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Abstract
To examine the molecular basis for the sensitivity of DP thymocytes to apoptosis and
to gain insight into some of the factors that affect selection, I investigated the regulation of
key bcl-2 family members in T-cell development.
A system of in vitro stimulation of
purified primary T-cells was used to investigate the regulation of key family members at the
message and protein levels. I propose that differences in expression or post-translational
modification of family members underlies the dramatically different reaction of immature
DP and mature SP T-cells to a high-avidity TCR stimulus; the former undergo apoptosis,
while the latter proliferate. This study shows that neither expression of the pro-apoptotic
family members bax and bak nor post-translational inactivation of bcl-XL seem to be
relevant to T-cell development at the DP stage. However, the pro-apoptotic family member
bad is expressed at a significant level in its putatively death-promoting form in DP
thymocytes, but in its putatively inactive form in mature SP cells. Hence, investigation of
the bcl-2 family members in T-cell populations indicates that bad may play a major role in
T-cell development via an unusual molecular mechanism.
Introduction
T-cells are an essential component of the adaptive immune system; they are the cells necessary
for mounting an immune response to “altered-self” cells (transformed or virally infected cells).
T-cells recognize, via their receptor (TCR), peptides bound to a transmembrane protein, the major
histocompatibility complex (MHC) (diagram 1). To generate the diversity of receptors necessary to
recognize the vast array of antigen an altered-self cell may potentially present, T-cells undergo
random rearrangement of the TCR genes early in development to generate TCRs unique to each
cell. In order to eliminate cells possessing either self-reactive TCRs or TCRs that cannot recognize
the body's MHC, T-cells undergo two rounds of selection in the thymus (where they are presented
with a vast array of self-peptides). Positive selection drives T-cells that recognize MHC to mature,
and negative selection induces apoptosis in those cells that recognize self-MHC/peptide complexes
too strongly (1).
Development of T-cells can be followed by their expression of two co-receptors, CD4 and
CD8. Immature stem cells in the bone marrow express neither molecule, and are called double
negative (DN) cells. These cells migrate to the thymus, where they express their unique TCR and
both CD4 and CD8; these are called double positive (DP) cells, and it is at this stage that selection
occurs. These cells mature and either downregulate CD8 to become TH cells (CD4+), or
downregulate CD4 to become TC cells (CD8+); these are collectively called single positive (SP) cells.
The mechanisms that drive these selection and maturation events are not well understood. In vitro
stimulation has shown that TCR stimulation is insufficient in itself to initiate developmental events;
costimulation with other molecules is necessary (2, 3). In vitro, co-engagement of the TCR and CD2
on a DP thymocyte will drive it to mature (3); intriguingly, co-engagement of the TCR and CD28,
which drives a SP to proliferate, will drive a DP to undergo apoptosis in a mimicry of negative
selection (2, diagram 2). The sensitivity of DPs to apoptosis, which seems to underlie selection
events, has been the focus of a great deal of investigation.
The bcl-2 family members, a class of proteins involved in the regulation of apoptosis, have
been shown to play a role in T-cell development (6, 25). Bcl-2 was initially discovered as a
dysregulated gene in a B-cell lymphoma; it is the mammalian homologue of the C. elegans gene ced-9
(4). An entire family of homologues has been discovered since, and its numbers are still growing.
Interestingly, this family consists of both pro- and anti-apoptotic members (diagram 3). The almost
ubiquitous expression of members of this family in mammalian cells coupled to the key role of
apoptosis in T-cell development leads to the hypothesis that regulation of these family members
may be a key factor in development. The discovery that DPs express bcl-2 at a substantially lower
level than DNs or SPs (5) was believed to be an explanation for the sensitivity of DPs to apoptosis;
however, DPs express bcl-XL, an equally potent inhibitor of cell death, at much higher levels than
DNs or resting SPs (6, 7). Hence, I became interested in possible mechanisms whereby bcl-XL’s
anti-apoptotic action might be regulated in DPs. Investigators have shown that anti-apoptotic
family members may be regulated by a balanced expression by pro-apoptotic members (8), or by
post-translational modification - such as phosphorylation or proteolytic cleavage (9-17). I therefore
hypothesized that expression of a pro-apoptotic member or post-translational modification of
bcl-XL in DPs may abrogate its protective effects.
I initially investigated expression of the pro-apoptotic family members bax and bak at the
mRNA level via RT-PCR. I chose these members because they have been shown to play a
significant role in the regulation of death in other in vivo systems (such as neuronal development and
cancers) (8, 18, 19) and can act to promote apoptosis in the more controlled system of yeast, which
does not normally express any bcl-2 members (20). I then moved to investigation of family
members at the protein level, as this is the level at which the family members exert their effects. I
chose to study the more recently discovered family member bad; it has been shown to specifically
interact with bcl-XL to promote cell death (21), and has a rapid and direct method of regulation via
phosphorylation (22).
Materials and Methods
Cells and culture. Primary cells were isolated from thymuses or spleens of C57/Bl6 mice and filtered over nylon mesh. Thymocytes
were panned on anti-CD8 to isolate the DPs; splenic cells were either ACK treated and negatively panned on RAMIG or positively
panned on anti-CD8 with no ACK treatment to isolate SP T-cells. SP thymocytes were isolated from mice injected IP with
dexamethasone 36 hours earlier by filtration over nylon mesh. Cells were cultured at 37oC in RPMI complete medium.
Tissues. Tissues were dissected from C57/B6 mice and lysed by homogenization in lysis buffer, as below.
Stimulation. Cells were stimulated by culture on plates coated with H57 (anti-TCR) or H57 and anti-CD28. Cells were cultured for
four hours or for four and eight hours and then harvested, as indicated.
RNA isolation and RT-PCR. RNA was isolated from cells via RNA-zol treatment, as per manufacturer's instructions. 1 g of RNA
(as determined by OD260) was subjected to RT using MMLV-RT (Gibco), and PCR performed using Taq polymerase (Gibco).
Aliquots were pulled at varying cycles, and products run on an agarose gel and visualized with EtBr.
Lysis and Western blotting. Cells were lysed in a buffer containing 0.1% Triton X-100 and 150mM NaCL, as well as the phosphatase
inhibitor sodium orthovanadate and the denaturing agent 2-me. Lystaes were stored at –20oC and run on a 13% polyacrylamide gel
(loading equalized, where indicated, by use of the Bio-Rad protein assay). The gel was transferred and the membrane subjected to
Western blotting for bcl-XL (Transduction), bad (Transduction), or actin (Santa Cruz). A HRP-conjugated secondary antibody
(anti-mouse IgG for Transduction primaries; anti-goat IgG for Santa Cruz primaries) was used for chemiluminescent detection.
Densitometrey was done on an LKB Laser Densitometer.
Phosphatase assay. Lysate not containing sodium orthovanadate or 2-me were incubated with 200U of -phosphatase for 2 hours at a
1:2 dilution. 2-me was then added, and sample stored at –20oC.
Results and Discussion
Utility of RT-PCR to examine differntial gene expression; lack of evidence for a role for bax
and bak in T-cell development.
RT-PCR IS APPLICABLE TO THE INVESTIGATION OF DIFFERENTIAL GENE REGULATION IN
T-CELLS. Expression patterns of bcl-2 and bcl-XL in T-cells confirms previous data (5, 6), thus
proving that RT-PCR can be used as a semi-quantitative assay of differential gene expression in this
system.
BCL-2 AND BCL-XL EXPRESSION PATTERNS IN RESTING T-CELLS CONFIRMS PREVIOUS DATA;
SIMULATIONS REVEAL POSSIBLE ROLES FOR REGULATION IN DEVELOPMENT. RT-PCR data showed
that bcl-XL is expressed at a high level in DP thymocytes, while bcl-2 is barely expressed (figure 1).
This expression pattern is reversed in SP T-cells; resting cells express low bcl-XL and high bcl-2.
TCR/CD28 stimulation (a proliferative stimulation, for these cells) upregulates bcl-XL while leaving
bcl-2 expression unchanged (figure 1). This data is in agreement with previous investigation (7, 24);
the bcl-2 expressed in a resting state is thought to be necessary for the survival of naïve T-cells, and
the bcl-XL upregulation thought to be necessary for SP proliferation and differentiation.
The data for resting DPs is in agreement with data seen by other investigators; however,
expression of these family members has only been assayed in a purified DP population by one other
group (1). This data therefore represents an important survey of the expression of anti-apoptotic
genes in a purified DP subset. The apoptotic TCR/CD28 stimulus, interestingly enough, had no
effect on bcl-XL expression in the four-hour time course assayed; however, bcl-2 expression
increased significantly within that time period. This is an interesting finding, counterintuitive to the
apoptotic nature of the TCR/CD28 stimulus; it is reasonable to expect that an apoptotic stimulus
would result in the downregulation of anti-apoptotic genes. There are several possible explanations
for the bcl-2 upregulation seen. It may be an artifact of the assay; an apoptotic stimulus may enrich
for a bcl-2hi population by selectively targeting the bcl-2lo cells for death. However, previous assays
have shown that an eight hour stimulus is necessary to induce the morphological signs of apoptosis,
and that significant death does not occur at four hours; therefore, such an enrichment is not a
reasonable explanation for the results seen. Bcl-2 has been shown to be upregulated by the neutral
TCR and the maturative TCR/CD2 stimuli (1, figure 2). One hypothesis consistent with these
results is that bcl-2 upregulation is one of many changes induced in maturation, and is induced by
TCR ligation. Other events, mediated by costimulation, would then determine the response of the
cell. Apoptotic components would be induced by CD28 costimulation, and additional maturative
components induced by CD2 or CD4 costimulation. It is also possible that TCR ligation induces
bcl-2 message upregulation, but that the CD28 costimulation affects translation of that message.
Flow cytometry to assay bcl-2 expression in TCR/CD28 thymocytes would distinguish between
these two possible scenarios; this assay has been hampered by difficulties with stimulation that have
recently occurred, but once those are rectified, the experiment will be done. It is also possible that
bcl-2 is present as an inactive isoform in stimulated DPs. Western blotting for bcl-2 in DPs would
address this issue.
The fact that bcl-XL expression remains unchanged in TCR/CD28 stimulated DPs is unusual,
and contradicts previous data (6) which states that negative selection signals result in the
downregualtion of bcl-XL. However, this researcher was looking at a gross thymocyte population
on a negatively selecting TCR-transgenic background. It is possible that the kinetics of DP
turnover on this background were so rapid that there were fewer of the bcl-XLhi DP thymocytes and
more of the bcl-XLlo DN and SP cells than in a nonselecting or positively selecting background.
This would result in equal thymocyte numbers showing reduced bcl-XL expression in a blot, even
though the DP thymocytes would still be expressing bcl-XL. Flow cytometry for bcl-XL protein in
saponin-permeabilized cells would address this issue in Korsemeyer’s system; a triple stain for
CD4/CD8 would allow the DPs specifically to be assayed for bcl-XL expression, while a quadruple
stain that included CD5 would allow the expression to be correlated with selection. Our in vitro
assay has two advantages over this in vivo system: a purified population and equalized kinetics (our
population has all received approximately the same stimulus at approximately that same time). It is
therefore reasonable to assume that the data taken from our model is more indicative of
physiological events. This data indicates, interestingly enough, that an apoptotic stimulus does not
induce downregulation of a potent anti-apoptotic family member. It is possible that this lack of
downregulation at the message level is not reflected in the protein level; costimulation may affect
translation or turnover. Once again, intracellular staining and flow cytometry would resolve the
issue of post-transcriptional regulation; we await only an appropriate secondary antibody to perform
the assay. An inactivating post-translational modification, such as cleavage or phosphorylation, is
also a possibility; Western blotting for bcl-XL in DPs would address this. From other systems,
however (18, 19, 20), it seems most reasonable that the mechanism of action of this apoptotic
stimulus is expression or activation of pro-apoptotic members. The next step was therefore to
assay the expression of pro-apoptotic members in development.
BAX AND BAK EXPRESSION DO NOT APPEAR TO BE RELEVANT TO DP DEVELOPMENT.
RT-PCR results showed that bax and bak message are not differentially expressed in DP versus SP
T-cells, and that their expression is not altered in cells given developmental stimuli - whether to
proliferate or to undergo apoptosis (figure 1). Some caveats must be taken into account when
interpreting this data. This assay was done on a heterogeneous population of CD5lo, -int, and -hi
DPs; it is possible that differential regulation across development was lost when all populations were
averaged for expression. FACS sorting of DP populations by CD5 expression or a four-color stain
for CD4, CD8, CD5, and bax or bak would address this issue. Although RT-PCR is sensitive and
can amplify a very small difference in message, is not as quantitative as such methods as Northerns
and RNAase protection assays. Some resolution may be lost in amplification. RT-PCR is also
problematic in that it only addresses expression at the RNA level, not giving data on any
post-transcriptional regulation or modification that may occur. Additionally, it was not established
that the stimulations given to these cells actually had the intended effect. This issue can be
addressed in future assays by verifying the stimulation via flow cytometry for ethidium bromide
exclusion or Annexin V (for DPs), or IL-2R flow cytometry, 3H uptake, or IL-2 ELISA (for SPs).
With these caveats in mind, these data strongly indicate that bax and bak are not key players in the
regulation of T-cell development; the expression patterns seen do not support a model in which
differential regulation of either gene is responsible for the sensitivity of DPs to apoptosis. As noted
above, RT-PCR has shown definite changes in other family members in T-cells at different stages in
development and in reaction to developmental stimuli (figure 1); hence, it is likely that data acquired
accurately reflects the regulation of bax and bak message. Intracellular staining and flow cytometry
(to quantitate expression at the protein level) or protein blots for bax and bak (which would address
post-translational modification) would more strongly support or possibly contradict this model;
these are therefore the next logical steps. Protein blots have been hampered by lack of a good
blotting antibody; flow cytometry has been similarly hampered. St. Clair et. al. have already
acquired some preliminary data showing that bax protein is expressed at a lower level by thymocytes
than splenocytes (25), indicating that post-transcriptionalr regulation may be a factor. This group
was looking at gross, rather than purified, populations, which may have affected their results.
Hence, these genes should be assayed at the protein level in purified populations to more definitlvely
answer this question.
Bad and bcl-XL isoforms detected by Western blotting; a possible role for bad in T-cell
development
MULTIPLE ISOFORMS OF BAD AND BCL-XL EXPRESSED IN MURINE TISSUES GIVE CLUES TO
POSSIBLE MECHANISMS FOR ACTION. Given the unpromising results from bax and bak, I tuned to
the family member bad to investigate at the protein level. It binds to bcl-XL with a much higher
affinity than bax or bak (26), and hence is a good candidate to block bcl-XL’s protective effects in
DP T-cells. I also moved to investigation of bcl-XL at the protein level to address the possibility
that post-translational modification of bcl-XL inactivates it in DP T-cells.
I used Western blotting to detect post-translational regulation of bad and bcl-XL. I began
with a tissue blot to investigate possible isoforms of these proteins. Bcl-XL appears as a doublet at
approximately 29kD, although it is not well resolved (figure 3). What initially appeared to be a
lower isoform of bcl-XL at approximately 26kD seems to be a contamination, as it does not appear
in purified populations or in the company-supplied positive control, mouse macrophage lysate. The
doublet is intriguing; it could represent phosphorylation, as bcl-XL posesses a loop domain similar to
that of bcl-2, which has been shown to be phosphorylated in vitro and in vivo, with both positive and
negative effects on acitivity (9, 14-17). The upper band might also be the recently discovered
anti-apoptotic splice variant bcl-X (28), which migrates at a slightly slower rate than bcl-XL (figure
8). The identity of the putative doublet could be addressed by phosphatase treatment, which would
cause a mobility shift if the upper bad is phosphorylated, or by blotting with an antibody that
specifically binds the unusual ankyrin-like C-terminus of bcl-X. The issue of which variant is
responsible for the doublet is relevant, as the phosphorylated form of bcl-XL is hypothesized to be a
pro-apoptotic factor (9) while bcl-X has been shown to be an anti-apoptotic factor (28). Bcl-XL
was expressed in intestine, heart, lymph node, and thymus, and at a low level in liver, but not in SP
T-cells, brain, lung, or kidney (figure 3). The lack of expression in SP T-cells and high lymph node
and thymus expression are in agreement with previous data (6,7); the high expression in the
high-turnover tissues, such as intestine and liver, is not surprising. The low expression in long-lived
brain cells is unusual, and contradicts previous data (8). It is probable that lanes were not equally
loaded; an actin reblot or OD280 reading of the lysates would resolve this.
Bad appeared as a doublet at approximately 24kD across a variety of tissues (figure 3). Bad
has been shown to be phosphorylated in vivo in response to anti-apoptotic stimuli (14, 15, 16), so I
initially assumed that the doublet represents phosphorylation. Phosphatase treatment to prove this
has not been successful (figure 7); however, this does not disprove the hypothesis, since we are
having difficulties with the positive control (anti-phosphotyrosine) for phosphatase action. Since it
is possible that a component of lysis buffer is inactivating the phosphatase, the next step is to
immunoprecipitate bad for phosphatase treatment. However, there are three reasons why it is
reasonable to assume that the lower band is the death-promoting bad and that the upper form is the
inactive phospho-bad. One, the blotting antibody has been shown to bind bad and phospho-bad
equally well (company communication); two, bad is known to be phosphorylated in vivo (11, 12,
21-23, 27); and three, researchers working with in vitro phosphorylation and un-phosphorylated
mutants saw a similar doublet using the same percentage gel (22). I therefore proceeded on the
assumption that the upper bad is phosphorylated, inactive bad, while the lower band is
dephosphorylated, active bad. The upper form of bad was predominant in intestine, heart, liver,
brain, and lung, while the lower form was predominant in kidney, lymph node, and thymus (figure
3). The expression pattern in murine tissues is intriguing, in light of the assumption that the upper
band is inactivated bad. This putatively inactive bad was almost completely dominant in intestine,
heart, liver, brain and lung. Brain and heart are not normally apoptotic tissues, and would be
expected to express a death promoter in its inactive form, if at all. As the intestine, lung, and liver
are tissues that are constantly undergoing turnover, however, expression of an inactive form of a
death promoter suggests an unusual mechainsm for regulationg apoptosis. A change in regulation
of a phosphatase or kinase could result in rapid switch to active bad, resulting in rapid apoptosis - an
interesting mechanism by which a hardy cell could rapidly turn over. Hence, tissue expression of
bad in well-characterized tissues gives clues as to how bcl-2 family members may regulate apoptosis.
BCL-XL ISOFORMS DO NOT APPEAR TO BE RELEVANT TO DP DEVELOPMENT. Bcl-XL is
constitutively expressed in DP thymocytes; relative expression of the two putative isoforms does not
appear to vary across a range of stimuli given to DP thymocytes (figure 4). These data indicate that
early events of maturation and selection do not affect the ratio of the two forms. One experiment
that could prove enlightening is to blot for bcl-XL in stimulated SPs to compare isoform expression
of induced bcl-XL to that expressed constitutively by DPs. Differences in bcl-XL isoform
expression may reflect a functional difference between bcl-XL in SPs and DPs. Clem et. al.
indicated that bcl-XL is cleaved into a death-promoting form by caspase-1 (10); however, no band
was seen at the predicted ~17kD molecular weight of the cleavage product in any of our assays. As
our antibody is directed at the C-terminus of bcl-XL, and the N-terminus is cleaved in vitro, such a
cleavage product should be detected by this assay; therefore, the lack of such a cleavage product in
any of our assays indicates that bcl-XL cleavage is unimportant in our system. Our experiments
should be repeated, however, using an antibody that recognizes a different bcl-XL epitope, or a
polyclonal antiserum, to determine if the lack of isoforms seen is due to antibody specificity rather
than lack of isoform expression. It might also be worthwhile to assay the effect of specific caspase
inhibitors - it may be possible to block DP apoptosis without blocking bcl-XL cleavage, allowing the
claeavage product to be seen, if it does exist and is relevant. However, Clem et. al. did their
experiemnts in vitro, and they have yet to prove that this cleavage event occurs in vivo. In light of the
lack of in vivo confirmation of this cleavage, it is reasonable to assume that it does not play a role in
DP development.
THE PUTATIVELY ACTIVE FORM OF BAD IS EXPRESSED IN DP THYMOCYTES. Only the lower
isoform of bad was seen in DP thymocytes given a variety of stimuli - none, TCR, TCR/CD2, or
TCR/CD28 - over a four and eight hour timecourse; the higher, putatively inactive form did not
appear with any of the stimuli (figure 4). This lack of expression of the upper isoform is consistent
across several experiments utilizing several DP preparations. This expression pattern is intriguing,
in light the strong affinity of bad for bcl-XL. Expression of the active form of bad may abrogate
the protective effects of bcl-XL expression in DP thymocytes, rendering them sensitive to apoptosis.
The lack of expression of the putative phospho-bad in cells stimulated with TCR/CD2 may be
developmentally significant; in light of the data on bad expression on SPs (see below), it is
reasonable to hypothesize that the TCR/CD2 stimulation does not result in bad phosphorylation or
downregulation, and that this loss of active bad is one of the events necessary for complete
maturation to an effector T-cell (which cannot yet be induced in vitro).
THE PHOSPHORYLATION STATE OF BAD IN SP T-CELLS REVEALS A POSSIBLE ROLE FOR BAD IN
DEVELOPMENT. Initial data on bad expression in SP thymocytes was inconsistent. Two different
preparations of splenic SP T-cells (both done using the ACK/panning protocol described in
Materials and Methods) gave different results in a blot for bad. One showed predominantly the
lower form, while the other showed a significant amount of the upper form (figure 5). I
hpothesized that this inconsistency may be an artifact of the procedure used to purify SP cells. It is
not a gentle procedure, and results in a high background death when SPs are cultured overnight;
such a traumatic isoaltion may affect the regulation of survival proteins, especially such a rapid event
as dephosphorylation. I therefore assayed two gentler methods of SP isolation. One takes
advantage of the fact that DPs apoptose when treated with dexamethasone - harvesting a thymus
from a dex-injected mouse yields a predominately SP population. Another method is a positive pan
of splenic cells on anti-CD8, whch also results in a SP-enriched population. Both of these
populations expressed the putatively inactive bad at significantly higher levels than the putatively
active bad (figure 6). In agreement with our hypothesis that stress affects bad regualtion, culture of
the SP thymocytes outside of their supportive thymic environment (effectively, growth factor
withdrawal) resulted in an increase in the lower form of bad. One caveat that must be taken into
account is that both of these blots involved a contaminating population of non-SP cells. It can be
argued that this is not significant - the contaminants would be expected to be different for each
environment (thymus and spleen), and one major contaminant, mouse macrophage, was shown to
express only the lower isoform of bad. However, a FACS sort of SP cells would resolve this
question definitively. The first attempt at a FACS sort yielded too little protein to detect, but
another attempt gave two intriguing results: bad does not appear to be expressed in CD4 + cells, and
although it is expressed in CD8+ cells, it is present predominately as the putatively inactive form.
These experiments show that the lower isoform of bad is assocaited with an apoptotic state in
T-cells, and support a model where the expression and the phosphorylation state of bad is a crucial
difference between SP and DP T-cells. Active bad, expressed in DPs, may result in their apoptotic
response to the stimulus that causes SP cells, which seem to express no bad or inactive bad, to
proliferate.
These experiments represent a novel approach to the fascinating and relevant question of
T-cell development. They are the first experiments to use a controlled and specific approach to
assay bcl-2 family member regulation in T-cell development. They are uniquie in their use of
purified T-cell subpopulations given in vitro stimuli with known developmental consequences previous assays to determine the role of these genes in T-cell development have looked at gross
populations, which contain many subpopulations committing or committed to myriad
developmental fates. Data obtained from these experiments are exciting. From them, I propose a
model in which the response of a T-cell to a high-avidity TCR ligation is mediated in part by bad
expression (diagram 4). Future directions are clear. Experiments done in this study on the
phosphorylation status of bad have attempted to address whether or not bad is active in SP versus
DP T-cells. Immunoprecipitation experiments would address bad’s functionality more directly, by
showing whether bad is associated with 14-3-3 (inactive) or bcl-XL (active). As all family members
act in combination with each other, expression of other family members in development should be
determined at the message and protein levels. Given the demonstrated applicability and ease of the
semi-quantitative RT-PCR used in this study, it would be possible to assay all known bcl-2 family
members in these populations within a short time. However, it is not necessary to assay all family
members in order to answer the question that arises from these data - “Is bad a necessary
component of T-cell development?” To address this question, a bad knockout can be constructed.
According to the hypothesis advanced, this mouse should have defects in negative selection or death
by neglect, with possible consequences for autoimmunity. This knockout would be most
enlightening if combined with a TCR transgene on a negatively selecting background. A more
informative experiment would be to construct a knock-in mouse, in which endogenous bad is
replaced with a mutant that cannot be phosphorylated (23). According to the hypothesis I have
advanced, this mouse should have defects in positive selection, resulting in a significant reduction in,
or complete loss of, SP cells. Such transgenic experiments would more conclusively address the
role of bad in T-cell development. It is possible that a bad knockout or the proposed knock-in
would be inviable; in that case the experiment could still be done by creating a chimera with a RAG
knockout. Such a mouse would have lymphocytes derived exclusively from the bad knockout or
knock-in stem cells.
This novel set of experiments has revealed tantalizing possibilities for the role of the
pro-apoptotic protein bad in T-cell development. Future directions to more accurately characterize
this role and to assay the proposed model are clear. Such experiments will prove revealing about
the role of the bcl-2 family members in the crucial and poorly understood system of T-cell
development. Such an understanding of the mechanisms of development is both of great
intellectual interest and has significant potential for practical benefit. It potentiates control of
development and the immune repertoire; furthermore, knowledge of the upstream signals that drive
development make possible in vitro development of T-cells, useful for research purposes and
disease therapy.
References
1) Cibotti R., Punt J., Dash K., Sharrow S., Singer A. “Surface molecules that drive T cell
development in vitro in the absence of thymic epithelium and in the absence of
lineage-specific signals.” Immunity 6(3):245-255 (1997).
2) Punt J., Osborne B., Takahama Y., Sharrow S., Singer A. “Negative selection of CD4+CD8+
thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be
provided by CD28.” Journal of Experimental Medicine 179(2):709-713 (1994).
3) Sprent J., Gao E., Kanagawa O., Webb S. “T-cell selection in the thymus.” Princess Takamatsu
Symposium 19:127-136 (1988).
4) Nunez G., Seto M., Seremetis S., Ferrero D., Grignani F., Korsmeyer S., Dalla-Favera R.
“Growth- and tumor-promoting effects of deregulated BCL2 in human B-lymphoblastoid
cells.” Proceedings of the National Academy of Science USA 86(12):4589-4593 (1989).
5) Gratiot-Deans J., Ding L., Turka L.A., Nunez G. “Bcl-2 proto-oncogene expression during
human T cell development. Evidence for biphasic regulation.” Journal of Immunology 151(1):
83-91 (1993).
6) Chao D.T., Korsmeyer S.J. “Bcl-XL-regulated apoptosis in T cell development.” International
Immunology 9(9): 1375-1384 (1997).
7) Boise L., Gonzalez-Garcia M., Postema C., Ding L., Lindsten T., Turka L., Mao X., Nunez G.,
Thompson C.
“Bcl-x, a bcl-2-related gene that functions as a dominant regulator of
apoptotic cell death.” Cell 74(4):597-608 (1993).
8) Vekrellis K., McCarthy M.J., Watson A., Whitfield J., Rubin L.L., Ham J. “Bax promotes
neuronal cell death and is downregulated during the development of the nervous system.”
Development 124(6): 1239-1249 (1997).
9) Chang B., Minn A., Muchmore S., Fesik S., Thompson C. “Identification of a novel regulatory
domain in bcl-XL and bcl-2.” EMBO Journal 16(5):968-977 (1997).
10) Clem R., Cheng E., Karp C., Kirsch D., Ueno K., Takahashi A., Kastan M., Griffin D.,
Earnshaw W., Veliuona M., Hardwick J. “Modulation of cell death by Bcl-XL through
caspase interaction.” Proceedings of the National Academy of Science, USA 95(2):554-559 (1998).
11) Datta S., Dudek H., Tao X., Masters S., Fu H., Gotoh Y., Greenberg M. “Akt phosphorylation
of BAD couples survival signals to the cell-intrinsic death machinery.” Cell 91(2):231-241
(1997).
12) del Peso L., Gonzalez-Garcia M., Page C., Herrera R., Nunez G. “Interleukin-3-induced
phosphorylation of BAD through the protein kinase Akt.” Science 278(5338):687-689 (1997).
13) Wang H., Rapp U., Reed JC. “Bcl-2 targets the protein kinase Raf-1 to mitochondria.” Cell
87(4):629-638 (1996).
14) Maundrell K., Antonsson B., Magnenat E., Camps M., Muda M., Chabert C., Gillieron C.,
Boschert U., Vial-Knecht E., Martinou J., Arkinstall S. “Bcl-2 undergoes phosphorylation by
c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively
active GTP-binding protein Rac1.” Journal of Biological Chemistry 272(40):25238-25242 (1997).
15) Ito T., Deng X., Carr B., May W. “Bcl-2 phosphorylation required for anti-apoptosis function.”
Journal of Biological Chemistry 272(18):11671-11673 (1997).
17) Haldar S., Jena N., Croce C. “Inactivation of Bcl-2 by phosphorylation.” Proceedings of the
National Academy of Science, USA 92(10):4507-4511 (1995).
18) Krajewska M., Moss S.F., Krajewski S., Song K., Holt P.R., Reed J.C. “Elevated expression of
Bcl-X and reduced Bak in primary colorectal adenocarcinomas.” Cancer Research 56(10):
2422-2427 (1996).
19) White F.A., Keller-Peck C.R., Knudson C.M., Korsmeyer S.J., Snider W.D. “Widespread
elimination of naturally occurring neuronal death in Bax-deficient mice.” Journal of
Neuroscience 18(4): 1428-1439 (1998).
20) Tao W., Kurschner C., Morgan J.I. “Modulation of cell death in yeast by the Bcl-2 family of
proteins.” Journal of Biological Chemistry. 272(24): 15547-15552 (1997).
21) Kelekar A. Chang B.S., Harlan J.E., Fesik S.W., Thompson C.B. “Bad is a BH3
domain-containing protein that forms an inactivating dimer with Bcl-XL.” Molecular Cell
Biology 17(12): 7040-7046 (1997).
22) Zha J., Harada H., Yang E., Jockel J., Korsmeyer S.J. “Serine phosphorylation of death agonist
BAD in response to survival factor results in binding to 14-3-3 not BCL-X.” Cell 87(4):
619-628 (1996).
23) Hsu S., Kaipia A., Zhu L., Hsueh A. “Interference of BAD (Bcl-XL/Bcl-2-associated death
promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11.” Molecular
Endocrinology 11(12):1858-1867 (1997).
24) Boise L., Minn A., Noel P., June C., Accavitti M., Lindsten T., Thompson C. “CD28
costimulation can promote T cell survival by enhancing the expression of Bcl-XL.” Immunity
3(1):87-98 (1995).
25)
St. Clair E., Anderson S., Oltvai Z.
“Bcl-2 counters apoptosis by Bax
heterodimerization-dependent and -independent mechanisms in the T-cell lineage.” Journal of
Biological Chemistry 272(46):29347-29355 (1997).
26) Yang E., Zha J., Jockel J., Boise L., Thompson C., Korsmeyer S. “Bad, a heterodimeric partner
for bcl-XL and bcl-2, displaces bax and promotes cell death.” Cell 80(2):285-291 (1995).
27) Zha J., Harada H., Yang E., Jockel J., Korsmeyer S. “Serine phosphorylation of death agonist
bad in response to survival factor results in binding to 14-3-3 not bcl-X.” Cell 87(4):619-628
(1996).
28) Yang X., Weber G., Cantor H. “A novel Bcl-x isoform connected to the T cell receptor
regulates apoptosis in T cells.” Immunity 7(5):629-639 (1997).
Motoyama N., Wang F., Roth K., Sawa H., Nakayama K., Nakayama K., Negishi I., Senju S., Zhang Q., Fujii
S., et. al. "Massive cell death of immature hematopoietic cells and neurons in Bcl-X-deficient mice."
Science 267(5203):1506-1510 (1995).
Shindler K., Latham C., Roth K. "Bax deficiency prevents the increased cell death of immature neurons in
bcl-x-deficient mice." Journal of Neuroscience 17(9):3112-3119 (1997).