Analysis of Apoptosis by Flow-cytometry

Analysis of Apoptosis by Flow-cytometry Marie-Lise GOUGEON Antiviral Immunity, Biotherapy and Vaccine Unit 2nd training course on Concepts and Methods in Programmed Cell Death «Genetic Pathways and Techniques for Detection of Cell Death» Budapest, October 1, 2005

Apoptosis Changes in morphology Cell shrinking Chromatin condensation Alteration of membrane integrity PS exposure Drop in ψ m Membrane alteration caspase activation Cell fragmentation Chromatin condensation DNA fragmentation Apoptotic bodies and phagocytosis

Alteration of Morphology Granularity Living cells Late apoptotic Early apoptotic Thymocytes Dead cells 24h DXM FACS acquisition Size Petit et al. J Cell Biology, 130:157-167

Membrane integrity Staining of apoptotic cells with : YO-PRO-1 7-AAD Staining of end staged dead cells with PI

Membrane integrity- Costaining with YO-PRO-1 and PI PI Treated Untreated Jurkat cells 4hrs Camptothecin 10mM YO-PRO-1 Co-staining with YO-PRO-1 and PI

Lecoeur et al. J Imm Meth, 209:111-123 7-AAD staining of Apoptotic Cells QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image.

QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. Lecoeur et al. J Imm Meth, 209:111-123

Phosphatidylserine 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. PI Jurkat + camptothecin Annexin-V

Two Examples (Left and Right) of Triple Stained (HO-AnnexFITC and PI) DHD Cells UV light Blue light Broken nucleus Intact cells are blue, apoptotic cells are green, late apoptotic necrotic cells are multilabelled.

Multiparametric Analysis of Apoptosis: FSC/7-AAD/ Annexin-V/ISNT QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. Lecoeur et al. J Imm Meth, 209:111-123

Two Major Apoptotic Pathways in Mammalian Cells Death receptor pathway Mitochondrial pathway DNA damage CD95L CD95 Bcl-xl DyM p53 c-flip FADD Procaspase-8 Bcl-2 Bax Caspase-8 caspase-8 Bid Procaspase-3 truncated bid cyto c AIF Apoptosome Apaf-1 Pro-casp-9 Type I cells caspase-3 IAPs Type II cells Apoptotic substrates Smac/DIABLO

Caspase activation Jurkat cells 4hrs Camptothecin 10mM Co-staining with FLICA and PI FLICA: caspase inhibitor, binds to the reactive cysteins on 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.

PI Detection of active caspase-8 Untreated Jurkat cells PI +, caspase - ( Dead) Camptothecin-treated cells PI -, caspase + ( Apoptotic) Caspase-8 reagent

Two Major Apoptotic Pathways in Mammalian Cells Death receptor pathway Mitochondrial pathway DNA damage CD95L CD95 Bcl-xl Dy DyM m p53 c-flip FADD Procaspase-8 Bcl-2 Bax Caspase-8 caspase-8 Bid Procaspase-3 truncated bid cyto c AIF Apoptosome Apaf-1 Pro-casp-9 Type I cells caspase-3 IAPs Type II cells Apoptotic substrates Smac/DIABLO

Mitochondrial Structure and Function Dy m assessment using DIOC 6 (3) and JC-1 dyes: DIOC 6 (3) accumulates in the mitochondrial matrix under the influence of the ψ m JC-1 forms aggregates under high mitoch ψ m, which fluoresce in red, whereas the monomeric form fluoresces in green. Mitochondrial structure analysis:nonyl Acridine Orange incorporation. Reveals alteration of cardiolipids in mitoch membrane. 1 cardiolipid molecule binds 2 NAO molecules=> decreased fluorescence means decreased or altered cardiolipids.

Thymocytes stained with JC1 following incubation : Living 4 C 37 C Apoptotic DXM 1mM Petit et al. J Cell Biology, 130:157-167

Kinetics of mitochondrial alterations in thymocytes treated with DXM DIOC 6 (3) NAO DNA fragmentation Petit et al. J Cell Biology, 130:157-167

Confocal microscopy analysis of apoptotic thymocytes DIOC 6 (3) JC-1 J-aggregates Monomeres PI/NAO Extinction of mito NAO Fluo/PI+ nuclei Living thymocytes Apoptotic thymocytes Petit et al. J Cell Biology, 130:157-167

Time course analysis of early events in DXM-apoptosis Dy m Fragmented DNA Trypan blue staining Petit et al. J Cell Biology, 130:157-167

Strategies for Phenotyping Apoptotic Lymphocytes in 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 cells within a complex population (peripheral lymphocytes) 2- To identify the phenotype of apoptotic cells

QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. Lecoeur et al. J Imm Meth, 209:111-123

QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. Lecoeur et al. J Imm Meth, 209:111-1

ISNT does not allow the proper detection of surface molecules on apop cells QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. Lecoeur et al. J Imm Meth, 209:111-123

Impact of detergents on cell morphology QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. Lecoeur et al. J Imm Meth, 217:11-26

EtOH is inappropriate for the phenotyping of apoptotic cells QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. Lecoeur et al. J Imm Meth, 217:11-26

Strategy for identification of both surface and intracellular molecules on apop cells QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. Lecoeur et al. J Imm Meth, 217:11-26

Intracellular cytokine detection in apoptotic cells PBMC PPI Brefeldin A Multiparametric FACS analysis (staining with CD3-,CD4-,CD8-, Cytokine-mAbs and 7-AAD) CD3 gated log 7-AAD log 7-AAD log IFN-γ % 23 % log 7-AAD contrôle log caspase-3

Intracellular cytokine detection in apoptotic cells QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. Lecoeur et al. J Imm Meth, 217:11-26

The example of apoptosis measurement in the context of HIV infection

Regulatory Pathway of Mature T Lymphocyte Apoptosis Resting T Cell Ag/HIV Activation Low or no Ag Ag/HIV Activated T Cell IL-2 Secondary Ag stimulation Bcl-2 Cycling Fas FasL or TNF Cytokine deprivation death - Antigen-independent - Induced by withdrawal of IL-2 - Prevented by Bcl-2, Bcl-xl - Eliminates excessive T cells after antigen clearance. Proliferating T cell Memory T cell TNFR Antigen-dependent death - Antigen-dependent - Mediated by FasL or TNF - Blocked by FLIPs - Limits response to repeated Ag stimulation

Apoptotic pathways triggered by HIV ML Gougeon, Nature Rev Immunology 2003

Gougeon et al. J Immunology, 156:3509 HIV-infection primes T lymphocytes for spontaneous and activation-induced apoptosis

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 HIV Lysis of infected cells perforin, granzymes, CD95/CD95L Naive HIV-CD4 Th DC IL-12 cytokines Poor cytotoxicity Cytokines Interferon-g TNF-a IL-2 CD4 Th1 IFNg CD8 CTL Chemokines MIP-1a, MIP-1ß RANTES HIV-CD4 Th Inhibition infection

Impact of premature apoptosis at the level of CD8 cytotoxic T cells

IV disease evolution is associated with progressive accumulation of low Bcl-2 CD8 T cells Bcl-2 Low Bcl-2 CD8 High Bcl-2 CD8 Boudet et al.j. Immunol.156:2580

Down-regulation of Bcl-2 expression primes patients CD8 T cells for apoptosis Boudet et al.j. Immunol.156:2580

In vivo Characteristics of low Bcl-2 CD8 T cells in patients blood and Lymph nodes T CD8 HLA-DR IL-2, IL-15 CD38 Bcl-2 IL-2R TiA1 Perforin 5-15% in blood, > 60% in LN Characteristics of activated CTL: HLA-DR+, CD38+, CD28-, express cytotoxic granules Boudet et al.j. Immunol.156:2580

Impact of premature apoptosis at the CD4 T cell level

Ex-vivo quantification of cytokine-producing precursors PBMCs PMA + PHA +Ionomycin Brefeldin A Gated on T CD3 33 33 % % % 30 % Triple staining with mabs: surface CD3/CD8 intracellular cytokines nuclear 7-AAD (apoptosis) FACS analysis log TNF-α 23 23 % % log 7-AAD Lecoeur H, Ledru E, Gougeon M-L J Imm Methods 217:11

Decrease of the frequency of IL-2 and TNF-α -producing T cell precursors 60 IL-2 60 IFN-γ 60 TNF-α IL-4 IL-13 60 60 % positive cells 50 40 30 20 10 * * Homme 50 40 30 20 10 50 40 30 20 10 * * 50 40 30 20 10 50 40 30 20 10 * 0 0 0 0 0 controls HIV+ CD4 > 29 % 13 % < CD4 < 28 % CD4 < 13 % * p < 0.05 vs contrôles CD3 + log IL-2 control HIV+ 39 % 14 % Ledru et al. J. Immunol. 160:3194 log CD3 +

Decreased frequency of IL-2 and TNF-α T cell producers is related to their priming for activation-induced apoptosis Controls HIV+ Patients CD4+ CD8+ CD4+ CD8+ 100 100 % Apoptose 80 60 40 20 % Apoptose 80 60 40 20 % Apoptosis within the subset 60 40 20 0 0 IL-2 IFN-γ 0 20 IL-2 TNF -α IL-2 p < 0.0001 r = -0.58 40 IL-2 60 IFN-γ TNF -α 60 40 20 0 0 20 NS r = O.10 40 60 IL-2 IFN-γ 0 TNF -α IL-2 * * * 60 40 20 0 0 20 IFN-γ IFNγ TNFα 40 TNF -α p < 0.02 r = -0.33 60 Ledru et al. J. Immunol. 160:3194 % cytokine-producing CD3 + T cells

T-cell priming for apoptosis contributes to: 1- Quantitative and qualitative defect in CD4 Thelper cells 2- Defect in CTL CD8 maturation 3- Altered type-1 cytokine expression Defective HIV-specific immunity Uncontrolled viral replication Collapse of the Immune system =>AIDS

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