Critical Reviews in Oncology/Hematology 66 (2008) 52–64

Concepts of activated T cell death

Dirk Brenner, Peter H. Krammer, Rüdiger Arnold ∗
Tumor Immunology Program, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
Accepted 16 January 2008

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2. Shut-down of the immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3. Regulation of AICD by cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4. NF-␬B is essential for T cell survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5. HPK1 controls the onset of AICD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
6. Suppression of NF-␬B induces AICD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
7. AICD can involve death receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
8. AICD and CD95 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
9. AICD independent of death receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
10. Activated T cell death without TCR restimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Abstract
Lymphocytes of the adaptive immune system play a crucial role in defending the organism against pathogens. Initial stimulation via antigen
receptors induces activation and proliferation of lymphocytes to generate effector cells that clear the pathogen from the body. During the
shut-down of the immune response activated lymphocytes are removed by two mechanisms. T cells that are restimulated during the end of the
immune response die by activation-induced cell death (AICD), whereas activated lymphocytes which are not restimulated die by activated cell
autonomous death (ACAD). Here, we discuss the regulation of AICD and ACAD in T cells and review the role of cytokines, T cell receptor
(TCR) proximal signaling mediators like hematopoietic progenitor kinase 1 (HPK1) and the NF-␬B pathway. We distinguish between AICD
dependent on or independent of death receptor ligation, and discuss caspase-independent death of T cells.
© 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Apoptosis; T cells; AICD; ACAD; Immune response; HPK1; NF-␬B

1. Introduction
Abbreviations: AICD, activation-induced cell death; ACAD, activated
Apoptosis is involved in T cell development, T cell recep-
T cell autonomous death; Bcl-2, B cell leukemia-2; BCR, B cell recep-
tor; CD95L, CD95-ligand; DISC, death-inducing signaling complex; FLIP, tor (TCR) repertoire selection and T cell homeostasis. T
FLICE-inhibitory protein; HPK1, hematopoietic progenitor kinase 1; HPK1- cells develop from common lymphocyte progenitors in the
C, C-terminal fragment of HPK1; IL-2, interleukin-2; IKK, I␬B kinase bone marrow and migrate to the thymus. In the thymus
complex; MAPK, mitogen-activated protein kinase; NF-AT, nuclear factor developing T cells pass through a series of distinct phases
of activated T cells; NF-␬B, nuclear factor regulating ␬-chain expression in
which are characterized by differential expression of cell
B cells; PKB/PKC, protein kinase B/C; TCR, T cell receptor; tg, transgenic.
∗ Corresponding author. Tel.: +49 6221 423769; fax: +49 6221 411715. surface receptors, like TCR, CD4 and CD8. The thymus
E-mail address: r.arnold@dkfz.de (R. Arnold). fosters the development of functional competent, but not

1040-8428/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.critrevonc.2008.01.002
 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64
self-reactive mature T cells by positive and negative selec-
tion. Positive selection ensures that the TCR is capable
to recognize its cognate ligand, the peptide binding major
histocompatibility complex (MHC). T cells whose TCR is
unable to bind to MHC do not gain survival signals and
undergo apoptosis in a process called ‘death by neglect’. Dur-
ing negative selection T cells are eliminated which respond
to MHC bound self-antigens presented within the thymus.
Therefore, T cell apoptosis during negative selection ensures
central tolerance. Maturation of T cells within the thymus
leads to the development of CD4+ and CD8+ single posi-
tive T cells which have diverse functions in the peripheral
immune system. CD4+ T cells develop into T helper cells
(Th) which can be further subdivided into Th1 and Th2
cells. The Th1 cells initiate cellular immune responses and
secrete, e.g. IFN-␥. The Th2 cells are important for humoral
immunity and secrete, e.g. IL-4, IL-5, IL-10 and TGF-␤.
Besides Th1 and Th2 cells several other subpopulations with
distinct cytokine profiles and functions have been identi-
fied. For example CD4+ CD25+ regulatory T cells (Treg),
which express high levels of IL-10 and TGF-␤, but little IL-
2 and IL-4 and suppress T cell functions in order to avoid
chronic activation or autoreactivity of T cells in the periph-
ery. Another immunosuppressive group of CD4+ T cells are
Th3 cells which seem to act via TGF-␤ as the dominant
cytokine.
In contrast to CD4+ T cells whose effector mechanisms
are mainly based on cytokine secretion, CD8+ T cells serve
as cytotoxic T cells which directly kill target cells. CD8+ T
cells are critical in controlling viral infections and infections
by intracellular bacteria. In addition CD8+ T cells are cru-
cial in host defense against some protozoa. Once a foreign
antigen is detected by CD8+ T cells on the surface MHC I
of an infected cell this cell is primed for death. Cytotoxic
T cells kill their target cells by inducing apoptosis mainly
by the release of lytic granules like perforin and granzymes
or in some cases by the ligation of the death receptor CD95
(APO1/Fas).
Naı̈ve T cells are found mainly in the peripheral lym-
phoid organs until they are activated by foreign antigens
presented by antigen presenting cells (APC). Upon activa-
tion, T cells enter a clonal proliferation phase and invade the
sites of infection or inflammation. After antigen clearance
the clonally expanded activated T cell population is removed
by apoptosis, which is essential for T cell homeostasis and
prevents the development of autoimmunity and lymphomas
[1]. Only a few T cells that have previously been exposed
to antigen survive and develop into memory T cells which
respond more rapidly to the same antigen than resting naive
T cells.
This review focuses on the mechanisms of apoptosis
induction which are critical for the removal of activated T
cells. We discuss the concepts of activation-induced cell death
(AICD) and activated T cell autonomous death (ACAD) and
summarize the involvement of various apoptosis pathways
and their molecular regulators.
53
Fig. 1. Scheme of a T cell immune response. Upon antigen challenge, T cells
are activated and proliferate. In the early expansion phase T cells are resistant
towards activation-induced cell death (AICD) or activated cell autonomous
death (ACAD). After reaching the peak of the immune response T cells
become sensitive towards cell death and T cell numbers decline during the
contraction phase. While ACAD is independent of secondary stimulation,
induction of AICD depends on restimulation via the T cell receptor.
2. Shut-down of the immune response
The immune system defends the organism against infec-
tions. Serving as the first line of defense the innate immune
system recognizes evolutionary conserved structures on dif-
ferent pathogens. Defenses by the adaptive immune system,
in contrast, are based on clonal selection of a diverse lym-
phocyte repertoire. After initiation of the immune response
lymphocytes proliferate and differentiate into effector cells.
This process is called expansion phase and is needed to gen-
erate sufficient numbers of effector T cells specific for the
pathogen (Fig. 1). Following the immune response activated
lymphocytes have to be eliminated. The high proliferative
capacity of antigen-activated lymphocytes requires an effec-
tive control of their life span in order to maintain lymphocyte
homeostasis. Programmed cell death (apoptosis) allows for
the decline of lymphocyte numbers during the contraction
phase of an immune response. Two concepts for the induction
of T cell death during the shut-down of an immune response
have been described: activation-induced cell death (AICD)
and activated T cell autonomous death (ACAD). In this con-
text AICD describes the induction of cell death in already
activated T cells by restimulation of their T cell receptors
(TCR) (Fig. 1, lower panel). During the expansion phase
of the immune response T cells possess an AICD-resistant
phenotype, whereas they gradually shift towards AICD sen-
sitivity during the contraction phase of the immune response
[2]. AICD can depend on or be independent of death recep-
tor engagement and therefore involves extrinsic or intrinsic
cell death pathways (see Sections 7–9). In contrast to AICD,
activated T cell death by ACAD during the contraction phase
54 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64
does not require restimulation of the TCR (Fig. 1, upper
panel). ACAD is not mediated by death receptors, depends
on the intrinsic cell death pathway and involves the mito-
chondria (see Sections 10 and 11) [3]. Most likely AICD
and ACAD cooperate during the shut-down of an immune
response. While AICD seems to be most important for elimi-
nation of chronically activated and potentially autoreactive T
cells [4], ACAD most prominently contributes to the down-
regulation of T cell numbers during the contraction phase
[5].
3. Regulation of AICD by cytokines
Upon initial TCR stimulation T cells are activated to elicit
an immune response. TCR restimulation of already acti-
vated T cells triggers AICD [6,7]. It has become evident that
AICD is an important mechanism to eliminate autoreactive
T cells and to ensure tolerance in the periphery [8]. Sensi-
tivity towards AICD is strictly controlled according to the
activation status of T cells [9–12].
One essential factor for the proliferation of T cells and sen-
sitization towards AICD is the cytokine interleukin-2 (IL-2)
[13]. Activation of naive AICD-resistant T cells triggers the
expression of IL-2 and its high affinity receptor subunit IL-
2R␣ (CD25) [14,15]. IL-2 is essential for T cell proliferation,
serves as survival factor and ensures T cell effector functions
to allow the development of a protective immune response
[16]. T cell survival mediated by IL-2 involves various sig-
naling pathways including proteins like signal transducer and
activator of transcription (STAT), mitogen-activated protein
kinase (MAPK) and protein kinase B (PKB/AKT). Signal-
ing via the IL-2R␤ chain and the SH2 domain-containing
transforming protein (SHC) induces PKB/AKT-mediated
expression of the anti-apoptotic factor Bcl-2, which protects
the cell against the induction of apoptosis [16]. PKB/AKT
and MAPK have been implicated in phosphorylation of the
Bcl-2 family members, either supporting their anti-apoptotic
function or inhibiting their pro-apoptotic function [17].
Besides its function as a survival factor, IL-2 is also
required for the sensitization towards AICD upon expansion
of T cells [18]. This sensitization is not found in T cells of
IL-2 deficient or IL-2R␣ deficient mice [19]. The molecu-
lar mechanisms by which IL-2 sensitizes T cells to AICD
are not completely understood. Increased expression of the
death receptor ligand CD95L (Fas-L/Apo-1L) (see Section
6) and decreased expression of the anti-apoptotic molecule
c-FLIP (see Section 7) are suggested to be IL-2 dependent
[19]. The IL-2 inducible T cell kinase (Itk) which belongs
to the Tec family of tyrosine kinases may play an important
role in this process [20]. Members of this kinase family have
an impact on TCR-mediated calcium (Ca2+ ) mobilization and
are known to activate phospholipase C␥1 (PLC␥1) [21]. Ca2+
signaling, however, is a prerequisite for expression of CD95L,
another molecule involved in AICD [8]. In accordance with
these studies Itk-deficient mice show reduced TCR-induces
Ca2+ mobilization as well as a reduced expression of CD95L
[22]. In addition, Itk-deficient T cells lack Egr2 and Egr3
expression. Egr2 and Egr3 are two transcription factors which
have been implicated in TCR-induced CD95L expression
[23]. It is not clear how exactly IL-2 and TCR-stimulation
cooperate in CD95L expression as both stimuli use differ-
ent pathways to induce the CD95L gene [24]. Sensitization
towards AICD is accompanied by T cell proliferation [25]
which is dependent on IL-2, and it is conceivable that other
mechanisms than expression of CD95L contribute to IL-2-
driven sensitization towards apoptosis.
The cytokine IL-15 shares several functions with IL-2. The
receptors for both cytokines share the beta (IL-2R␤) chain
and the common gamma (␥c ) chain and differ only in their
alpha chains (IL-2R␣ vs. IL-15R␣). Decreased IL-2 levels
in IL-15 transgenic mice were reported to interfere with sur-
vival and death during AICD resulting in elevated levels of
CD8+ , CD44+ memory T cells [26]. Increased survival of
CD8+ memory T cells was also found in wild type mice upon
repeated IL-15 injection [27,28]. Therefore, both cytokines
stimulate T cell proliferation and act as a survival factor, but
IL-15 can inhibit the IL-2 dependent sensitization towards
AICD.
Two other cytokines which positively influence AICD by
different mechanisms are IL-4 and interferon-␥ (IFN-␥). In T
cells derived from IL-4 deficient mice, AICD is significantly
reduced [29]. Interestingly, IL-4 sensitizes towards AICD via
an IL-2-dependent mechanism which involves transcriptional
upregulation of the IL-2 receptor. As a consequence, AICD
resistance of IL-4 deficient T cells could be overcome by
exogenous addition of IL-2. On the molecular level IL-4 and
IL-2 seem to be necessary for degradation of the apoptosis
inhibitor cellular FLICE-inhibitory protein (c-FLIP) which
correlates with AICD sensitivity [29]. T cells from IFN-
␥-deficient mice are also resistant to AICD [30]. In CD4+
T cells, IFN-␥ controls caspase-8 expression in a STAT-
dependent manner. Consequently, rescue of IFN-␥-deficient
T cells with exogenous IFN-␥ restores caspase-8 levels and
susceptibility to AICD.
4. NF-␬B is essential for T cell survival
It is presently still unsolved how the TCR signal is inte-
grated to provide activation, proliferation and survival upon
initial stimulation and cell death by AICD upon restimula-
tion. TCR stimulation can trigger gene expression of proteins
with anti-apoptotic functions, like c-FLIP, Bcl-2, Bcl-XL ,
Bcl-2A1, IAP/XIAP [31] or GADD45␤ [31–33] and proteins
with pro-apoptotic functions like CD95L [34] and Nur77
[35] (Fig. 2). The balance between expression of pro- and
anti-apoptotic proteins determines T cell fate and is largely
dependent on the type of signal received by the cell, e.g.
strength of TCR stimulation and costimulation and the pres-
ence of intracellular signaling modulators downstream of the
TCR, e.g. adapter molecules and kinases [36].
 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64
Fig. 2. Pro- and anti-apoptotic genes are induced by TCR stimulation. Stim-
ulation of the T cell receptor (TCR) by its cognate ligand induces the
expression of pro-apoptotic as well as anti-apoptotic genes. A shift of the
equilibrium induces activation and survival on the one side or death by
AICD on the other side. TCR stimulation can induce gene expression of
the anti-apoptotic molecules c-FLIP, Bcl-2, Bcl-XL , Bcl-2A1, IAP/XIAP,
GADD45␤ and the pro-apoptotic molecules CD95L, and Nur77.
Naı̈ve and activated T cells interpret the same signal
in two different ways. Recent studies emphasize that dif-
ferential regulation of NF-␬B influences the transition of
AICD-resistant to AICD-sensitive T cells [33,37,38]. Regu-
lation of lymphocyte fate and execution of immune functions
are connected with transcription factors of the NF-␬B fam-
ily [31,39,40]. NF-␬B determines life and death decisions
in developing lymphocytes and provides important signals
in T cells to ensure cell survival [41,42]. NF-␬B activa-
tion in response to antigen receptor ligation is mediated
by a high molecular weight I␬B kinase (IKK) complex
[43]. The IKK complex comprises two enzymatic subunits,
IKK␣ and IKK␤, and the regulatory subunit IKK␥ (NEMO)
[44]. Activation of the IKK complex upon stimulation of
the T cell receptor (TCR) depends on the caspase recruit-
ment domain (CARD)-containing signaling adapter protein
Carma1 (CARD11) [45]. TCR stimulation leads activation
of protein kinase C (PKC)␪ which in turn phosphorylates
Carma1 within its linker region between the coiled-coil and
the PDZ domains [46–48]. Subsequently, Carma1 interacts
with the CARD domain of Bcl10, which recruits the adaptor
protein Malt1 [49]. The Malt1-interacting E3 ubiquitin ligase
TRAF6 triggers Lys63-linked polyubiquitinylation of IKK␥
and induces activation of the IKK complex in T cells [50].
NF-␬B/Rel proteins are dimeric, sequence-specific tran-
scription factors which are rendered inactive within the
cytoplasm by interaction with inhibitory I␬B proteins [43].
Activation of the IKK complex results in phosphorylation and
subsequent ubiquitination and degradation of the inhibitor
of NF-␬B (I␬B). Liberated NF-␬B dimers translocate to the
nucleus where they bind and transactivate promoter regions
of NF-␬B-regulated genes [44].
55
Contrary to activation of NF-␬B, inhibition of NF-␬B
is considered to fulfill an important pro-apoptotic function
[51,52]. It has been shown that the onset of AICD correlates
with reduced NF-␬B activity in the nucleus [33]. One molecu-
lar regulator of NF-␬B involved in AICD is the hematopoietic
progenitor kinase 1 (HPK1) which is discussed in the next
section.
5. HPK1 controls the onset of AICD
Recently it was shown that the TCR-proximal kinase
hematopoietic progenitor kinase 1 (HPK1) is involved in the
regulation of cell death in primary T cells [37,53]. HPK1
is a serine–threonine kinase which is exclusively expressed
in hematopoietic tissues and activates the c-Jun N-terminal
kinase (JNK) pathway [54] and the NF-␬B pathway [55].
HPK1 is activated upon stimulation of immunoreceptors
in T and B cells [56,57] and is recruited to the immune
synapse [58,59]. During the expansion phase of an immune
response, AICD-resistant T cells contain full-length HPK1,
which is an essential mediator of TCR-induced NF-␬B sig-
naling (Fig. 3). Transient knockdown of HPK1 in T cell lines
blocks activation of IKK and NF-␬B [37]. In highly prolif-
erating and viable T cells during expansion HPK1 is cleaved
into its N- and C-terminal fragments [37]. Despite of HPK1
being a caspase target [55,60] this cleavage is not associ-
ated with apoptosis induction [61], but it is crucially needed
for the induction of AICD sensitivity during T cell expan-
sion [62]. The presence of the C-terminal cleavage product
of HPK1 (HPK1-C) sensitizes T cells towards AICD [37,53]
Fig. 3. HPK1-C sensitizes T cells to AICD by suppression of NF-␬B activity.
Role of hematopoietic progenitor kinase 1 (HPK1) in T cell receptor (TCR)-
mediated AICD. HPK1 is cleaved into HPK1-N and HPK1-C in T cells.
HPK1-C renders T cells AICD-sensitive. HPK1-mediated I␬B Kinase (IKK)
activation in AICD-resistant T cells leads to NF-␬B activation by liberation
of the NF-␬B heterodimer (p65/p50) from its inhibitor I␬B. The presence of
HPK1-C blocks IKK activation downstream of the TCR, thereby blunting
nuclear translocation of NF-␬B and suppressing NF-␬B target gene expres-
sion. Therefore, full length HPK1 confers NF-␬B activation and resistance
towards AICD while HPK1-C is blocking NF-␬B and sensitizes towards
AICD.
56 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64
HPK1-C inhibits NF-␬B activation [55] and sequesters the
inactive IKK complex by binding to IKK␣ and IKK␤ (Fig. 3)
[37].
Presence of HPK1 as a full-length or a truncated molecule
leads to different results upon TCR stimulation. Full-length
HPK1 supports activation of NF-␬B, resistance towards
AICD and T cell proliferation during the expansion phase.
Presence of HPK1-C results in inhibition of NF-␬B acti-
vation, sensitization towards AICD and shut-down of an
immune response. As TCR stimulation results in expres-
sion of proteins with pro- and anti-apoptotic functions,
HPK1-C-mediated blocking of the anti-apoptotic NF-␬B
pathway shifts the equilibrium towards apoptosis and sen-
sitizes towards AICD (Fig. 3).
It is tempting to speculate that conversion of HPK1 to
HPK1-C shifts the outcome of the very same TCR signal
simply by modulation of gene expression. A similar conclu-
sion was drawn by a recent report correlating loss of nuclear
location of the NF-␬B proteins p65/RelA with induction of
AICD [33]. Therefore, the HPK1/HPK1-C ratio might serve
as a molecular timer for the T cell to sense age and limit its
life span.
6. Suppression of NF-␬B induces AICD
Several other groups have linked NF-␬B-suppression
to susceptibility towards cell death [31,63,64]. The NF-
␬B-dependent inhibition of the expression of the tumor
suppressor p73 is required for the survival of antigen-
stimulated T cells [38], while expression of p73 has been
shown to be essential for AICD [65]. Furthermore, transgenic
expression of the IKK-inhibitory protein I␬B␣ increases
TCR-induced AICD [38,66].
Another mechanism depending on NF-␬B-inhibition and
considered to exhibit important pro-apoptotic function is the
generation of reactive oxygen species (ROS). TCR stimula-
tion leads to fast generation of ROS [34]. A recent study has
shown that the core components of TCR signaling includ-
ing ZAP70, LAT, SLP76 and PLC␥1 are crucial for ROS
induction [67]. ROS production is mediated by the mito-
chondrial respiratory chain complex I involving NADPH
oxidase II [67,68]. Interestingly, PKC␪ which is activated
by TCR stimulation translocates to the mitochondria and
is required for activation-induced generation of ROS [67].
Increasing ROS levels suppress Bcl-2 expression and thereby
initiate the mitochondrial death pathway [69]. Furthermore
ROS has been shown to be essential for CD95L-induction
in response to stimulation of the TCR [34]. Recent studies
have shown that ROS production alone is not sufficient to
efficiently induce expression of CD95L, but requires Ca2+
mobilization in addition [34]. Stimulation of the TCR results
in increased intracellular Ca2+ levels depending on PLC-␥1
and possibly also on ROS itself [70]. Therefore, the com-
bined action of Ca2+ and ROS leads to efficient induction of
CD95L expression and sensitizes towards AICD.
Recently it has been reported that HIV-1-expressed
trans-activator of transcription (HIV-1 Tat) can enhance or
substitute for ROS signaling in T cells [34]. This might
explain strongly enhanced AICD of T cells from AIDS
patients [71,72]. During progression of AIDS, HIV-1 Tat
and CD4+ -dependent Ca2+ mobilization induce expression
of CD95L and lead to T cell depletion by AICD.
7. AICD can involve death receptors
It is commonly believed that AICD involves the engage-
ment of death receptors, like CD95 (Apo-1/Fas) and the
TNF receptor. Apoptosis mediated by CD95 is essential
for T cell homeostasis since defects within this pathway
cause abnormal T cell accumulation, lymphadenopathy and
autoimmunity [73]. Three naturally occurring mutations have
been widely used to study the impact of the CD95/CD95L-
system in T cell AICD. In lpr mice a retrotransposon is
inserted into intron 2 of the CD95 gene leading to a dramatic
decrease in CD95 surface expression [74]. An additional
mutation which results in a phenotype congenic to lpr is
lprcg . The lprcg phenotype is caused by a single point
mutation within the death domain of CD95 which abro-
gates apoptotic signaling [75]. The gld mutation results in
the generation of a defective CD95L [76]. T cells from
mice with these mutations show reduced but clearly evi-
dent AICD in the presence of IL-2 [77]. Until now it is
unclear which CD95/CD95L-independent mechanism medi-
ates AICD under these conditions.
Although it has been suggested that tumor necrosis factor
(TNF) might be involved, its role in AICD is controversial.
While TNF and TNF receptor deficient mice show normal
AICD, blocking of TNF reduces TCR-induced cell death in
vitro and in vivo [78,79]. TCR stimulation can induce expres-
sion of TNF and its role might be comparable to the role
of the CD95/CD95L system in the early phase of AICD.
TNF, however, might be important in a late phase of AICD
[80].
Another death receptor which has been described to medi-
ate AICD of CD8+ T cells depending on CD4+ T cell help
is TNF-related apoptosis-inducing ligand (TRAIL) recep-
tor [81]. CD8+ T cells which have been primed in the
presence of CD4+ T cells acquire the ability to enter a sec-
ond, restimulation-dependent round of clonal proliferation.
In contrast to that, CD8+ T cells which have been initially
primed without CD4+ T cell help (helpless T cells) do not
respond with a second round of clonal proliferation, but
undergo AICD mediated by TRAIL. Therefore it is thought
that regulation of TRAIL expression by CD4+ T cells is
an important mechanism for CD8+ memory generation and
adaptive immunity.
It has also been shown that redistribution of death
receptors into lipid rafts enhances the susceptibility to
apoptosis. TCR stimulation of CD4+ T cells results in
a redistribution of CD95 into lipid rafts [82]. Interest-
 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64 57

ingly, unlike antigen receptors which accumulate in lipid
rafts upon triggering, reorganisation of CD95 is indepen-
dent of CD95L-ligation. The TCR-induced translocation of
CD95 into lipid rafts increases sensitivity to CD95-induced
apoptosis and therefore may account for the clonotypic
specificity of restimulated T cell death during immune
responses.
The complexity is further increased by the fact that
blocking of death receptors alone does not completely res-
cue from TCR-induced T cell death. Caspase-independent
mechanisms have been described as well as a granzyme
B-dependent pathway [83]. It is important that T cells use
various mechanisms to execute AICD. The different path-
ways allow a fine-tuned regulation of AICD and might enable
different T cell subtypes to respond differently to TCR res-
timulation.
8. AICD and CD95
Stimulation of CD95 by its cognate ligand CD95L induces
the formation of the death-inducing signaling complex
(DISC) [84] (Fig. 4). The CD95 DISC contains the oligomer-
ized, most likely trimerized CD95 and a death domain (DD)
containing adaptor molecule Fas-associated death domain
(FADD). FADD is recruited to CD95 by homotypic DD
interactions and mediates interaction and dimerization of
procaspase-8a (FLICE, MACH-␣1, Mch5␤), procaspase-8b
(MACH-␣2), procaspase-10 and procaspase-2 [85–87]. The
precise molecular events of CD95-induced caspase activa-
tion according to the ‘induced proximity model’ are reviewed
elsewhere [87,85].
The cellular FLICE-inhibitory protein (c-FLIP) is a potent
inhibitor of caspase activation at the DISC [88]. c-FLIP
resembles a catalytic inactive procaspase with a tandem DED.
The ratio between c-FLIP and the procaspases at the DISC
determine whether apoptosis is initiated or not [85]. Several
different c-FLIP-isoforms are described on the protein level
including a long and a short isoform, c-FLIPL and c-FLIPS
[89,90], and an isoform which was recently found in Raji
cells, c-FLIPR [91,92].
c-FLIPL and c-FLIPS have been implicated in regulation
of AICD [19,93]. Upon initial T cell activation c-FLIPS is
strongly upregulated [94,95]. This increase in expression of
c-FLIP correlates with the resistance of freshly activated T
cells towards CD95-induced apoptosis. In expanded AICD-
and CD95-sensitive T cells c-FLIPS levels decline, suggest-
ing that c-FLIPS might be the switch that renders T cells
susceptible to CD95-dependent AICD [96]. Downregulation
of c-FLIP seems to depend on T cell proliferation and it is
declining in an IL-2 dependent manner most likely due to
the G1/S transition of c-FLIP protein expression [13,95,97].
The important role of c-FLIPS in AICD is underlined by
the observation that costimulation via CD28 rescues T cells

Fig. 4. Extrinsic CD95L-dependent AICD-pathways. Freshly activated T cells (left) form low amounts of death-inducing signaling complex (DISC). Under
this condition CD95 engagement by CD95L activates low amounts of caspase-8. Caspase-8 cleaves Bid to tBid which migrates to the mitochondria. In freshly
activated T cells the pro-apoptotic ability of tBid is blocked by elevated levels of anti-apoptotic Bcl-XL . The presence of c-FLIP blocks activation of caspase-8
at the DISC. As a consequence these T cells are resistant to CD95-dependent AICD. Long-term activated T cells form large amounts of DISC. This results
in robust activation of caspase-8 which subsequently leads to activation of caspase-3 and the execution of apoptosis. Additionally, long-term activated T cells
exhibit low Bcl-XL levels which allow activation of the mitochondrial death pathway by tBid.
58 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64
from TCR-induced apoptosis [42,94]. This costimulation is
followed by upregulation of c-FLIPS which abrogates CD95-
induced procaspase-8 activation at the DISC.
Another mechanism which influences CD95-induced
apoptosis sensitivity and which can be linked to AICD is
the capacity of differential DISC formation [95,98]. Freshly
activated T cells show low activation of caspase-8 and depend
on amplification of the apoptotic signal by the mitochondria.
In these cells low amounts of caspase-8 are activated which
cleave the cytosolic Bcl-2 family member Bid (Fig. 4). Trun-
cated Bid (tBid) migrates to the mitochondria, where it fulfils
its pro-apoptotic role and induces the release of cytochrome
c (cyt c) into the cytosol. This is followed by formation of the
apoptosome, caspase-9 activation and subsequent activation
of caspase-3 and execution of cell death.
The resistance of freshly activated T cells against CD95-
induced apoptosis is caused by c-FLIP and Bcl-XL which
are upregulated upon initial T cell activation. While c-FLIP
lowers the amount of caspase-8-activation Bcl-XL blocks the
mitochondria-dependent cell death pathway (Fig. 4) [95,96].
During the expansion phase of the immune response T cells
shift towards AICD-sensitivity. In these T cells triggering
of CD95 activates caspase-8 in sufficient amounts to exe-
cute apoptosis independently of the mitochondrial pathway.
In addition, T cells switch from a Bcl-XL high to a Bcl-XL
low state allowing activation of the mitochondrial cell death
pathway [95].
T cell activation itself influences the susceptibility to
CD95-induced apoptosis by two mechanisms: First, initial
T cell activation leads to upregulation of CD95 and second,
TCR-stimulation leads to CD95L-independent relocalization
of CD95 into lipids rafts and thereby increases the sensitivity
to clonotypic deletion [82]. In summary, CD95-dependent
cell death is an important mechanism in AICD. The regu-
lation of this pathway is complex and involves alterations
in signal transduction, caspase-8 activation at the DISC and
localization of CD95.
Several studies highlight the role of PKC␪ in CD95-
dependent AICD. PKC␪ can synergize with calcineurin to
provide robust CD95L expression upon TCR stimulation
[99]. This was further supported by a recent study show-
ing that T cells from PKC␪ knock out mice fail to induce
CD95L [100]. TCR-mediated induction of CD95L is depen-
dent on the transcription factors NF-AT, AP-1 and NF-␬B
[23,101–104]. In this scenario PKC␪ is crucial for full
NF-AT activation as mutations of the NF-AT binding sites
within the CD95L promoter abrogate the ability of PKC␪ to
induce CD95L. In addition, PKC␪ seems to influence CD95-
dependent cell death directly. CD4+ T cells from PKC␪
deficient mice are less sensitive towards CD95-induced apop-
tosis, which is supported by a weaker activation of caspase-8
and Bid compared to wild type T cells [105]. Deletion
of PKC␪ differentially affects CD8+ and CD4+ T cells by
impairing BclXL expression upon activation [106]. PKC␪
might also mediate cell survival by phosphorylation of Bad
[107]. In summary, PKC␪ confers activation and survival of
antigen-activated T cells, but is also required for expression
of CD95L during AICD.
9. AICD independent of death receptors
The initiation of an adaptive immune response is a strictly
regulated process and it is largely dependent on the nature of
the antigen. Besides Th1, Th2 and Th3 cells, recent stud-
ies have identified a new T helper population which was
named Th17. Production of IL-17 and also of IL-22, was
found to be characteristic for this subpopulation [108]. How-
ever in this review we will focus on Th1 and Th2 cells. The
decision between Th1 and Th2 depends on cytokines and
affinity of the antigen to the TCR. Th1 vs. Th2 cells direct
the outcome of the adaptive immune system towards cell-
based vs. antibody-based immunity, respectively. While Th1
cells enable macrophages to efficiently destroy intracellu-
lar microorganisms, Th2 cells activate the antibody-based
defense by driving B cell differentiation and eosinophil/mast
cell immune responses. In a physiological situation one of
the two subsets prevails. In a pathologic situation prevalence
of one of the subsets might induce autoimmunity, allergic
reactions or chronic infections.
AICD of Th1 and AICD of Th2 cells differs in several
points. It has been reported that Th1 cells are more suscepti-
ble to AICD compared to Th2 cells [109]. This is consistent
with various reports showing an increased CD95L sensitiv-
ity in Th1 cells. Consequently Th1 or Th2 cells can kill Th1
but not Th2 target cells [110–112]. A study showed that in
contrast to Th1, Th2 cells undergo AICD independently of
CD95/CD95L engagement, but dependent on granzyme B
[83]. The serine protease granzyme B, which is present in
intracellular lytic granules, was first defined to play a role
in the effector function of cytotoxic T lymphocytes [113].
Upregulation of granzyme B expression in response to TCR
stimulation was shown in Th2 cells and could be abrogated
by the Th2 survival factor vasocative interstine peptide [114].
In the presence of granzyme B AICD of Th2 cells could not
be prevented by blocking of CD95L and AICD of Th2 cells
from granzyme B-deficient mice was completely abrogated
[82,83]. This indicates that AICD of Th2 cells is completely
dependent on granzyme B. Furthermore, accumulation of
Th2 cytokines in granzyme B deficient mice increased the
susceptibility to allergen-induced asthma [83]. This under-
lines the physiological significance of TCR-induced AICD
in controlling a balanced immune response.
Recently, we have shown that T and B lymphocytes from
HPK1-C transgenic mice undergo AICD independently of
the CD95/CD95L system, but involve caspase-9 [62]. Knock
down of HPK1/HPK1-C or Bim by siRNA showed that
CD95L-dependent and HPK1/HPK1-C-dependent cell death
pathways complement each other in AICD of primary T
cells. These results define HPK1-C as a mediator of CD95L-
independent AICD, which contributes to the removal of
activated lymphocytes.
 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64 59

10. Activated T cell death without TCR restimulation
Recently, a novel concept of activated T cell death has
been established which has been named activated T cell
autonomous death (ACAD) [39]. In contrast to AICD, ACAD
does not require TCR restimulation and is independent of
death receptor engagement. Several in vivo studies show
that the T cell contraction phase at the end of the immune
response, when ACAD takes place, may be largely indepen-
dent of CD95 signaling [5,115].
ACAD is also referred to as death by neglect, death by
cytokine withdrawal or passive cell death. These terms reflect
the common finding that deprivation of survival factors ini-
tiates T cell death. Nevertheless, ACAD is an active and
cell autonomous program [5]. Several studies suggest that
ACAD is regulated by the intrinsic cell death pathway and
involves members of the Bcl-2 family (Fig. 5). In general,
the Bcl-2 family members are known to have pro- and anti-
apoptotic functions within a cell [17]. On the structural level
the Bcl-2 family is divided into three subgroups: The first
group exhibits anti-apoptotic activities and shares the BH1-4
homology domains. Bcl-2 and Bcl-XL are members of this
group. The second group with its members Bax and Bak is
clearly pro-apoptotic and characterized by three BH domains
(BH1-3). The BH3-only proteins are the third group of the
Bcl-2 family proteins and share the BH3-domain, a nine
amino acid sequence. In contrast to the other groups, BH3-
only proteins are more regulators than executioners of death
and are localized in various organelles or in the cytoplasm
[17]. Under survival conditions they are held in an inactive
state. The pro-apoptotic BH3-only protein Bid for example
requires cleavage to tBid in order to migrate to the mito-
chondria and induce apoptosis. Therefore Bid might serve as
an amplifier of a low caspase or proteolytic activity within
the cell. Another example of a BH3-only protein controling
the health status of the cell is Bad. Bad is phosphorylated
by the kinase PKB/AKT [116,117]. PKB/AKT exhibits anti-
apoptotic functions and phosphorylation of Bad keeps the
molecule in an inactive conformation. Diminished PKB/AKT
activity is followed by a dephosphorylation and migration of
Bad to the mitochondria where it cooperates with Bax and
Bak to induce apoptosis [17,116,117]. The BH3-only protein
Bim also has to be modified, most likely phosphorylated, to
execute its apoptotic function [17]. In addition to posttransla-
tional modification, regulation of Bim transcription by TCR
stimulation has been described [118].
In general BH3-only proteins serve as stress sensors and
control the viability of a cell. In the absence or the pres-
ence of certain stimuli they get activated and fulfill their
pro-apoptotic function. From studies with Bax/Bak double
knock out mice we know that BH3-only proteins need Bax
and Bak to execute apoptosis thereby following a defined
hierarchy [119]. There is an ongoing debate about how BH3-
only proteins induce apoptosis (details reviewed elsewhere)

Fig. 5. ACAD is mediated by Bim and declining Bcl-2 levels. Inactive Bim is sequestered by the dynein motor complex and is bound to microtubules. Bcl-2
levels are sufficient to inactivate the pro-apoptotic function of Bax and Bak (left). Induction of cell death, e.g. by growth factor deprivation, leads to the release
of Bim from microtubules and allows its migration to the mitochondrion. At the peak of an immune response Bcl-2 levels decline and activated Bim can now
cooperate with Bax and Bak to override the anti-apoptotic function of Bcl-2 and to induce the release of cytochrome c (cyt c) from the mitochondria. cyt c,
APAF1 and procaspase-9 form the apoptosome and activate caspase-9. As a consequence, caspase-9 activates caspase-3/7 which then induces cell death.
60 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64
[17]. The ratio between pro- and anti-apoptotic Bcl-2 family
members determines whether the cell dies or survives.
It is important to note that Bcl-2 proteins influence the
strength and the prolongation of the immune response.
Expression of a Bcl-2 transgene increases the T cell response
in vivo [120]. T cells from mice challenged with different
antigens show a decrease in Bcl-2 and Bcl-XL -levels at the
peak of the immune response compared to non-activated T
cells [5,120,121]. Interestingly, memory cells which are not
deleted in the T cell contraction phase show elevated Bcl-2
levels that might protect them from ACAD [122]. In a T cell
activation model in which the superantigen staphylococcus
enterotoxin B (SEB) is used to activate T cells, transgenic
expression of Bcl-2 inhibits ACAD. This is clearly not the
case in lpr-mice which are bearing a CD95-mutation [5].
This further supports the finding that Bcl-2 family members
and the CD95-system regulate different apoptosis pathways
within T cells. Several groups have reported a CD95/CD95L-
independent apoptosis pathway after application of SEB in
vivo [5,13,123,124]. Some studies, however, show exactly the
opposite effect [125,126]. These controversial findings might
arise from different doses of SEB or different experimen-
tal models used in the studies. Nevertheless, these findings
led to the hypothesis that T cell deletion in the presence of
low amounts of antigen might be preferentially mediated via
ACAD whereas high antigen load results in AICD depending
on CD95/CD95L engagement.
The complexity is further increased by the finding that
T cells which are deficient for the BH3-only protein Bim
persist after SEB-injection in vivo [5]. Interestingly, these
Bim-deficient T cells show the same downregulation of Bcl-2
as their wild-type counterparts, which indicates that down-
regulation of Bcl-2 alone – by a ROS-dependent mechanism
[69] – is insufficient to induce ACAD. This suggests a two-
step mechanism for the induction of ACAD involving Bcl-2
and Bim (Fig. 5). As Bim levels change only marginally upon
T cell activation it is tempting to speculate that Bim is some-
how activated, released from the dynein motor complex at
the microtubules and fulfills its pro-apoptotic functions when
Bcl-2 levels are decreasing. The molecular details of Bim
activation are currently not fully understood. In summary
ACAD is limiting T cell responses in vivo and is indepen-
dent of death receptor signaling. ACAD is executed via the
intrinsic, mitochondrial pathway where decreasing Bcl-2 lev-
els cannot compensate the pro-apoptotic activity of activated
Bim.
11. Conclusions
Two concepts of activated T cell death exist: AICD which
is dependent on TCR-restimulation and ACAD which is an
autonomous, intrinsic death program. Both death programs
control T cell homeostasis and it largely depends on the
antigen which program is activated. ACAD might be the
dominating mechanism at the end of the immune response
when foreign antigen is present only at very low concen-
trations. AICD, in contrast, is the prevailing mechanism if
antigens are present at higher concentrations for example
during chronic infections or in autoimmune diseases. Thus,
AICD might serve to eliminate autoreactive T cells and main-
tain peripheral tolerance towards self-antigens in the steady
state. Such self-antigens are systemic and mostly present
at higher concentrations. AICD controls T cell homeosta-
sis by balancing autoimmunity and the ability to mount a
proper immune response. The different thresholds of reactiv-
ity towards antigen concentrations are probably the central
discrimination between ACAD and AICD. In summary, two
different regulatory pathways may control T cell homeostasis
under different physiological conditions: ACAD in response
to foreign antigens and AICD in response to self-antigens.
Reviewers
Christian Widmann, Ph.D., Associate Professor, Lausanne
University, Department of Physiology, Bugnon 7/9, CH-1005
Lausanne, Switzerland.
Ingo Schmitz, Ph.D., University of Duesseldorf, Depart-
ment of Molecular Medicine, Universitätsstrasse 1, D-40225
Duesseldorf, Germany.
Klaus-Michael Debatin, M.D., University Children’s Hos-
pital, Erythstrasse 24, D-89070 Ulm, Germany.
Acknowledgements
We thank Inna Lavrik, Lucie Dörner and Karsten Gülow
for critically reading the manuscript and are grateful to Heidi
Sauter for expert secretary assistance. This work was sup-
ported by grants from the Deutsche Forschungsgemeinschaft,
the Wilhelm Sander-Stiftung, the Deutsche Krebshilfe and
the European Community.
References
[1] Krammer PH, Arnold R, Lavrik IN. Life and death in peripheral T
cells. Nat Rev Immunol 2007;7:532–42.
[2] Krammer PH. CD95’s deadly mission in the immune system. Nature
2000;407:789–95.
[3] Strasser A, Pellegrini M. T-lymphocyte death during shutdown of an
immune response. Trends Immunol 2004;25:610–5.
[4] Stranges PB, Watson J, Cooper CJ, et al. Elimination of antigen-
presenting cells and autoreactive T cells by Fas contributes to
prevention of autoimmunity. Immunity 2007;26:629–41.
[5] Hildeman DA, Zhu YN, Mitchell TC, et al. Activated T cell death in
vivo mediated by proapoptotic Bcl-2 family member Bim. Immunity
2002;16:759–67.
[6] Brunner T, Mogil RJ, LaFace D, et al. Cell-autonomous Fas
(CD95)/Fas-ligand interaction mediates activation-induced apoptosis
in T-cell hybridomas. Nature 1995;373:441–4.
[7] Dhein J, Walczak H, Baumler C, Debatin KM, Krammer PH.
Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature
1995;373:438–41.
 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64
[8] Krueger A, Fas SC, Baumann S, Krammer PH. The role of CD95
in the regulation of peripheral T-cell apoptosis. Immunol Rev
2003;193:58–69.
[9] Klas C, Debatin KM, Jonker RR, Krammer PH. Activation inter-
feres with the APO-1 pathway in mature human T cells. Int Immunol
1993;5:625–30.
[10] Owen-Schaub LB, Yonehara S, Crump III WL, Grimm EA. DNA
fragmentation and cell death is selectively triggered in activated
human lymphocytes by Fas antigen engagement. Cell Immunol
1992;140:197–205.
[11] Peter ME, Kischkel FC, Scheuerpflug CG, Medema JP, Debatin
KM, Krammer PH. Resistance of cultured peripheral T cells towards
activation-induced cell death involves a lack of recruitment of FLICE
(MACH/caspase 8) to the CD95 death-inducing signaling complex.
Eur J Immunol 1997;27:1207–12.
[12] Radvanyi LG, Shi YF, Vaziri H, et al. CD28 costimulation inhibits
TCR-induced apoptosis during a primary T cell response. J Immunol
1996;156:1788–98.
[13] Van Parijs L, Refaeli Y, Lord JD, Nelson BH, Abbas AK, Balti-
more D. Uncoupling IL-2 signals that regulate T cell proliferation,
survival, and Fas-mediated activation-induced cell death. Immunity
1999;11:281–8.
[14] Baumann S, Dostert A, Novac N, et al. Glucocorticoids inhibit
activation-induced cell death (AICD) via direct DNA-dependent
repression of the CD95 ligand gene by a glucocorticoid receptor
dimmer. Blood 2005;106:617–25.
[15] Schuh K, Twardzik T, Kneitz B, Heyer J, Schimpl A, Serfling E.
The interleukin 2 receptor alpha chain/CD25 promoter is a target
for nuclear factor of activated T cells. J Exp Med 1998;188:1369–
73.
[16] Benczik M, Gaffen SL. The interleukin (IL)-2 family cytokines: sur-
vival and proliferation signaling pathways in T lymphocytes. Immunol
Invest 2004;33:109–42.
[17] Strasser A. The role of BH3-only proteins in the immune system. Nat
Rev Immunol 2005;5:189–200.
[18] Lenardo MJ. lnterleukin-2 programs mouse ␣␤ T lymphocytes for
apoptosis. Nature 1991;353:858–61.
[19] Refaeli Y, Van Parijs L, London CA, Tschopp J, Abbas AK. Biochem-
ical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis.
Immunity 1998;8:615–23.
[20] Liao XC, Littman DR. Altered T cell receptor signaling and dis-
rupted T cell development in mice lacking Itk. Immunity 1995;3:757–
69.
[21] Sommers CL, Rabin RL, Grinberg A, Tsay HC, Farber J, Love PE.
A role for the Tec family tyrosine kinase Txk in T cell activation and
thymocyte selection. J Exp Med 1999;190:1427–38.
[22] Miller AT, Berg LJ. Defective fas ligand expression and activation-
induced cell death in the absence of IL-2-Inducible T cell kinase 5. J
Immunol 2002;168:2163–72.
[23] Li-Weber M, Laur O, Krammer PH. Novel Egr/NF-AT composite
sites mediate activation of the CD95 (APO-1/Fas) ligand promoter in
response to T cell stimulation. Eur J Immunol 1999;29:3017–27.
[24] Xiao S, Matsui K, Fine A, et al. FasL promoter activation by IL-
2 through SP1 and NFAT but not Egr-2 and Egr-3. Eur J Immunol
1999;29:3456–65.
[25] Radvanyi LG, Shi YF, Mills GB, Miller RG. Cell cycle progression
out of G1 sensitizes primary-cultured nontransformed T cells to TCR-
mediated apoptosis. Cell Immunol 1996;170:260–73.
[26] Marks-Konczalik J, Dubois S, Losi JM, et al. IL-2-induced activation-
induced cell death is inhibited in IL-15 transgenic mice. PNAS
2000;97:11445–50.
[27] Ku CC, Murakami M, Sakamoto A, Kappler J, Marrack P. Control of
homeostasis of CD8+ memory T cells by opposing cytokines. Science
2000;288:675–8.
[28] Zhang X, Sun S, Hwang I, Tough DF, Sprent J. Potent and selective
stimulation of memory-phenotype CD8+ T cells in vivo by IL-15.
Immunity 1998;8:591–9.
61
[29] Zhang J, Bardos T, Shao Q, et al. IL-4 potentiates activated
T cell apoptosis via an IL-2-dependent mechanism. J Immunol
2003;170:3495–503.
[30] Refaeli Y, Van Parijs L, Alexander SI, Abbas AK. Interferon [Gamma]
is required for activation-induced death of T lymphocytes. J Exp Med
2002;196:999–1005.
[31] Karin M, Lin A. NF-␬ B at the crossroads of life and death. Nat
Immunol 2002;3:221–7.
[32] De SE, Zazzeroni F, Papa S, et al. Induction of gadd45beta by
NF-kappaB downregulates pro-apoptotic JNK signalling. Nature
2001;414:308–13.
[33] Mittal A, Papa S, Franzoso G, Sen R. NF-␬ B-dependent regulation
of the timing of activation-induced cell death of T lymphocytes. J
Immunol 2006;176:2183–9.
[34] Gulow K, Kaminski M, Darvas K, Suss D, Li-Weber M, Krammer PH.
HIV-1 trans-activator of transcription substitutes for oxidative signal-
ing in activation-induced T cell death. J Immunol 2005;174:5249–
60.
[35] Cui H, Matsui K, Omura S, et al. Proteasome regulation of activation-
induced T cell death. PNAS 1997;94:7515–20.
[36] Arnold R, Brenner D, Becker M, Frey C, Krammer PH. How
T lymphocytes switch between life and death. Eur J Immunol
2006;36:1654–8.
[37] Brenner D, Golks A, Kiefer F, Krammer PH, Arnold R. Activation or
suppression of NFkB by HPK1 determines sensitivity to activation-
induced cell death. EMBO J 2005;24:4279–90.
[38] Wan YSY, DeGregori J. The survival of antigen-stimulated T cells
requires NF␬ B-mediated inhibition of p73 expression. Immunity
2003;18:331–42.
[39] Hildeman DA, Zhu YN, Mitchell TC, Kappler J, Marrack P. Molecular
mechanisms of activated T cell death in vivo. Curr Opin Immunol
2002;14:354–9.
[40] Ruland J, Mak TW. From antigen to activation: specific signal trans-
duction pathways linking antigen receptors to NF-␬ B. Sem Immunol
2003;15:177–83.
[41] Kane LP, Lin J, Weiss A. It’s all relative: NF-␬ B and CD28
costimulation of T-cell activation. Trends Immunol 2002;23:413–
20.
[42] Schmitz ML, Dienz O, Bacher S. NF-␬ B activation pathways induced
by T cell costimulation. FASEB J 2003;17:2187–93.
[43] Ghosh S, Karin M. Missing pieces in the NF-␬ B puzzle. Cell
2002;109:S81–96.
[44] Hayden MS, Ghosh S. Signaling to NF-␬ B. Genes Dev 2004;
18:2195–224.
[45] Thome M. CARMA1, BCL-10 and MALT1 in lymphocyte develop-
ment and activation. Nat Rev Immunol 2004;4:348–59.
[46] Matsumoto R, Wang D, Blonska M, et al. Phosphorylation of
CARMA1 plays a critical role in T cell receptor-mediated NF-␬ B
activation. Immunity 2005;23:575–85.
[47] Rueda D, Thome M. Phosphorylation of CARMA1: the link(er) to
NF-␬ B activation. Immunity 2005;23:551–3.
[48] Sommer K, Guo B, Pomerantz JL, et al. Phosphorylation of
the CARMA1 linker controls NF-␬ B activation. Immunity
2005;23:561–74.
[49] Rawlings DJ, Sommer K, Moreno-Garcia ME. The CARMA1 sig-
nalosome links the signalling machinery of adaptive and innate
immunity in lymphocytes. Nat Rev Immunol 2006;6:799–812.
[50] Sun LJ, Deng L, Ea CK, Xia ZP, Chen ZJJ. The TRAF6 ubiquitin lig-
ase and TAK1 kinase mediate IKK activation by BCL10 and MALT1
in T lymphocytes. Mol Cell 2004;14:289–301.
[51] Kamata H, Honda S, Maeda S, Chang LF, Hirata H, Karin M.
Reactive oxygen species promote TNF␣-induced death and sus-
tained JNK activation by inhibiting MAP kinase phosphatases. Cell
2005;120:649–61.
[52] Pham CG, Bubici C, Zazzeroni F, et al. Ferritin heavy chain upreg-
ulation by NF-␬ B inhibits TNF␣-induced apoptosis by suppressing
reactive oxygen species. Cell 2004;119:529–42.
62 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64
[53] Schulze-Luehrmann J, Santner-Nanan B, Jha MK, Schimpl A, Avots
A, Serfling E. Hematopoietic progenitor kinase 1 supports apoptosis
of T lymphocytes. Blood 2002;100:954–60.
[54] Kiefer F, Tibbles LA, Anafi M, et al. HPK1, a hematopoi-
etic protein kinase activating the SAPK/JNK pathway. EMBO J
1996;15:7013–25.
[55] Arnold R, Liou J, Drexler HCA, Weiss A, Kiefer F. Caspase-mediated
cleavage of hematopoietic progenitor kinase 1 (HPK1) converts
an activator of NF␬ B into an inhibitor of NF␬ B. J Biol Chem
2001;276:14675–84.
[56] Liou J, Kiefer F, Dang A, et al. HPK1 is activated by lympho-
cyte antigen receptors and negatively regulates AP-1. Immunity
2000;12:399–408.
[57] Liu SK, Smith CA, Arnold R, Kiefer F, McGlade CJ. The adaptor
protein Gads (Grb2-related adaptor downstream of Shc) is implicated
in coupling hemopoietic progenitor kinase-1 to the activated TCR. J
Immunol 2000;165:1417–26.
[58] Arnold R, Patzak IM, Neuhaus B, et al. Activation of hematopoi-
etic progenitor kinase 1 involves relocation, autophosphorylation,
and transphosphorylation by protein kinase D1. Mol Cell Biol
2005;25:2364–83.
[59] Le Bras S, Foucault I, Foussat A, Brignone C, Acuto O, Deckert M.
Recruitment of the actin-binding protein HIP-55 to the immunological
synapse regulates T cell receptor signaling and endocytosis. J Biol
Chem 2004;279:15550–60.
[60] Chen YR, Meyer CF, Ahmed B, Yao ZB, Tan TH. Caspase-mediated
cleavage and functional changes of hematopoietic progenitor kinase
1 (HPK1). Oncogene 1999;18:7370–7.
[61] Arnold R, Frey CR, Muller W, Brenner D, Krammer PH, Kiefer F.
Sustained JNK signaling by proteolytically processed HPK1 medi-
ates IL-3 independent survival during monocytic differentiation. Cell
Death Differ 2007;14:568–75.
[62] Brenner D, Golks A, Becker M, et al. Caspase-cleaved HPK1 induces
CD95L-independent activation-induced cell death in T and B lym-
phocytes. Blood 2007;110:3968–77.
[63] Campbell KJ, Rocha S, Perkins ND. Active repression of anti-
apoptotic gene expression by ReIA(p65) NF-␬ B. Mol Cell
2004;13:853–65.
[64] Shishodia S, Aggarwal BB. Guggulsterone inhibits NF-␬ B and
I kappa B alpha kinase activation, suppresses expression of anti-
apoptotic gene products, and enhances apoptosis. J Biol Chem
2004;279:47148–58.
[65] Lissy NA, Davis PK, Irwin M, Kaelin WG, Dowdy SF. A com-
mon E2F-1 and p73 pathway mediates cell death induced by TCR
activation. Nature 2000;407:642–5.
[66] Vallabhapurapu S, Ryseck RP, Malewicz M, Weih DS, Weih F. Inhi-
bition of NF-kappa B in T cells blocks lymphoproliferation and
partially rescues autoimmune disease in gld/gld mice. Eur J Immunol
2001;31:2612–22.
[67] Kaminski M, Kiessling M, Suess D, Krammer PH, Gulow K. Novel
role for mitochondria: protein kinase C theta dependent oxidative
signalling organelles in activation induced T cell death. Mol Cell Biol
2007;27:3625–39.
[68] Jackson SH, Devadas S, Kwon J, Pinto LA, Williams MS. T cells
express a phagocyte-type NADPH oxidase that is activated after T
cell receptor stimulation. Nat Immunol 2004;5:818–27.
[69] Hildeman DA, Mitchell T, Aronow B, Wojciechowski S, Kappler J,
Marrack P. Control of Bcl-2 expression by reactive oxygen species.
PNAS 2003;100:15035–40.
[70] Schieven GL, Mittler RS, Nadler SG, et al. ZAP-70 tyrosine
kinase, CD45, and T cell receptor involvement in UV- and H2 O2 -
induced T cell signal transduction. J Biol Chem 1994;269:20718–
26.
[71] Baumler CB, Bohler T, Herr I, Benner A, Krammer PH, Debatin
KM. Activation of the CD95 (APO-1/Fas) system in T cells from
human immunodeficiency virus type-1-infected children. Blood
1996;88:1741–6.
[72] Debatin KM, Fahrig-Faissner A, Enenkel-Stoodt S, Kreuz
W, Benner A, Krammer PH. High expression of APO-1
(CD95) on T lymphocytes from human immunodefi-
ciency virus-1-infected children. Blood 1994;83:3101–
3.
[73] Bidere N, Su HC, Lenardo MJ. Genetic disorders of programmed cell
death in the immune system. Annu Rev Immunol 2006;24:321–52.
[74] Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA,
Nagata S. Lymphoproliferation disorder in mice explained by defects
in Fas antigen that mediates apoptosis. Nature 1992;356:314–7.
[75] Nagata S. Mutations in the Fas antigen gene in lpr mice. Sem Immunol
1994;6:3–8.
[76] Takahashi T, Tanaka M, Brannan CI, et al. Generalized lymphoprolif-
erative disease in mice, caused by a point mutation in the Fas ligand.
Cell 1994;76:969–76.
[77] Radvanyi LG, Raju K, Spaner D, Mills GB, Miller RG. Interleukin-
2 reverses the defect in activation-induced apoptosis in T cells from
autoimmune lpr mice. Cell Immunol 1998;183:1–12.
[78] Gorak-Stolinska P, Truman JP, Kemeny DM, Noble A. Activation-
induced cell death of human T-cell subsets is mediated by Fas and
granzyme B but is independent of TNF-␣ 21. J Leukocyte Biol
2001;70:756–66.
[79] Sytwu HK, Liblau RS, McDevitt HO. The roles of Fas/APO-1 (CD95)
and TNF in antigen-induced programmed cell death in T cell receptor
transgenic mice. Immunity 1996;5:17–30.
[80] Green DR, Droin N, Pinkoski M. Activation-induced cell death in T
cells. Immunol Rev 2003;193:70–81.
[81] Janssen EM, Droin NM, Lemmens EE, et al. CD4(+) T-cell help con-
trols CD8(+) T-cell memory via TRAIL-mediated activation-induced
cell death. Nature 2005;434:88–93.
[82] Muppidi JR, Siegel RM. Ligand-independent redistribution of Fas
(CD95) into lipid rafts mediates clonotypic T cell death. Nat Immunol
2004;5:182–9.
[83] Devadas S, Das J, Liu C, et al. Granzyme B is critical for T
cell receptor-induced cell death of type 2 helper T cells. Immunity
2006;25:237–47.
[84] Peter ME, Krammer PH. The CD95 (APO-1/Fas) DISC and beyond.
Cell Death Differ 2003;10:26–35.
[85] Bentele M, Lavrik I, Ulrich M, et al. Mathematical modeling
reveals threshold mechanism in CD95-induced apoptosis. J Cell Biol
2004;166:839–51.
[86] Lavrik IN, Golks A, Baumann S, Krammer PH. Caspase-2 is activated
at the CD95 death-inducing signaling complex in the course of CD95-
induced apoptosis. Blood 2006;108:559–65.
[87] Lavrik I, Golks A, Krammer PH. Death receptor signalling. J Cell Sci
2005;118:265–7.
[88] Thome M, Tschopp J. Regulation of lymphocyte proliferation and
death by flip. Nat Rev Immunol 2001;1:50–8.
[89] Krueger A, Schmitz I, Baumann S, Krammer PH, Kirchhoff S. Cellu-
lar FLICE-inhibitory protein splice variants inhibit different steps of
caspase-8 activation at the CD95 death-inducing signaling complex.
J Biol Chem 2001;276:20633–40.
[90] Scaffidi C, Schmitz I, Krammer PH, Peter ME. The role of c-
FLIP in modulation of CD95-induced apoptosis. J Biol Chem
1999;274:1541–8.
[91] Djerbi M, Darreh-Shori T, Zhivotovsky B, Grandien A. Characteri-
zation of the human FLICE-inhibitory protein locus and comparison
of the anti-apoptotic activity of four different FLIP isoforms. Scand
J Immunol 2001;54:180–9.
[92] Golks A, Brenner D, Fritsch C, Krammer PH, Lavrik IN. c-FLIPR,
a new regulator of death receptor-induced apoptosis. J Biol Chem
2005;280:14507–13.
[93] Schmitz I, Weyd H, Krueger A, et al. Resistance of short term activated
T cells to CD95-mediated apoptosis correlates with de novo protein
synthesis of c-FLIPshort. J Immunol 2004;172:2194–200.
[94] Kirchhoff S, Muller WW, Krueger A, Schmitz I, Krammer PH.
TCR-mediated up-regulation of c-FLIPshort correlates with resis-
 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64
tance toward CD95-mediated apoptosis by blocking death-inducing
signaling complex activity. J Immunol 2000;165:6293–300.
[95] Schmitz I, Krueger A, Baumann S, Schulze-Bergkamen H, Kram-
mer PH, Kirchhoff S. An IL-2-dependent switch between CD95
signaling pathways sensitizes primary human T cells toward CD95-
mediated activation-induced cell death. J Immunol 2003;171:2930–
6.
[96] Fas SC, Baumann S, Krueger A, et al. In vitro generated
human memory-like T cells are CD95 type II cells and resistant
towards CD95-mediated apoptosis. Eur J Immunol 2006;36:2894–
903.
[97] Budd RC, Yeh WC, Tschopp J. cFLIP regulation of lymphocyte acti-
vation and development. Nat Rev Immunol 2006;6:196–204.
[98] Scaffidi C, Schmitz I, Zha J, Korsmeyer SJ, Krammer PH, Peter ME.
Differential modulation of apoptosis sensitivity in CD95 type I and
type II cells. J Biol Chem 1999;274:22532–8.
[99] Villalba M, Kasibhatla S, Genestier L, Mahboubi A, Green DR, Alt-
man A. Protein kinase C␪ cooperates with calcineurin to induce Fas
ligand expression during activation-induced T cell death. J Immunol
1999;163:5813–9.
[100] Manicassamy S, Sun Z. The critical role of protein kinase C-␪
in Fas/Fas ligand-mediated apoptosis. J Immunol 2007;178:312–
9.
[101] Holtz-Heppelmann CJ, Algeciras A, Badley AD, Paya CV. Transcrip-
tional regulation of the human FasL promoter-enhancer region. J Biol
Chem 1998;273:4416–23.
[102] Kasibhatla S, Genestier L, Green DR. Regulation of Fas-ligand
expression during activation-induced cell death in T lymphocytes via
nuclear factor kappa B. J Biol Chem 1999;274:987–92.
[103] Latinis KM, Norian LA, Eliason SL, Koretzky GA. Two NFAT tran-
scription factor binding sites participate in the regulation of CD95
(Fas) ligand expression in activated human T cells. J Biol Chem
1997;272:31427–34.
[104] Li-Weber M, Laur O, Hekele A, Coy J, Walczak H, Krammer PH.
A regulatory element in the CD95 (APO-1/Fas) ligand promoter
is essential for responsiveness to TCR-mediated activation. Eur J
Immunol 1998;28:2373–83.
[105] Manicassamy S, Gupta S, Huang Z, Sun Z. Protein kinase C-␪-
mediated signals enhance CD4+ T cell survival by up-regulating
Bcl-xL. J Immunol 2006;176:6709–16.
[106] Saibil SD, Jones RG, Deenick EK, et al. CD4+ and CD8+ T cell
survival is regulated differentially by protein kinase C␪, c-Rel, and
protein kinase B. J Immunol 2007;178:2932–9.
[107] Villalba M, Bushway P, Altman A. Protein kinase C-theta medi-
ates a selective T cell survival signal via phosphorylation of BAD.
J Immunol 2001;166:5955–63.
[108] Weaver CT, Harrington LE, Mangan PR, Gavrieli M, Murphy KM.
Th17: an effector CD4 T cell lineage with regulatory T cell ties.
Immunity 2006;24:677–88.
[109] Varadhachary AS, Perdow SN, Hu CG, Ramanarayanan M, Salgame
P. Differential ability of T cell subsets to undergo activation-induced
cell death. Proc Natl Acad Sci USA 1997;94:5778–83.
[110] Roberts AI, Devadas S, Zhang XR, et al. The role of activation-
induced cell death in the differentiation of T-helper-cell subsets.
Immunol Res 2003;28:285–93.
[111] Zhang XR, Zhang LY, Devadas S, Li L, Keegan AD, Shi YF. Recip-
rocal expression of TRAIL and CD95L in Th1 and Th2 cells: role
of apoptosis in T helper subset differentiation. Cell Death Differ
2003;10:203–10.
[112] Zhang XH, Brunner T, Carter L, et al. Unequal death in T helper cell
(Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid
Fas/FasL-mediated apoptosis. J Exp Med 1997;185:1837–49.
[113] Lord SJ, Rajotte RV, Korbutt GS, Bleackley RC. Granzyme B: a
natural born killer. Immunol Rev 2003;193:31–8.
[114] Sharma V, Delgado M, Ganea D. VIP protects Th2 cells by downreg-
ulating granzyme B expression. Vip Pacap Rel Peptides: Gene Ther
2006;1070:540–4.
63
[115] van Parijs L, Peterson DA, Abbas AK. The Fas/Fas ligand pathway
and Bcl-2 regulate T cell responses to model self and foreign antigens.
Immunity 1998;8:265–74.
[116] Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD
couples survival signals to the cell-intrinsic death machinery. Cell
1997;91:231–41.
[117] Zha JP, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phospho-
rylation of death agonist BAD in response to survival factor results in
binding to 14-3-3 not BGL-X(L). Cell 1996;87:619–28.
[118] Bouillet P, Purton JF, Godfrey DI, et al. BH3-only Bcl-2 family mem-
ber Bim is required for apoptosis of autoreactive thymocytes. Nature
2002;415:922–6.
[119] Zong WX, Lindsten T, Ross AJ, MacGregor GR, Thompson CB.
BH3-only proteins that bind pro-survival Bcl-2 family members fail
to induce apoptosis in the absence of Bax and Bak. Genes Dev
2001;15:1481–6.
[120] Strasser A, Harris AW, Cory S. bcl-2 transgene inhibits T cell death
and perturbs thymic self-censorship. Cell 1991;67:889–99.
[121] Mitchell T, Kappler J, Marrack P. Bystander virus infection prolongs
activated T cell survival. J Immunol 1999;162:4527–35.
[122] Grayson JM, Zajac AJ, Altman JD, Ahmed R. Cutting edge: increased
expression of Bcl-2 in antigen-specific memory CD8(+) T cells. J
Immunol 2000;164:3950–4.
[123] Gonzalo JA, Tarazona R, Schuurman HJ, et al. A single injection
of Staphylococcus aureus enterotoxin B reduces autoimmunity in
MRL/lpr mice. Clin Immunol Immunopathol 1994;71:176–82.
[124] Hildeman DA, Mitchell T, Teague TK, et al. Reactive oxygen species
regulate activation-induced T cell apoptosis. Immunity 1999;10:
735–44.
[125] Ettinger R, Panka DJ, Wang JK, Stanger BZ, Ju ST, Marshak-
Rothstein A. Fas ligand-mediated cytotoxicity is directly responsible
for apoptosis of normal CD4+ T cells responding to a bacterial super-
antigen. J Immunol 1995;154:4302–8.
[126] Renno T, Hahne M, Tschopp J, MacDonald HR. Peripheral T cells
undergoing superantigen-induced apoptosis in vivo express B220 and
upregulate Fas and Fas ligand. J Exp Med 1996;183:431–7.
Biographies
Dirk Brenner is a junior post-doc at the DKFZ, Heidel-
berg, Germany, in the division of Peter H. Krammer in the
subgroup of Rüdiger Arnold. He studied biochemistry at the
universities of Bonn and Witten/Herdecke (Germany) and
Stanford and Harvard (USA). He joined the division of Peter
H. Krammer for his Ph.D. thesis and is working since then
on signal transduction pathways influencing sensitivity and
resistance to activation-induced cell death in lymphocytes.
Peter H. Krammer is head of the Division of Immuno-
genetics and speaker of the Tumor Immunology Program
at the DKFZ, Heidelberg, Germany. He studied medicine at
the universities of Freiburg, Germany, St. Louis, USA, and
Lausanne, Switzerland. After his M.D. he joined the Basel
Institute of Immunology, Switzerland, where he focused on T
cell biology. In 1976, he moved to the DKFZ to continue his
work on T cell function and characterized the death recep-
tor CD95 (APO-1/Fas). Peter H. Krammer is interested in
the general mechanisms influencing life and death decisions
in lymphocytes. The research in his division is focused on
molecular mechanisms involving CD95, regulatory T cells,
cytokine gene expression (e.g. IL-4) and T cell receptor sig-
naling in health and disease.
64 D. Brenner et al. / Critical Reviews in Oncology/Hematology 66 (2008) 52–64

Rüdiger Arnold is group leader at the DKFZ, Heidel-
berg, Germany, in the division of Peter H. Krammer. He
studied biology at the universities of Darmstadt, Germany,
and Giessen, Germany, and joined the department of Rainer
Renkawitz, Giessen, Germany to undertake his Ph.D. on
mechanisms of gene regulation. He performed his postdoc-
toral studies at the Max-Planck-Institute for Physiological
and Clinical Research, Bad Nauheim, Germany, where he
studied signal transduction mechanisms in hematopoietic
cells in the group of Friedemann Kiefer. He moved to Hei-
delberg in 2002 to continue his work on signal transduction,
where his group studies mechanisms of activation, prolifera-
tion and differentiation of leukocytes.