Standardized and flexible eight-color flow cytometry panels harmonized between different laboratories to study human NK cell phenotype and function

Kalpana Singh¹, John P. Veluchamy²³, María Delso-Vallejo¹, Nina Kok³, Fenna Bohme³, Ruth Seggewiss-Bernhardt⁴⁵, Hans J. van der Vliet², Tanja D. de Gruijl², Volker Huppert¹, and Jan Spanholtz³¹Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; ²Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, Amsterdam, The Netherlands; ³Glycostem Therapeutics, Oss, The Netherlands; ⁴Department of Hematology and Medical Oncology, University Hospital of Würzburg, Würzburg, Germany; ⁵Department of Hematology and Medical Oncology, Sozialstiftung Bamberg, Bamberg, Germany

IntroductionAdvancements in flow cytometry led to the development of multicolor antibody panels, which have found application in numerous fields. However, comparability of data obtained from different laboratories is still a major challenge. Harmonization of flow cytometry panels and protocols for data analysis will be essential to compare data from various centers. Therefore, we developed standardized flow cytometry panels and inde-pendently established acquisition protocols for three different flow cytometers (BD™ FACSCanto™ II, BD LSRFortessa™, and

MACSQuant® Analyzer 10), with compatible lasers and filter settings. Eight-color flow cytometry protocols were designed to study natural killer (NK) cell receptor phenotype and function (e.g. antibody dependent cell mediated cytotoxicity (ADCC)) of NK cells within peripheral blood mononuclear cells (PBMCs). Using our established SOPs and harmonized flow cytometry panels, we compared the effects of freezing and thawing of PBMCs on NK cell phenotype and function.

• Comparable and reproducible data from three different flow cytometers were obtained using optimized 8-color flow cytometry panels for the analysis of NK cells.

• Channels corresponding to drop-in antigens can be flexibly used to obtain novel phenotypic or functional information.

• Cryopreserved PBMCs might be preferably used for studies on NK cell phenotype and function in multicenter trials.

• The presented NK flow cytometry panels set a precedent for future harmonization of multicolor panels focussed on other immune cell types.

These data are part of the following publication: Veluchamy, J. P. et al. (2017) Sci. Rep. 7: 43873.

Uniform gating strategy to generate reproducible data between flow cytometers1

Fluorescence intensities of defined fluorochrome-conjugated antibodies are comparable between three flow cytometers2

Materials and methods

Results

Conclusion and outlook

Instruments and reagents 1

Figure 1

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Detectors Filter settings: band pass and long pass (LP) filters PMT voltages

BD FACSCanto II BD LSRFortessa MACSQuant Analyzer 10 BD FACSCanto II BD LSRFortessa

MACSQuant Analyzer 10

FSC 488/10 488/10 488/10 319 489 328

SSC 488 /10 488/10 488/10 462 266 476

FL1 530/30 and 502LP 530/30 and 505LP 525/50 496 484 417

FL2 585/42 and 556LP 575/26 and 550LP 585/40 435 473 416

FL3 780/60 and 735LP 780/60 and 750LP 750LP 540 549 487

FL4 670LP and 655LP 695/40 and 685LP 655–730 507 681 594

FL5 660/20 670/14 and 655LP 655–730 588 484 522

FL6 780/60 and 735LP 780/60 and 750LP 750LP 474 451 579

FL7 450/50 450/50 450/50 375 423 444

FL8 510/50 and 502LP 525/50 and 505LP 525/50 403 453 560

Instrument settings of the three different flow cytometers used in this study.Table 1

Panel Laser Antibody Fluorochrome Clone Titration Manufacturer Order no.

Violet 405 nm CD45 VioGreen™ 5B1 1:11 Miltenyi Biotec 130-096-906

CD3 VioBlue® BW264/56 1:11 Miltenyi Biotec 130-094-363

TCRγ/δ VioBlue 11F2 1:11 Miltenyi Biotec 130-101-557

CD14 VioBlue TÜK4 1:11 Miltenyi Biotec 130-094-364

CD19 VioBlue LT19 1:11 Miltenyi Biotec 130-098-598

SYTOX® Blue Dead cell marker 1:1000 Thermo Fisher Scientific S11348

Blue 488 nm KIR2D FITC NKVFS1 1:11 Miltenyi Biotec 130-098-689

NKG2A PE-Vio® 770 REA110 1:11 Miltenyi Biotec 130-105-647

NKG2C PE REA205 1:11 Miltenyi Biotec 130-103-635

NKG2D PerCP-Cy™5.5 1D11 1:11 BioLegend 320818

Red 633 nm CD16 APC VEP13 1:11 Miltenyi Biotec 130-091-246

CD56 APC-Vio 770 REA196 1:11 Miltenyi Biotec 130-100-694

Violet 405 nm CD45 VioGreen 5B1 1:11 Miltenyi Biotec 130-096-906

CD14 VioBlue TÜK4 1:11 Miltenyi Biotec 130-094-364

CD19 VioBlue LT19 1:11 Miltenyi Biotec 130-098-598

SYTOX® Blue Dead cell marker 1:1000 Thermo Fisher Scientific S11348

Blue 488 nm CD25 VioBright™ FITC 4E3 1:11 Miltenyi Biotec 130-104-274

CD107a PE H4A3 1:11 Miltenyi Biotec 130-095-515

NKp44 PE-Vio 770 2.29 1:11 Miltenyi Biotec 130-104-195

CD3 PerCP-Vio 700 BW264/56 1:11 Miltenyi Biotec 130-097-582

TCRγ/δ PerCP-Vio 700 11F2 1:11 Miltenyi Biotec 130-103-784

Red 633 nm CD16 APC VEP13 1:11 Miltenyi Biotec 130-091-246

CD56 APC-Vio 770 REA196 1:11 Miltenyi Biotec 130-100-694 Antibodies detected based on the violet and red lasers (indicated in dark blue) represent the panel backbone, antibodies detected based on the blue laser (indicated in light blue) represent the drop-in antigens.Table 2

NK

ph

enot

ype

pan

elN

K fu

nct

ion

pan

el

A solid backbone concept enabled exclusion of non-NK lympho-cyte populations and dead cells, followed by gating on viable NK cells (CD45+CD3–CD56+ cells). Flexible addition of further

antibodies allowed the detailed analysis of NK cell receptors CD16, NKG2A, NKG2C, NKG2D, and KIR2D (A) and NK cell func-tionality markers CD25, NKp44, and CD107a (B).

Figure 2

Compatible instrument settings and optimized protocols resulted in comparable data sets as reflected by expression lev-els on single-stained cells for each backbone and drop-in anti-gen of the two panels. One representative set of histograms from three samples obtained from a single healthy donor is shown for each antibody-fluorochrome conjugate tested using three dif-ferent flow cytometers. Data collected from individual experi-

ments were analyzed using the pre-defined gating strategy with KALUZA® software. No significant differences could be observed between centers, resulting in highly reproducible data sets for the characterization of lymphocyte subsets and NK cell pheno-type and function. This result provides a strong rationale for combining data sets for analysis in multicenter studies.

NK cell phenotypes and functions are highly preserved after cryopreservation3Additionally, we aimed to identify the most suitable conditions to study NK cell phenotype and function in a multicenter setting. Both freshly isolated and corresponding cryopreserved PBMC samples (n = 12) were either activated with cytokines (IL-2/IL-15) overnight or left untreated. NK cell phenotype and cytotoxicity

against A431 cells alone or coated with cetuximab antibody (CET) were similar in fresh and cryopreserved PBMCs, suggesting that the use of cryopreserved PBMCs is appropriate for NK cell stud-ies in a multicenter setting.

NK

phe

noty

pe

pan

elN

K fu

ncti

on p

anel

Gated on PBMCs Gated on singlets Gated on CD45+ cells Gated on NK cells

Gated on NK cells Gated on NK cells Gated on NK cells Gated on NK cells

Gated on PBMCs Gated on singlets Gated on CD45+ cells Gated on NK cells

Gated on NK cells Gated on NK cells Gated on NK cells

15.93 83.84

0.00 0.20

60.40 39.60

0.00 0.00

90.40 9.60

0.00 0.00

8.09 91.91

0.00 0.00

45.44 54.56

0.00 0.00

87.09 12.91

0.00 0.00

1.33 98.67

0.00 0.00

92.51 7.49

0.00 0.00

22.36 77.64

0.00 0.00

BD FACSCanto II

BD LSRFortessa

MACSQuant Analyzer 10

BD FACSCanto II

BD LSRFortessa

MACSQuant Analyzer 10

CD45-VioGreen

CD3-PerCP-Vio 700 CD3-VioBlue

Anti-TCRγ/δ-PerCP-Vio 700

Anti-TCRγ/δ-VioBlue CD14-VioBlue CD19-VioBlue SYTOX Blue

CD56-APC-Vio 770

CD16-APCAnti-NKG2A-

PE-Vio 770CD25-

VioBright FITCAnti-NKp44-

PE-Vio 770 CD107a-PEAnti-KIR2D-

FITCAnti-

NKG2C-PENKG2D-

PerCP-Cy5.5

Gat

ed o

n P

BMC

sG

ated

on

NK

cel

ls

Fresh Cryopreserved Fresh Cryopreserved Fresh Cryopreserved Fresh Cryopreserved

This project was funded by the ITN EU project NaturImmun.

% N

KG2A

+ N

K ce

lls

% N

KG2C

+ N

K ce

lls

% C

D10

7a+ N

K ce

lls

% C

D16

+ N

K ce

lls

FSC-

H

FSC-A CD45-VioGreen CD45-VioGreen CD16-APC

CD3-

, Ant

i-TCR

γδ-,

CD14

-, CD

19-V

ioBl

ue /

SYTO

X B

lue

CD56

-APC

-Vio

770

CD56

-APC

-Vio

770

% N

KG2D

+ N

K ce

lls

% P

an K

IR2D

+ N

K ce

lls

% N

Kp44

+ N

K ce

lls

% C

D25

+ N

K ce

lls

CD56

-APC

-Vio

770

CD56

-APC

-Vio

770

CD56

-APC

-Vio

770

CD56

-APC

-Vio

770

Anti-NKG2A-PE-Vio 770 Anti-NKG2C-PE NKG2D-PerCP-Cy5.5 Anti-KIR2D-FITC

FSC-A CD45-VioGreen CD16-APCCD3-, Anti-TCRγδ-PerCP-Vio 700

CD25-VioBright FITC Anti-NKp44-PE-Vio 770 CD107a-PE

FSC-

H

CD14

-, CD

19-V

ioBl

ue /

SYTO

X B

lue

CD56

-APC

-Vio

770

CD56

-APC

-Vio

770

CD56

-APC

-Vio

770

CD56

-APC

-Vio

770

CD56

-APC

-Vio

770

A

B

Figure 3 PBNK control PBNK + A431 PBNK + A431 + CET