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Analysis of Apoptosis by FlowAnalysis of Apoptosis by Flow--cytometrycytometry
• Antiviral Immunity, Biotherapy and Vaccine Unit
Marie-Lise GOUGEON
2nd training course onConcepts and Methods in Programmed Cell Death
« Genetic Pathways and Techniques for Detection of Cell Death »Budapest, October 1, 2005
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Cell shrinkingChromatin condensation
Cell fragmentation
Apoptotic bodies and phagocytosis
Apoptosis
Changes in morphology
Alteration of membraneintegrity
PS exposure
Drop in ∆ψm
caspase activation
Membrane alteration
Chromatin condensation
DNA fragmentation
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Alteration of Morphology
Size
Granularity
Living cells
Early apoptoticLate apoptotic
Dead cells
Thymocytes
FACS acquisition
DXM24h
Petit et al. J Cell Biology, 130:157-167
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Membrane integrity
Staining of apoptotic cells with :
YO-PRO-17-AAD
Staining of end staged dead cells with PI
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Membrane integrity- Costaining with YO-PRO-1 and PI
YO-PRO-1
PI
Jurkat cellsCamptothecin 10mM
Co-staining with YO-PRO-1 and PI
UntreatedTreated
4hrs
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7-AAD stainingof ApoptoticCells
Lecoeur et al. J Imm Meth, 209:111-123
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Lecoeur et al. J Imm Meth, 209:111-123
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Phosphatidylserine exposurePhosphatidylserine exposure
PS, normally located on the cytoplasmic surface of the cell membrane,becomes exposed to the extracellular environment. Annexin V (human vascular anticoagulant with high affinity for PS), allows the detection of PS.
Annexin-V
PIJurkat + camptothecin
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Two Examples (Left and Right) of Triple Stained(HO-AnnexFITC and PI) DHD Cells
Blue light
UV light
Intact cells are blue, apoptotic cells are green, late apoptotic necroticcells are multilabelled.
Broken nucleus
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Multiparametric Analysis of Apoptosis:FSC/7-AAD/Annexin-V/ISNT
Lecoeur et al. J Imm Meth, 209:111-123
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Death receptor pathway
Apoptosome
IAPs
Bid
truncatedbid
CD95LCD95
FAD
D
Procaspase-8
caspase-8
Procaspase-3
caspase-3
Apoptotic substrates
c-FLIP
Two Major Apoptotic Pathways in Mammalian Cells
Type I cells
AIFcyto c
Apaf-1
Pro-casp-9
DyM
DNA damage
Mitochondrial pathway
p53
Bax
Bcl-xl
Bcl-2
Smac/DIABLO
Type II cells
Caspase-8
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Caspase activation
FLICA: caspase inhibitor, binds to the reactive cysteinson active caspases and inhibits further enzymatic activity.Unbound FLICA diffuses out of the cell and is washed away;The green fluorescent signal measures the amount of active caspase that was present at the time FLICA was added.
Jurkat cellsCamptothecin 10mM
Co-staining with FLICA and PI4hrs
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Untreated Jurkat cells
Camptothecin-treated cells
Caspase-8 reagent
PI
PI+, caspase- (Dead)
PI-, caspase+ (Apoptotic)
Detection of active caspase-8
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Death receptor pathway
Apoptosome
IAPs
Bid
truncatedbid
CD95LCD95
FAD
D
Procaspase-8
caspase-8
Procaspase-3
caspase-3
Apoptotic substrates
c-FLIP
Two Major Apoptotic Pathways in Mammalian Cells
Type I cells
AIFcyto c
Apaf-1
Pro-casp-9
DyM
DNA damage
Mitochondrial pathway
p53
Bax
Bcl-xl
Bcl-2
Smac/DIABLO
Type II cells
Caspase-8
∆ψm
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Mitochondrial Structure and Function
∆ψm assessment using DIOC6(3) and JC-1 dyes:
DIOC6(3) accumulates in the mitochondrial matrix underthe influence of the ∆ψm
JC-1 forms aggregates under high mitoch ∆ψm, which fluoresce in red, whereas the monomeric form fluoresces in green.
Mitochondrial structure analysis:Nonyl Acridine Orangeincorporation. Reveals alteration of cardiolipids in mitochmembrane. 1 cardiolipid molecule binds 2 NAO molecules=>decreased fluorescence means decreased or altered cardiolipids.
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Thymocytes stained with JC1 following incubation :
4 C
37 C
DXM 1µM
Living
Apoptotic
Petit et al. J Cell Biology, 130:157-167
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Kinetics of mitochondrial alterations in thymocytes treated with DXM
DIOC6(3)
NAO
DNA fragmentation
Petit et al. J Cell Biology, 130:157-167
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Confocal microscopy analysis of apoptotic thymocytes
DIOC6(3)JC-1
J-aggregatesMonomeres
Living thymocytes Apoptotic thymocytes
PI/NAO Extinctionof mito NAOFluo/PI+ nuclei
Petit et al. J Cell Biology, 130:157-167
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Time course analysis of early events in DXM-apoptosis
∆ψm
Fragmented DNA
Trypan blue staining
Petit et al. J Cell Biology, 130:157-167
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Strategies for Phenotyping Apoptotic Lymphocytesin a complex population
Method for the simultaneous Detection of Both Surface
and Intracellular Molecules on apoptotic cells
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Objectives:
1- To determine the frequency of apoptotic cellswithin a complex population (peripheral lymphocytes)
2- To identify the phenotype of apoptotic cells
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Lecoeur et al. J Imm Meth, 209:111-123
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Lecoeur et al. J Imm Meth, 209:111-123
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ISNT does not allow the proper detection of surface molecules on apop cells
Lecoeur et al. J Imm Meth, 209:111-123
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Impact of detergents on cell morphology
Lecoeur et al. J Imm Meth, 217:11-26
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EtOH is inappropriate for the phenotyping of apoptotic cells
Lecoeur et al. J Imm Meth, 217:11-26
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Strategy for identification of both surface and intracellular molecules on apop cells
Lecoeur et al. J Imm Meth, 217:11-26
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Intracellular cytokine detection in apoptotic cells
log
7-A
AD
log
7-A
AD
log caspase-3contrôle
PBMC
PPIBrefeldin A
Multiparametric FACS analysis(staining with CD3-,CD4-,CD8-,Cytokine-mAbs and 7-AAD)
%
23 %
log 7-AAD
log
IFN
-γ
CD3 gated
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Intracellular cytokine detection in apoptotic cells
Lecoeur et al. J Imm Meth, 217:11-26
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The example of apoptosis measurement in the context of HIV infection
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Regulatory Pathway of Mature T Lymphocyte Apoptosis
RestingT Cell
ActivatedT Cell IL-2
Activation
ProliferatingT cell
Low or no Ag Secondary Ag stimulation
Memory T cell
Cytokine deprivation death Antigen-dependent death- Antigen-independent- Induced by withdrawal of IL-2- Prevented by Bcl-2, Bcl-xl- Eliminates excessive T cells
after antigen clearance.
- Antigen-dependent- Mediated by FasL or TNF- Blocked by FLIPs- Limits response to repeated Ag stimulation
Ag/HIV
Cycling
Ag/HIV
FasL or TNFBcl-2
Fas
TNFR
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Apoptotic pathways triggered by HIV
ML Gougeon, Nature Rev Immunology 2003
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HIV-infection primes T lymphocytes for spontaneous and activation-induced apoptosis
Gougeon et al. J Immunology, 156:3509
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Quantification of apoptosis in CD4 and CD8 T cells with 7-AAD staining
Gougeon et al. J Immunology, 156:3509
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CD4 T-cell dependence of CTL. HIV-dependent impairment
NaiveNaiveHIVHIV--CD4 ThCD4 Th
DCDC
HIV
IL-12
CD4 CD4 Th1Th1
IL-2IFNγ
cytokines
Lysis of infected cellsperforin, granzymes, CD95/CD95L
CD8 CD8 CTLCTL
HIV-CD4 Th
Cytokines• Interferon-γ• TNF-α
ChemokinesMIP-1a, MIP-1ßRANTES
Inhibitioninfection
Poor cytotoxicityPoor cytotoxicity
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Impact of premature apoptosis at the levelof CD8 cytotoxic T cells
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Low Bcl-2 CD8 High Bcl-2 CD8
HIV disease evolution is associated with progressive accumulation of low Bcl-2 CD8 T cells
Bcl-2
Boudet et al.J. Immunol.156:2580
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Down-regulation of Bcl-2 expression primes patients’ CD8 T cells for apoptosis
Boudet et al.J. Immunol.156:2580
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In vivo Characteristics of low Bcl-2 CD8 T cells in patients’blood and Lymph nodes
T CD8
HLA-DR
TiA1
Perforin
Characteristics of activated CTL: HLA-DR+, CD38+, CD28-, express cytotoxic granules
CD38
Bcl-2IL-2R
IL-2, IL-15
5-15% in blood, > 60% in LN
Boudet et al.J. Immunol.156:2580
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Impact of premature apoptosis at theCD4 T cell level
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PBMCs
Triple staining with mAbs: surface CD3/CD8intracellular cytokines nuclear 7-AAD (apoptosis)
PMA + PHA +IonomycinBrefeldin A
33 % %
23 %
33 % 30 %
23 %
log 7-AAD
log
TNF-
α
Ex-vivo quantification of cytokine-producing precursors
Gated on T CD3
Lecoeur H, Ledru E, Gougeon M-LJ Imm Methods 217:11
FACS analysis
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Decrease of the frequency of IL-2 and TNF-α -producing T cell precursors
Homme
log CD3+
log
IL-2
control HIV+39 % 14 %
CD3+
60
50
40
30
20
10
0
IL-2
**
60
50
40
30
20
10
IFN-γ
**
60
50
40
30
20
10
0
TNF-α IL-4 IL-13
*
% p
ositi
ve c
ells
0
controls HIV+ CD4 > 29 % 13 % < CD4 < 28 % CD4 < 13 % *p < 0.05 vs contrôles
60
50
40
30
20
10
0
60
50
40
30
20
10
0
Ledru et al. J. Immunol. 160:3194
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Decreased frequency of IL-2 and TNF-α T cell producers is related to their priming for activation-induced apoptosis
CD8+CD4+
0
20
40
60
80
100
CD8+CD4+
0
20
40
60
80
100
Controls HIV+ Patients
%A
popt
ose
% A
popt
ose
IL-2
IFN
-γTN
F-α
IL-2
IFN
-γTN
F-α
IL-2
IFN
-γTN
F-α
IL-2
IFN
-γTN
F-α
* * *
6040200
60
40
20
0
p < 0.0001 r = -0.58
6040200
60
40
20
0
NS r = O.10
6040200
60
40
20
0
p < 0.02 r = -0.33
% A
popt
osis
with
in t
he s
ubse
t
% cytokine-producing CD3+ T cells
IL-2
IL-2 IFN-γ
TNF-α
Ledru et al. J. Immunol. 160:3194
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1- Quantitative and qualitative defect in CD4 Thelper cells
2- Defect in CTL CD8 maturation
3- Altered type-1 cytokine expression
TT--cell priming for apoptosis contributes to:cell priming for apoptosis contributes to:
Defective HIVDefective HIV--specific immunityspecific immunityUncontrolled Uncontrolled viral replicationviral replicationCollapse of the Immune system =>AIDSCollapse of the Immune system =>AIDS
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References
P.X.PETIT,LECOEUR H, ZORN E.DAUGUET C.MIGNOTTE B.,GOUGEON ML Alterations in mitochondrial structure and function are early events of dexamethasone-induced apoptosis. J. Cell Biology. (1995) 130: 157-167
F. BOUDET, LECOEUR H, GOUGEON M-LApoptosis associated with ex-vivo down-regulation of Bcl-2 and up-regulation of Fas in potential cytotoxic CD8+ T lymphocytes during HIV infection. J. Immunol. (1996),156:2282
M-L. GOUGEON, LECOEUR H, DULIOUST A, ENOUF M. G., CROUVOISIER M., GOUJARD C. DEBORD T., MONTAGNIER L. Programmed cell death in peripheral lymphocytes from HIV-infected persons : the increased susceptibility to apoptosis of CD4 and CD8 T cellscorrelates with lymphocyte activation and with disease progression;J. Immunol. (1996), 156:3509
H. LECOEUR and M-L. GOUGEON.Comparative analysis of flow cytometric methods for apoptosis quantitation in thymocytes and human peripheral bloodlymphocytes of controls and HIV+ persons. Evidence forinterferences of granulocytes and erythrocytes. J. Immunol. Methods (1996)198:87-99
M-L GOUGEON, LECOEUR H., BOUDET F., LEDRU E., MARZABAL S., BOULLIE, S., ROUE R., NAGATA S., HEENEY J. Lack of chronic immune activation in HIV-infected chimpanzees correlates with the resistance of T cells to Fas/Apo-1 (CD95)-induced apoptosis and preservation of a Th1 phenotype. J. Immunol. (1997) 158:2964
H. LECOEUR, LEDRU E., PREVOST M-C., GOUGEON M-L. Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT,Annexin-V and 7-AAD cytofluorometric methods. J. Immunol. Methods (1997) 209:11-20
E. LEDRU, LECOEUR H, GARCIA S, DEBORD T, GOUGEON M-L.Differential susceptibility to activation-induced apoptosis among peripheral Th1subsets.Correlation with Bcl-2 expression and consequences for AIDS pathogenesis. J. Immunology (1998), 160: 3194-3206
H. LECOEUR, LEDRU E, GOUGEON M-L.A cytofluorometric method for the simultaneous detection of both intracellular and surfaceantigens on apoptotic peripheral lymphocytes. J. Immunol. Methods (1998), 217:11-26
E LEDRU, N CHRISTEFF, O PATEY, J-C MELCHIOR, M-L GOUGEON. Alteration of TNFa T cell homeostasis following HAART. Implication in thedevelopment of HIV-associated lipodystrophy syndrome.BLOOD (2000) 95:3191-98
H LECOEUR, MC PRÉVOST AND M-L GOUGEON. Oncosis is associated to exposure of phosphatidylserine residues on the outside layer of plasma membrane. A reconsideration of the specificity of the AnnexinV-Propidium Iodide assay. Cytometry 2001, 44:65-72
M-L GOUGEON. Apoptosis as an HIV strategy to escape immune attack. Nature Review Immunology, 2003
M-L GOUGEON. To kill or be killed: how HIV exhausts the immune system. Cell Death Diff. 2005, i n press