and SE of three independent experiments. B, CD4 and CD8 T cells from Bim+/+ mice that expressed homozygously or lacked a human bcl-2 transgene were purified by high-speed sorting. Real-time PCR was used to measure the levels of Bim mRNA relative to actin mRNA in each type of cell. Results shown are the mean and SE of three independent measurements. C, Cells were analyzed as in A, but stained for expression of mBcl-2 protein using an Ab that reacts with mBcl-2 but not hBcl-2. Results shown are the mean and SE of three independent experiments. Probabilities represent the likelihood that the samples shown are the same and are calculated using a two-tailed Student t test of the data.

To find out whether this change in the level of Bim protein reflected a change in the amount of Bim mRNA per cell, we isolated naive CD4 or CD8 T cells from hBcl-2 transgenic and control mice. RNA was prepared from the sorted cells, and the amount of Bim mRNA was measured, by comparison with an actin control, using real-time PCR. Cells overexpressing Bcl-2 because of the human transgene did indeed express greatly elevated amounts of Bim mRNA (Fig. 5⇑B). Therefore in this case, the increase in Bim protein in the hBcl-2 transgenic cells was probably caused by a greatly increased level of Bim mRNA and, consequently, increased production of the protein.

Bcl-2 affects its own expression

Finally, we checked whether overexpression of Bcl-2, via the human bcl-2 transgene, would affect expression of endogenous mBcl-2. To check this affect, we measured mBcl-2 in T cells using an anti-mouse Bcl-2 Ab that does not react with hBcl-2. As shown in Fig. 5⇑C, overexpression of Bcl-2 via a transgene drastically reduced expression of the endogenous protein.

The effect of overexpression of Bcl-2 on Bim is not due to cell adaptation during development

Almost all the phenomena described so far involve cells that have developed under continuous pressure from gene deficiency or gene overexpression. Therefore the effects we observed may have been caused by selection during development of the cells, rather than being immediate and continuous responses to the abnormal environment inside the cell. To find out whether the phenomena could be induced by short-term changes in the cell, we isolated activated T cells, transduced them with retroviruses expressing just Thy1.1 or Thy1.1 and Bcl-2 (35), and studied the effects of this transduction on Bim expression in the cells. Acute overexpression of Bcl-2 raised the amount of endogenous Bim per cell (Fig. 6⇓A). Thus, the ability of Bcl-2 to regulate the amount of Bim protein per cell is not caused by selection during development but rather is an immediate response of the cell to its intracellular conditions.

FIGURE 6.
FIGURE 6.

Induction of Bim and reduction in endogenous Bcl-2 by overexpression of Bcl-2 occurs acutely and does not require cell development with high levels of Bcl-2. Vβ8+ T cells in B6 mice were activated in vivo with staphylococcal enterotoxin B. A, Two days later, T cells were purified from the animals and transduced with retroviruses expressing Thy1.1 (MIT) or Thy1.1 and Bcl-2 (MIT-Bcl-2). Two days later, live cells were purified and stained with anti-CD4 or anti-CD8, lysed, and then stained with anti-Bcl-2 or anti-Bim. Results shown are the mean and SE of triplicate cultures and are representative of two independent experiments. B, Two days later, T cells were purified from the animals and transduced with retroviruses expressing GFP (MiG) or GFP and hBcl-2 (MiGh-Bcl-2). At 2–3 days later, live T cells were purified and stained with anti-CD4 or anti-CD8, lysed, and then stained with anti-mouse Bcl-2. Results shown are the mean and SE of three to four cultures. Probabilities represent the likelihood that the samples shown are the same and were calculated using a two-tailed Student t test of the data.

To check this idea in a different way, activated T cells were transduced with a retrovirus coding for GFP, or for GFP and hBcl-2. At 2–3 days later, the T cells were harvested, and live cells were stained for their intracellular amounts of mBcl-2 and hBcl-2. The results in Fig. 6⇑B show that an increase in hBcl-2 induced by retroviral transduction led to a rapid decrease in intracellular levels of mBcl-2. Thus, overexpression of Bcl-2 via transduction led to a rapid decrease in the amount of endogenous Bcl-2 per cell.

These results, together with those in Fig. 3⇑, indicated that cells could respond acutely to changes in the amount per cell of endogenous Bcl-2 and mirror the changes with alterations in the amount of Bim. In addition, cells adapt to acute artificially driven changes in Bcl-2 by altering their levels of endogenous Bcl-2 protein. Hence, the effects described in this study do not seem to be due to long-term adaptation of cells to abnormal Bcl-2 levels throughout development.

Discussion

We show that Bcl-2 and Bim mutually control the amount of protein expressed by the other in T cells. Conceptually this result is not surprising. T cell life and death is to a large extent controlled by these two proteins, with Bcl-2 protecting T cells from death (8, 35) and Bim involved in their death (4, 28). Bcl-2 is not altogether a positive protein, and Bim is not altogether a negative protein, for T cells, however, because overexpression of Bcl-2 (49) and, conversely, under expression of Bim (our unpublished observations) inhibit lymphocyte proliferation. Therefore one might predict that the balance between these two proteins would be carefully controlled.

The fact that lack of Bim affects Bcl-2 levels is not confined to T cells, we have observed a similar decrease in Bcl-2 levels in B220+ (presumably B cells) lacking Bim.

Surprisingly, Bim deficiency affects the amount of Bcl-2 per cell at the level of the protein. This is demonstrated by the fact that mRNA transcripts of both the endogenous mBcl-2 and human bcl-2 transgene were unaffected by the absence of Bim. However, the amount of endogenous mBcl-2 and transgenic hBcl-2 was reduced about 2-fold in Bim−/− cells. This unexpected result could be caused by inhibition of Bcl-2 mRNA translation by lack of Bim, and in fact, proteins that bind to the AU-rich 3′ end of Bcl-2 mRNA and inhibit translation of the mRNA have been reported (50). However, we think this explanation is unlikely for the results observed in this study because the human bcl-2 transgene is affected by the absence of Bim, and the reported AU-rich sequence of the Bcl-2 mRNA is absent from the transgene (8, 46, 47).

Alternatively, the Bcl-2 protein might be turned over more rapidly in the absence of Bim. Two phenomena could explain this thought. In normal T cells some of the Bcl-2 is normally bound to Bim (11). This binding might stabilize Bcl-2 and protect it from degradation, but it seems unlikely that this explanation is the only one for the finding because only ∼17% of Bcl-2 in normal T cells is thus engaged (data not shown). Probably an even lesser proportion of the total Bcl-2 in the hBcl-2 transgenic cells is bound in this way because the transgene is overexpressed and, although the amount of Bim per cell rises, it only doubles. Therefore it seems that there is not enough Bim in the cell to protect as much as 50% of the endogenous Bcl-2 and transgenic Bcl-2 from degradation.

More probably, lack of Bim has downstream effects on the phenomena that lead to degradation of Bcl-2. It has been reported that the Bcl-2-related protein, Mcl-1, is controlled in this way (51). The E3 ubiquitin ligase MULE/LASU1/ARF-BP1, which attacks Mcl-1, is affected by Bim because the BH3 region of Bim competes with the BH3 region of MULE for binding to Mcl-1 (52). Some similar sequence of events may apply to ubiquitinylation and degradation of Bcl-2. Equilibrium of this kind could also explain how overexpression of the human bcl-2 transgene leads to lower amounts of endogenous Bcl-2 per cell (Fig. 5⇑C).

Results similar to those described in this study have been reported for the interaction between Bim and another Bcl-2-related antiapoptotic protein, A1 (53). The report showed that the C-terminal end of A1 affected the half-life of the protein, reducing it to ∼15 min in a B cell lymphoma line via ubiquitinylation. Surprisingly, overexpression of Bim in the same cells stabilized the A1 protein, via inhibition of ubiquitinylation. We have attempted similar experiments in this study with Bcl-2 in the mature T cells used, and have not obtained an interpretable result. For example, coculture of mature T cells in cycloheximide does not result in a lowered amount of Bcl-2 per cell over the time range that such an experiment can be done (<24 h). This result may reflect the fact that Bcl-2 has a much longer half-life than A1 under any circumstances (53).

There is also the question of how increased expression of Bcl-2, via the human bcl-2 transgene, causes increased amounts of Bim mRNA per cell (Fig. 5⇑A). Bim transcription is induced by factors such as FOXO1, FOXO3a, and FOXO4 (19, 54). There is, however, no current evidence that transcription of any of these factors is induced by Bcl-2.

The increase in Bcl-2 that is needed to induce Bim may not be very great. Because the anti-human and anti-mouse Bcl-2 Abs are different, the increase in Bcl-2 caused by the human bcl-2 transgene cannot be measured directly. However, transduction of mBcl-2-expressing retrovirus (Fig. 6⇑A) caused an approximate doubling in the amount of Bcl-2 per cell. This increase is about that seen when T cells are activated by Ag, before their levels of Bcl-2 start to drop and the cells die (32). It is interesting to note that although the rise in Bcl-2 caused by transduction caused an increase in the amount of Bim protein per cell, a similar increase in Bim is not induced in vivo during the expansion phase of T cell responses. Perhaps this finding indicates an additional level of control of Bim expression needed to keep T cells alive while they are responding productively to Ag. If so, this control must be absent in our short-term in vitro cultures, which lack Ag and costimulatory factors.

One could argue that the effects observed in these experiment, most of which are in the range of 2-fold, are quite subtle and unlikely to affect the fate of the cell. However, we and others have previously shown that Bim is needed for activated and resting T cells to die efficiently (4, 28). Activated T cells die much more rapidly than resting T cells. The amount of Bim per cell is similar in activated and resting T cells, but this similarity is not true for Bcl-2. The amount of Bcl-2 per cell is approximately halved in activated vs resting T cells (30). We have argued that this quite subtle change in the amount of Bcl-2 per cell is sufficient to unleash Bim and allow the protein to drive T cell death. Therefore the changes observed may be sufficient to kill or rescue the cell under the right circumstances.

Acknowledgments

We thank the members of the Affymetrix Gene Array Facility at the University of Colorado Health Sciences Center, Bill Townend, Shirley Sobus, and Josh Loomis in the Flow Cytometry Facility at National Jewish Medical and Research Center, and Tibor Vass very much for help with these experiments. We also thank Drs. Stanley Korsmeyer, Andreas Strasser, Philippe Bouillet, Suzanne Cory, and Jerry Adams for the gifts of mice and reagents.

Disclosures

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 in part by a National Institute of Child Health and Human Development Award K12-HD00850 (to M.W.). This work was also supported by Grants AI-17134, AI-18785, and AI-22295 from the U.S. Public Health Service, by Grant AI-002 from the Autoimmunity Center of Excellence, and by Grant CA-046934 from the Cancer Center Core Funding (to P.M. and J.W.K.). This work is supported by Grant CA-117802 from the U.S. Public Health Service and by a Translational Research Award from the Leukemia and Lymphoma Society (to Y.R.). M.W. is a National Institute of Child Health and Human Development Fellow of the Pediatric Scientist Development Program.

  • ↵2 T.N.J., A.M., and M.W. contributed equally to the work.

  • ↵3 Address correspondence and reprint requests to Dr. Philippa Marrack, Integrated Department of Immunology, Howard Hughes Medical Institute, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: marrackp{at}njc.org

  • ↵4 Abbreviations used in this paper: hBcl-2, human Bcl-2; mBcl-2, mouse Bcl-2; MMTV, mouse mammary tumor virus.

  • Received June 1, 2007.
  • Accepted June 11, 2007.

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