Apoptosis Regulators Fas and Bim Cooperate in Shutdown of Chronic Immune Responses and Prevention...

Immunity

Article

Apoptosis Regulators Fas and Bim Cooperatein Shutdown of Chronic Immune Responsesand Prevention of AutoimmunityPeter D. Hughes,1,2 Gabrielle T. Belz,1 Karen A. Fortner,3 Ralph C. Budd,3 Andreas Strasser,1,4,* and Philippe Bouillet1,4,*1Molecular Genetics of Cancer, The Walter and Eliza Hall Institute of Medical Research, Melbourne, 3050, Australia2Department of Medical Biology, The University of Melbourne, Melbourne 3010, Australia3Immunobiology Program, The University of Vermont College of Medicine, Burlington, VT 05405-0068, USA4These authors contributed equally to this work.

*Correspondence: [email protected] (A.S.), [email protected] (P.B.)

DOI 10.1016/j.immuni.2007.12.017

SUMMARY

Apoptotic death of T lymphocytes is critical for shut-down of immune responses and hemopoietic cell ho-meostasis. Both death receptor (Fas) activation andmitochondrial apoptosis triggered by the BH3-onlyprotein Bim have been implicated in the killing of anti-gen-stimulated T cells. We examined mice lacking thegene encoding Bim (Bcl2l11) and with the inactivatinglpr mutation in the gene encoding Fas (Fas), desig-nated Bcl2l11�/�Faslpr/lpr mice. Shutdown of an acuteT cell response to herpes simplex virus involvedonly Bim with no contribution by Fas, whereas bothpathways synergized in killing antigen-stimulatedT cells in chronic infection with murine g-herpesvirus.Bcl2l11�/�Faslpr/lpr mice developed remarkably en-hanced and accelerated fatal lymphadenopathy andautoimmunity compared to mice lacking only one ofthese apoptosis inducers. These results identify criti-cal overlapping roles for Fas and Bim in T cell deathin immune response shutdown and prevention of im-munopathology and thereby resolve a long-standingcontroversy.

INTRODUCTION

In response to infection or immunization, T cells that express

antigen-specific T cell receptors (TCR) become activated and

proliferate, and some differentiate into effector cells (Sprent

and Tough, 2001). Activated T cells then produce cytokines that

help coordinate the immune response aimed at eliminating the

pathogen. Clearance of the antigen is accompanied by the shut-

down of T cell immune responses and involves apoptosis of

a large fraction of antigen-activated T cells. This prevents accu-

mulation of no-longer-needed and potentially dangerous effector

cells and thereby precludes immunopathology. Two distinct

but ultimately converging pathways control apoptosis initiation

in mammalian cells (Strasser et al., 1995). Ligation of ‘‘death re-

ceptors,’’ members of the tumor necrosis factor receptor (TNF-

R) family with an intracellular ‘‘death domain’’ (e.g., Fas or the

tumor necrosis factor receptor 1 [TNF-R1]) triggers formation

of the ‘‘death-inducing signaling complex’’ (DISC), which pro-

motes cell demolition through activation of caspase-8 (Ashkenazi

and Dixit, 1998; Krammer, 2000; Wallach et al., 1998). Con-

versely, the ‘‘mitochondrial’’ (also called ‘‘intrinsic’’ or ‘‘B cell

lymphoma [Bcl-2]-regulated’’) apoptotic pathway, activated by

developmental cues or cytotoxic stimuli, such as cytokine depri-

vation, is independent of caspase-8 and Fas-associated death

domain protein (FADD), but instead involves mitochondrial re-

lease of cytochrome c, which promotes Apaf-1-mediated activa-

tion of caspase-9 (Green and Kroemer, 2004; Wang, 2001). This

pathway is controlled by the Bcl-2 protein family, which consists

of three subgroups: the Bcl-2-like prosurvival proteins essential

for cell survival; the Bax and Bak-like proteins required for mito-

chondrial outer membrane disruption, release of apoptogenic

proteins (e.g., cytochrome c), and activation of the caspase cas-

cade (Green and Kroemer, 2004; Wang, 2001); and the proteins

containing only the Bcl-2 homology domain 3 (BH3-only pro-

teins, e.g., Bim), which are activated by distinct death stimuli

and are essential for initiation of apoptosis signaling (Huang

and Strasser, 2000).

Both death receptor signaling and the mitochondrial apoptotic

pathway have been shown to be critical for lymphocyte homeo-

stasis (Krammer, 2000; Sprent and Tough, 2001). Mice (Wata-

nabe-Fukunaga et al., 1992) or humans (ALPS, autoimmune lym-

phoproliferation syndrome patients) (Rieux-Laucat et al., 1995)

defective for Fas (Faslpr/lpr mice) or its ligand, FasL (Faslgld/gld

mice), develop progressive lymphadenopathy and splenomeg-

aly resulting from accumulation of excess mature T and B cells

as well as ‘‘unusual’’ abTCR+CD4�CD8�B220+ T cells. Bcl-2-

deficient mice (Veis et al., 1993) and mice lacking myeloid cell

leukemia 1 (Mcl-1) in the hematopoietic system (Opferman

et al., 2005) exhibit profound lymphopenia, whereas mice over-

expressing a Bcl-2 transgene throughout the hematopoietic

compartment show marked lymphadenopathy (Ogilvy et al.,

1999). Mice lacking the BH3-only protein Bim also accumulate

excess lymphocytes and myeloid cells and these cells are abnor-

mally resistant to cytokine deprivation, deregulated calcium flux,

and ER stress (Bouillet et al., 1999; Puthalakath et al., 2007).

Moreover, Bim is critical for apoptosis during negative selection

of autoreactive thymocytes (Bouillet et al., 2002), mature T cells

(Davey et al., 2002), and B cells (Enders et al., 2003).

The relative contributions of the two distinct apoptotic path-

ways in the termination of T cell immune responses has been

Immunity 28, 197–205, February 2008 ª2008 Elsevier Inc. 197

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Bim and Fas Cooperate in Chronic Immune Responses

a matter of controversy for a long time. FasL and Fas have been

implicated because activation-induced cell death (AICD), an

in vitro model in which mitogen-activated T cell blasts are killed

by TCR restimulation, which causes FasL upregulation, is in-

hibited by FasL or Fas inactivation (Brunner et al., 1995; Dhein

et al., 1995; Russell et al., 1993). Moreover, clearance of Staph-

ylococcus Enterotoxin B (SEB)-activated TCR Vb8+ T cells was

reported to depend partially on Fas (Mogil et al., 1995; Van Parijs

et al., 1996). The mitochondrial pathway has been implicated in

immune response shutdown, because Bcl-2 overexpression

(Strasser et al., 1991), loss of Bim (Hildeman et al., 2002; Pelle-

grini et al., 2003), or Bax and Bak deficiency (Rathmell et al.,

2002) inhibited death of T cells stimulated in vitro or in vivo by

a single dose of SEB or in vivo after infection with human herpes

simplex virus (HSV-1). It is conceivable that Fas and Bim may

have overlapping roles in T cell killing, with their relative contribu-

tion determined by the nature of the immune response (acute

versus chronic). In acute immune responses, apoptosis is trig-

gered by the drop in cytokine amounts, after clearance of the

pathogen or injected immunogen (Hildeman et al., 2002; Kram-

mer, 2000; Sprent and Tough, 2001). A single administration of

SEB, which is known to be eliminated quickly from the body

(Vabulas et al., 1996), would therefore mimic an acute infection.

In contrast, TCR restimulation of activated T cells in vitro (AICD)

or repeated administration of SEB to mice is more likely to imitate

the repeated TCR activation that is thought to occur during

chronic immune responses, such as persistent infections or

stimulation with self-antigens (Krammer, 2000; Sprent and

Tough, 2001).

The removal of effector T lymphocytes prevents immunopa-

thology, but the mechanisms for apoptosis induction remain

contentious (Krammer, 2000; Sprent and Tough, 2001). In the

present study, we have generated mice lacking Bim and harbor-

ing the inactivating lpr mutation in the Fas gene, and we used

acute and chronic viral infection models to decipher the indi-

vidual and combined roles of Fas and Bim in the shutdown of

T cell immune responses and in immunopathology.

RESULTS

Bcl2l11�/�Faslpr/lpr Lymphoid Cells Are Resistantto Both Fas-Transduced as well as Bim-DependentApoptotic StimuliTo investigate the functional interaction of Bim and Fas in the

control of lymphocyte homeostasis, we generated mice lacking

both of these apoptosis inducers (with both parental strains

backcrossed to C57BL/6 >20 generations prior to intercrossing).

Bim is required for the killing of lymphocytes induced by growth

factor withdrawal, deregulated calcium flux, DNA damage, or

treatment with dexamethasone (Bouillet et al., 1999). Bcl2l11�/�

thymocytes are, however, normally sensitive to treatment with

phorbol myristyl acetate (PMA, a death stimulus that can be

countered by Bcl-2 overexpression) or FasL, which triggers the

death receptor pathway (Bouillet et al., 1999). To determine the

consequences of the combined mutations on cell survival, we

sorted several lymphocyte populations and subjected them in

culture to cytokine deprivation, treatment with FasL, the calcium

ionophore ionomycin, PMA, or the DNA damage-inducing drug

etoposide. CD4+CD8+ thymocytes (Figure 1A) as well as mature

198 Immunity 28, 197–205, February 2008 ª2008 Elsevier Inc.

B cells (see Figures S1E–S1G available online) and T cells (CD4+,

CD8+, and abTCR+CD4�CD8�B220+; Figures 1B and 1C;

Figure S1) from Bcl2l11�/�Faslpr/lpr mice were resistant to both

FasL as well as Bim-dependent apoptotic stimuli, such as

growth factor withdrawal or treatment with ionomycin. Thymo-

cytes remained, however, normally sensitive to PMA (Figure 1),

which was shown to require the BH3-only protein Puma for cell

killing (Villunger et al., 2003). These results demonstrate that

Fas signaling and Bim exert nonredundant roles in the apo-

ptosis of hemopoietic cells.

Clearance of HSV-Specific CD8+ T Cells in AcuteInfection Requires the BH3-Only Protein Bimbut Is Fas IndependentAlthough the importance of Fas and Bim in lymphocyte homeo-

stasis is widely accepted, their respective roles in immune re-

sponse shutdown remain controversial (Hildeman et al., 2002;

Krammer, 2000; Sprent and Tough, 2001). We addressed the ex-

isting controversy by measuring the numbers of antigen-stimu-

lated CD8+ T cells in spleen and lymph nodes during acute and

chronic immune responses in Bcl2l11�/�Faslpr/lpr as well as

Bcl2l11+/�Faslpr/lpr, Faslpr/lpr, Bcl2l11�/�, and WT mice (Figures

2A–2D and 3A–3D). Consistent with the acute nature of an

HSV-1 (HSV1 KOS) infection and the ensuing immune response

(Pellegrini et al., 2003), all mice were able to clear the virus rapidly

(not shown). Previous studies have shown that the number of

HSV-specific CD8+ T cells in the spleen of WT and Faslpr/lpr

mice peaks at day 7, after which the majority of these effectors

are eliminated by cell death (Pellegrini et al., 2003). Consistent

with this, flow cytometric analysis showed that the spleens of

WT and Faslpr/lpr mice contained only low numbers (<106) of

HSV-specific CD8+ T cells on days 17 and 35 after HSV infection

(Figures 2A and 2B). In contrast, in Bcl2l11�/�mice, the numbers

of HSV-specific CD8+ T cells in the spleen did not fall substan-

tially after pathogen clearance (Figure 2E). Notably, the addi-

tional loss of Fas (in Bcl2l11�/�Faslpr/lpr mice) did not further

increase the accumulation of viral antigen-stimulated CD8+ T

cells (Figure 2E). HSV-specific CD8+ cell numbers were also sim-

ilar in nonlymphoid tissues (lungs and liver) of Bcl2l11�/� and

Bcl2l11�/�Faslpr/lpr mice 17 and 35 days after infection with

HSV-1 (Figure S2). These results demonstrate that Fas activa-

tion, which requires repeated TCR stimulation to upregulate

FasL in T cells, is not involved in the shutdown of acute immune

responses; instead, this death is mediated predominantly by Bim.

Bim and Fas Cooperate to Eliminate MHV-OVA-SpecificT Cells in Chronic InfectionBoth mitochondrial as well as Fas-mediated apoptosis have been

hypothesized to mediate the death of CD4+ as well as CD8+ T

cells during chronic antigenic stimulation (Krammer, 2000;

Sprent and Tough, 2001). We therefore examined the individual

and combined roles of Bim and Fas in the T cell immune response

to mouse g-herpes virus (MHV) transgenically expresssing

ovalbumin (MHV 68-OVA) (Figure 3). This virus causes a chronic

infection predominantly in the lung and B cells, with persistent an-

tigen presentation in the lung (Figure S3; Rajcani and Kudelova,

2005). Total numbers of leukocytes as well as CD8+ T cells

were determined 14, 40, and 60 days after infection (Figures

3A–3D). Surface staining for viral antigen (ovalbumin)-specific

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Figure 1. Leukocytes from Bcl2l11�/�Faslpr/lpr Mice Are Resistant to

Both FasL as well as to Bim-Dependent Apoptotic Stimuli

(A) CD4+8+ thymocytes from mice of the genotypes indicated were purified by

FACS after staining with antibodies to CD4 and CD8. Cells were challenged in

TCR (Figures 3E and 3F) or intracellular staining for IFN-g

production after brief antigenic (ovalbumin) stimulation in culture

(Figures 3G and 3H) demonstrated that in spleens and draining

lymph nodes of WT mice, MHV-OVA-specific CD8+ T cells

(undetectable prior to infection) were greatly expanded on

days 14 and 40 after infection, followed by a loss of nearly

50% of these cells by day 60. Loss of Bim resulted in a significant

(p < 0.05), albeit relatively small, increase in MHV-OVA-specific

CD8+ T cells on days 40 and 60, which was particularly promi-

nent in the spleen (Figures 3E and 3G). Fas-deficient mice con-

tained similarly increased numbers of these cells (Figures 3E

and 3G; p < 0.05). Remarkably, the combined absence of both

apoptosis inducers had dramatic effects, causing a 43- or 63-

fold increase of MHV-OVA-specific CD8+ T cells in the spleen

compared to WT mice on days 40 and 60, respectively. The

increase of MHV-OVA-specific CD8+ T cells in the mediastinal

lymph nodes was even more spectacular, with an 83- or 845-

fold difference compared to WT mice observed on days 40

and 60, respectively (Figures 3F and 3H). This preferential

increase of antigen-specific CD8+ T cells in the draining lymph

nodes might be a consequence of the ongoing antigen release

from the lungs resulting in lymphocyte homing to this site. These

results demonstrate that Fas-mediated death receptor signaling

and the Bim-mediated mitochondrial apoptotic pathway co-

operate to prevent deregulated expansion of activated T cells

during chronic antigenic stimulation.

Bcl2l11�/�Faslpr/lpr and Bcl2l11+/�Faslpr/lpr Mice DevelopExtreme LymphadenopathyLoss of either Bim or Fas both lead to marked lymphadenopathy.

Whereas Bcl2l11�/� mice show an increase in most lymphoid

and myeloid cell populations in the periphery (Bouillet et al.,

1999), lymphadenopathy in Faslpr/lpr mice is due in large part to

the accumulation of unusual abTCR+CD4�CD8�B220+ T cells

(Watanabe-Fukunaga et al., 1992). These unusual T cells are

not found in WT or Bcl2l11�/� mice. Bcl2l11�/�Faslpr/lpr and

even Bcl2l11+/�Faslpr/lpr mice developed lymphadenopathy

and splenomegaly at a dramatically increased magnitude and

rate compared to mice lacking either apoptosis initiator alone

(Figure 4; Figure S5). In particular, the abTCR+CD4�CD8�B220+

T cell population was increased 4–5 times in the spleen of

Bcl2l11�/�Faslpr/lpr mice compared to Faslpr/lpr mice. Precise

quantification in the lymph nodes was made impossible by

huge variations between individual mice (one particular

Bcl2l11�/�Faslpr/lpr mouse was found to have 12.7 g of lymph no-

des; Figure S6), but very large lymph nodes were a hallmark of all

Bcl2l11�/�Faslpr/lpr and Bcl2l11+/�Faslpr/lpr mice (Figures S5A

and S5B). Conventional CD4+ and CD8+ T cells, B cells, and, un-

expectedly, granulocytes were also markedly increased in spleens

and lymph nodes of Bcl2l11�/�Faslpr/lpr and Bcl2l11+/�Faslpr/lpr

mice compared to WT mice or animals lacking only one of these

apoptosis inducers (Figure 4). This shows that the Fas-activated

culture with the apoptotic stimuli indicated and survival assessed daily by

propidium iodide staining and FACS analysis.

(B and C) Similar experiments were performed with CD4+CD8� and

CD4�CD8+ T cells (B) and abTCR+CD4�CD8�B220+ T cells (C) purified from

lymph nodes of mice of the genotypes indicated. Data represent the mean ±

SEM of 3–7 mice for each genotype.


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death receptor and the Bim-mediated mitochondrial apoptotic

pathways act synergistically to control homeostasis of all mature

lymphocyte subpopulations and granulocyte turnover.

The thymi of Bcl2l11�/�Faslpr/lpr, Bcl2l11+/�Faslpr/lpr, Bcl2l11�/�,

Faslpr/lpr, and WT mice were all similar in size and cellularity. The

distribution of the four major cellular subsets (defined by the

expression of CD4 and CD8) within the thymus of Bcl2l11�/�

Faslpr/lpr mice was comparable to that of Bcl2l11�/�mice (Bouillet

et al., 1999), with a reduction in CD4+CD8+ cells and a 2- to 3-fold

increase in CD4�CD8� as well as mature (CD4+CD8� and

CD4�CD8+) T cells (Figure S4). Thus, loss of Bim and Fas

do not cooperate to cause abnormalities in the thymus, demon-

strating that the enhanced lymphadenopathy and spleno-

megaly seen in Bcl2l11�/�Faslpr/lpr and Bcl2l11+/�Faslpr/lpr

mice are a consequence of defects in peripheral lymphoid

organs.

Lymphadenopathy in Bcl2l11�/�Faslpr/lpr

and Bcl2l11+/�Faslpr/lpr Mice Is Not Associatedwith Lymphoid MalignancyDeregulation of apoptosis is thought to contribute to the devel-

opment of many, perhaps all, types of cancer (Hanahan and

Figure 2. Accumulation of Leukocytes, Total CD8+ T Cells,

and HSV-1-Specific CD8+ T Cells in Bcl2l11�/�Faslpr/lpr,

Bcl2l11�/�, Faslpr/lpr, or WT Mice after HSV-1 Infection

(A–D) Total numbers of leukocytes (A, C) and total numbers of

CD8+ T cells (B, D) from spleen and popliteal lymph nodes of

HSV-1-infected mice were quantified by cell counting and staining

with CD8 mAbs followed by FACS analysis. Data from spleens

represent the mean ± SEM from 3–4 mice of each genotype (2

for Bcl2l11+/�Faslpr/lpr). Lymph node samples from mice of the

same genotype (3–4 mice of each genotype except 2 for

Bcl2l11+/�Faslpr/lpr) were pooled and analyzed together 17 and

35 days after infection.

(E and F) Mice of the indicated genotypes were injected subcuta-

neously with HSV-1 in each hindfoot, and HSV-specific CD8+ T

cells in spleen (E) and popliteal lymph nodes (F) were enumerated

after 17 and 35 days by immunofluorescent staining with anti-

bodies to CD8 plus PE-streptavidin-conjugated Kb tetramers

loaded with the immunodominant HSV-1 epitope followed by

FACS analysis. Lymph node samples from mice of the same

genotype were pooled. Data points represent the mean ± SEM

of 3–4 mice (2 for Bcl2l11+/�Faslpr/lpr).

Weinberg, 2000). We have previously shown that loss

of Bim accelerates lymphoma development in mice ex-

pressing a Myc transgene in their B cell compartment

(Egle et al., 2004). Because the dramatic expansion of

the lymphocyte compartment in Bcl2l11�/�Faslpr/lpr

and even the Bcl2l11+/�Faslpr/lpr mice was due to the

disruption of apoptotic pathways, we examined

whether the T cells accumulating in these animals

were monoclonal and hence most likely lymphoma-

tous. However, immunofluorescent staining with

a panel of monoclonal antibodies to different TCR-Vb

subtypes demonstrated that T cells from multiple

lymph nodes of different Bcl2l11�/�Faslpr/lpr mice had

a normal polyclonal repertoire of TCR specificities (Fig-

ure 5; Figure S7). To confirm the absence of hemato-

logical (or other) malignancy in Bcl2l11�/�Faslpr/lpr mice, we ver-

ified that C57BL/6 mice injected with 2 3 106 lymph node cells

from several Bcl2l11�/�Faslpr/lpranimals did not develop tumors

over a 12 month observation period (data not shown). These re-

sults show that the extreme lymphadenopathy and splenomegaly

in Bcl2l11�/�Faslpr/lpr and Bcl2l11+/�Faslpr/lpr mice are caused by

the polyclonal expansion of nontransformed lymphocytes.

Autoimmune Pathology in Bcl2l11�/�Faslpr/lpr

and Bcl2l11+/�Faslpr/lpr MiceBim (Davey et al., 2002) as well as Fas (Van Parijs et al., 1998)

have been implicated in the killing of T cells chronically activated

by self-antigens, so it appears predictable that loss of both may

synergize to cause severe immunopathology. Although Faslpr/lpr

(Watanabe-Fukunaga et al., 1992) as well as Bcl2l11�/� (Bouillet

et al., 1999) mice generally produce autoantibodies, they de-

velop immune-complex deposits and fatal SLE-like autoimmune

disease only on particular genetic backgrounds (e.g., MRL for lpr

mice or mixed C57BL/6x129SV for Bcl2l11�/� mice). On the

C57BL/6 background, Bcl2l11�/�mice develop lymphocyte infil-

trates in lung, liver, and kidneys around 1 year of age, but these

have no discernible consequences for their health. All Faslpr/lpr


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and Bcl2l11�/� mice examined between 16 and 42 weeks were

healthy, and histological examination did not show any signs of

severe autoimmunity. In contrast, all Bcl2l11�/�Faslpr/lpr and

even the Bcl2l11+/�Faslpr/lpr mice had become moribund by

250 days of age, with a median survival of 114.5 days for

Bcl2l11�/�Faslpr/lpr mice and 159 days for Bcl2l11+/�Faslpr/lpr

mice (Figure 6). Almost all of these animals had to be sacrificed

because they presented with labored breathing resulting from

filling of the chest cavity by large lymph nodes. Histological

examination revealed the presence of massive lymphocyte infil-

trates in the lungs, kidneys, and liver of Bcl2l11�/�Faslpr/lpr and

Bcl2l11+/�Faslpr/lpr mice (Figure 6A). All moribund mice also

tested positive for the presence of anti-DNA autoantibodies

(not shown). 20%–30% of the Bcl2l11�/�Faslpr/lpr and �10% of

the Bcl2l11+/�Faslpr/lpr mice also presented with urticarial rash

between the ears and in the neck area. In contrast, all Faslpr/lpr,

Bcl2l11�/�, and WT mice survived well beyond 250 days without

symptoms. Theseobservations demonstrate that Bim-dependent

mitochondrial apoptosis and Fas death receptor signaling co-

operate in hematopoietic cell homeostasis and prevention of im-

munopathology.

Figure 3. After Intranasal MHV-OVA Infection, Bcl2l11�/�Faslpr/lpr

Mice Accumulate More Viral Antigen-Specific CD8+ T Cells than

Bcl2l11�/�, Faslpr/lpr, or WT Mice

Mice were infected intranasally with MHV-OVA and sacrificed after 14, 40, or

60 days, and mediastinal lymph nodes and spleen were analyzed.

(A–D) Total leukocytes (A, B) and total numbers of CD8+ T cells (C, D) from

spleen and mediastinal lymph nodes were quantified by cell counting and

staining with CD8 mAbs followed by FACS analysis. Data represent the

mean ± SEM from 3–4 mice of each genotype. Lack of data at day 14 for

the Bcl2l11+/�Faslpr/lpr genotype is indicated with an asterisk.

(E–H) MHV-OVA-specific CD8+ T cells were measured by immunofluorescent

staining with PE-streptavidin-conjugated Kb/SIINFEKL tetramers plus anti-

bodies to CD8 (E, F), or, after brief antigenic stimulation in vitro, by intracellular

staining for IFN-g (G, H), both followed by FACS analysis. Lymph node

samples from mice of the same genotype were pooled. Data points represent

the mean ± SEM of 3–4 mice (2 mice at some time points for Bcl2l11+/�Faslpr/lpr

and Faslpr/lpr mice).

Figure 4. Loss of Bim and Fas Synergize in the Development of

Lymphadenopathy and Splenomegaly

Mice (WT, Bcl2l11�/�, Faslpr/lpr, Bcl2l11+/�Faslpr/lpr, and Bcl2l11�/�Faslpr/lpr)

were analyzed between the ages of 6 and 10 weeks. Spleen and lymph

node cells were counted and analyzed by immunofluorescent staining with

surface marker-specific antibodies and FACS analysis. Data represent

mean ± SEM from 3–6 mice of each genotype.


Immunity


DISCUSSION

Immune responses to an infectious pathogen or injection of a for-

eign substance result in the clonal expansion of antigen-specific

T cells and, upon pathogen or antigen clearance, this is followed

by contraction of this T cell population (Sprent and Tough, 2001).

The shutdown of T cell immune responses is mediated by their

programmed death (Kawabe and Ochi, 1991), but the mecha-

nisms for apoptosis induction remain controversial (Hildeman

et al., 2002; Krammer, 2000). This is (at least in part) due to the

use of different model systems. Culturing antigen- or mitogen-

activated T cells with IL-2 and restimulating them through the

TCR or with PMA plus ionomycin has long been used as the stan-

dard in vitro model to mimic the removal of activated T cells

in vivo (Krammer, 2000). Experiments with blocking antibodies

or FasL- or Fas-deficient mutant mice demonstrated that in

this setting, TCR restimulation causes the expression of FasL,

which induces autocrine and/or paracrine killing of Fas-express-

ing T cells (Brunner et al., 1995; Dhein et al., 1995; Russell et al.,

1993). This led to the conclusion that FasL-Fas signaling is re-

sponsible for the shutdown of T cell immune responses. Some

studies showed that deletion of SEB-activated T cells in mice

was in part FasL-Fas dependent (Mogil et al., 1995; Renno

et al., 1996), whereas others found that Fas deficiency (in

Faslpr/lpr mutant mice) had no impact but that loss of Bim pro-

vided profound protection (Hildeman et al., 2002). These

discrepancies might be due to differences in the regimes of

SEB injection. This is supported by the finding that effects of

Fas deficiency on deletion of activated T cells in vivo were ob-

served most consistently when mice were administered several

doses of SEB (Strasser et al., 1995). Interestingly, the death of

antigen-activated CD8+ T cells during shutdown of and acute

immune response to a viral (HSV-1) infection was substantially

inhibited by loss of Bim but was not affected by Fas deficiency

(Pellegrini et al., 2003). We therefore reasoned that depending

on the nature of an immune response—acute versus chronic—

Fas-mediated death receptor signaling and Bim-dependent

mitochondrial apoptosis might contribute to different extents to

the killing of activated T cells (Strasser and Pellegrini, 2004). Ac-

cordingly, a single low-dose challenge with SEB would mimic

a response to an acute infection, whereas high-dose and/or

repeated challenge with this superantigen would be expected

to resemble the response to a chronic infection.

Figure 5. Bcl2l11�/�Faslpr/lpr Mice Have a Normal Polyclonal Usage

of TCR-Vb Chains in Lymph Node T CellsTCR-Vb chain expression on lymph node T cells from Bcl2l11�/�Faslpr/lpr and

WT mice was analyzed by FACS after staining with mAbs to Thy-1 plus mAbs

to Vb3, Vb4, Vb5, Vb6, Vb7, Vb8, or Vb10. The percentages of T cells express-

ing each TCR-Vb chain and the mean are represented.


Our analysis of Bcl2l11�/�Faslpr/lpr mice demonstrates that kill-

ing of activated T cells during termination of a response to acute

infection relies predominantly on Bim with no apparent contribu-

tion by Fas death receptor signaling. In contrast, these two path-

ways synergize in the killing of T cells during shutdown of an

immune response to chronic infection, and they also cooperate

tomaintainhomeostasisof T cells, B cells, aswell asgranulocytes,

and to prevent autoimmune pathology. These results help recon-

cile current controversies regarding the relative contributions of

the death receptor versus the mitochondrial apoptotic pathway

to the shutdown of T cell immune responses. They are consistent

with the notion that during shutdown of an acute immune re-

sponse, killing of activated T cells is not elicited by repeated

TCR ligation, which would have been required for activation of

the Fas pathway (Hildeman et al., 2002; Pellegrini et al., 2003). In-

stead, apoptosis requires the BH3-only protein Bim, which is

thought to be activated by the drop in cytokine levels that occurs

after pathogen clearance (Strasser and Pellegrini, 2004). This fits

with the observations that Bim is critical for cytokine depriva-

tion-induced apoptosis (Bouillet et al., 1999) and transcriptionally

(Dijkers et al., 2000) as well as post-translationally (reviewed in Ley

et al., 2005) activated by this death stimulus. It is possible that

a drop in the levels of prosurvival Bcl-2 family members, particu-

larly Bcl-2 (Hildeman et al., 2002), and/or the induction of the

BH3-only protein Puma (encoded by the Bbc3 gene), which is

also involved in growth factor withdrawal-induced apoptosis of

hemopoietic cells (Jeffers et al., 2003; Villunger et al., 2003), are

also involved in the killing of antigen-activated T cells during shut-

down of an acute immune response. Indeed, Bim and Puma dou-

bly deficient T cells survive better in culture in the absence of cy-

tokines than those lacking only Bim or Puma (Erlacher et al., 2006).

Analysis of HSV-1-infected Bcl2l11�/�Puma�/� mice could help

determine whether these two BH3-only proteins have overlapping

functions in the shutdown of acute T cell immune responses.

The finding that Fas-mediated death receptor signaling and the

Bim-dependent mitochondrial apoptosis both contribute to shut-

down of chronic immune responses is consistent with the obser-

vations that loss of Bim (Grayson et al., 2006), Bcl-2 overexpres-

sion (Strasser et al., 1995), or loss of Fas (Kawabe and Ochi,

1991), each on their own, enhanced (to a relatively small extent)

survival of T cells chronically stimulated by repeated SEB injection

orLCMV infection.These results are consistentwith the notion that

the death receptor and the mitochondrial apoptotic pathways are

distinct and that they can cooperate in the removal of a cell popu-

lation (Strasser et al., 1995). Our results are reminiscent of the

effects observed upon the overexpression of Bcl-2 in Faslpr/lpr

mice. It is highly probable that Bcl-2 might have countered Bim

function in these mice. The importance of the genetic background

on the severity of the Faslpr/lpr and Bcl2l11�/� phenotypes is well

established. Direct comparison of the Bcl2-tg Faslpr/lpr mice and

our present study is limited by the fact that the mice used in the for-

mer study were on the C3H/HeJ background, whereas the

Bcl2l11�/�Faslpr/lpr mice are on the C57BL/6 background. It is,

however, possible that other BH3-only proteins may be involved

in the killing of chronically activated T cells, and lymphadenopathy

and splenomegaly may therefore be even more severe in Bcl2-tg

Faslpr/lpr mice than in Bcl2l11�/�Faslpr/lpr mice.

It appears likely that repeated TCR stimulation causes activa-

tion of the FasL-Fas signaling pathway in T cells during chronic

Immunity


infection (Brunner et al., 1995; Dhein et al., 1995), but how Bim is

induced in this setting is less clear. This might be a consequence

of a drop in the amounts of cytokines, similar to shutdown of

acute immune responses. Alternatively, the calcium flux elicited

by repeated TCR ligation might activate Bim (Bouillet et al.,

Figure 6. Accelerated Immunopathology and Premature Death

in Bcl2l11�/�Faslpr/lpr and Bcl2l11+/�Faslpr/lpr Mice

(A) Lungs (i, iv), kidney (ii, v), and liver (iii, vi) of a 260-day-old Bcl2l11+/+Faslpr/lpr

mouse (i, ii, iii) and a 114-day-old Bcl2l11�/�Faslpr/lpr mouse (iv, v, vi) showing

large lymphocyte infiltrates in Bcl2l11�/�Faslpr/lpr tissues. Scale bars represent

1 mm.

(B) Bcl2l11�/�Faslpr/lpr (n = 8) and Bcl2l11+/�Faslpr/lpr (n = 10) mice were found

dead in their cages at the indicated times. No Bcl2l11+/+Faslpr/lpr, Bcl2l11�/�

or WT mice (n = 10) were found dead by 250 days. p < 0.001 for both

Bcl2l11+/�Faslpr/lpr and Bcl2l11�/�Faslpr/lpr mice.

(C) Model for induction of apoptosis in T lymphocytes during shutdown of an

acute or chronic immune response. Single stimulation of the TCR in an acute

response triggers activated T cell death via Bim, whereas repeated stimulation

in a chronic infection recruits Fas to contract the activated T cell population.

2002), and, pertinently, Bim is essential for deregulated calcium

flux-induced apoptosis (Bouillet et al., 1999).

Combined loss of Fas and Bim also synergized in causing

lymphadenopathy, demonstrating that these two pathways co-

operate not only in the killing of T cells chronically activated by

pathogen-borne antigens but also those stimulated by self-anti-

gens. The unusual abTCR+CD4�CD8�B220+ T lymphoid cells

are seen only in Faslpr/lpr but not in Bcl2l11�/� mice, consistent

with the view that they are descendants of chronically activated

‘‘conventional’’ T cells, which previously expressed CD4 or CD8

(Renno et al., 1996) and would normally be killed by FasL-Fas

signaling (Brunner et al., 1995; Dhein et al., 1995). These unusual

T cells are as sensitive as conventional T cells to cytokine depri-

vation but can be protected by loss of Bim, providing a likely ex-

planation as to why their numbers are greatly increased in

Bcl2l11�/�Faslpr/lpr and even Bcl2l11+/�Faslpr/lpr mice compared

to animals lacking only Fas. Although loss of either Fas or Bim

causes fatal autoimmune disease on certain genetic back-

grounds (e.g., MRL for Faslpr/lpr [Watanabe-Fukunaga et al.,

1992] or mixed C57BL/6x129SV for Bcl2l11�/� [Bouillet et al.,

1999]), such pathology is not apparent for either mutation

on an inbred C57BL/6 background. The immunopathology

seen in Bcl2l11�/�Faslpr/lpr and Bcl2l11+/�Faslpr/lpr mice on this

background therefore demonstrates that these two apoptotic

pathways have overlapping functions in the prevention of

autoimmune disease.

These findings have implications for clinical medicine. For ex-

ample, abnormally increased levels of Bim and enhanced sensitiv-

ity toFasL (Gougeon,2003) havebeendetected in leukocytes from

patients infected with human immunodeficiency virus (HIV) or

other pathogens. Thus, Bim- and Fas-mediated apoptosis may

cooperate to cause the excessive killing of antigen-specific T cells

that is thought to impair clearance of the infectious microbes in

these settings. Conversely, the inability tokill T cells that are chron-

ically stimulated by self-antigens might underlie some autoim-

mune conditions. For example, because loss of one allele of Bim

synergizes with Fas deficiency to cause immunopathology, it is

possible that combinationsof mutantalleles of Bim and Fas, which

by themselves do not cause readily identifiable abnormalities, may

underlie certain autoimmune diseases in humans.

In summary, two major pathways regulate the life and death of

lymphocytes. The death receptor pathway involves membrane

receptors of the TNFR1 family, whereas the mitochondrial path-

way is regulated by proteins of the Bcl-2 family. Here, we show

that both pathways synergize to kill activated T cells in chronic

immune responses, whereas death of activated T cells in acute

immune responses relies only on the mitochondrial pathway.

EXPERIMENTAL PROCEDURES

Mice

All animal experiments were performed in accordance with the guidelines of the

Melbourne Directorate Animal Ethics Committee. Bcl2l11�/� and Faslpr/lpr mice

had been backcrossed onto a C57BL/6 genetic background for >20 genera-

tions prior to intercrossing and were housed in specific pathogen-free (SPF)

conditions prior to infectious challenge.

Immunofluorescent Staining, FACS Analysis, Cell Sorting, Tissue

Culture, and Cell-Survival Assays

Single-cell suspensions were prepared from thymus, spleen, and lymph nodes

(axillary, brachial, and inguinal) of 6- to 10-week-old mice and total leukocyte


Immunity


numbers counted. Leukocyte subpopulations were identified by immunofluo-

rescent staining with surface marker-specific mAbs coupled to FITC, R-PE,

APC, Cy5, or biotin (the latter revealed by staining with streptavidin coupled

to R-PE or Tricolor; Caltag) followed by flow cytometric analysis in a FACScan

(Becton Dickinson) or sorting in a FACS DIVA (Becton Dickinson) or MoFlo (Cy-

tomation). Sorted cells were resuspended in DMEM supplemented with 10%

FCS, 50 mM 2-mercaptoethanol (2-ME), and 100 mM asparagine and cultured

at 37�C in 96-well flat-bottom plates. Cells were incubated in medium alone

(no treatment, i.e., cytokine deprivation) or treated with FLAG-tagged human

recombinant FasL 10 ng/mL (Alexis) crosslinked with 1 mg/mL M2 FLAG mono-

clonal antibody (Sigma), ionomycin (1 mg/mL), PMA (10 ng/mL), etoposide

1 mg/mL, or g-irradiated (1.25 Gy). Cell survival was quantified daily by staining

with propidium iodide (PI; 2 mg/mL) and FACS analysis.

HSV Infection and Quantification of HSV Antigen-Specific

CD8+ T Cells

Mice were injected subcutaneously (s.c.) in each hind foot with 4 3 105 plaque-

forming units (PFU) of HSV-1 KOS strain under general anesthesia. After 17

and 35 days, mice were killed, and popliteal lymph nodes, spleen, liver, lungs,

and feet were removed. Single-cell suspensions were prepared from these tis-

sues, and those from the lymph nodes of mice of the same genotypes were

pooled. Lymphocytes were purified from liver and lung on Percoll density gra-

dients. To measure the percentages of viral antigen-specific T cells, single-cell

suspensions from spleen and lymph nodes were first enriched for CD8+ T cells

with magnetic bead depletion by staining with M1/70 (anti-Mac-1), F4/80

(macrophage marker), Ter 119 (erythroid marker), RB6-8C5 (anti-Gr-1),

M5/114 (anti-class II MHC), and GK 1.5 (anti-CD4) antibodies followed by

incubation with goat anti-rat IgG antibody-coupled magnetic beads (Dynal,

Oslo and QIAGEN). Enriched CD8+ cells were washed and stained for 45

min at room temperature with PE-streptavidin-conjugated Kb-gB tetramers

containing the immuno-dominant HSV gB498-505 peptide. Cells were then

stained for 30 min on ice with APC-conjugated CD8 mAb (53-6.7) in balanced

salt solution containing 2% FBS. PI staining was used to exclude dead cells

and data were acquired on at least 104 viable cells with a FACSCalibre flow

cytometer and analyzed with CellQuest Pro (BD Biosciences).

MHV Infection and Quantification of Viral Antigen-Specific

CD8+ T Cells by Staining with PE-Streptavidin-Conjugated

Kb-OVA Tetramers

Mice were infected under brief general anesthesia intranasally with 3 3 104

PFU of recombinant murine g-herpesvirus that coexpressed ovalbumin

(OVA) from an intergenic expression cassette driven by an ectopic MHV-68

lytic promoter (MHV 68-OVA). Infected mice were killed after 14, 40, or 60

days, and lungs, mediastinal lymph nodes, and spleen were collected. Sin-

gle-cell suspensions were prepared and leukocytes within the lymph nodes

and spleen were counted. Cells collected from the lymph nodes from mice

of the same genotype were pooled. Total numbers of CD4+ and CD8+ T cells

were measured by staining cells for 30 min on ice with PE-coupled anti-CD4

mAb (CT-CD4) and FITC-coupled anti-CD8 mAb (CT-CD8), and analysis was

performed as described above. To enumerate viral antigen-specific T cells,

suspensions from lymph nodes and spleen were enriched for CD8+ T cells

as described above, labeled for 30 min on ice with FITC-coupled anti-CD8

mAb, washed, stained for 45 min at RT with PE-coupled streptavidin conju-

gated to Kb class I MHC tetramers loaded with SIINFEKL peptide, and then

analyzed as described above.

Intracellular FACS Analysis of Cytokine Production

Approximately 1 3 106 cells from lymph nodes or spleen of Bcl2l11�/�Faslpr/lpr,

Bcl2l11+/�Faslpr/lpr, Faslpr/lpr, Bcl2l11�/�, and WT mice were incubated for 5 hr

at 37�C in RPMI 1640 supplemented with 50 mM 2-ME, 10% FBS, 5 mg/mL bre-

feldin A (which was also provided in all subsequent washes and incubations),

and 10 U/mL recombinant mouse IL-2 plus either 1 mM HSV gB498-505 pep-

tide for mice infected with HSV or 1 mM Ovalbumin257-265 (SIINFEKL) peptide

for mice infected with MHV-OVA or, as a control, with no peptide. Cells were

harvested, washed, stained for 30 min on ice with FITC-coupled anti-CD8

mAb, washed again, fixed for 15 min at 4�C with 1% formalin, permeabilized

for 10 min at RT in PBS containing 0.5% saponin and 1% BSA, stained for

30 min on ice with PE-coupled rat anti-IFN-g mAb (C1 XMG1.2), washed,


and analyzed on a LSR or FACSCalibre flow cytometer. The frequency of

virus-specific CD8+ T cells was calculated by subtracting the frequency of

CD8+ IFN-g+ T cells in samples stimulated with no peptide from the frequency

of CD8+ IFN-g+ T cells in samples with the antigenic peptide.

MHV-OVA Viral Titer Assays

Titers of infectious MHV-OVA virus were determined by plaque assay. In brief,

10-fold dilutions of (virus containing) spleen cell suspensions from infected

mice were incubated for 2 hr on BHK-21 cell monolayers. The inoculum was

then removed and the monolayers overlaid with Dulbecco’s modified Eagle

medium containing 0.3% carboxymethylcellulose. After 5 days, the mono-

layers were fixed in 10% formaldehyde and stained with 10% Giemsa (Merck)

for 30 min before rinsing with water. Monolayers were dried at room tempera-

ture overnight before plaques were counted with a plate microscope.

Analysis of Persisting Antigen Presentation

during MHV-OVA Infection

Persistent antigen presentation was evaluated by analyzing the induction of

proliferation of CFSE-labeled OVA-specific CD8+ T cells (from OT-I TCR trans-

genic mice) after injection into mice that had been infected with MHV-OVA for

various lengths of time. In brief, lymphocyte suspension from lymph nodes (in-

guinal, axillary, sacral, cervical, and mesenteric) of OT-I TCR transgenic mice

were enriched for CD8+ T cells by incubation with antibodies specific for Mac-1

(M1/70), Mac-3 (F4/80), erythrocytes (Ter-119), B220 (RA3-6B2), MHC class II

(M5/114), and CD4 (GK1.5). Antibody-coated cells were removed with goat

anti-rat IgG antibody-coated magnetic beads (QIAGEN). Purity was verified

by FACS analysis and was routinely found to be �95%. Enriched CD8+ T

cell preparations were resuspended at 107 cells/mL in PBS containing 0.1%

BSA and incubated for 10 min at 37�C with 5 mM CFSE stock solution (5 mM

in DMSO, Molecular Probes). 1 3 106 CFSE-labeled OT-I TCR transgenic T

cells were injected into naive or MHV-OVA-infected mice, and proliferation

was analyzed by FACS analysis after 3 days to determine the reduction in

CFSE staining intensity.

TCR-Vb Expression Analysis

TCR-Vb expression was analyzed by preparing cell suspensions from multiple

separate lymph nodes from Bcl2l11�/�Faslpr/lpr with massive lymphadenopa-

thy (aged approximately 12 weeks). Cells were stained with FITC-coupled

Thy-1 mAb (T24.31.2) and biotinylated mAbs specific for TCR Vb3, 4, 5, 6, 7,

8, or 10 (BD PharMingen), followed by R-phycoerythrin (PE)-streptavidin. PI

was used to exclude dead cells and cells were analyzed in a FACScan.

Supplemental Data

Seven figures are available at http://www.immunity.com/cgi/content/full/28/2/

197/DC1/.

ACKNOWLEDGMENTS

We are grateful to J. Adams, S. Cory, A. Harris, and D. Vaux for advice and dis-

cussions. We thank J. Coughlin, G. Siciliano, J. Allen, and S. Shehata for expert

animal care; B. Helbert and C. Young for genotyping; M. Robati for technical

assistance; F. Battye, V. Lapatis, and C. Tarlinton for cell sorting; and S. Mihaj-

lovic, E. Tsui, and A. Hasanein for histology. This work was supported by fel-

lowships and grants from the National Health and Medical Research Council

(Australia; #257502, #461263, and #356214), the Charles and Sylvia Viertel

Foundation, the Leukemia and Lymphoma Society (New York; SCOR grant

#7015), the Wellcome Trust (UK), the Howard Hughes Medical Institute, and

the National Cancer Institute (NIH, US; CA 80188 and CA 43540).

Received: October 24, 2007

Revised: December 9, 2007

Accepted: December 10, 2007

Published online: February 14, 2008

REFERENCES

Ashkenazi, A., and Dixit, V.M. (1998). Death receptors: signaling and modula-

tion. Science 281, 1305–1308.

http://www.immunity.com/cgi/content/full/28/2/197/DC1/

http://www.immunity.com/cgi/content/full/28/2/197/DC1/

Immunity


Bouillet, P., Metcalf, D., Huang, D.C.S., Tarlinton, D.M., Kay, T.W.H., Kontgen,

F., Adams, J.M., and Strasser, A. (1999). Proapoptotic Bcl-2 relative Bim

required for certain apoptotic responses, leukocyte homeostasis, and to

preclude autoimmunity. Science 286, 1735–1738.

Bouillet, P., Purton, J.F., Godfrey, D.I., Zhang, L.-C., Coultas, L., Puthalakath,

H., Pellegrini, M., Cory, S., Adams, J.M., and Strasser, A. (2002). BH3-only Bcl-

2 family member Bim is required for apoptosis of autoreactive thymocytes.

Nature 415, 922–926.

Brunner, T., Mogil, R.J., LaFace, D., Yoo, N.J., Mahboubi, A., Echeverri, F.,

Martin, S.J., Force, W.R., Lynch, D.H., Ware, C.F., et al. (1995). Cell-autono-

mous Fas (CD95)/Fas-ligand interaction mediates activation-induced apopto-

sis in T-cell hybridomas. Nature 373, 441–444.

Davey, G.M., Kurts, C., Miller, J.F., Bouillet, P., Strasser, A., Brooks, A.G.,

Carbone, F.R., and Heath, W.R. (2002). Peripheral deletion of autoreactive

CD8 T cells by cross presentation of self-antigen occurs by a Bcl-2-inhibitable

pathway mediated by Bim. J. Exp. Med. 196, 947–955.

Dhein, J., Walczak, H., Baumler, C., Debatin, K.-M., and Krammer, P.H. (1995).

Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature 373,

438–441.

Dijkers, P.F., Medema, R.H., Lammers, J.J., Koenderman, L., and Coffer, P.J.

(2000). Expression of the pro-apoptotic Bcl-2 family member Bim is regulated

by the forkhead transcription factor FKHR-L1. Curr. Biol. 10, 1201–1204.

Egle, A., Harris, A.W., Bouillet, P., and Cory, S. (2004). Bim is a suppressor

of Myc-induced mouse B cell leukemia. Proc. Natl. Acad. Sci. USA 101,

6164–6169.

Enders, A., Bouillet, P., Puthalakath, H., Xu, Y., Tarlinton, D.M., and Strasser,

A. (2003). Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim

inhibits BCR stimulation-induced apoptosis and deletion of autoreative B cells.

J. Exp. Med. 198, 1119–1126.

Erlacher, M., Laabi, V., Manzl, C., Bock, G., Tzankov, A., Haecker, G., Strasser,

A., and Villunger, A. (2006). Puma cooperates with Bim, the rate-limiting

BH3-only protein in cell death during lymphocyte development, in apoptosis

induction. J. Exp. Med. 203, 2939–2951.

Gougeon, M.L. (2003). Apoptosis as an HIV strategy to escape immune attack.

Nat. Rev. Immunol. 3, 392–404.

Grayson, J.M., Weant, A.E., Holbrook, B.C., and Hildeman, D. (2006). Role of

Bim in regulating CD8+ T-cell responses during chronic viral infection. J. Virol.

80, 8627–8638.

Green, D.R., and Kroemer, G. (2004). The pathophysiology of mitochondrial

cell death. Science 305, 626–629.

Hanahan, D., and Weinberg, R.A. (2000). The hallmarks of cancer. Cell 100,

57–70.

Hildeman, D.A., Zhu, Y., Mitchell, T.C., Bouillet, P., Strasser, A., Kappler, J.,

and Marrack, P. (2002). Activated T cell death in vivo mediated by pro-apopto-

tic Bcl-2 family member, Bim. Immunity 16, 759–767.

Huang, D.C.S., and Strasser, A. (2000). BH3-only proteins—essential initiators

of apoptotic cell death. Cell 103, 839–842.

Jeffers, J.R., Parganas, E., Lee, Y., Yang, C., Wang, J., Brennan, J., MacLean,

K.H., Han, J., Chittenden, T., Ihle, J.N., et al. (2003). Puma is an essential

mediator of p53-dependent and -independent apoptotic pathways. Cancer

Cell 4, 321–328.

Kawabe, Y., and Ochi, A. (1991). Programmed cell death and extrathymic

reduction of Vb8+CD4� T cells in mice tolerant to Staphylococcus aureus

enterotoxin B. Nature 349, 245–248.

Krammer, P.H. (2000). CD95’s deadly mission in the immune system. Nature

407, 789–795.

Ley, R., Ewings, K.E., Hadfield, K., and Cook, S.J. (2005). Regulatory phos-

phorylation of Bim: sorting out the ERK from the JNK. Cell Death Differ. 12,

1008–1014.

Mogil, R.J., Radvanyi, L., Gonzalez-Quintial, R., Miller, R., Mills, G., Theofilo-

poulos, A.N., and Green, D.R. (1995). Fas (CD95) participates in peripheral T

cell deletion and associated apoptosis in vivo. Int. Immunol. 7, 1451–1458.

Ogilvy, S., Metcalf, D., Print, C.G., Bath, M.L., Harris, A.W., and Adams, J.M.

(1999). Constitutive bcl-2 expression throughout the hematopoietic compart-

ment affects multiple lineages and enhances progenitor cell survival. Proc.

Natl. Acad. Sci. USA 96, 14943–14948.

Opferman, J., Iwasaki, H., Ong, C.C., Suh, H., Mizuno, S., Akashi, K., and Kors-

meyer, S.J. (2005). Obligate role of anti-apoptotic MCL-1 in the survival of

hematopoietic stem cells. Science 307, 1101–1104.

Pellegrini, M., Belz, G., Bouillet, P., and Strasser, A. (2003). Shut down of an

acute T cell immune response to viral infection is mediated by the pro-apopto-

tic Bcl-2 homology 3-only protein Bim. Proc. Natl. Acad. Sci. USA 100,

14175–14180.

Puthalakath, H., O’Reilly, L.A., Gunn, P., Lee, L., Kelly, P.N., Huntington, N.D.,

Hughes, P.D., Michalak, E.M., McKimm-Breschkin, J., Motoyama, N., et al.

(2007). ER stress triggers apoptosis by activating BH3-only protein Bim. Cell

129, 1337–1349.

Rajcani, J., and Kudelova, M. (2005). Murine herpesvirus pathogenesis:

a model for the analysis of molecular mechanisms of human gamma herpes-

virus infections. Acta Microbiol. Immunol. Hung. 52, 41–71.

Rathmell, J.C., Lindsten, T., Zong, W.-X., Cinalli, R.M., and Thompson, C.B.

(2002). Deficiency in Bak and Bax perturbs thymic selection and lymphoid

homeostasis. Nat. Immunol. 3, 932–939.

Renno, T., Hahne, M., Tschopp, J., and MacDonald, H.R. (1996). Peripheral T

cells undergoing superantigen-induced apoptosis in vivo express B200 and

upregulate Fas and Fas ligand. J. Exp. Med. 183, 431–437.

Rieux-Laucat, F., Le Deist, F., Hivroz, C., Roberts, I.A.G., Debatin, K.M.,

Fischer, A., and de Villartay, J.P. (1995). Mutations in Fas associated with

human lymphoproliferative syndrome and autoimmunity. Science 268,

1347–1349.

Russell, J.H., Rush, B., Weaver, C., and Wang, R. (1993). Mature T cells of

autoimmune lpr/lpr mice have a defect in antigen-stimulated suicide. Proc.

Natl. Acad. Sci. USA 90, 4409–4413.

Sprent, J., and Tough, D.F. (2001). T cell death and memory. Science 293,

245–248.

Strasser, A., and Pellegrini, M. (2004). T-lymphocyte death during shutdown of

an immune response. Trends Immunol. 25, 610–615.

Strasser, A., Harris, A.W., and Cory, S. (1991). Bcl-2 transgene inhibits T cell

death and perturbs thymic self-censorship. Cell 67, 889–899.

Strasser, A., Harris, A.W., Huang, D.C.S., Krammer, P.H., and Cory, S. (1995).

Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.

EMBO J. 14, 6136–6147.

Vabulas, R., Bittlingmaier, R., Heeg, K., Wagner, H., and Miethke, T. (1996).

Rapid clearance of the bacterial superantigen staphylococcal enterotoxin B

in vivo. Infect. Immun. 64, 4567–4573.

Van Parijs, L., Ibraghimov, A., and Abbas, A.K. (1996). The roles of costimula-

tion and Fas in T cell apoptosis and peripheral tolerance. Immunity 4, 321–328.

Van Parijs, L., Peterson, D.A., and Abbas, A.K. (1998). The Fas/Fas ligand

pathway and Bcl-2 regulate T cell responses to model self and foreign anti-

gens. Immunity 8, 265–274.

Veis, D.J., Sorenson, C.M., Shutter, J.R., and Korsmeyer, S.J. (1993). Bcl-2-

deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys,

and hypopigmented hair. Cell 75, 229–240.

Villunger, A., Michalak, E.M., Coultas, L., Mullauer, F., Bock, G., Ausserlech-

ner, M.J., Adams, J.M., and Strasser, A. (2003). p53- and drug-induced apo-

ptotic responses mediated by BH3-only proteins Puma and Noxa. Science

302, 1036–1038.

Wallach, D., Kovalenko, A.V., Varfolomeev, E.E., and Boldin, M.P. (1998).

Death-inducing functions of ligands of the tumor necrosis factor family:

a Sanhedrin verdict. Curr. Opin. Immunol. 10, 279–288.

Wang, X. (2001). The expanding role of mitochondria in apoptosis. Genes Dev.

15, 2922–2933.

Watanabe-Fukunaga, R., Brannan, C.I., Copeland, N.G., Jenkins, N.A., and

Nagata, S. (1992). Lymphoproliferation disorder in mice explained by defects

in Fas antigen that mediates apoptosis. Nature 356, 314–317.


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