Development of a 22-color Panel Measuring Myeloid Derived ...
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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: [email protected] Phone: 1-609 252-6827