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 cells - chemicals - radiation - immune cells * Cytotoxic T cells * Natural Killer (NK) cells * Lymphokine Activated Killer (LAK) cells

Cytotoxic T cells kill target cells bearing certain antigens Target cells : - infected cells - tumor cells Immunobiology, 5th edition, Janeway et al.

Lytic granules in cytotoxic T cells migrate to the point of contact Non-specific binding Cytoskeleton Lytic granules Specific binding by T cell receptor Release of granules Immunobiology, 5th edition, GA=Golgi MTOC=Microtubuli-organizing center Janeway et al.

Proteins involved in cell killing by cytotoxic T cells Perforin 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 Granzymes Family of serine proteases, which can activate/cleave caspases (“executioner proteins”)  activates DNases  apoptosis of the target cell Granzyme A / B / H / K / M Lopez et al, Trends in Immunol, 2012, p406 – 412

Proteins involved in cell killing by cytotoxic T cells Fas ligand Fas ligand is expressed as a trimer on T cells, it trimerizes the FasReceptor present on the target cell  activation of caspases  apoptosis

Killing of an Influenza infected target cell by a cytotoxic T cell movie Liver cells blebbing and undergoing apoptosis (Memorial Sloan Kettering Cancer Center) B16 melanoma tumor cells

Killing of an Influenza infected target cell by a cytotoxic T cell

In vitro killing of melanoma tumor cells by TCR transduced T cells B16-A2-gp100 tumor cells 20,000 tumor cells seeded / overnight incubation with T cells no T cells B16-A2-gp100 tumor cells antigen negative B16 tumor cells + TCR T cells + TCR T cells

How to quantify cytotoxicity of T cells? Target cell * 51 Chromium release assay * Fluorochrome release assay * Cellular viability assay (WST-1) T cell * CD107 mobilization assay

Target cell * 51 Chromium release assay (gammacounter) * Fluorochrome release assay (fluorimeter) - Calcein-acetoxymethyl (AM) Comparable sensitivity Higher spontaneous release of Calcein-AM (up to 30%) as compared to 51 Cr.

51 Chromium release assay Active uptake of 51 Cr by target cells Incubation 4-6 hr target cell T cell Add T cells Measure released 51 Cr in medium

Target : Melanoma cell Effector : cytotoxic T cell ( high Protocol TCR specific for Melanoma antigen: (HLA-A2 positive) gp100/A2) • 0.5x10 6 Melanoma target cells • Count T cells • Add 2 Mbq 51 Cr • Add decreasing number to the target cells • Incubate 2 hrs at 37°C / 5% CO 2 • e.g. Effector:Target ratio 40-5 • Wash 2x = 100.000 - 12.500 per well (100 µl) • Incubate 15 min with/without gp100 peptide E:T ratio 40 20 10 5 . . . . . . . . . . . . . . . . . . • In 96-wells plate : 2500 cells/well in triplo (in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . no peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100µl) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +gp100 targets only (bkg) targets + 1% triton (max) Controls: • Targets + medium (background release of 51 Cr) • Targets + 1% triton (max. release) Centrifuge briefly Incubate 4-6 hrs at 37°C / 5% CO 2 Harvest supernatants and count 51 Cr.

Protocol Count tubes for 51 Cr in gamma-counter (2 min / tube) Data (CPM)  1. Subtract background 2. Relate to max control (%) 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

51 Chromium release assay 120 +gp100 peptide no peptide 100 % Cr51 release 80 60 40 20 0 40 20 10 5 2,5 1,25 0,625 E:T ratio

51 Chromium release assay Dose response curve E:T = 10 T cell-1 T cell-2 T cell-3 C1Rwt + wt peptide 100 90 80 70 % Cr51 release 60 50 40 30 20 10 0 0 10^-6 10^-5 10^-4 10^-3 10^-2 10^-1 1 -10 uM peptide

Cellular viability assays used to quantify cell killing MTT / WST-1 proliferation assay In living cells: Measure activity of mitochondrial enzymes 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 OD 450 nm incubate 4 hours OD Medium = 0% killing WST-1 substrate Formazan (dark yellow) OD Triton = 100% killing WST-1 killing assay (E:T 4) WST-1 killing assay 100% 100% TCR T cells 80% Mock T cells 80% % Killing 60% % Killing 60% 40% 40% 20% 20% 0% 8 4 2 1 0,5 0,25 0% E:T ratio number of T cells B16-gp100 tumor cells

How to quantify cytotoxicity of T cells? Target cell * 51 Chromium release assay * Fluorochrome release assay * Cell proliferation assay T cell * CD107 mobilization assay

CD107 Mobilization assay Detection (and isolation) of viable cytolytic T cells based on surface expression of CD107 Lysosomal Associated Membrane Protein (LAMP) LAMP-1 = CD107a LAMP-2 = CD107b  Upon degranulation CD107a and -b are highly glycosylated expressed on the cell surface  Linear relationship between CD107 expression and cytolytic activity (Rubio et al. Nature Med. 9 (2003)

CD107 Mobilization assay Internalization of Cytolytic T-cell Cytolytic T-cell antibodies (surface CD107-negative) (surface CD107-positive) M. Uhrberg , Leukemia 19 (2005)

CD107 mobilization assay Protocol 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. -/+ gp100 peptide (1 µM) 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)

CD107mobilization assay E:T ratio 30:1 No CD107 staining of 1hr no peptide 1hr + 1µM gp100-wt control cells (4 hrs) targets only T cells only 4hr no peptide 4hr + 1µM gp100-wt

CD107 mobilization assay Mouse T cells vs. B16 mouse melanoma tumor cells Quadruple staining 4 hours (at 37°C): 1. CD107-PE then 30 min (at 4°C): 2. CD3-PerCP 3. CD8-APC 4. VB3-FITC (TCR) TCR positive T cells

Cytotoxicity assays Pros and cons 51 Cr release assay 1. Radioactive 2. Gammacounter required High sensitivity, however dependent on target viability for good 51 Cr-labelling 3. efficiency 4. Possible to finish the assay (end of the day) and measure radioactivity later (e.g. next day) MTT/WST-1 cellular viability assay 1. Indirect measurement of killing (lack of viability) 2. Added T cells can result in higher background MTT/WST-1 signal

Cytotoxicity assays 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