Title: Apoptosis is a highly regulated process that plays critical role in many cellular functions:
1Apoptosis is a highly regulated process that
plays critical role in many cellular
functions 1 Cell differentiation and
development (deletion of caspases leads to organ
malformations) 2 T cell Homeostasis (AICD)
which is a process by which unwanted activated T
cells Are eliminated. This is mainly mediated by
Fas, TNFR and in some cases perforin and
granzymes 3 killing virally infected cells
and tumors CTL mediated apoptosis.
2Ag-responsive T cells expand, die, or become
memory cells
Survivin
Survivin
IL-2-Driven
Expansion/
Effector Cell
Continued IL-2
Survival/
(short-lived)
driven expansion
Differentiation
IL-2
Naive T cell
Activation-Induced
AgAPC
Death (FasL/Fas)
Death by Cytokine
Deprivation
AgAPC
?
Memory Cell
Stromal
(long lived)
Survival Factor
Survivin
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4Survivin
- In mammalian cells, apoptosis is modulated by two
protein families the BCL2 and inhibitor of
apoptosis (IAP) families. - Survivin is a unique member of the IAP family. It
is associated with several subcellular
compartments and its expression is regulated by
many signalling pathways. - The survivin pathway interfaces with both the
cell-death machinery and mechanisms of cell-cycle
progression and microtubule stability. - Survivin expression is undetectable in most
normal adult tissues, but is overexpressed in
virtually every human tumour that has been
studied. Several mechanisms have been proposed to
account for this overexpression, one of which is
loss of wild-type p53. - Indispensable (proliferation and apoptosis)
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8Ag-responsive T cells expand, die, or become
memory cells
Survivin
Survivin
IL-2-Driven
Expansion/
Effector Cell
Continued IL-2
Survival/
(short-lived)
driven expansion
Differentiation
IL-2
Naive T cell
Activation-Induced
AgAPC
Death (FasL/Fas)
Death by Cytokine
Deprivation
AgAPC
?
Memory Cell
Stromal
(long lived)
Survival Factor
Survivin
9Structures of the B7-1/B7-2CD28/CTLA-4
superfamily members. CD28 family members are
IMMUNOGLOBULIN SUPERFAMILY members with a single
immunoglobulin V-like domain. CD28 and CTLA-4
have a MYPPPY motif that is essential for binding
B7-1 and B7-2, whereas ICOS has a FDPPPF motif
and binds ICOSL but not B7-1 and B7-2. PD-1 is a
receptor for both PD-L1 and PD-L2, which might
also bind to other, as yet unidentified,
receptors on T cells (indicated by the dotted
arrows and the question mark).
10- ICOS engagement promotes T-helper-cell
differentiation and effector function, and is
particularly important for interleukin 10 (IL-10)
production but has a modest role in regulating
T-cell expansion and IL-2 production. ICOS
engagement can upregulate CD40L, and this pathway
has a key role in promoting immunoglobulin
isotype switching. Little is known about
signalling pathways downstream of ICOS. ICOS has
a phosphatidylinositol 3-kinase motif in its
cytoplasmic tail.
11- PD-1 contains two tyrosines in its cytoplasmic
tail, forming ITIM and ITSM motifs. Mutation of
the ITSM tyrosine but not the ITIM tyrosine
abolishes PD-1-mediated inhibitory activity. The
signalling pathways by which PD-1 exerts its
effects are just beginning to be understood.
Ligation of TCR and PD-1 can lead to tyrosine
phosphorylation (P) and activation of SHP-2,
resulting in dephosphorylation of signalling
molecules and reduced cytokine mRNA synthesis.
Ligation of both PD-1 and the BCR can inhibit
tyrosine phosphorylation of effector signalling
molecules.
12Hypothetical model of TNFR control of primary
T-cell responses. As herpes-virus entry
mediator (HVEM) and CD28 are constitutively
expressed by naive T cells, and their ligands are
expressed by resting antigen-presenting cells
(APCs) or rapidly induced, they might control
initial activation and cell division. CD27 might
also function to regulate clonal expansion.
Expression of OX40, 4-1BB and CD30 and their
ligands is induced by T cells and APCs with
delayed kinetics and might function later. OX40
and 4-1BB, and possibly CD30, prevent apoptosis
and regulate the ability of effector cells to
persist at the peak of the response. The timeline
depicted is based on both in vitro and in vivo
data from several studies. Most, but not all,
data fit this model. A great deal of overlap in
the timing of action of tumour-necrosis factor
receptor (TNFR) molecules is probable and will
vary depending on the nature of the antigen, its
dose and pro-inflammatory factors.
13Structural organization of the co-stimulatory
TNFRTNF-family members. The tumour-necrosis
factor (TNF) ligands (top) are shown as
homotrimeric type II transmembrane proteins. The
TNF receptor (TNFR)-family molecules (bottom) are
depicted as type I transmembrane monomers that
are thought to associate in trimers when
interacting with their ligands. The commonly used
names are in bold, with alternate names and
TNF/TNFR superfamily (SF) designations in
parentheses. The total amino-acid length and
number of intracellular amino acids (parentheses)
are also indicated. Web access to TNF/TNFR
nomenclature and sequences can be found at
14Generalized time course of expression of
co-stimulatory TNFR-family members. The
transition of naive T cells after activation to
effector and memory stages is accompanied by the
upregulation or downregulation of expression of
tumour-necrosis factor receptor (TNFR)-family
members. Few molecules have been studied at the
same time or in similar model systems, therefore
this generalized model is based on separate,
mostly in vitro, studies, some of which represent
different situations. So, expression levels are
relative and kinetics depicted might vary
depending on the system. Herpes-virus entry
mediator (HVEM) and CD27 are constitutively
expressed by naive T cells and some memory T
cells. OX40, 4-1BB and CD30 are not expressed by
naive T cells and the peak level of expression
occurs after encounter with antigen, before and
at the height of the effector response in both
primary and secondary immune reactions.
15Signalling intermediates in TNFR-family
co-stimulation. Each co-stimulatory
tumour-necrosis factor receptor (TNFR) binds
TNFR-associated factors (TRAFs) that function as
adaptor proteins for kinases. TRAF2 might be used
by all TNFR molecules and link to several common
signalling pathways, including those that involve
JUN N-terminal kinase (JNK) and the AP1 (FOS/JUN)
transcriptional complex nuclear factor- B (NF-
B) and phosphatidylinositol 3-kinase (PI3K)
and protein kinase B (PKB, also known as AKT).
Signalling through OX40 and 4-1BB upregulate the
expression of anti-apoptotic members of the BCL2
family, and might negatively regulate
pro-apoptotic members, such as BAD
(BCL2-antagonist of cell death) and BIM
(BCL2-interacting mediator of cell death).
Although most co-stimulatory TNFR molecules can
activate AP1 and NF- B, direct links to T-cell
effector function, cell division or survival
await further investigation (indicated by dashed
arrows).
16Hypothesis Co-stimulation and Signaling Thru
OX40 (TNFRSF) leads to activation of survivin
(survival and proliferation)
Control of antigen-specific T-cell numbers by
TNFR-family co-signals. According to this
model, in the absence of tumour-necrosis factor
receptor (TNFR) co-signals, little expansion in
the number of antigen-reactive T cells will occur
in the primary response, and the frequency of
effector T cells will be low. The number of
antigen-specific memory T cells that develop is
small. If memory T cells do not receive clonal
expansion and survival signals from TNFR
molecules during secondary responses to recall
antigen, the frequency of secondary effector T
cells will be markedly reduced. Preventing a
single TNFRTNF interaction might have a less
pronounced effect, although suppressive effects
have been seen in several primary and secondary
responses. It is probable that blocking several
interactions at once inhibits either stage of a
response more markedly.
17Figure 1. Defective Survivin Expression in the
Absence of OX40
- Sustained proliferation of recently activated T
cells is dependent on OX40 - Survivin expression is lower OX40 KO and is
specific (cell cycle proteins and aurora) - Survivin expression is dependent on OX40
- CD4 T cells from wt or OX40 KO AND TCR transgenic
mice were stimulated in vitro with APCs and
peptide.(A) Naive T cell proliferation over 7
days. Data are mean thymidine incorporation SD
in triplicate cultures and are representative of
three experiments.(B and C) Naive T cell
Survivin expression after stimulation of wt or KO
T cells (B) or wt T cells in the presence of
agonist anti-OX40 (C). Relative levels of
Survivin over time are shown compared to peak
levels in wt T cells (value of 1) based on
densitometric scans. Similar data were obtained
in two to four experiments.
18- Re-expression of survivin in effector T cells
was also impaired - The number of survivin positive dividing cells
was reduced especially at d3 - CD28KO mice show deficiency in early
proliferation of T cells. However, this was
accompanied by defects in genes involved in
proliferation (Cyclin E, Cyclin D, CDK2, and
Aurora). - Naive wt or OX40 KO T cells were stimulated with
anti-CD3 and CD28 for 7 days. The primed T cells
were restimulated with Ag and APCs, and Survivin,
Cyclin E, Cyclin D, CDK2, and Aurora B
visualized. Similar data were obtained in three
experiments.(E and F) Survivin intracellular
staining, after labeling naive wt or OX40 KO T
cells with CFSE and stimulating with Ag/APCs (E).
Quantification of percent Survivin-positive cells
in each division on day 5 (F). Data are
representative of three experiments. Data are
mean SD from triplicate cultures.
19What signaling pathway link OX40 to up-regulation
of apoptosis?
- Active PKB fully restore survivin expression in
the absence of OX40 signaling - IP experiments showed that survivin is
phorsphorylated Thr34 as seen in cancer cells - Dominant negative mutant of PKB inhibited
survivin expression. - Figure 2. PKB Regulates Survivin
- Naive CD4 cells from wt or OX40 KO AND mice were
stimulated with peptide and APCs. On day 2 and 3,
T cells were transduced with retroviral vectors
expressing GFP alone (Mig), GFP with
myristoylated PKB (Mig-myr-PKB) (A and B, wt and
OX40 KO), or GFP with dominant-negative PKB
(Mig-dn-PKB) (C, wt). - (A and C) On day 6 of primary culture, GFP CD4
cells were sorted, and lysates were analyzed for
Survivin and PKB. - (B) On day 6 of primary culture, GFP CD4 cells
were sorted, lysates were immunoprecipitated with
anti-Survivin and analyzed for phosphorylated
threonine.
20- Blocking PI3K with specific inhibitor inhibited
survivin - Blocking PI3Kinase in effector T cells after
re-expression suppressed survivin
expression - Sustained or periodic PKB activation is
nceccessary to maintain survivin. - (D) Naive wt T cells were stimulated with peptide
and APCs, and LY294002 was added for the last 24
hr of culture (days 23 or 34). Survivin
expression was determined at the end of the 24 hr
period. - (E) Activated wt T cells, taken after 7 days of
primary culture, were restimulated with
peptide/APCs in the presence of DMSO, LY294002,
or Wortmannin added at time 0
21- Figure 3. Survivin Is Expressed in G1 and Is
Regulated by OX40 Signals - (A) Naive CD4 T cells from wt AND mice were
stimulated with Ag and APCs in the presence of
DMSO (control), Hydroxyurea, Aphidicolin, or
Nocodazole added at time 0 hr. After 2 days,
Survivin expression was analyzed in various
phases of the cell cycle after BrdU pulsing and
7-AAD staining. Left cell cycle analysis shown
in red with Survivin expression overlaid in blue,
after gating on CD4 cells. Right total percent
Survivin expression in CD4 cells.
- Rationale report suggested that survivin is only
expressed in mitosis and is predominantly
involved in the induction of proliferation. - Survivin is expressed in all phases
- Inhibitors of S phase progression did not
influence survivin expression
22- Figure 3. Survivin Is Expressed in G1 and Is
Regulated by OX40 Signals - (BD) Survivin induction in effector T cells. Wt
effector T cells taken after 711 days of primary
culture were stimulated for 24 hr with agonist
anti-OX40 alone or control IgG in the presence of
DMSO, Hydroxyurea, Aphidicolin, or Nocodazole (B
and C) or DMSO, ethanol, cyclohexamide (CHX), or
LY294002 (D), added at time 0 hr. Survivin
expression by intracellular staining (B) and
RT-PCR (C and D). Similar data were obtained in
one repeat experiment. Errors are mean SD from
triplicate PCR wells.
- OX40 had direct effect on survivin expressoion
- OX40 regulate survivin expression thru AKT
signaling pathway.
23- Figure 4. Survivin Rescues Proliferation and
Death of OX40-Deficient T Cells In Vitro - CD4 T cells from wt or OX40 KO AND mice were
stimulated with peptide and APCs, and transduced
on days 2 and 3 with retroviral vectors
expressing GFP (Mig) or GFP with wt Survivin
(Mig-Survivin). - (A) Primary T cell survival on day 8 after
reculturing unsorted T cells without further
stimulation on day 3. Data show percent recovery
calculated based on assigning the input number of
GFP cells in each culture as 100. Data are mean
SD from three experiments. Cell lysates were
analyzed by immunoblotting for Survivin (top). - (B) Primary proliferation on day 4 and day 8
measured in unseparated cultures by pulsing with
thymidine for the last 20 hr. Data are mean SD
from triplicate cultures and are representative
of three experiments. - (C) Primary apoptosis on day 6 measured in
unseparated cultures by gating on CD4 cells
(GFP and GFP-) and staining with annexin V. Open
histograms are unstained cells. Data are
representative of three experiments.
- Data showed that in primary T cell culture,
introducing survivin leads - Recovery of T cells in OX40 KO
- Sustained division of OX40 deficient T cells
- Reduced apoptosis
24Recall T cell survival after sorting GFP T cells
on day 6 and re-stimulating with peptide and APC
in secondary response. Results OX40 cells
transduced with wt survivin showed enhanced
recover. Increase accumulation of T cells in the
S phase
25antigen
PBS
- Figure 5. Survivin Restores Defective Expansion
of OX40-Deficient T Cells In Vivo - CD4 T cells from wt or OX40 KO OT-II mice were
stimulated with peptide in vitro and transduced
on days 2/3 with retroviral vectors expressing
GFP (Mig) or GFP with wt Survivin (Mig-Survivin).
After 6 days, GFP CD4 cells were sorted, labeled
with PKH26, and adoptively transferred to
syngeneic recipients. Mice were challenged with
OVA in PBS (closed histograms) or PBS without
antigen (open histograms). - (A) T cell expansion after 3 and 7 days, with the
number of GFPVß5CD4 cells enumerated in the
spleen and lymph nodes. Data are mean response
SD from three individual mice per group and are
representative of three experiments.
- OX40 T cells expanded less after 3 days and it
was rescued by survivin - The defect was more evident at day 7
-
26- Figure 5. Survivin Restores Defective Expansion
of OX40-Deficient T Cells In Vivo - (B) Division of transferred T cells was monitored
for 3 days after challenge by determining either
the extent of dilution of PKH26 (left) or the
incorporation of BrdU after an in vivo pulse for
2 days (middle). Apoptosis was measured by
annexin V staining after gating on Vß5CD4 (GFP
and GFP-) cells (right). Bold line, Mig-Survivin
group dashed line, Mig-control group. Shaded
histograms are recipients of Mig-Survivin T cells
injected with PBS. Data are representative of
three experiments.
Increase in cell division and less apoptosis in
wt transgenic mice. Overall, Sustained survivin
expression thru OX40OX40L interaction is
required to maintain T cell division overtime.
27- Figure 6. Survivin Does Not Sustain Long-Term CD4
T Cell Survival - CD4 T cells from wt or OX40 KO AND mice were
stimulated with peptide and transduced with
retroviral vectors on day 2/3. - (A) Expression of total (human/mouse) and
endogenous (mouse) Survivin and Bcl-2-like
proteins on day 6 in lysates of GFPCD4-sorted
cells. - (B) In vitro recall T cell recovery after 12 days
of secondary culture after sorting GFPCD4 cells
and restimulating with peptide. Data show mean
percent recovery SD calculated based on the
input number of cells. - (C) In vivo T cell recovery 14 days after
immunization, after sorting GFPCD4 cells,
adoptively transferring to syngeneic recipients,
and challenging with PCC in PBS (closed
histograms) or PBS alone (open histograms). Data
are mean number of GFPVß3CD4 cells SD
enumerated in the spleen and lymph nodes from
three individual mice per group. Results
representative of two or three experiments.
A Survivin did induce expression of BCl-2 B In
vitro 12 day secondary immune response, survivin
was not able to recover specific T cells C In
vivo secondary immune response gave the same
resuts
28- Figure 7. Dominant-Negative Survivin Inhibits T
Cell Expansion - CD4 T cells from wt OT-II TCR transgenic mice
were stimulated with peptide/APCs and transduced
with retroviral vectors expressing GFP (Mig) or
GFP with dominant-negative Survivin
(Mig-T34A-Survivin). - (A) Expression of total (human and mouse) and
endogenous (mouse) Survivin, Bcl-xL, and Bcl-2,
in GFP/CD4-sorted cells on day 6. - (BD) In vitro response. T cell expansion (B)
based on enumerating GFP cells and expressed as
percent of input number, proliferation (C) based
on incorporation of thymidine, and cell cycle
progression (D) based on BrdU/7-AAD staining, all
after in vitro restimulation of GFP/CD4-sorted
cells. Data are representative of two or three
experiments. Errors indicate mean SD of
triplicate cultures.
29- Figure 7. Dominant-Negative Survivin Inhibits T
Cell Expansion -
- (E and F) In vivo response. GFPCD4 T cells were
sorted, labeled with PKH26, and adoptively
transferred into naive syngeneic recipients. Mice
were challenged with OVA in PBS (closed
histograms) or PBS alone (open histograms). (E) T
cell expansion after 3 or 7 days to assess the
number of GFP/Vß5/CD4 cells in combined spleen
and lymph nodes. Data are mean SD from three
individual mice per group and are representative
of three experiments. (F) Cell division or
apoptosis after 3 days by determining the extent
of dilution of PKH26 (top) and incorporation of
BrdU after a 2 day pulse (middle) after gating on
Vß5GFP cells and staining for annexin V after
gating on Vß5CD4 (GFP and GFP-) cells. Bold
line, Mig control dashed line,
Mig-T34A-Survivin. Shaded histograms are profiles
from recipients of Mig-T34A-Survivin T cells
injected with PBS. Data are representative of
three experiments.
30Hypothetical model of TNFR control of primary
T-cell responses. As herpes-virus entry
mediator (HVEM) and CD28 are constitutively
expressed by naive T cells, and their ligands are
expressed by resting antigen-presenting cells
(APCs) or rapidly induced, they might control
initial activation and cell division. CD27 might
also function to regulate clonal expansion.
Expression of OX40, 4-1BB and CD30 and their
ligands is induced by T cells and APCs with
delayed kinetics and might function later. OX40
and 4-1BB, and possibly CD30, prevent apoptosis
and regulate the ability of effector cells to
persist at the peak of the response. The timeline
depicted is based on both in vitro and in vivo
data from several studies. Most, but not all,
data fit this model. A great deal of overlap in
the timing of action of tumour-necrosis factor
receptor (TNFR) molecules is probable and will
vary depending on the nature of the antigen, its
dose and pro-inflammatory factors.
31Current approaches for survivin targeting in
cancer therapy. a Generation of
antigen-specific cytolytic T cells against
survivin peptides is being considered for
vaccination strategies. b Molecular antagonists
of survivin, including antisense, ribozymes and
expression of dominant-negative mutants, have
been consistently associated with induction of
apoptosis, enhancement of cell-death stimuli and
inhibition of tumour growth in vivo. c
Interference of mitotic phosphorylation of
survivin by CDC2 by sequential administration of
cyclin-dependent kinase inhibitors flavopiridol
or Purvalanol A after chemotherapy-induced
mitotic arrest has resulted in Strong anticancer
activity in vitro and in vivo. A similar response
has been observed by adenoviral over expression
of a phosphorylation-deficient survivin Thr34?Ala
mutant (pAd-T34A).