Cytotoxicity assay Cor Berrevoets Lab Tumor Immunology Dept. - - PowerPoint PPT Presentation
Course on Biomedical Research Techniques 2018 Cytotoxicity assay Cor Berrevoets Lab Tumor Immunology Dept. Medical Oncology Erasmus MC Cancer Institute c.berrevoets@erasmusmc.nl Cytotoxicity The degree to which something is toxic to living
Lab Tumor Immunology
Erasmus MC Cancer Institute c.berrevoets@erasmusmc.nl
Course on Biomedical Research Techniques 2018
Immunobiology, 5th edition, Janeway et al.
Target cells : - infected cells
Non-specific binding Specific binding by T cell receptor
Immunobiology, 5th edition, Janeway et al. GA=Golgi MTOC=Microtubuli-organizing center
Release of granules
Cytoskeleton Lytic granules
a) Initially thought to polymerize to form a pore in target membrane b) Enables release of lytic complexes from endocytic vesicles of the target cell, where granzymes would otherwise be trapped
Granzyme A / B / H / K / M
Lopez et al, Trends in Immunol, 2012, p406–412
B16 melanoma tumor cells Liver cells blebbing and undergoing apoptosis
(Memorial Sloan Kettering Cancer Center)
B16-A2-gp100 tumor cells
20,000 tumor cells seeded /
no T cells + TCR T cells
B16-A2-gp100 tumor cells antigen negative B16 tumor cells
+ TCR T cells
target cell T cell
Target : Melanoma cell
(HLA-A2 positive)
Effector : cytotoxic T cell (high
TCR specific for Melanoma antigen: gp100/A2)
100µl)
E:T ratio 5 10 20 40
no peptide
+gp100 targets only (bkg) targets + 1% triton (max)
= 100.000 - 12.500 per well (100 µl) Centrifuge briefly Incubate 4-6 hrs at 37°C / 5% CO2
Harvest supernatants and count 51Cr.
Controls:
Protocol
Protocol
CPM CPM CPM avg CPM avg-bkg % of max no peptide 250 200 190 213 113 5,5 +peptide 1400 1800 1550 1583 1483 71,8 bkg 110 85 105 100 max 2500 1900 2100 2167 2067 100,0
peptide = antigen
20 40 60 80 100 120
40 20 10 5 2,5 1,25 0,625
E:T ratio
% Cr51 release
+gp100 peptide no peptide
C1Rwt + wt peptide
10 20 30 40 50 60 70 80 90 100 10^-6 10^-5 10^-4 10^-3 10^-2 10^-1 1 uM peptide
% Cr51 release
T cell-1 T cell-2 T cell-3 E:T = 10
MTT / WST-1 proliferation assay
Formazan (dark yellow)
In living cells:
WST-1 substrate
WST-1 killing assay
0% 20% 40% 60% 80% 100% 8 4 2 1 0,5 0,25 E:T ratio number of T cells % Killing
WST-1 killing assay (E:T 4)
0% 20% 40% 60% 80% 100%
B16-gp100 tumor cells
% Killing
TCR T cells Mock T cells
Day 1 : seed tumor cells (96 well plate) Day 2 : add T cells (incl. medium/ triton control) Day 3 : add MTT/WST-1 reagent (10 µl/well) Measure OD450 nm incubate 4 hours ODMedium = 0% killing ODTriton = 100% killing
Measure activity of mitochondrial enzymes
(Rubio et al. Nature Med. 9 (2003)
Cytolytic T-cell (surface CD107-negative) Cytolytic T-cell (surface CD107-positive)
Internalization of antibodies
1. 100.000 T cells per well (in 50 µl) 2. 3.333 target cells per well (in 50 µl), total volume of 100 µl 3.
4. Add anti-CD107a (and CD107b), FITC or PE labelled (20 µl each per ml) 5. Spin 2 min, 200xg 6. Incubate 1-4 hrs at 37°C/ 5% CO2 7. Wash in plate 1x 200 µl PBS 8. Fix in 200 µl PFA1% and analyze in flowcytometer
Betts et al., J.Immunol.Meth. 281 (2003)
Protocol
1hr no peptide 1hr + 1µM gp100-wt 4hr no peptide 4hr + 1µM gp100-wt
E:T ratio 30:1
T cells only targets only
No CD107 staining of control cells (4 hrs)
4 hours (at 37°C):
then 30 min (at 4°C):
Quadruple staining Mouse T cells vs. B16 mouse melanoma tumor cells
TCR positive T cells
Pros and cons
51Cr release assay
1. Radioactive 2. Gammacounter required 3. High sensitivity, however dependent on target viability for good 51Cr-labelling efficiency 4. Possible to finish the assay (end of the day) and measure radioactivity later (e.g. next day)
1. Indirect measurement of killing (lack of viability) 2. Added T cells can result in higher background MTT/WST-1 signal
Pros and cons CD107 mobilization assay 1. Non-radioactive 2. Flowcytometer required 3. Monitoring of individual T cells based on costaining patterns 4. Degranulation is not always correlated with cytolysis ! 5. Time-consuming when handling large number of samples and co-stainings