Cytotoxicity assays. Rory D. de Vries, PhD 1. Viroscience lab, Erasmus MC, Rotterdam, the Netherlands

Transcription

1 Cytotoxicity assays Rory D. de Vries, PhD 1 1 Viroscience lab, Erasmus MC, Rotterdam, the Netherlands

2 Anti-influenza immunity Humoral / CD4+ / CD8+ / NK?

3 Function of CTL Elimination of virus-infected cells? Function of CTL is to eliminate virus-infected cells By lysis and induction of apoptosis through release of perforin, granzyme and FasLexpression Release of cytokines, e.g. IFNg and TNFa Recognition by CTL is MHC class I restricted

4 Anti-influenza CTL CTL as correlate of protection in humans

5 Detection of CTL Virus-specific CTL detection Proliferation of virus-specific CTL 3H-thymidine incorporation CFSE dilution assay Functional properties of virus-specific CTL Lytic activity ( 51 Cr classic) Cytokine production Activation markers Epitope specificity Peptides Multimers of MHC class I / peptide complex All of the above

6 Detection of CTL Virus-specific CTL detection Proliferation of virus-specific CTL 3H-thymidine incorporation CFSE dilution assay Functional properties of virus-specific CTL Lytic activity Cytokine production Activation markers Epitope specificity Peptides Multimers of MHC class I / peptide complex All of the above

7 CFSE dilution assay In vitro proliferation of antigen-specific T cells 5,6-CarboxyFluorecein diacetate Succinimidyl Ester (CFSE) labelling

8 CFSE dilution assay Advantages and disadvantages Advantages Easy to perform Flow cytometry Allows identification of cells Functional profile and differentiation No use of isotopes Disadvantages Identifies proliferating cells only Bystander activation?

9 Detection of CTL Virus-specific CTL detection Proliferation of virus-specific CTL 3H-thymidine incorporation CFSE dilution assay Functional properties of virus-specific CTL Lytic activity ( 51 Cr classic) Cytokine production Activation markers Epitope specificity Peptides Multimers of MHC class I / peptide complex All of the above

10 Alternatives to 51 Cr Classical 51 Cr release assay has only disadvantages Since the 51 Cr release assay has only disadvantages, alternatives: Flow cytometric methods CD107a degranulation assay Label target cells with fluorescent dyes, add effectors, measure target cell viability FATT-CTL Viral clearance assay

11 Lytic activity of CTL CD107a expression, marker for degranulation

12 Lytic activity of CTL FATT-CTL Fluorescent-antigen-transfected target (FATT) cells CTL assay Nucleofection into target cell by electroporation Target cell expressing antigen of interest detectable by flow cytometry 488nm Antigen-encoding gene in eukaryotic expression vector including GFP

13 Lytic activity of CTL FATT-CTL Cytotoxicity (target cell destruction) TCR 488 nm 635 nm TOPRO-3 MHC-I FITC detector APC detector

14 Lytic activity of CTL FATT-CTL TP3 % eliminated from live gate GFP Effectors: Targets: Antigen: Flu NP CD8+ T cell clone HLA-matched PBMC pnp01-gfp / ptat-gfp

15 Lytic activity of CTL FATT-CTL ex vivo without prior expansion!

16 FATT CTL assay Advantages and disadvantages Advantages No use of isotopes Endogenous antigen processing No use of peptides Endogenous antigen presentation No autologous cell lines required PBMC as target cells Advantages Sensitive Prolonged incubation times allow detection of lytic activity without prior expansion of T cells Frequency must be high Chronic infections No need for HLA typing Use of easy to prepare plasmids No viral vectors required

17 Alternative: Viral clearance / suppression assay Set-up for measles expressing reporters, could be adapted for influenza Many publications on influenza viruses expressing reporter genes Advantage over FATT-CTL: No construction of plasmids Context of live viral infection Easier to perform Disadvantage over FATT-CTL: Less standardization friendly

18 Alternative: Viral clearance assay

19 Alternative: Viral clearance assay

20 Alternative: Viral clearance assay

21 Considerations Different assays available, perform which assay? Many different techniques available: Considerations: Prior expansion / stimulation in vitro allowed YES / NO Antigen used for stimulation PEPTIDE / PROTEIN / VIRUS Immunodominant epitopes NO / YES, which HLA type KNOWN / UNKNOWN Exposure history KNOWN / UNKNOWN Phenotyping of cells required YES / NO

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