Apoptosis is a highly regulated process that plays critical role in many cellular functions: - PowerPoint PPT Presentation

1 / 31
About This Presentation
Title:

Apoptosis is a highly regulated process that plays critical role in many cellular functions:

Description:

This is mainly mediated by Fas, TNFR and in some cases perforin and granzymes ... Web access to TNF/TNFR nomenclature and sequences can be found at ... – PowerPoint PPT presentation

Number of Views:74
Avg rating:3.0/5.0
Slides: 32
Provided by: fournie
Category:

less

Transcript and Presenter's Notes

Title: Apoptosis is a highly regulated process that plays critical role in many cellular functions:


1
Apoptosis 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.
2
Ag-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
3
(No Transcript)
4
Survivin
  • 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)

5
(No Transcript)
6
(No Transcript)
7
(No Transcript)
8
Ag-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
9
Structures 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.

12
Hypothetical 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.
13
Structural 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
14
Generalized 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.
15
Signalling 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).
16
Hypothesis 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.
17
Figure 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.

19
What 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

24
Recall 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
25
antigen
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.

30
Hypothetical 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.
31
Current 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).
Write a Comment
User Comments (0)
About PowerShow.com