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The Journal of Experimental Medicine
J. Exp. Med.
©
The Rockefeller University Press • 0022-1007/2004/10/1189/7 $8.00
Volume 200, Number 9, November 1, 2004 1189–1195
http://www.jem.org/cgi/doi/10.1084/jem.20041328
Brief Definitive Report
1189
Loss of Bim Increases T Cell Production and Function in
Interleukin 7 Receptor–deficient Mice
Marc Pellegrini, Philippe Bouillet, Mikara Robati, Gabrielle T. Belz,
Gayle M. Davey, and Andreas Strasser
The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia
Abstract
Interleukin (IL)-7 receptor (R) signaling is essential for T and B lymphopoiesis by promoting
proliferation, differentiation, and survival of cells. Mice lacking either IL-7 or the IL-7R
chain have abnormally low numbers of immature as well as mature T and B lymphocytes.
Transgenic expression of the apoptosis inhibitor Bcl-2 rescues T cell development and function
in IL-7R
–deficient mice, indicating that activation of a proapoptotic Bcl-2 family member
causes death of immature and mature T cells. BH3-only proteins such as Bim, which are distant
proapoptotic members of the Bcl-2 family, are essential initiators of programmed cell death and
stress-induced apoptosis. We generated Bim/IL-7R
double deficient mice and found that loss
of Bim significantly increased thymocyte numbers, restored near normal numbers of mature
T cells in the blood and spleen, and enhanced cytotoxic T cell responses to virus infection in
IL-7R
/
mice. These results indicate that Bim cooperates with other proapoptotic proteins
in the death of IL-7–deprived T cell progenitors in vivo, but is the major inducer of this pathway
to apoptosis in mature T cells. This indicates that pharmacological inhibition of Bim function
might be useful for boosting immune responses in immunodeficient patients.
Key words: apoptosis • Bim • Bcl-2 • IL-7 • T cells
Introduction
T and B lymphopoiesis depend on IL-7 and its cognate
receptor, composed of the IL-7R
chain and the
c
chain,
which also forms part of the receptors for IL-2, IL-4, IL-9,
and IL-15 (1). Repeated injection of IL-7 into mice or
IL-7
transgene expression promotes progressive lymphadenopathy
(2, 3). Conversely, mice lacking IL-7 or IL-7R
have ab-
normally reduced numbers of immature as well as mature T
and B lymphocytes (4, 5). Mice lacking the
c
chain have
an even more severe lymphopoenia than
IL-7
/
or
IL-
7R
/
mice (6), most likely because cytokines in addition
to IL-7, which require
c
for signaling, also contribute to
lymphopoiesis.
Cytokine signaling promotes cell proliferation, cell differ-
entiation, and cell survival, but in many settings it is unclear
which one of these processes is the most critical. In the case
of IL-7R signaling, it appears that in T cell development,
inhibiting apoptosis is the most critical function because
expression of a
Bcl-2
transgene restored normal numbers of
thymocytes and peripheral T cells in
IL-7R
/
mice (7, 8).
In the B lymphoid lineage,
Bcl-2
transgene expression caused
an approximately threefold increase in peripheral B cells in
IL-7R
/
mice, but B cell precursors in bone marrow re-
mained abnormally low (9). This indicates that for B lym-
phopoiesis, IL-7R signaling is more important for cell pro-
liferation and differentiation than for inhibiting apoptosis.
BH3-only proteins are a proapoptotic subgroup within
the Bcl-2 protein family that share with their relatives only
the short BH3 (Bcl-2 homology) domain, which is required
for protein–protein interaction and proapoptotic activity.
In mammals this group includes Bad, Bik/Blk/Nbk, Hrk/
DP5, Bid, Bim/Bod, Noxa, Bmf, and Puma/Bbc3 (10). All
of these proteins can bind to at least some of their antiapop-
totic Bcl-2–like relatives (10). Biochemical and genetic ex-
periments have demonstrated that BH3-only proteins ini-
tiate apoptosis signaling, whereas Bax/Bak-like proteins, the
second proapoptotic subgroup within the Bcl-2 family, are
required further downstream for cell death (10).
Experiments with knockout mice have identified the
essential functions of BH3-only proteins as well as those of
M. Pellegrini and P. Bouillet contributed equally to this work.
The online version of this article contains supplemental material.
Address correspondence to Andreas Strasser, The Walter and Eliza
Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria
3050, Australia. Phone: 61-3-9345-2624; Fax: 61-3-9347-0852; email:
strasser@wehi.edu.au
M. Pellegrini’s present address is Dept. of Immunology, Faculty of
Medicine, University of Toronto, 1 King’s College Circle, Toronto, On-
tario, M5S 1AB, Canada.
Bim Loss Rescues T Lymphopenia
1190
Bax/Bak. Bim is required for cytokine withdrawal–
induced apoptosis of lymphoid and myeloid cells (11), for
negative selection of autoreactive T and B cells (12–14),
and for termination of T cell immune responses (15, 16).
Although cells lacking single BH3-only proteins have spe-
cific resistance to certain apoptotic stimuli but not others,
lymphocytes from
Bax
/
Bak
/
mice are refractory to all
Bcl-2–regulated apoptosis signaling (17), consistent with
the model that BH3-only proteins function in distinct sig-
naling pathways that converge upon Bax/Bak.
Because Bcl-2 overexpression rescues T cell develop-
ment and function in
IL-7R
/
mice (7, 8), it appears
likely that activation of one or several BH3-only proteins is
involved in the death of T lymphoid cells in IL-7R
/
mice. Bim is a candidate for this function because its loss
inhibits IL-7 deprivation–induced apoptosis in resting (11)
as well as activated T cells (16). Therefore, we generated
Bim
/
IL-7R
/
mice and found that in the absence of
IL-7R
, loss of Bim could slightly augment T cell produc-
tion in the thymus and restore near normal numbers of ma-
ture T cells in the spleen, which were capable of mounting
a cytotoxic response to viral infection. These results indi-
cate that Bim cooperates with other proapoptotic proteins
in the death of IL-7–deprived T cell progenitors in vivo,
but is the major inducer of this pathway to apoptosis in ma-
ture T cells.
Materials and Methods
Mice and In Vivo Challenge with HSV.
The generation of
Bim
/
(11) and
IL-7R
/
mice (The Jackson Laboratory; refer-
ence 4) has been described. Protocols for genotyping of the ani-
mals by PCR will be provided upon request. Experiments in
which mice were infected with HSV-1 to measure CTL re-
sponses in vivo were performed as described previously (16), ac-
cording to the guidelines of the Melbourne Directorate Animal
Ethics Committee.
Immunofluorescent Staining and Cell Sorting.
Immunofluores-
cent staining, FACS analysis, and cell sorting were performed as
described previously (18).
Tissue Culture and Mitogenic T Cell Activation In Vitro.
Activa-
tion of T cells in culture was performed as described previously (9).
Online Supplemental Material.
Fig. S1 shows FACS analysis of
thymocytes from 6-wk-old WT,
IL-7R
/
,
Bim
/
, and
Bim
/
IL-7R
/
mice stained with fluorochrome-conjugated antibod-
ies to CD4 and CD8. Fig. S1 is available at http://www.jem.org/
cgi/content/full/jem.20041328/DC1.
Results and Discussion
To examine the role of Bim in the death of T and B cells
in
IL-7R
/
mice, we generated
Bim
/
IL-7R
/
mice
by intercrossing of the parental strains. To minimize the
risk of having background genes affecting the phenotype,
both parental strains were first backcrossed for
12 genera-
tions onto the C57BL/6 background. 6–10-wk-old WT,
IL-7R
/
,
Bim
/
, and
Bim
/
IL-7R
/
mice were
killed to measure their thymus and spleen cellularity, and
subcellular composition was determined by immunofluo-
rescent staining with surface marker–specific antibodies and
flow cytometric analysis. All
IL-7R
/
mice had an ab-
normally small thymus containing between 2 and 10% of
the number of cells found in WT animals (Fig. 1). The
Bim
/
IL-7R
/
mice had between three- to fivefold
more thymocytes than the
IL-7R
/
littermates (Fig. 1).
All four major thymocyte subsets, CD4
8
pro-T,
CD4
8
immature thymocytes, and CD4
8
as well as
CD4
8
mature T cells, were present in
IL-7R
/
mice,
Figure 1. Loss of Bim increases thymus cellularity and numbers of
splenic T and B cells in IL-7R/ mice. 6–10-wk-old (A, C, and D) or
newborn (B) WT, Bim/, IL-7R/, Bim/ IL-7R/, and Bim/
IL-7R/ mice were killed to determine total leukocyte numbers in the
thymus (A, B, and C) and spleen (D). Cell subset composition was deter-
mined by immunofluorescent staining with surface marker–specific anti-
bodies (CD4 and CD8 for T cells and B220, CD19, and sIg for B cells)
followed by flow cytometric analysis. Data are presented as means SD
of at least five mice of each genotype.
Pellegrini et al. Brief Definitive Report
1191
albeit all at abnormally low numbers (Fig. 1 and Fig. S1,
which is available at http://www.jem.org/cgi/content/
full/jem.20041328/DC1), consistent with the notion that
IL-7R signaling is not essential for T cell differentiation,
but required for normal magnitude of T cell production.
Loss of Bim increased the numbers of all four thymocyte
subsets in
IL-7R
/
mice by
3- (for CD48) to 10-
fold (for mature CD48 and CD48 thymocytes; Fig. 1).
IL-7R/ mice also have a severe deficit (5–10-fold fewer
than WT animals) in mature CD48 and CD48 T cells
in the spleen and other peripheral lymphoid organs. Al-
though loss of Bim increased thymic cellularity only by a
relatively small extent, Bim/ IL-7R/ mice had normal
or near normal numbers of mature CD48 and CD48
T cells in their spleen (Fig. 1) and blood (not depicted).
This indicates that Bim plays a role in the apoptosis of IL-
7R signaling–deprived T cells during development in the
thymus and in peripheral lymphoid organs. Because Bim/
IL-7R/ mice had almost normal numbers of splenic T
cells, it appears that no other proapoptotic factor can com-
pensate for Bim in their death. In contrast, Bim/ IL-
7R/ mice had much fewer thymocytes than WT or Bcl-2
transgenic IL-7R/ mice, indicating that IL-7R signal-
ing blocks the proapoptotic function not only of Bim, but
also of other BH3-only proteins. Puma and Bad are candi-
dates for this function because Puma loss renders certain he-
mopoietic cells refractory to cytokine withdrawal (19, 20)
and because Bad function has been reported to be regulated
by growth factors (10). Most likely, all BH3-only protein–
induced apoptosis elicited by loss of IL-7R signaling re-
quires Bax and Bak because partial rescue of T cell produc-
tion in IL-7R/ mice was achieved by Bax deficiency
(21). It is interesting to speculate why Bim/ IL-7R/
mice have almost normal numbers of splenic T cells despite
having an abnormally small thymus. Because we have not
observed any evidence for enhanced proliferation of mature
T cells in Bim/ IL-7R/ mice, increased accumulation
of mature T cells compared with IL-7R/ mice is probably
a consequence of enhanced thymic output plus increased
survival in peripheral lymphoid organs.
IL-7R signaling plays a critical role at the pro–T2 and
pro–T3 stages of T cell differentiation (4, 5), and the defect
caused by IL-7R loss can be rescued by Bcl-2 overexpres-
sion (8). We investigated the impact of Bim deficiency on
early T lymphopoiesis in IL-7R/ mice by immuno-
fluorescent staining of thymocytes with antibodies to
CD25 and CD44 to identify pro–T1 (CD2544), pro–
T2 (CD2544), pro–T3 (CD2544), and pro–T4
(CD2544) subsets. As reported previously (4), adult and
neonatal IL-7R/ mice had a striking deficit (40–80-
fold reduction compared with WT mice) in pro–T2 and
pro–T3 cells (Fig. 2, A and B). Loss of Bim increased the
numbers of pro–T2 and pro–T3 cells in newborn IL-
7R/ mice by approximately fivefold (Fig. 2 B), provid-
ing a likely explanation for the increase in total thymocyte
numbers found in Bim/ IL-7R/ mice compared with
IL-7R/ mice (Fig. 1). Adult Bim/ IL-7R/ mice
had no significant increase in pro–T2 and pro–T3 cells
compared with IL-7R/ littermates (Fig. 2 A). Remark-
ably, adult Bim/ mice had approximately fourfold fewer
pro–T2 and pro–T3 cells compared with WT animals (Fig.
2 A). For the following reasons we speculate that accumu-
lation of mature T cells suppresses T lymphocyte produc-
tion in the thymus through an unknown negative feedback
Figure 2. Effect of loss of Bim on the numbers of pro–T cells
(CD348) in the thymus of IL-7R/ mice. 6–10-wk-old (A) or
newborn (B) WT, IL-7R/, Bim/, and Bim/ IL-7R/ mice
were killed and thymocytes were stained with Tricolor streptavidin and a
cocktail of biotinylated antibodies to exclude immature CD48 thy-
mocytes, mature CD48 and CD48 T cells, B cells, myeloid and ery-
throid cells, plus a Cy-5–labeled antibody to Thy1 to gate on T lymphoid
cells. Pro–T cells were visualized by staining with a PE-labeled anti-
CD25 antibody and a FITC-labeled anti-CD44 antibody. Total numbers
of thymocytes are listed above the dot plots and the percentages of cells in
each of the quadrant are indicated. Data shown are representative of at least
five mice of each genotype.
Bim Loss Rescues T Lymphopenia
1192
mechanism. We have previously found that not only
pro–T cell numbers, but also the numbers of immature
CD48 thymocytes were abnormally low in Bim/ mice
(11) and similar effects were seen by others in Bax/ Bak/
double knockout mice (17). In both knockout strains, re-
duced thymic T cell production was only seen in adult, but
not newborn animals, indicating that this negative feedback
is activated by the build-up of excess mature T cells. Be-
cause we observed low numbers of pro–T2 and pro–T3
cells in adult Bim/ IL-7R/ mice, which have more
mature T cells than IL-7R/ mice, but not more than
WT mice, it might be the ratio of mature T cells to pro–T
cells and not the absolute numbers of mature T cells that
elicits this proposed negative feedback.
IL-7R signaling is critical for the survival of resting as
well as antigen-activated mature T lymphocytes (4, 5), and
defects in mature T cell function caused by IL-7R defi-
ciency can be rescued by Bcl-2 overexpression (7, 8). Be-
cause loss of Bim restored near normal numbers of mature
CD48 and CD48 T cells in IL-7R/ mice, we in-
vestigated the ability of these T cells to function in an im-
mune response. First, we stimulated purified T cells in cul-
ture with plate-bound mitogenic antibodies to CD3 alone
or with anti-CD28 antibodies and measured proliferation
by [3H]thymidine incorporation. As reported previously
(4), IL-7R/ CD48 and CD48 T cells could prolif-
erate in culture, but to a lesser extent than WT T cells, par-
ticularly when low concentrations of anti-CD3 antibodies
were used. Loss of Bim enhanced proliferation of IL-
7R/ T cells significantly under all stimulatory condi-
tions studied (not depicted).
The impact of IL-7R deficiency on physiological T cell
immune responses in vivo has not yet been studied in de-
tail. Therefore, we infected WT, Bim/, IL-7R/,
Bim/ IL-7R/, and Bim/ IL-7R/ mice with
HSV-1 by injection into both hind feet. CTL responses
were measured after 2, 7, and 14 d by two methods that
Figure 3. Total numbers of leukocytes and CD48 splenic T cells in
HSV-infected WT, Bim/, IL-7R/, Bim/ IL-7R/, and Bim/
IL-7R/ mice. Control WT, Bim/, IL-7R/, Bim/ IL-7R/,
and Bim/ IL-7R/ mice were infected with HSV by injection into
both hind feet. On days 2, 7, and 14 after infection, total numbers of leu-
kocytes (A) and CD48 T cells (B) in the spleen were determined by
cell counting combined with immunofluorescent staining with surface
marker–specific antibodies and FACS analysis. Data shown represent
means SD of at least three mice of each genotype.
Figure 4. Loss of Bim enhances CTL immune responses to HSV in IL-
7R/ mice. Control WT, Bim/, IL-7R/, Bim/ IL-7R/,
and Bim/ IL-7R/ mice were infected with HSV by injection into
both hind feet. (A) On days 2, 7, and 14 after infection, HSV-specific
CTLs in the spleen were enumerated by stimulating cells in culture with
the gB498–505 peptide (the major epitope from HSV recognized by CTLs
in C57BL/6 mice) or as a control with Kb-OVA257–264 peptide SIIN-
FEKL, followed by intracellular staining for IFN-. Total numbers of
HSV-specific CTLs are listed above the dot plots and the percentages of
CD8 IFN- T cells are indicated in the top right quadrants. (B) The
means SD of such analyses using at least three mice of each genotype at
each time point are shown.
Pellegrini et al. Brief Definitive Report
1193
yielded comparable results. Spleen cells were stained with
antibodies to CD8 plus PE-conjugated MHC class I tet-
rameric complexes incorporating the gB498–505 HSV glyco-
protein peptide (SSIEFARL), the major epitope from HSV
recognized by CD8 T cells (16). Alternatively, splenic T
cells were stimulated in culture with the gB498–505 peptide
(or as a control with Kb-OVA257–264 peptide SIINFEKL)
followed by intracellular staining for IFN- (16). Efficiency
of viral clearance in these animals was tested by measuring
HSV titers in their hind feet. Fig. 3 shows that at all time
points after infection, total numbers of leukocytes, and
CD48 T cells were abnormally low in IL-7R/ mice.
In contrast, Bim/ IL-7R/ mice had normal numbers
of these cells and, remarkably, even loss of a single Bim al-
lele caused an increase of these populations (Figs. 3, A and
B). As shown previously (16), infected WT mice produced
substantial numbers of HSV-specific CTLs that were found
first in the draining popliteal lymph nodes and later also in
the spleen and other lymph nodes. The numbers of these
CTLs peaked on day 7 after infection and by day 14, de-
clined to 15% of peak levels (Figs. 4 and 5). Only a very
small number (20-fold fewer compared with WT mice)
of HSV-specific CTLs could be found on day 7 in IL-
7R/ mice and by day 14, no signal above background
was detected (Figs. 4, A and B, and 5, A and B). In con-
trast, readily identifiable numbers of HSV-specific CTLs,
reaching 50% of those seen in WT animals, were found on
day 7 after infection in Bim/ IL-7R/ mice (Figs. 4, A
and B, and 5, A and B). Unlike in WT mice, numbers of
HSV-specific CTLs did not decline in the Bim/ IL-
7R/ mice in which peak levels were still found on day
14 (Fig. 4 B). It is likely that this is a consequence of ex-
tended survival of antigen-activated CTLs because pro-
longed CTL survival was also seen in HSV-infected Bim/
mice (16), but protracted antigenic stimulation cannot be
ruled out as a cause (see below). Loss of even a single allele
of Bim enhanced the CTL response to HSV in IL-7R/
mice (Fig. 4 B), consistent with the increased basal num-
bers of T cells found in Bim/ IL-7R/ mice (Fig. 1).
We have shown previously that loss of Bim can rescue T
cell production and function in Bcl-2–deficient mice (22).
Because of these observations and because IL-7R signaling
increases Bcl-2 expression (23), we speculate that Bim loss
promotes T cell accumulation and function in IL-7R/
mice by allowing cells with abnormally low Bcl-2 levels,
which would otherwise be killed by a Bim-dependent
mechanism, to survive. When we analyzed HSV titers in
the hind feet of infected mice, we found that only WT an-
imals cleared the infection rapidly (by day 7), whereas ani-
mals of all other genotypes needed 14 d (Fig. 5 C). This
raises two questions. How do IL-7R/ mice clear HSV
and why do Bim/ IL-7R/ mice take the same time for
viral clearance as IL-7R/ mice, even though they have
much higher numbers of HSV-specific CTLs? First, IL-
7R/ T cells can proliferate in response to mitogenic
stimulation, albeit less well than WT T cells (see above),
and a small number of HSV-specific CTLs can be found in
infected IL-7R/ mice (Fig. 4, A and B). Therefore, we
speculate that this diminished CTL response is still capable
of eradicating HSV in this infection model. The alternative
explanation, that cells other than lymphocytes surmount
the infection, appears unlikely in view of the observation
that B– and T cell–deficient RAG-1/ mice die from this
infection (Carbone, F., personal communication). Second,
Figure 5. Loss of Bim enhances CTL immune responses to HSV in IL-
7R/ mice, but does not accelerate viral clearance. Control WT,
Bim/, IL-7R/, Bim/ IL-7R/, and Bim/ IL-7R/ mice
were infected with HSV by injection into both hind feet. (A) On day 7
after infection, HSV-specific CTLs in the spleen were enumerated by
staining with antibodies to CD8 plus PE-conjugated MHC class I tet-
rameric complexes incorporating the gB498–505 HSV glycoprotein peptide
SSIEFARL, the major epitope from HSV recognized by CD8 T cells.
Total numbers of HSV-specific CTLs are listed above the dot plots and
the percentages of CD8 T cells binding to gB498–505 HSV presented by
MHC class I are indicated in the top right quadrants. (B) The means
SD of such analyses using at least three mice of each genotype are shown.
(C) The kinetics of viral clearance in the infected mice as determined by
viral plaque assays on extracts from feet are shown. Data shown represent
means SD of viral PFUs for both feet from at least three mice of each
genotype for each time point.
Bim Loss Rescues T Lymphopenia
1194
we believe that the severe reduction in lymph node cellu-
larity and abnormal architecture not only found in IL-
7R/, but also in Bim/ IL-7R/ mice (4 and not de-
picted), explains why HSV clearance is slow in the latter
despite production of substantial numbers of specific CTLs
in the spleen. It appears likely that for rapid HSV eradica-
tion in the feet, activation of a CTL response in the local
lymph nodes is critical.
An approximately threefold increase in mature splenic B
cells was observed in Bim/ IL-7R/ mice compared
with IL-7R/ mice (Fig. 1), but similar deficits in B cell
precursors were found in the bone marrow of animals of
both genotypes (not depicted). This phenotype is similar to
that observed in the B lineage of Bcl-2 transgenic IL-7R/
mice (9), indicating that the critical function of IL-7R sig-
naling in B lymphopoiesis is to promote cell division and
inhibiting apoptosis is subsidiary. The accumulation of ma-
ture B cells in Bim/ IL-7R/ mice is probably due to
increased survival of cells at later stages of development.
In summary, our results show that Bim plays a role in the
death of T lymphoid cells deprived of IL-7R signaling in
vivo during development in the thymus, and even more
prominently at the mature stage in peripheral lymphoid or-
gans and during immune responses. This may indicate that
the ability of IL-7 therapy to restore immune responses in
certain experimental models of immunodeficiency (24)
may, at least in part, be due to inhibition of Bim-mediated
T cell apoptosis. Consequently, like the proposed IL-7
therapy (24), therapies designed to lower Bim expression
levels, for example by using RNA interference based tech-
nology, might be a useful approach for treating immunode-
ficient patients.
We are grateful to Profs. J. Adams and S. Cory for the use of Bim-
deficient mice. We thank J. Morrow and K. Pioch for animal care,
Drs. F. Battye, V. Lapatis, C. Tarlinton, D. Kaminaris, and C. Clark
for cell sorting, Profs. J. Adams and S. Cory, and Drs. A. Harris, D.
Huang, D. Vaux, and L. O’Reilly for insightful discussions.
This work was supported by fellowships and grants from the Na-
tional Health and Medical Research Council (Canberra), the
Charles and Sylvia Viertel Charitable foundation, the Wellcome
Trust (Senior Overseas Fellowship to G.T. Belz), the Virtual Re-
search Institute of Ageing (Boehringer Japan), the Leukemia and
Lymphoma Society of America, and the National Institutes of Health.
The authors have no conflicting financial interests.
Submitted: 2 July 2004
Accepted: 27 August 2004
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