University:
Boston University
Course:
CAS BI 108 | Biology 2
Academic year:

2021
Views:

183

Pages:

18
Author:

Maddison Melton

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 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 The example of apoptosis measurement in the context of HIV infection Regulatory Pathway of Mature T Lymphocyte Apoptosis Resting T Cell Ag/HIV Low or no Ag Ag/HIV Bcl-2 Activation Activated T Cell IL-2 Secondary Ag stimulation Fas FasL or TNF Cycling TNFR Cytokine deprivation death Proliferating T cell - Antigen-independent - Induced by withdrawal of IL-2 - Prevented by Bcl-2, Bcl-xl - Eliminates excessive T cells after antigen clearance. Memory T cell Antigen-dependent death - Antigen-dependent - Mediated by FasL or TNF - Blocked by FLIPs - Limits response to repeated Ag stimulation Apoptotic pathways triggered by HIV HIV-infection primes T lymphocytes for spontaneous and activation-induced apoptosis Quantification of apoptosis in CD4 and CD8 T cells with 7-AAD staining CD4 T-cell dependence of CTL. HIV-dependent impairment HIV Lysis of infected cells perforin, granzymes, CD95/CD95L Naive HIVHIV -CD4 Th DC IL-12 Poor cytotoxicity Cytokines • Interferon-γ • TNF-α cytokines IL-2 CD4 Th1 IFNγ CD8 CTL Chemokines MIP-1a, MIP-1ß RANTES Inhibition infection HIV-CD4 Th Impact of premature apoptosis at the level of CD8 cytotoxic T cells HIV disease evolution is associated with progressive accumulation of low Bcl-2 CD8 T cells Bcl-2 Low Bcl-2 CD8 High Bcl-2 CD8 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 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 Impact of premature apoptosis at the CD4 T cell level Ex-vivo quantification of cytokine-producing precursors Gated on T CD3 PBMCs 33 33 % % Triple staining with mAbs: surface CD3/CD8 intracellular cytokines nuclear 7-AAD (apoptosis) FACS analysis log TNF-α PMA + PHA +Ionomycin Brefeldin A % 30 % 2323 %% log 7-AAD Decrease of the frequency of IL-2 and TNF-α -producing T cell precursors IL-2 60 % positive cells 60 50 40 30 * * 20 10 Homme 0 IFN-γ 50 50 40 40 30 30 20 20 * * 10 10 0 0 controls HIV+ TNF-α 60 CD4 > 29 % 13 % < CD4 < 28 % control log IL-2 CD3+ 39 % log CD3+ HIV+ 14 % IL-4 IL-13 60 60 50 50 40 40 30 30 20 20 10 10 0 0 CD4 < 13 % * * p < 0.05 vs contrôles Decreased frequency of IL-2 and TNF-α T cell producers is related to their priming for activation-induced apoptosis HIV+ Patients Controls CD8+ CD4+ % Apoptose 80 60 40 20 80 60 40 20 % Apoptosis within the subset IL-2 2 IFN - 40 40 20 20 0 0 0 40 p < 0.02 r = -0.33 NS r = O.10 20 20 * TNFα 60 60 IL-2 40 ** IFNγ p < 0.0001 r = -0.58 60 IL - IL - 2 IFN - γ TN F -α IL 2 IFN -γ TN F -α 0 γ TN F -α IL 2 IFN -γ TN F -α % Apoptose 100 0 0 CD8+ CD4+ 100 60 0 20 40 60 0 % cytokine-producing CD3+ T cells 20 40 60 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