gougeon - ecdo documents/gougeon.pdf · analysis of apoptosis by flow-cytometry • antiviral...

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

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

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

Membrane integrity

Staining of apoptotic cells with :

YO-PRO-17-AAD

Staining of end staged dead cells with PI

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

QuickTime™ et undécompresseur TIFF (non compressé)

sont requis pour visionner cette image.

7-AAD stainingof ApoptoticCells

Lecoeur et al. J Imm Meth, 209:111-123

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

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



Multiparametric Analysis of Apoptosis:FSC/7-AAD/Annexin-V/ISNT


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

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

Untreated Jurkat cells

Camptothecin-treated cells

Caspase-8 reagent

PI

PI+, caspase- (Dead)

PI-, caspase+ (Apoptotic)

Detection of active caspase-8

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

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.

Thymocytes stained with JC1 following incubation :

4 C

37 C

DXM 1µM

Living

Apoptotic


Kinetics of mitochondrial alterations in thymocytes treated with DXM

DIOC6(3)

NAO

DNA fragmentation


Confocal microscopy analysis of apoptotic thymocytes

DIOC6(3)JC-1

J-aggregatesMonomeres

Living thymocytes Apoptotic thymocytes

PI/NAO Extinctionof mito NAOFluo/PI+ nuclei


Time course analysis of early events in DXM-apoptosis

∆ψm

Fragmented DNA

Trypan blue staining


Strategies for Phenotyping Apoptotic Lymphocytesin a complex population

Method for the simultaneous Detection of Both Surface

and Intracellular Molecules on apoptotic cells

Objectives:

1- To determine the frequency of apoptotic cellswithin a complex population (peripheral lymphocytes)

2- To identify the phenotype of apoptotic cells



ISNT does not allow the proper detection of surface molecules on apop cells




Impact of detergents on cell morphology




EtOH is inappropriate for the phenotyping of apoptotic cells




Strategy for identification of both surface and intracellular molecules on apop cells


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



Intracellular cytokine detection in apoptotic cells


The example of apoptosis measurement in the context of HIV infection

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

Apoptotic pathways triggered by HIV

ML Gougeon, Nature Rev Immunology 2003

HIV-infection primes T lymphocytes for spontaneous and activation-induced apoptosis

Gougeon et al. J Immunology, 156:3509

Quantification of apoptosis in CD4 and CD8 T cells with 7-AAD staining

Gougeon et al. J Immunology, 156:3509

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

Impact of premature apoptosis at the levelof CD8 cytotoxic T cells

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

Down-regulation of Bcl-2 expression primes patients’ CD8 T cells for apoptosis


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


Impact of premature apoptosis at theCD4 T cell level

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

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

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

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

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

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