cytometry-based cytotoxicity analysis for natural killer
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Cytometry-based cytotoxicityanalysis for
Natural Killer cell based immunotherapy
Alar Aints, PhDTartu University and
Competence Centre for Cancer Research
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•Large granuar lymphocytes•5-15% of PBMC•Develop in BM•No rearranging receptors•CD56+CD16+CD3−NKp46+
NK (Natural Killers)
PBMC, hematoxylin/eosin
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NK function regulationMechanism NK receptor Ligand
Ab-dependent cytotoxicity
CD16 Antibodies
Stressmarkers NKG2D MICA, MICB, ULBP
Missing self KIR MHC
Pathogen-associated patterns
TLR3, TLR9 dsRNA, CpG DNA
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Clinical indications/conditions• FHL1(? 9q22) (Familial hemophagocytic lymphohistiocytosis)
• FHL2 (PRF1) • FHL3 (munc13-4) • FHL4 (syntaxin-11)• Chediak-Higashi syndrome (CHS1/LYST) • Griscelli syndrome (Rab27a)• XLPD (SAP)• Cell therapy applications
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NK cells can improve HSCTNK cell effect Biological
mechanismRejection X pt T-cells
Relapse X pt tumor cells
GvHD X pt dendritic cells
Infections Faster immune recovery
NK cells can enable more safe use of haploidentical donors
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Cell culture
• CellGroSCGM, Human serum, IL-2 and OKT3 (Carlens et al, Human Immunology 2001)
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Cell culture
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All eventsCellsNK cellsT cells
All eventsCellsNK cells
T cells
NK cells
T cells T cells
NK cells
T cells
NK cells
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Cytox methods - release assays
• Na251CrO4 Chromium release assay,
• Enzyme release: phosphatases, LDH• Fluorescent dye release (MTT [3-(4,5-
dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide] , MUH (4-methylumbellipheryl heptanoate), Alamar blue , CFDA (carboksyfluoresceine diacetate) , BCECF (2',7'-bis-carboksyethyl-carboxyfluoresceine) , Hoechst 33342 , lanthanides , calcein AM
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Double labeling
• CFDA (carboxyfluorescein diacetate) , DiO18 (dioctadecyl-oxacarbocyanin perchlorate) , PKH2 (green fluorescent Cell Linker) , FITC, calcein AM, CFSE
• PI, 7AAD, Annexin5
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CFSE/7AAD
• CFSE (~1µM) “universal marker“• non-toxic• stable• 7AAD (~3µg/ml) 647nm - CFSE non-
overlapping• fixable, resistant to permeabilization
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Compensation &
biexponentialdisplay
Effector cells
Target cellsTarget cells
Target cellsTarget cells
Effector cells
Effector cells Effector cells
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Setting the gatesA B C
DE
F
R1 - Cells R2-Target cells R3- Permeabilisedtarget cells
R4-Effector cells
CFSE and 7-AAD target cells
Tx-100 lysed target cells
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Setting gates/Dot plot viewA B C
D E
F
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CalculationsR2-Target cells
R3- Permeabilisedtarget cells
R4-Effector cells
CellsTarget Cells
Permeabilized Target CellsEfector Cells
All
• L=R3• C=(Lexp-Lspont)/(Lmax-
Lspont)x100• N=R4/R2 x
NK%/100
7AAD
CFSE
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N
Law of mass action
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Different sensitivity, same shape
K562
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 150
102030405060708090
100
R2=0.97
N
C
721.221
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 150
102030405060708090
100
R2=0.93
N
C
Namalwa
0 2 4 6 8 10 12 14 16 18 20 220
102030405060708090
100
R2=0.96
N
C
A B C
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Sensitivity and HLA-C
721.221-wt
0 2 4 6 8 100
20
40
60
80
100
R2=0.97
N
C
721.221-1202
0 2 4 6 8 100
20
40
60
80
100
R2=0.99
N
C
A
C
721.221-0401
0 2 4 6 8 100
20
40
60
80
100
R2=0.99
N
C
721.221-1403
0 2 4 6 8 100
20
40
60
80
100
R2=0.97
N
C
B
D
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Range of analysis
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Conclusions• Non-radioactive• Single-cell based• Can be integrated with surface analysis• Sensitive• Flexibility of post-collection analysis
• Strong lysis can lead to disappearance of cells
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Alar AintsHele EverausAnna StserbakovaHelen Lust
Marcel G.J. TilanusJennifer Schellekens
Evren Alici Mari GilljamBirgitta Stellan
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•Suured granulaarsed lümfotsüüdid•5-15% perifeerse veremononukleaarsetestrakkudest•Arenevad luuüdis•Loomuliku immuunsuse osa, NK rakkudel ei toimu retseptorite geenide ümberkorraldust•CD56+CD16+CD3−ja CD3-/NKp46+
NK rakud (Natural Killers) ehkloomulikud tappurrakud
Perifeerse vere lümfotsüütidepreparaat- rakud on värvitud hematoksüliin-eosiiniga