Cytometry-based cytotoxicityanalysis for

Natural Killer cell based immunotherapy

Alar Aints, PhDTartu University and

Competence Centre for Cancer Research

•Large granuar lymphocytes•5-15% of PBMC•Develop in BM•No rearranging receptors•CD56+CD16+CD3−NKp46+

NK (Natural Killers)

PBMC, hematoxylin/eosin

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

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

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

Cell culture

• CellGroSCGM, Human serum, IL-2 and OKT3 (Carlens et al, Human Immunology 2001)

Cell culture

All eventsCellsNK cellsT cells

All eventsCellsNK cells

T cells

NK cells

T cells T cells

NK cells

T cells

NK cells

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

Double labeling

• CFDA (carboxyfluorescein diacetate) , DiO18 (dioctadecyl-oxacarbocyanin perchlorate) , PKH2 (green fluorescent Cell Linker) , FITC, calcein AM, CFSE

• PI, 7AAD, Annexin5

CFSE/7AAD

• CFSE (~1µM) “universal marker“• non-toxic• stable• 7AAD (~3µg/ml) 647nm - CFSE non-

overlapping• fixable, resistant to permeabilization

Compensation &

biexponentialdisplay

Effector cells

Target cellsTarget cells

Target cellsTarget cells

Effector cells

Effector cells Effector cells

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

Setting gates/Dot plot viewA B C

D E

F

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

N

Law of mass action

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

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

Range of analysis

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

Alar AintsHele EverausAnna StserbakovaHelen Lust

Marcel G.J. TilanusJennifer Schellekens

Evren Alici Mari GilljamBirgitta Stellan

•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