Clinical Flow Cytometry, Translational Sciences-TM

Development of a 22-color Panel Measuring Myeloid Derived Suppressor Cells (MDSC) Subsets in Blood using Spectral Cytometer Aurora to Support a Clinical Biomarker Study

Tania Nevers, Doli Patel, Yongliang Sun and Michael HedrickClinical Flow Cytometry, Bristol-Myers Squibb Co., 3551 Lawrenceville Rd

Princeton, NJ 08540

1

BACKGROUND

Myeloid cells, a highly diverse cellular population, have evolved to

play an important role in tumor progression. Recent evidence

demonstrates that myeloid derived suppressor cells (MDSCs) are

implicated in tumor angiogenesis, drug resistance, promotion of

tumor metastasis, and suppression of various cells of the immune

system. Therefore, targeting these cells may provide an attractive

anti-cancer therapeutic opportunity. To support a clinical

biomarker study, a 22-color panel for extensive phenotyping of the

rare MDSC population in human blood using the Cytek Aurora

spectral cytometer was developed and validated.

RESULTS

EXPERIMENTAL DESIGN

Antibody Fluorochrome Clone Concentration (μL) Purpose

CD45 AF532 HI30 5 All Leukocytes

Lineage (CD3, 19, 20) AF488OKT3,

HIB19,2H7

0.3125, 1.25,

0.3125T and B Lymphocytes

CD11b SB436 ICRF44 2.5 Myeloid DC, B cell subset

CD80 PE Cy5 L307.4 20 DC Phenotying

CD16 eF450 3G81.25

Monocytes, NK, and

neutrophils

HLA-DR APC-Fire750 G46-6 (L243) 5B-cells, monocytes

CD11c BB515 B-ly65

Monocytes, myeloid DC

CD123 BV785 6H62.5

Plasmacytoid DC, myeloid DC,

Basophils

CD141 BV605 M80 2.5 DC subset

CD56 BV711 NCAM16.2 2.5 NK cells

CD66b APC G10F5 5 Granulocytic cells

CCR2 BV480 LS132.1D9

5

Monocytes, Macrophages and

activated T cells, Chemokine

receptor

CD7 PerCP-Cy5.5 CD7-6B72.5

T and NK Cells

CD15 BV650 W6D30.3125

Granulocytes (Neutrophils,

Eosinophils)

CD14 PerCP eF710 M5E25

monocytes

CD303 BV421 201A 5 Plasmacytoid DC

CD86 BB700 FUN-15

DC phenotyping

CCR5 PE-Cy7 2D7 0.625 Chemokine Receptor

CD33 BV570 WM53 2.5 Myeloid Cells

PD-L1 PE eF610 MIH10.3125

Immune Checkpoint

CD1c BUV805 F10/21A3 5 Myeloid DC

Zombie NIR NA 1 : 6000 Dead cell exclusion

Selection of individual flow cytometry antibody concentration

CD3 AF488

CD

3

T8 T7 T6 T5 T4 T3 T2 T1FMO 0

50

100

150

0

20

40

60

80

100

CD3 AF488

Titration

Sep

era

tio

n In

dex

%P

ositiv

e

Seperation Index

Stain Index

FMO T8 T7 T6 T5 T4 T3 T2 T1

FSC-A

SS

C-A

FSC-A

FS

C-H

FSC-A

Zo

mb

ie

CD45

SS

C-A

Lin

SS

C-A

Leukocytes Singlets Live CD45+

Lineage -

General Gating Strategy

SUMMARY AND CONCLUSIONS

Figure 1. Antibody titration and selection of titer. Titrations were performed on healthy human whole blood

using an eight point, two fold serial dilution starting with twice the manufacturer’s recommended

concentration. Representative general gating strategy used (Figure 1A) as a backbone before titrating

individual antibodies in the panel. Red squares depict the chosen concentration (Figure 1B and 1C), which

was either saturating or had an optimal separation/spillover spreading (Figure 1D). Individual FCS files

were concatenated for representation (Figure 1C). Note. The separation index – separation between the

positive and negative populations. The Staining index – Allows for the comparison of the relative

brightness of fluorochromes.

• Human whole blood (WB) was collected in cyto chex tubes andused 24 hours post collection

• RBCs (red blood cells) were lysed, stained with viability dye toexclude dead cells, blocked with Fc blocking buffer andincubated with antibody for 30 mins on ice.

• Note: To detect activation markers, activated vericells were usedor PBMCs were activated with PMA and ionomycin cell stimulationcocktail

• The antibodies were first titered with an 8 point, 2 fold serialdilution starting with twice the manufacturer’s recommendedconcentration in 100 µL of stain buffer

• Samples were stained and resuspended in 200 µL of 1x BDStabilizing Buffer before collection on Aurora.

• Once the correct titer was identified, full panel staining and FMOcontrols were done to optimize the panel

A.

FMO T8 T7 T6 T5 T4 T3 T1T2

CD3

SS

C-A

Gated on Zombie NIR+CD45+ cells

CD3 AF488 Titration (Lin)B.

C. D.

Taken together, we have demonstrated that the developed MDSC

assay is robust, and meets the fit-for-purpose criteria. It can be

used to measure levels of monocytic MDSC, granuocytic MDSC,

classical/non-classical/intermediate monocytes and dendritic cell

subsets, as well as activation markers on these subsets in support of

clinical trials. Studying these major players of the immune system in

one single panel provides valuable biomarker information in the

clinical development of novel immunotherapies and will give us a

broader view of the role of MDSCs in immune suppression in cancer.

Antibodies used in the optimized multicolor immunofluorescence panel

Example gating strategy for a deep dive phenotyping of MDSCs in human blood.

Figure 2. Example gating strategy for visualization of MDSCs, monocytes, dendritic cells, and natural killer

cells in health human blood 24h post collection. A. General gating strategy, B. NK cells, C. Monocytes, D,

MDSCs, and E. DC subsets

Normal healthy controls

Fluoroscent Labeled

Lymphocytes Lysed RBCBlood

Acquired on Cytek

Aurora Spectral

Cytometer

Analyzed using Flowjo

Software

CD14-

CD56-CD7-

CD11c+CD123+

CD1c

cDCpDC+

SS

C-A

B.

C.

A.

D.

E.

NK cells

MonocytesMDSCs

DCs

CD

123 B

V785

CD

66b A

PC

Contact: Tania Nevers Email: tania.nevers@bms.com Phone: 1-609 252-6827