TRENDING NOWCYTOF® FLOW CYTOMETRY PUBLICATIONS

Welcome to the March issue of Trending Now, a quarterly
anthology of recent impactful publications by researchers
using CyTOF technology.

March 2022

This month’s edition features work using the Maxpar Direct
Immune Profiling Assay and demonstrates its contribution
to accelerating translational and clinical research aroundthe world.

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WHAT’S NEW IN
BEST-IN-CLASSCYTOMETRICIMMUNE PROFILING

Immunophenotyping made simple in recent researchusing the Maxpar Direct Immune Profiling Assay
Immune profiling is an extremely powerful tool for understanding an individual’s unique immunologic response
to disease or therapeutic interventions. When performed longitudinally on the same patient, or cohort ofpatients, a timeline of immune response is generated. This timeline provides researchers and clinicians withrich immunologic information that can be used to better understand disease pathogenesis, progression andresolution, or even to predict response to therapeutics.

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In translational and clinical research, immune profiling studies often require collaborations involving multiple
sites, possibly around the globe. This presents challenges in study design to ensure harmonized sample
processing and data analysis, as well as sample storage and shipping, if needed. Much time and energy isspent prior to the start of every study in the design and testing of an immune profiling approach, as well as in
how to preserve valuable samples and generate reproducible high-quality data.
The Maxpar® Direct™ Immune Profiling System provides a standardized, high-parameter cytometric assay withproven run-to-run and site-to-site performance ideal for multi-site and longitudinal studies. The 30-marker
backbone panel comes in a dry, single-tube format and can be used to analyze whole blood, PBMC or evencultured cells. At least 37 immune cell populations can be quantified in less than 5 minutes with Maxpar
Pathsetter™ software. An additional bonus is that stained samples can be stored for extended periods of timeand even shipped for analysis at distant sites if needed.
This CyTOF® based system is the first complete sample-to-answer solution for high-dimensional immune
profiling and has now been successfully used in a variety of settings. The publications and National Clinical
Trials listed here exemplify the range of research areas and applications to which the assay is being applied.

Dendritic
cells

Monocytes

Myeloid,
plasmacytoid

Classical,
transitional,
nonclassicalGranulocytes

Neutrophils,
CD66b– neutrophils,
basophils,
eosinophils

Lymphocytes

Maxpar tubes
CD3
CD4
CD8
CD11c
CD14
CD16
CD19
CD20
CD25
lyophilizedCD27

CD28
CD38
CD45
CD45RA
CD45RO
CD56
CD57
CD66b
CD123
CD127
drops

CD161
CD294
CCR4
CCR6
CCR7
CXCR3
CXCR5
HLA-DR
IgD
TCRγδ
pointed

30 unique markers
1 sample tube

Naive,
central memory,effector memory,
terminal
effector

CD3+

CD4+
T
regulatory
cells

Th1, Th2,
Th17

NK

Early, lateB cellsγδ
T cellNaive, memory,
plasmablasts

CD8+

MAIT/
NKT

blood
5-minute data analysis

Naive,
central memory,effector memory,
terminal
effector

37 immune cell
populations quantified

Figure 1. Maxpar Direct Immune Profiling System

3Simple standardization for clinical trials
Since the Maxpar® Direct™ Immune Profiling Assay™ includes a dried 30-marker panel designed to identify
all major circulating immune cell subsets, it is an easy go-to assay for clinical trials that require a high level ofstandardization, run-to-run and site-to-site. In addition, the specificity of metal-tagged antibodies facilitatespanel modifications and the ability to customize the assay for expanded mass cytometry panels, as recently
described in the latest edition of Methods in Molecular Biology (Petes et al.).

IMPACC (Immunophenotyping Assessment in a COVID-19 Cohort) Clinical Study
The Immunophenotyping Assessment in a COVID-19
Cohort (IMPACC) longitudinal study is underway. This
study is designed to identify immunologic, virologic,
proteomic, metabolomic and genomic featuresof COVID-19-related susceptibility, severity anddisease progression in 1,000 hospitalized patientswith COVID-19 (NCT04378777). The goal is to detailin depth the relationship of pathogen dynamics
and immune response with disease severity andpathogenesis, as well as identify biomarkers andinitiate effective therapeutic development.
CyTOF technology and bulk RNA transcriptomics
are being used to identify distinct immune cell

Figure 2. IMPACC study overview (Science Immunology 2021)

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populations and quantify changes in gene
expression and activation markers. The CyTOF
workflow employs the Maxpar Direct Immune
Profiling Assay, which allows samples to be fixed,
cryopreserved and shipped to the IMPACC corelabs for barcoded batched processing. This
includes labeling with a supplemental panel of 14
additional antibodies targeting fixation-resistantepitopes to resolve additional dynamic changes
in cell phenotype. The Maxpar Direct assay waschosen specifically for this study to reduce sample
usage, streamline sample processing and minimize
experimental variability.

COntAGIouS (COvid-19 Advanced Genetic and Immunologic Sampling) Trial
The COntAGIouS trial is a prospective study
aimed at characterizing clinical and immunologicalfeatures of severe COVID-19 infection compared
to mild cases (NCT04327570). The study could
contribute to our understanding of the underlying
pathophysiology of the disease and help to identify
at-risk patients, guiding better treatment strategies
for this group.
The transdisciplinary approach identifies
host factors resulting in hypersusceptibility to
SARS-CoV-2 infection that can more directly
support targeted medical interventions. The teamperforms multiparametric analysis using mass
cytometry with the Maxpar Direct Immune Profiling
Assay for longitudinal immune profiling.

Watch a webinar in which Frederik De Smetreports on use of the Maxpar Direct Immune
Profiling System in the COntAGIouS COVID-19 trial.
A Complete Immune Monitoring Solution with
CyTOF | Frederik De Smet, MSc, PhD

Antidepressant Trial with P2X7 Antagonist JNJ-54175446 (ATP)An ongoing clinical trial investigating the relationshipbetween inflammation and depression is set tobe completed later this year (NCT04116606).
Since depression is a significant cause of disability
and treatments are only moderately effective,
The Cambridge Clinical Trials Unit (CCTU)-Corein collaboration with Janssen Pharmaceuticals is
testing whether the anti-inflammatory drug
JNJ-54175446, which blocks the activity of
the P2X7 receptor, could have antidepressanteffects on patients with moderate to severe
depressive symptoms.

The group conducts immune profiling by mass
cytometry, using the Maxpar Direct Immune Profiling
Assay, to monitor the immune system before, during
and after treatment. With any observed response,
further study could identify associated immune
biomarkers. Patients will also complete additional
blood tests, questionnaires and magnetic resonanceimaging brain scans throughout the trial to morecomprehensively understand anti-inflammatory
effects on the immune system and the brain.

Read more about this trial from first author
Lori Turner in this customer spotlight.

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COVID-19: Pediatric Research Immune Network on SARS-CoV-2 and MIS-C (PRISM)
A prospective, multicenter, observational cohort
study, Pediatric Research Immune Network on
SARS-CoV-2 and MIS-C (PRISM), is evaluating
the short- and long-term health outcomes of
SARS-CoV-2 infection and multisystem inflammatory
syndrome in children (MIS-C) by characterizing
associated immunologic pathways (NCT04588363).

The study goals are to determine the proportion ofchildren with severe or fatal infections and identify
immune signatures associated with the disease
spectrum over the course of a one-year followup. Using mass cytometry with the Maxpar Direct
Immune Profiling Assay supports the cross-sitecollection and processing of samples, maintaining
a standardized protocol that allows for directcomparison of data.

Profiling infectious disease
The onset of an infection is typically a rapid process, meaning that any research aimed at studying diseaseprogression must keep pace. Ready-to-use assays like the Maxpar Direct Immune Profiling System offer aquick approach that can be adopted immediately to collect, stain and store samples, enabling rapid response
without compromising quality results.

Molecular and cellular immune features of aged patients with severe COVID-19 pneumonia
Immunity is known to wane in the elderly; thus,
aging is a major risk factor in the developmentof severe COVID-19. Immune system remodelingvia immunosenescence and inflammaging cancause predispositions to infection, including by
SARS-CoV-2. Work led by the University of Modena
and Reggio Emilia (Lo Tartaro et al.) profiled themain immune characteristics in older patients withsevere SARS-CoV-2 infection, compared to subjects
younger than 60 years of age.

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The team used a 38-parameter mass cytometry
panel, including the Maxpar Direct Immune Profiling
Assay plus 6 drop-in Standard BioTools™ catalog
antibodies and 2 custom-conjugated mAbs,
focusing on the T lymphocyte, B cell and monocyte
compartments. Results showed that immune
response in older patients was characterized byhigher plasma levels of pro-inflammatory cytokines
and a profound dysregulation in several immune
compartments.

Underlying co morbidity reveals unique immune signatures in type II diabetes patients
infected with SARS-CoV-2
Given that SARS-CoV-2 infection in patients withcomorbidities can be a major challenge, scientists
at the Institute of Life Science in India compared
immune response, viral loads and clinical parameters
among COVID-19 patients with and without type
II diabetes as well as in healthy controls to better
understand their clinical pathology (Sengupta et al.).
Whole blood immunophenotyping was performed
using the Maxpar Direct Immune Profiling Assay inaddition to plasma cytokine, chemokine, antibody
isotyping and viral load determination.

The study established that type II diabetes patients
exhibited higher inflammatory markers and adysregulated immune response versus patients
without diabetes. Analysis of these results suggeststhat chronic low-grade inflammation and an
associated dysregulated immune response might
be responsible for the higher level of inflammatory
response in COVID-19 patients that leads tosevere infection.

A monocyte/dendritic cell molecular signature of SARS-CoV-2-related multisystem
inflammatory syndrome in children with severe myocarditis
Multisystem inflammatory syndrome in children(MIS-C) develops as an acute pathology of SARSCoV-2 infection, though a better understanding of
disease pathophysiology is needed. Researchersat the Imagine Institute in Paris combined cytokine
measurements, deep immune cell phenotyping
and transcriptomics analyses at the single-cell level
to investigate SARS-CoV-2-related conditions inchildren (de Cevins et al.).

The team compared acute infection to post-acute
hyperinflammation in order to analyze pathways
and molecular signatures characteristic of themost severe form of MIS-C with myocarditis.
Single-cell analysis by the Maxpar Direct Immune
Profiling System and RNA sequencing correlated
low monocyte and dendritic cell frequencies with
post-acute hyperinflammatory illness, creating an
inflammatory profile for severe cases in children.

Figure 3. A multi-omics analytical strategy (Carapito et al.)7

Identification of driver genes for critical forms of COVID-19 in a deeply phenotyped
young patient cohort
Are drivers of severe COVID-19 different for the
younger population? A collaboration from Universitéde Strasbourg and Harvard Medical School used
multi-omics analysis and artificial intelligence to finda unique gene signature and characteristic virusinduced molecular changes that differentiate criticalfrom noncritical patients (Carapito et al.).

The team used whole genome sequencing,whole blood RNA sequencing, plasma and bloodmononuclear cell proteomics, cytokine profiling andhigh-throughput immunophenotyping with machinelearning. The Maxpar Direct Immune Profiling Assay
was used, where cells were stained and then stored
at –80 °C until acquisition. The results detail thesystemic immune response, define a transcriptomic
signature that differentiates critical patients and offer
potential diagnostic, prognostic and therapeutictargets to combat COVID-19.

Changes in immune response to cancer progression and immunotherapy
The longitudinal assessment of biospecimens can be crucial to identifying key players in the progressionof cancer or immune response to treatment. With sample collection, staining, storage and shipping easily
enabled with the Maxpar Direct Immune Profiling Assay, immune monitoring studies retain data integrity
over the course of the disease or treatment period.

Immune profiling mass cytometry assay harmonization: multicenter experiencefrom CIMAC-CIDC
The goal of the Cancer Immune Monitoring andAnalysis Centers – Cancer Immunologic Data
Commons (CIMAC-CIDC) Network is to identify
relevant biomarkers in response to cancer
immunotherapies for use in clinical trials. Mass
cytometry is a primary platform used in thesestudies and is performed at all CIMAC laboratories.
Multistep cross-site harmonization for this research
was an important quality control step to ensure
CyTOF data could be directly compared across sites(Sahaf et al.). The Maxpar Direct Immune Profiling
Assay supported this effort with a standardized
assay and protocol that could easily be adapted to
the needs of the study—using the base 30-antibody
panel that can be augmented with multiple
additional antibodies of interest to particularstudies within the program.

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Watch this presentation in which Holden Maecker
describes the results and lessons learned
from the study’s harmonization efforts, as well
as plans for using the Maxpar Direct Immune
Profiling Assay in future CIMAC studies.
CyTOF in the Cancer Immune Monitoring andAnalysis Centers (CIMAC) | Holden Maecker, PhD

30-marker*
panel
clonesand metals
30-marker* panel
withwithclonesand metals
Antibody
CD45
Live/dead indicator
CD196 (CCR6)
CD123
CD19
CD4
CD8a
CD11c
CD16
CD45RO
CD45RA
CD161
CD194 (CCR4)
CD25
CD27
CD57

Clone
HI30
N/A
G034E3
6H6
HIB19
RPA-T4
RPA-T8
Bu15
3G8
UCHL1
HI100
HP-3G10
L291H4
BC96
O323
HCD57

Metal
89Y
103Rh
141Pr
143Nd
144Nd
145Nd
146Nd
147Sm
148Nd
149Sm
150Nd
151Eu
152Sm
153Eu
154Sm

Antibody
CD183 (CXCR3)
CD185 (CXCR5)
CD28
CD38
CD56 (NCAM)
TCRγδ
CD294
CD197 (CCR7)
CD14
CD3
CD20
CD66b
HLA-DR
IgD
CD127

18 open channels for panel customization

Clone
G025H7
J252D4
CD28.2
HB-7
NCAM16.2
B1
BM16
G043H7
63D3
UCHT1
2H7
G10F5
LN3
IA6-2
A019D5

Metal
156Gd
158Gd
160Gd
161Dy
163Dy
164Dy
166Er
167Er
168Er
170Er
171Yb
172Yb
173Yb
174Yb
176Yb

155Gd

* 31 markers including the 103Rh live/dead indicator
2

Figure 4. Maxpar Direct Immune Profiling Assay setup (from Holden Maecker)

Humoral and cellular correlates of a novel immune-related adverse event and its treatment
A group at the University of Texas Southwestern
Medical Center examined whether humoral andcellular immune biomarkers may inform toxicity
monitoring and management in patients receiving
immune checkpoint inhibitor therapies (Gonugunta
et al.). Using high-dimensional mass cytometry
with the Maxpar Direct Immune Profiling Assay, theteam performed longitudinal immune cell profilingto compare patients with immune-related adverse
events after treatment to those with no toxicity andhealthy controls. Their CyTOF panel contained the
30 standard markers plus 6 additional markers
customized to relevant cell types.

They determined that preceding any toxic response,
patients can develop broad increases in cytokines,
autoantibodies, CD8 T cells and plasmablasts.
Additionally, the study captured the biologic
effects of corticosteroids on these parameters,
showing profound decreases and indicating
effective administration.

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Mass cytometry: a robust platform for the comprehensive
immunomonitoring of CAR-T-cell therapies
To address the challenge of defining the immune
mechanisms that underlie the success of CAR T celltherapy, scientists (Corneau et al.) at the Hôpital
Pitié-Salpêtrière presented the first application ofmass cytometry to the comprehensive immune
monitoring of CAR T cell therapies.

Watch a recent webinar on this publication:
Mass cytometry: A robust platform for thecomprehensive immunomonitoring of CAR
T cell therapies | Jonathan Caron, PhD

The team customized the standardized Maxpar
Direct Immune Profiling Assay panel by developing
metal-tagged antibodies that identify CAR T
cells. The panel was then validated to support
comprehensive immune monitoring after CAR T celladministration. The combination of antibodies in thecustomized panel allowed characterization of CAR
and non-CAR immune cells and their functional states,in a single pass and over time.

CD3
CD4

CD56
CD57
CD66b
CD11c
CD123
CD14
CD127
CD16
CD161
CD19
CD294
CD20
CCR4
CD25
CCR6
CD27
CCR7
CD28
CXCR3
lyophilizedCD38 drops
CXCR5
CD45
HLA-DR
CD45RA
IgD
CD45ROTCRγδ

CD8
Maxpar tubes

empty

liquid1
Blood collection Collect
CAR T
whole
blood orcell-specificisolatePBMC.
staining

2

blood

3

Stain
Maxpar staining/ Fix
Freezeandprocessing
process
samples inready-to-use
assay tube.
Figure 5. Immune profiling by cytometry workflow

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pointedand freeze
samples.

4

5

6

Ship
Data acquisition

Collect
Pathsetter data

Reportfrozen samples
to core lab.

thawedanalysissamples
on CyTOF XTor Helios™.

populatio
statistics
plot dispand QC mwith Max
PathsetteReferences

National Clinical Trials

Carapito, R. et al. “Identification of driver genes forcritical forms of COVID-19 in a deeply phenotyped young
patient cohort.” Science Translational Medicine 14 (2021):
doi:10.1126/scitranslmed.abj7521.

Antidepressant Trial with P2X7 Antagonist JNJ-54175446 (ATP)(NCT04116606) Sponsor: CCTU-Core

Corneau, A. et al. “Mass cytometry: a robust platform for thecomprehensive immunomonitoring of CAR-T-cell therapies.”
British Journal of Haematology 194 (2021): 788–792.
De Cevins, C. et al. “A monocyte/dendritic cell molecularsignature of SARS-CoV-2-related multisystem inflammatory
syndrome in children with severe myocarditis.” Med 2 (2021):
1,072–1,092.e7.

COntAGIouS (COvid-19 Advanced Genetic and Immunologic
Sampling) Trial (NCT04327570) Sponsor: Universitaire
Ziekenhuizen Leuven
COVID-19: Pediatric Research Immune Network on SARS-CoV-2
and MIS-C (PRISM) (NCT04588363) Sponsor: National Institute ofAllergy and Infectious Diseases (NIAID)
IMPACC (Immunophenotyping Assessment in a COVID-19
Cohort) Clinical Study (NCT04378777) Sponsor: National Instituteof Allergy and Infectious Disease (NIAID)

Gonugunta A.S. et al. “Humoral and cellular correlates of a novel
immune-related adverse event and its treatment.” Journal for
ImmunoTherapy of Cancer 9 (2021): e003585.
Günther, A. et al. “Complicated long term vaccine induced
thrombotic immune thrombocytopenia—a case report.” Vaccines
9 (2021): 1344.
Lo Tartaro et al. “Molecular and cellular immune features of aged
patients with severe COVID-19 pneumonia.” Research Square(2022): doi:10.21203/rs.3.rs-1318107/v1.
Maxpar Direct Immune Profiling System: Publications, Preprints,
and National Clinical Trials, March 2022.
Petes, C. et al. “Customizing Maxpar Direct Immune Profiling
Assay with Additional Surface Marker and Intracellular Cytokine
Staining Workflows for Expanded Mass Cytometry Panels.”
Single-Cell Protein Analysis (2021): 129–145.
Sahaf, B. et al. “Immune profiling mass cytometry assay
harmonization: multicenter experience from CIMAC-CIDC.”
Clinical Cancer Research 27 (2021): 5,062–5,071.
Sengupta, S. et al. “Underlying co morbidity reveals unique
immune signatures in Type II diabetes patients infected with
SARS-CoV2.” medRxiv (2021): doi:10.1101/2021.12.03.21267282.

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