Application NoteTM

Maxpar Direct Immune Profiling Assay Expanded
to Explore Antigen-Specific Immune Responses
Introduction
Monitoring the immune response to disease andassessing clinical recovery are critical steps towards
identifying effective targets for therapies. The complex
and heterogeneous nature of the immune system
requires deep interrogation at the single-cell level.1
Mass cytometry, which uses CyTOF® technology,
utilizes antibodies tagged with monoisotopic metals,
resulting in discrete signals that enable highly
multiplexed characterization on a single-cell level.
CyTOF can currently enable cellular phenotypingof over 50 single-cell parameters simultaneously,
including phenotypic and functional markers, thereby
significantly increasing the ability to comprehensively
evaluate immune responses.2 The Maxpar® Direct™
Immune Profiling Assay™ is a pre-titrated, dried-down,
30-marker antibody cocktail for immune profiling of
human whole blood and peripheral blood mononuclear
cells (PBMC) by CyTOF (Figure 1). When coupled with
Maxpar Pathsetter™ software for post-acquisition
data analysis, single-cell events can be automatically
identified as one of 37 immune populations including
major lineages and their subsets by using probability
state modeling. The software eliminates the variabilityof manual gating and provides an automated report
including population statistics, stain assessments, andrelevant data plots. The Maxpar Direct Assay has been
applied in numerous publications and clinical trials.
Maxpar tubes

With 18 open channels for panel expansion, theMaxpar Direct Assay was designed with customizationin mind. This application note describes the use of
the Maxpar Direct T Cell Activation Expansion Paneland sample multiplexing with live-cell barcoding to
add 15 parameters to the backbone Maxpar Direct
panel. However, other specific markers can be chosen
to address a variety of research applications.
The array of key surface and intracellular functionalparameters in this expanded panel enables
comprehensive analysis of immune cell activation andantigen-specific recall responses to cells stimulatedwith a pool of immunodominant microbial peptides.
Furthermore, the existing Maxpar Pathsetter model
was modified to automate analysis of the expanded
panel and report on new populations and additional
functional parameters such as cytokine production.
Taken together, this powerful technique unlocked
deeper immunophenotyping and functional
profiling of activated immune cells and provided
insight into the cell-mediated adaptive immune
response to foreign targets through detection andcharacterization of antigen-specific T cells. Suchphenomena are hallmarks for research on infection,
vaccine development, and immunotherapy.

Maxpar tubes

CD3liquid

Dried

drops

pointedpointed

blood

empty

1
liquidDried

drops 2pointedpointedStainand process PBMC orwhole blood samples
in ready-to-use
assay tube.
4–5 hours* hands-on

AcquireCryodata with customized
acquisition template
on a CyTOF system.
~20 minutes perempty
sample* Staining includes an overnight incubation

3bloodReport

CD56
CD57
CD8
CD66b
CD11c
CD123
CD14
CD127
CD16
CD161
CD19
CD294
CD20
CCR4
CD25
CCR6
CD27
CCR7
CD28
CXCR3
CXCR5
lyophilizedCD38
drops
CD45
HLA-DR
CD45RA
IgD
CD45ROTCRγδ

CD4
Maxpar tubes

Cryoof population
frequencies, dataplot displays, and QC
metrics with
MaxparliquidPathsetter software
5 minutes per file

30 unique markers
37 	immune cell populations
18+ open channels forpanel customization

pointedblood

Figure 1. The Maxpar Direct Immune Profiling System. The Maxpar Direct Immune Profiling System is a complete sample-to-answer solution
for high-dimensional immune profiling of human PBMC and whole blood. Designed as a simple single-tube workflow, the system brings together
CyTOF technology, a dry 30-marker antibody panel, and automated analysis with Maxpar Pathsetter software for quantification of 37 immune cell
populations. Flexible by design, the assay allows for the addition of 18-plus additional antibodies for further panel customization.

Study Design

Objectives• 	Demonstrate the flexibility of the Maxpar Direct
Assay to incorporate additional functional markers
and automatically report on new populations using
a customized Maxpar Pathsetter model template•	Showcase combined applications in a single CyTOF
experiment, including immunophenotyping with
Maxpar Direct, intracellular cytokine profiling, andsample multiplexing with live-cell barcoding•	Illustrate the utility of the Maxpar Direct T Cell
Activation Expansion Panel to identify antigenspecific immune responses in the context of
infectious disease

A 47-parameter panel was designed for this applicationnote (Figure 2). Specifically, the Maxpar Direct Immune
Profiling Panel, consisting of 30 markers, was usedas the backbone for immune cell phenotyping. An
11-marker panel comprised of both surface andintracellular markers, the Maxpar Direct T Cell Activation
Expansion Panel (Cat. No. 201409), was selected fordeeper phenotyping and functional characterizationof activated immune cells. This panel enables themeasurement of immune cell activation, frequencyof antigen-specific T cells after natural infection or
vaccination, cytotoxic potential in T and natural killer(NK) cells, and identification of activated helper T cell
populations. The diverse phenotypic and functionalfeatures of this panel allow for in-depth immune cellcharacterization in one tube.
A schematic of the stimulation and multi-step staining
experiment carried out in this application note is shownin Figure 3. Human PBMC from 4 different donors wererested overnight and either left untreated or stimulatedwith phorbol myristate acetate (PMA) and ionomycin ora mix of immunodominant microbial peptides (Step 1 inFigure 3). Brefeldin A and monensin transport inhibitors
were added to all samples 1 hr post-stimulation andincubated for an additional 4 hr.

(BC
)

IL2

89Y
103Rh

209Bi

106
Cd
CD1
110
07a
Cd
11
CD
1C
45
d
(BC
11
CD
)
2C
45
d

CD45

GrzB

110Cd

4P

111Cd

t

3
11

Metals

113Cd

u

d
4C

114Cd

1

d
16C

γ
IFN

116Cd

173Yb

Yb

172Yb

171171Yb
Yb

168Er
168Er

167Err
167E
166Er
Er
166
o
94
5H
CD2
16
y
4D
0
16
Dy
IL-1
163Dy
63
δ
1
162Dy
Rγ
C
T
161Dy
56
160Gd
159Tb
CD

141Pr

148Nd

2D
y

1 5 8 Gd
156Gd

151Eu

156Gd

155Gd

154Sm

152Sm

153Eu

CD19
CD4

Target (clone)

Isotope

Staining

CD107a (H4A3)
CD45 (HI30)
CD45 (HI30)
IL-2 (MQ1-17H12)
CD69 (FN50)
TNFα (Mab11)
IFNγ (B27)
IL-4 (MP4-25D2)
CTLA-4 (14D3)
IL-10 (JES3-9D7)
IL-17A (BL-168)
CD45 (HI30)
CD45 (HI30)
Perforin (B-D48)
Granzyme B (Gb11)

106Cd
110Cd
111Cd
112Cd
113Cd
114Cd
116Cd
142Nd
162Dy
165Ho
169Tm
194Pt
195Pt
196Pt
198Pt

Surface
Surface (barcode)
Surface (barcode)
Intracellular
Surface
Intracellular
Intracellular
Intracellular
Intracellular
Intracellular
Intracellular
Surface (barcode)
Surface (barcode)
Intracellular
Intracellular

CD57

CD27

CD185
/C
XCR5
CD183/
CXCR3

158Gd

IL-4
CD123/
IL-3R

Nd
14
CD8
7S
a
m
14
8N
CD
14
d
11
9S
c
m
CD
16
149Sm

1D
y
160
Gd
159T
b

16

d

RO
45
CD
RA
45
CD
1
16
d
CD
C
0N
94/
15
CD14
1Eu
15
CR
Sm
152
CD25
u
153E

16

144Nd

14145Nd
5N

150Nd

155Gd

CD
28

144Nd

146Nd

1 5 4 Sm

LA
-4

143Nd
143Nd

147Sm

164Dy

6/
CD19
CCR6

142Nd

146

165Ho

CD
38

I*
127I
127

141Pr
142Nd

Maxpar Direct
Immune Profiling
Panel

170E
170Er
r
169Tm
169Tm

CT

Fα

TN

Cd

11

174Yb

Yb

69
CD

106Cd

112Cd

175Lu

b

103Rh

176Yb

5L

4Y

89Y

209Bi

b

172

17

CD14
7/
CD19 R7
CC

173

6Y

IL-17A

17

CD3

19

7

CD20

17

198Pt
t
196P
Pt
195

C)
5 (B

)
(BC

12
CD

CD6
6b

IgD

forin
Perr

CD4

45
CD

HL
A-D
R

196Pt198Pt
195Pt
194Pt

ViabilityMarkers

Figure 2. Building a 47-parameter panel for functional profiling of activated immune cells. (Left) Panel wheel illustrating cellular markers andtheir respective isotope channels. The base Maxpar Direct Immune Profiling Panel (Cat. No. 201334) is depicted by the orange boxes. The Maxpar
Direct T Cell Activation Expansion Panel (Cat. No. 201409) is depicted by the purple boxes. Cadmium- and platinum-labeled anti-CD45 antibodies
used for live-cell barcoding (Cat. Nos. 3110001B, 3111001B, 3194001B, 3195001B) are depicted by the blue boxes. Empty boxes indicate open
channels. (*IdU is the only 127I conjugate currently available.) (Right) Table detailing expansion panel antibody clones, live-cell barcoding reagents,and staining method required.

2

Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

Results

After stimulation, cells were added to the Maxpar
Direct dry antibody pellet and concurrently stainedwith additional surface markers from the Maxpar Direct
T Cell Activation Expansion Panel (Step 2 in Figure 3).
Furthermore, a unique 4-choose-3 anti-CD45 barcode
mix for each stimulation condition was added tocorresponding tubes during this surface staining step.

Optimizing Workflow and Data Quality with
Sample Multiplexing and Automatic Analysis
Live-cell barcoding
Live-cell barcoding (LCB) using Maxpar anti-human CD45
antibodies allowed for all stimulation conditions froma single PBMC donor to be stained with intracellular
antibodies simultaneously in a single tube. LCB effectively
eliminates staining variability between samples, reduces
reagent use, and streamlines the acquisition workflow.

Multiplexed samples from each PBMC donor werecombined into a single tube, fixed, permeabilized, andstained with the intracellular antibodies from the Maxpar
Direct T Cell Activation Expansion Panel (Step 3 inFigure 3). Samples were then incubated with Cell-ID™
Intercalator-Ir overnight for total cell identification.

Figure 4 shows a representative sample debarcoding
analysis from PBMC Donor 3 and illustrates efficient
sample separation between the positive and negative
barcode channels used. A clear visualization of barcode
channel intensities can be observed in the scatter plot
and resulted in a barcode yield of 81% (Figure 4A).

Barcoded samples were acquired on a Helios™
instrument (Step 4 in Figure 3).* Sample debarcoding
was carried out (Step 5 in Figure 3) and individual FCS
files were uploaded into Maxpar Pathsetter software for
automatic analysis using a customized model template(Step 6 in Figure 3; see section on Updating the Maxpar
Pathsetter Model in this application note).

After sample debarcoding, FCS files were further
evaluated for data quality and consistency. Specifically,cells were gated on live, singlet populations, andCD45 signal intensity was analyzed. All debarcoded
files exhibited a positive signal for the expectedbarcode channels (yellow histograms) and had minimal
signal overlap into negative barcode channels (black
histograms) (Figure 4B). This was also visualized in aheat map depicting median signal intensities for eachbarcode channel.

Additional details on this experiment and ordering
information for the reagents used in this study can befound below in the Methods section and Appendix A,
respectively.
* This assay can also be acquired on a CyTOF XT system.

Donor 1

Donor 3

Donor 2

Donor 4

(1) PBMC
stimulation

-

+

-

-

+

+

-

+

(4) Acquire on Helios(2) Maxpar Direct
surface staining andlive-cell barcoding(5) Sample debarcoding

Donor 1

(3) Maxpar Direct
intracellularcytoplasmic staining

Donor 3

Donor 2

Donor 4

(1) PBMC
stimulation

-

+

-

+

-

+

-

+

(4) Acquire on Helios(2) Maxpar Direct
surface staining andlive-cell barcoding(5) Sample debarcoding(3) Maxpar Direct
intracellularcytoplasmic staining(6) Maxpar Pathsetter analysis(6) Maxpar Pathsetter analysis

Figure 3. Study design. Experimental workflow illustrating the multi-step stimulation and staining procedures. Sample tubes are color-coded byhuman PBMC donor. Stimulations depicted by (–) for unstimulated, ( ) for microbial peptide pool, and (+) for PMA/ionomycin. Live-cell barcoding is
illustrated by gray (110Cd-CD45), black (111Cd-CD45), gold (194Pt-CD45), and yellow (195Pt-CD45) bars.

Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

3

A

B

A

B
Barcode yields with current filters: 81% assigned

110Cd
111Cd
194Pt
195Pt

Unstimulated
(s1)

s1
s2
s3

Microbial
peptides
(s2)

2.27

PMA/ionomycin
(s3)
110

Cd_CD45111

Cd_CD45

194

Pt_CD45

195

147.4

Pt_CD45

B

110Cd
111Cd
194Pt
195Pt

Figure 4. Debarcoding results. A) Single-cell Debarcoder3 software screen capture of sample deconvolution. Green histogram bars and barcode
separation curves are displayed for all 3 barcoded samples from PBMC Donor 3 as a representative donor (left panel). Barcode event counts
illustrate sample yields and event dot plots depict barcode channel intensities (right panel). Barcode 2 (s2) is shown as a representative sample.
UnstimulatedB) (s1)
Histograms and heat map depicting median signal intensities for 110Cd-CD45, 111Cd-CD45, 194Pt-CD45, and 195Pt-CD45 barcode channels.
Both plots display data from PBMC Donor 3 gated on the viable, singlet PBMC population.

Updating the Maxpar Pathsetter model

s1
s2
s3

Maxpar Pathsetter is a fast, reliable, and flexible data
analysis solution. The default Pathsetter model2.27
was 147.4
specifically designed to automatically identify 37
immune cell types in samples processed with Maxpar
Pt_CD45
Cd_CD45
Pt_CD45
Cd_CD45
Direct. For automated analysis of the 11 additional
markers introduced in the Maxpar Direct T Cell
Activation Expansion Panel, a customized model
template was created following the Method Develop:
Customize the Maxpar Direct Immune Profiling
Assay User Guide (FLDM-00151).

PMA/ionomycin
(s3)

110

111

194

195

The Maxpar T Cell Activation Pathsetter model template
incorporates new functional information based on theactivation markers and cytokines in this panel and resultsin >100 additional measurements within specified immune
cell subsets. All samples in this study were processedusing the updated model template and a summary of
the results are shown in the following sections.

Mapping the Functional Landscape of
Activated Immune Cells
Broad immune cell activation via PMA/ionomycin
stimulation typically results in a marked upregulation in
the frequency of CD4 and/or CD8 T cells expressing
functional activation markers and induces significant
cytokine production. PMA/ionomycin stimulated samples
were used in this study to showcase that when usedin tandem,
the base Maxpar Direct panel and T Cell
100
Activation
Expansion
Panel robustly capture activated90
Unstimulated
immunesubsets.
80% Parent population

Microbial
peptides
(s2)

35

70

Unstimulated
PMA/ionomycin

60
50
40
30
20

30
25
20
15
10
5

10
0

% Parent population% Parent population% Parent population% Parent population

80

60

Population
frequency reports, automatically generated by50
the 40Maxpar T Cell Activation Pathsetter model template,effectively
identified the anticipated upregulation30
of functional
markers across all PBMC samples tested
20
when
10 compared to unstimulated controls (Figure 5).
This0included activation markers CD107a and CD69,
CD107a+CD69+ IL-2+ CD4 IFNγ+ CD8 TNFα+CTLA-4+
intracellular
cytokines
IL-2,
IFNγ,Tand
TNFα,
as well
as
CD8 T
CD4 T
T Cells
CD4 T
Cells
CD3 T
Cells
Cells
Cells
Cells
the exhaustion
marker
CTLA-4.

100
90

PMA/ionomycin

70

Maxpar Pathsetter analysis of activated immune cells

CD107a+CD69+ IL-2+
IL-2+
IFNγ+
TNFα+
CTLA-4+
CD107a+CD69+
CD4 IFNγ+
CTLA-4+
CD8 TNFα+
CD8
CD4
T cells
CD4
T cells CD8
T cells CD3
T cells
CD4
T cells
CD8T cells
T
CD4
T
T Cells
CD4
T
T Cells
CD3
T
Cells
Cells
Cells
Cells

0

IL-4+
IL-4+
Th2
Th2
cells
Cells

IL-10+
IL-17A+
Perforin+
Granzyme B+
IL-10+
CD4 T IL-17A+
Th17 Perforin+
CD8 Granzyme
B+
CD4
T cells
Th17
cells
CD8
T cells
CD8TTCells
cells
Cells
Cells
T CellsCD8

opulation

Figure 5. Frequency of T cells expressing functional markers post stimulation. CD4 or CD8 T cells expressing markers from the Maxpar
Direct T Cell Activation Expansion Panel between unstimulated and PMA/ionomycin-stimulated samples were measured and reported by Maxpar
35
Pathsetter
software. Bar graphs depict mean % parent population frequencies ± standard error of the mean (SEM) from all 4 human PBMC donors.
430

Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

25
20

Cauchy enhanced nearest-neighbor stochastic
embedding (Cen-se′™)4 is a powerful dimensionality
reduction tool built into Pathsetter. This tool can be used
to visualize the phenotypic and functional landscapeacross various immune cell populations on a single plot.
This high-dimensional modeling correctly identified
various expansions or reductions in the cellular islands
corresponding to CD4 and CD8 T cells, NK cells, B cells,
and myeloid populations when stimulated with PMA/
ionomycin (Figure 6A). Additionally, the relative signal
intensity for each of the 11 expansion panel markers wasoverlaid as heat maps to monitor expression on immune

cell population clusters between unstimulated and PMA/
ionomycin pairs from the same donor (Figure 6B). These
heat maps revealed more intricacies of the cellularresponse to stimulation in the Cen-se′ plot such asubiquitously high expression of CD69 across the T cellislands, exhausted CTLA-4+ CD4 T cells, and increased
production of cytokines such as IFNγ in specific T cellclusters. Upregulation of CD107a across numerous
activated populations was also observed. This included
NK and CD8 T cell islands, indicating cytotoxic potentialof these cell subsets.

A

B

Figure 6. Visualization of immune cell activation via high-dimensional Cen-se′ analysis. Maxpar Pathsetter generated Cen-se′ maps ofunstimulated and PMA/ionomycin-stimulated samples from PBMC Donor 2 are shown. A) Clusters are colored by cell types and subsets. CD8 T cells,
CD4 T cells, B cells, NK cells, and myeloid cells are labeled. B) Heat maps of signal intensities illustrating relative expression across immune cell
populations for each of the 11 functional markers from the Maxpar Direct T Cell Activation Expansion Panel are shown for both unstimulated and
PMA/ionomycin stimulated samples from the same donor.
Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

5Cytokine profiling of CD4 T cell subsets
The addition of specific activation and intracellularmarkers can also complement the phenotyping carriedout by the base Maxpar Direct panel. For example,
the helper T cell (Th) subsets Th1, Th2, and Th17 aredefined by expression of specific chemokine receptors
in the base Maxpar Direct assay. Investigation ofcytokine profiles in this work confirmed the definition ofthese subsets (Figure 7). As expected, PMA/ionomycinstimulated samples revealed that while IL-2 and TNFα
were ubiquitously expressed across all 3 Th subsets,
IFNγ, IL-4, and IL-17A were predominantly expressed in
Th1, Th2, and Th17 populations, respectively.5
Taken together, these results demonstrate the abilityof the Maxpar Direct Assay combined with the T
Cell Activation Expansion Panel to effectively andreliably identify and phenotypically profile activatedimmune cells.

Exploring T Cell-Mediated Host Immune
Responses to Foreign Antigens
Quantification of antigen-specific T cell responses iscritical for understanding immunity and pathogenesis
to infectious disease. Furthermore, clinical research
applications, such as vaccine development, aim torecapitulate the response to natural infection, and theenumeration of vaccine-induced T cells is frequently
used to assess immunogenicity. Importantly, the Maxpar
Direct T Cell Activation Expansion Panel, combined with
Maxpar Pathsetter, allows for the robust assessment ofthese antigen-specific responses through measurementof cytokines and markers of activation and cytotoxicity.

Representative bivariate plots portraying the expressionof IFNγ, TNFα, and CD107a in CD8 T cells depict anantigen-specific immune response, as shown by a clear
upregulation and positive correlation of these functional
markers in peptide-stimulated samples when comparedto paired unstimulated samples from the same donor(Figure 8, top panel). These data plots are conveniently
color-coded based on CD8 T cell memory subsets:naive/central memory (Nv/Cm), effector memory (Em),and terminal effector (TEff). The majority of the antigenspecific recall responses were found in CD8 effector
cells, which are poised to protect the host from invading
pathogens. Expression profile plots, which are uniqueto Pathsetter, visualize individual functional marker
expression across the CD8 T cell memory subsets for
additional granularity on reactive T cell populations.
A clear antigen-specific upregulation of cytokines anddegranulation markers in CD8 Em and TEff cells wasobserved here (Figure 8, bottom panel). IFNγ, TNFα,CD107a, and IL-2 are shown as representative plots.
However, all 8 functional markers within CD8 T cells areavailable in the full customized Pathsetter report.
IL-4

IFNγ

IL-17A

68.98%

70.82%

2.38%

28.59%

0.00%

64.02%

77.68%

1.90%

5.78%

1.55%

170Er_CD3

112Cd_IL-2

114Cd_TNFα

116Cd_IFNγ

170Er_CD3

0.15%

170Er_CD3

7.77%

170Er_CD3

73.48%

170Er_CD3

79.57%

Th-1-like cells

53.51%

Th-2-like cells

TNFα

Investigating the spectrum of antigen-specific CD8
memory subsets

Th-17-like cells

IL-2

In this study, cells stimulated with a pool of
immunodominant microbial peptides were used as anin vitro model of infectious disease and were processedwith the Maxpar Direct Assay and Maxpar Direct T
Cell Activation Expansion Panel. A detailed report
page focusing on antigen-specific CD4 and CD8 T cell
recall and functionality is provided following Pathsetter
analysis, as depicted in Figure 8 and Figure 9A below.

142Nd_IL-4

169Tm_IL-17A

Figure 7. Cytokine profile of CD4 T-helper subsets after PMA/ionomycin stimulation. CD3 vs. IL-2, TNFα, IFNγ, IL-4, or IL-17A bivariate plots
are displayed for Th1-like, Th2-like, and Th17-like populations from PBMC Donor 3. Gate labels display % of cells positive for the corresponding
cytokine expression. Th1-like cells were gated on CD4+CXCR5-CCR4-CD45RA-CD45RO+CXCR3+CCR6- populations. Th2-like cells were gated on
CD4+CXCR5-CD45RA-CCR4+CXCR3-CCR6- populations. Th17-like cells were gated on CD4+CXCR5-CD45RA-CCR4+CXCR3-CCR6+ populations.
6

Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

TEff

TNFα

Em

10²

0
0

50

Progression (% events)

100

0

50

Progression (% events)

100

10³

0

Nv/Cm

Em

10²

10³

CD107a

10�

TEff

50

Progression (% events)
TEff

IL-2

Em

0

50

Progression (% events)
Nv/Cm

10³

Nv/Cm

100

Em

100

TEff

0

TEff

10�

0

0

Em

0

10�

Nv/Cm

100

10²

10²
-10¹

50

Progression (% events)

-10¹

TEff

10³

Em

0

IL-2

10�

Nv/Cm

100

CD107a50

Progression (% events)

10²

0

10³

CD107a

10³

Nv/Cm

0 10¹
0

10³

10²

TEff

10²

TNFα

10²

0 10¹

10²

TNFα

10³
10²

IFNγ

0 10¹

10�

10³

10³

IFNγ

Em

10²

CD107a

10²

Nv/Cm

10³

IFNγ
0

0

10²

0 10¹

10�

10³

TEff

10²

10³
0 10¹

10³

10²

Em

10²

CD107a

TNFα

10³

Nv/Cm

0

10³

10²

TNFα

10³
10²

IFNγ
10²

TNFα

0

IFNγ

0 10¹

CD107aMicrobial peptides

0 10¹

10²
0 10¹

IFNγ

10³

Unstimulated

050

Progression (% events)

100

0

50

Progression (% events)

100

Figure 8. Antigen-specific CD8 T cells identified using Maxpar Pathsetter. Bivariate plots from CD8 T cells showing IFNγ vs. TNFα, IFNγ vs.
CD107a, and TNFα vs. CD107a for unstimulated and peptide stimulated samples from PBMC Donor 2 (top panel). Upregulation and positive
correlation of functional markers highlight antigen-specific T cells identified in microbial peptide-stimulated samples. Expression profile plots of
IFNγ, TNFα, CD107a, and IL-2 across naive/central memory (Nv/Cm), effector memory (Em), and terminal effector (TEff) CD8 T cell memory subsets(bottom panel). All plots were generated by Maxpar Pathsetter automatic analysis and are color-coded by CD8 T cell memory subsets.

Quantification of polyfunctional antigen-specific
CD8 T cell populations
The frequency of functional marker expression within
antigen-specific T cells, defined as IFNγ+ CD8+ T
cells, was also automatically reported in a summary
table for additional quantification and phenotypic
analysis (Figure 9A). As expected, greater frequenciesof activation markers and cytokine production canbe observed in antigen-specific CD8 T cells when

A A

Maxpar Pathsetter Reportcompared to global CD8 T cell populations across all
PBMC donors tested (Figure 9B). These pathogenspecific T cells were polyfunctional, as assessed byco-expression of numerous activation and cytolytic
markers, a correlate of T cell efficacy.6
Overall, endogenous antigen-specific CD8 T cells
across various PBMC donors were successfully
identified and characterized in response to microbial
peptide stimulation using Maxpar Direct panels andautomated Pathsetter analysis.

BB% Parent population

90
80

Total CD8 T cells

70

Antigen-specific
IFNγ+ CD8 T cells

60
50
40
30
20
10
0

CD107aCD69

CTLA-4 Granzyme B

Perforin

TNFα

IL-2

Figure 9. Profiling antigen-specific CD8 T cells. A) Screen capture of the customized Maxpar Pathsetter report page for peptide-stimulated
CD8 T cells from PBMC Donor 2. Frequency of CD8 T cells expressing CD107a, CD69, CTLA-4, Granzyme B, Perforin, TNFα, and IFNγ is shown.
Additional breakdown of these frequencies on antigen-specific CD8 T cells, defined as IFNγ+, is reported. B) Bar graphs depicting frequency offunctional markers expressed on total CD8 T cells compared to antigen-specific IFNγ+ CD8 T cells across all four PBMC donors (mean % parent
population frequency ± SEM).
Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

7Summary

Conclusions

This application note highlights the flexibility of the
Maxpar Direct Immune Profiling Assay to accommodate
expansion panels and further facilitate comprehensive
immune profiling and functional analysis of cellularresponses. Several key takeaways from this study arelisted below:

The Maxpar Direct Immune Profiling System is acomplete and flexible sample-to-answer solution for
single-cell high-dimensional immune profiling. This study
demonstrated how to leverage the power of Maxpar
Direct Expansion Panels for clinically-relevant research
to interrogate antigen-specific T cells in an in vitro
model of infectious disease.

•	A 47-parameter CyTOF panel was developed by
combining the base Maxpar Direct Immune Profiling
Panel, the Maxpar Direct T Cell Activation Expansion
Panel, and Maxpar anti-CD45 LCB reagents.
•	The preconfigured expansion panel included 9
antibodies targeting cytoplasmic markers, thereby
introducing a novel workflow of intracellular staining
to the Maxpar Direct Immune Profiling Assay.
•	LCB enhanced efficiency of intracellular antibody
staining and sample acquisition thereby streamlining
the Maxpar Direct workflow.
•	The existing Pathsetter model was adapted toincorporate new functional immune markers resultingin >100 additional measurements on various
cell subsets•	Automatic analysis using Pathsetter successfullyclassified new populations such as antigen-specific
T cells in response to microbial peptide pools.
•	Immunophenotyping and functional profiling of these
populations were enabled by assessing expressionof key activation, cytokine, and cytotoxic markers,
thereby providing insight into potential cell-mediatedimmune protection from foreign pathogens.

8

Although this application note focused on recall
responses in CD8 T cell populations, antigen-specificCD4+ T cells can also be identified and characterized
automatically with this method. Furthermore, experimental
workflows and antibody panels similar to the one
described herein can easily be applied to other infectious
disease models such as SARS-CoV-2 infection7, response
to vaccination8, and immunotherapy research9.
Additional preconfigured Maxpar Direct Expansion
Panels are available, encompassing a broad selectionof surface and intracellular targets for studies requiring
more in-depth characterization of specific immune cell
populations or functional states in both human PBMC
and whole blood.
With robust high-parameter coverage, efficient andreliable workflows, and even more flexibility for panel
expansion, the Maxpar Direct Immune Profiling System
powered by CyTOF technology is an invaluable toolthat empowers users to carry out deep interrogationof the immune system and unravel its complexities ina single tube.

Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

Tips for Success

References•	Expression of functional markers may varydepending on stimulation or disease conditions.
We recommend titrating the Maxpar Direct Expansion
Panel antibodies with representative test samples.

1.	Chattopadhyay, P.K., Gierahn, T.M., Roederer, M. et
al. “Single-cell technologies for monitoring immune
systems.” Nature Immunology (2014)•	Heparin blocking of permeabilized cells prior to
intracellular staining is recommended to reduce
background staining in select immune cell
populations. Refer to the Maxpar Cytoplasmic/
Secreted Antigen Staining with Fresh Fix Protocol(400279) for additional details.
•	Titration of each metal-labeled anti-CD45 antibody
is essential prior to carrying out LCB to ensuresufficient signal intensities for sample debarcoding.
Concentrations often vary between sample types.
For example, whole blood samples may require
higher concentrations of anti-CD45 antibodies
compared to PBMC. Furthermore, downregulation of
CD45 may occur under some stimulation or diseased
sample conditions.
•	After pooling barcoded samples, scale-up buffers
and antibody volumes according to cell number as
described in the Cell-ID 20-Plex Barcoding Kit User
Guide (PRD023).
•	More information on how to customize the Maxpar
Pathsetter model can be found in the Method
Develop: Customize the Maxpar Direct Immune
Profiling Assay User Guide (FLDM-00151). For a copyof the model template used in this study, contact
your local Field Applications Specialist or TechnicalSupport Specialist, who can connect you with aregional Field Applications Specialist.
•	Contact your local Field Applications Specialist for
further guidance on any details described in thisapplication note.

2. Baskar, R., Kimmey, S.C., Bendall, S.C. “Revealing
new biology from multiplexed, metal-isotope-tagged,
single-cell readouts.” Trends in Cell Biology (2022)
3. Fread, K.I., Strickland, W.D., Nolan, G.P. et al. “An
updated debarcoding tool for mass cytometry withcell type-specific and cell sample-specific stringency
adjustment.” Pacific Symposium on Biocomputing
22 (2017). Software licensed under GNU General
Public License.
4.	Bagwell, C.B. “High-dimensional modeling forcytometry: Building rock solid models using
GemStone™ and Verity Cen-se′ high-definition t-SNE
mapping.” Methods in Molecular Biology (2018)
5.	Zhu, X. and Zhu, J. “CD4 T helper cell subsets
and related human immunological disorders.”
International Journal of Molecular Sciences (2020)
6.	Boyd, A., Almeida, J.R., Darrah, P.A. et al. “Pathogenspecific T cell polyfunctionality is a correlate of T cell
efficacy and immune protection.” PLoS One (2015)7.	
Keeton, R., Tincho, M.B., Ngomti, A. et al. “T cell
responses to SARS-CoV-2 spike cross-recognize
Omicron.” Nature (2022)
8.	
Mateus, J., Dan, J.M., Zhang, Z. et al. “Low-dosemRNA-1273 COVID-19 vaccine generates durable
memory enhanced by cross-reactive T cells.”
Science (2021)
9. Ramesh, P., Shivde, R., Jaishankar, D. et al. “A paletteof cytokines to measure anti-tumor efficacy of T cellbased therapeutics.” Cancers (2021)

Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

9

Methods
Human PBMC from 4 donors were obtained from
STEMCELL Technologies™ and thawed in complete
RPMI 1640 (cRPMI) medium (Thermo Fisher Scientific™)
containing Anti-Aggregate Wash™ Supplement (CellularTechnology Ltd.), following manufacturer instructions,and rested overnight at 37 °C and 5% CO2.
Cells were seeded in 24-well plates at a density of 6 x
106 cells/mL and were either left untreated or cultured
in the presence of PMA/ionomycin (Cell Activation
Cocktail; BioLegend®) or a mix of immunodominantmicrobial peptides (CEFX peptide pool; JPT Peptide
Technologies) according to manufacturer guidelines.
Cells were also stained with 106Cd-CD107a antibodies
during the stimulation. Brefeldin A (BioLegend) andmonensin (BioLegend) were added to all wells 1 hr into
the culture and were subsequently incubated for an
additional 4 hr prior to harvesting.
Cells were washed with Maxpar Cell Staining Buffer(CSB), blocked with Human TruStain FcX™ (BioLegend)
and stained with the Maxpar Direct Immune Profiling
Panel according to the Maxpar Direct Immune Profiling
Assay Cell Staining and Data Acquisition User Guide(400286). 113Cd-CD69 and corresponding CD45
antibody-based barcode mixtures were added as
drop-in antibodies during surface staining (unstimulated:
110
Cd-CD45, 111Cd-CD45, 194Pt-CD45; microbial peptides:
110
Cd-CD45, 111Cd-CD45, 195Pt-CD45; PMA/ionomycin:
110
Cd-CD45, 194Pt-CD45, 195Pt-CD45).
After surface and barcode staining, cells were washedwith CSB and fixed with 0.1X methanol-free Pierce™ 16%
Formaldehyde (w/v) (Thermo Fisher Scientific) diluted in
Maxpar PBS. Cells were washed with CSB and barcodedsamples corresponding to each individual donor werecombined into a single 5 mL round bottom tube.

10

Combined samples were permeabilized with Maxpar
Perm-S Buffer and blocked with 100 U/mL sodium
heparin salt (Sigma Aldrich™) in Perm-S Buffer. Samples
were then stained with the following intracellular
antibodies according to the Maxpar Cytoplasmic/
Secreted Antigen Staining with Fresh Fix Protocol(400279): 112Cd-IL-2, 114Cd-TNFα, 116Cd-IFNγ, 142Nd-IL-4,
162
Dy-CTLA-4, 165Ho-IL-10, 169Tm-IL-17A, 196Pt-Perforin,and 198Pt-Granzyme B. After intracellular staining, cells
were washed with CSB and fixed a second time. Finally,
samples were incubated with Cell-ID Intercalator-Ir(31.25 nM final concentration) in Maxpar Fix and Perm
Buffer overnight at 4°C.
The Helios instrument with CyTOF Software version
7.0.8493 was tuned using Tuning Solution. Note that thisassay can also be acquired on a CyTOF XT™ system.
Immediately prior to acquisition, the instrument wasconditioned with Maxpar Cell Acquisition Solution(CAS) Plus. Samples were washed and resuspended
to 1 x 106 cells/mL in 0.1X EQ™ Four Element Calibration
Beads as an internal standard diluted in CAS Plus.
Approximately 1.5 x 106 cellular events were acquired
per pooled sample at 250–350 events/sec on the
Helios instrument with the WB Injector.
Original FCS files were normalized using CyTOF
Software version 7.0.8493 and were subsequently
debarcoded using the Zunder Lab Single Cell
Debarcoder.3 For more information on LCB, refer to theapplication note: Enabling Live-Cell Barcoding with
Anti-CD45 Antibodies in Suspension Mass Cytometry(FLDM-00488).
The processed and debarcoded FCS files from eachexperiment were analyzed using Maxpar Pathsetter
software version 3.0.15 and Cytobank (Cytobank.org).

Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

Related Documents

Appendix A: Ordering Information

The following related documents can be found at
standardbiotools.com:

Standard BioTools™ Reagents•	
Maxpar Direct Immune Profiling Assay Cell Staining
and Data Acquisition User Guide (400286)

Name

Catalog
NumberMaxpar® Direct™ Immune Profiling Assay™,
30 Marker—25 Tests

201334

Maxpar Direct T Cell Activation Expansion
Panel, 11 Marker—25 Tests

201409•	Method Develop: Customize the Maxpar Direct
Immune Profiling Assay User Guide (FLDM-00151)

Anti-Human CD45 (HI30)-110Cd—100 or
25 Tests

3110001B/C•	Maxpar Cytoplasmic/Secreted Antigen Staining with
Fresh Fix Protocol (400279)

Anti-Human CD45 (HI30)-111Cd—100 or
25 Tests

3111001B/C•	Cell-ID 20-Plex Pd Barcoding Kit User Guide(PRD023)

Anti-Human CD45 (HI30)-194Pt—100 or
25 Tests

3194001B/C•	The Benefits of Palladium Barcoding on Data Quality
and Workflow Application Note (FLDM-00012)

Anti-Human CD45 (HI30)-195Pt—100 or
25 Tests

3195001B/C• E
 nabling Live-Cell Barcoding with Anti-CD45
Antibodies in Suspension Mass Cytometry
Application Note (FLDM-00488)

Maxpar Cell Staining Buffer

201068

Maxpar PBS

201058•	Approach to Bivariate Analysis of Data Acquired
Using the Maxpar Direct Immune Profiling Assay
Technical Note (400248)

Maxpar Perm S Buffer

201066• Helios, a CyTOF System User Guide (400250)

Maxpar Fix and Perm Buffer

201067• CyTOF XT User Guide (FLDM-00254)

EQ™ Four Element Calibration Beads

201078Tuning Solution

201072

Reagents from Other Suppliers
VendorName

Part Number

STEMCELL
Technologies

Human PBMC

70025

Cellular Technology CTL Anti-Aggregate
Limited (CTL)
Wash 20x Solution

CTL-AA-010

Thermo Fisher
Scientific

Pierce 16%
Formaldehyde (w/v),
Methanol-free

28906

BioLegend

Human TruStain FcX

422302

BioLegend

Cell Activation Cocktail
423301(without Brefeldin A)

BioLegend

Brefeldin A Solution

420601

BioLegend

Monensin Solution

420601

JPT Peptide
Technologies

CEFX Ultra
SuperStim Pool

PM-CEFX-1

Sigma AldrichHeparin Sodium Salt

H3149-10KU

Maxpar Direct Immune Profiling Assay Expanded to Explore Antigen-Specific Immune Responses

11Appendix B: Customer Support
For access to the CyTOF Software acquisition templateand custom Maxpar Pathsetter model template usedin this application note, contact your local Field
Applications Specialist.

Learn more: fluidigm.com/immuneprofile
Or contact: tech.support@fluidigm.com

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For Research Use Only. Not for use in diagnostic procedures.
Information in this publication is subject to change without notice. Limited Use Label License: The purchase of this Standard BioTools Instrument and/or Consumableconveys to the purchaser the limited, nontransferable right to use only with Standard BioTools Consumables and/or Instruments respectively except as approved inwriting by Standard BioTools Inc. (f.k.a. Fluidigm Corporation): www.fluidigm.com/legal/salesterms. Patents: www.fluidigm.com/legal/notices. Trademarks: Standard
BioTools, the Standard BioTools logo, Fluidigm, the Fluidigm logo, Cell-ID, CyTOF, CyTOF XT, Direct, EQ, Helios, Immune Profiling Assay, Maxpar and Pathsetter are
trademarks and/or registered trademarks of Standard BioTools Inc. or its affiliates in the United States and/or other countries. All other trademarks are the sole propertyof their respective owners. ©2022 Standard BioTools Inc. All rights reserved. 06/2022
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