A Rapid and Robust Solution for Cellular Response Characterization
App Note / Case Study
Last Updated: June 19, 2024
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Published: May 21, 2024
Credit: iStock
T cells are pivotal in the immune system's response to cancer, however, challenges such as poor tumor recognition and penetration into solid tumors hinder their efficacy.
Novel immunotherapies, including tumor infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells, have been developed to enhance T-cell responses against cancer.
This app note explores the use of an assay to facilitate rapid monitoring of T-cell function and simultaneous assessment of cell phenotype, activation markers, proliferation, viability and cytokine secretion.
Download this app note to discover insights into the:
- Concentration-dependent effects of therapies
- Impact of environmental factors on TILs
- Antigen specificity of CAR T cells
Application Note
Characterization of T Cell Activation and
Function in Cancer Immunotherapy Using
High-Throughput Cytometry
Author: Veronica Bruce¹, Kirsty McBain², Nicola Bevan²
1. Sartorius Corporation, 5700 Pasadena Avenue NE, Albuquerque, NM 87113 USA
2. Sartorius UK Ltd, Units 2 & 3 The Quadrant, Newark Close, Royston, Hertfordshire, SG8 5HL, UK
Correspondence
Email: AskAScientist@sartorius.com
Abstract
T cells are pivotal in the immune system's response to cancer, with their ability to recognize and eliminate tumor cells. However,
challenges such as poor tumor recognition and penetration into solid tumors hinder their efficacy. Novel immunotherapies,
including bispecific antibodies, tumor infiltrating lymphocytes (TILs), and chimeric antigen receptor (CAR) T cells, have been
developed to enhance T cell responses against cancer. To facilitate rapid monitoring of T cell function, a high-content, multiplexed
assay using high-throughput flow cytometry has been developed. The iQue® Human T Cell Activation Kit allows simultaneous
assessment of cell phenotype, activation markers, proliferation, viability, and cytokine secretion in a single well assay.
These studies present data on antibody-mediated T cell activation, TILs assay models, and CAR-T cell activation assays,
demonstrating the kit's ability to evaluate T cell responses to various stimuli. Results indicate that the iQue® platform can
effectively characterize T cell activation and function, providing insights into the concentration-dependent effects of therapies,
the impact of environmental factors on TILs, and the antigen specificity of CAR-T cells. The platform's high-throughput capability
and minimal sample volume requirements make it an ideal tool for developing new immunotherapies, with the potential to predict
on-target off-tumor effects and aid in the rapid identification of therapeutic candidates.
March, 2024
Keywords or phrases:
T Cell Activation, T Cells, Flow Cytometry, TILs, CAR-T,
BiTE
For further information, visit www.sartorius.com/ique
2
Introduction
T cells play a critical role in immunosurveillance and clearance
of cells infected with viruses and bacteria, as well as cancerous
cells. Upon recognition of tumor antigens and co-stimulatory
signals, T cells can become activated, proliferate, and consume
or secrete various cytokines that support T cell health and/or
effector functions. Recognition and killing of cancerous cells is
effectively performed by the actions of activated T cells, thus
making them an attractive therapy against this disease.
Despite the innate tumor cell killing ability of T cells,
research is ongoing to combat challenges such as poor
tumor recognition, targeting, and solid tumor penetration.
Discovery of novel immunotherapies that specifically target
and enhance the T cell response against cancer, for example,
bispecific antibodies, tumor infiltrating lymphocytes (TILs),
and chimeric antigen receptor (CAR) modified T cells,
have revolutionized cancer treatment paradigms and are
rapidly expanding research areas.1,2 Not limited to immunooncology, bispecific antibodies are valuable in making
the connection between immune cells and their target
cells, increasing the directed killing towards those targets.
This approach has yielded some success in the form of
bispecific T cell engager (BiTE) antibodies in the fight against
cancer. To overcome issues with tumor recognition, the
development and optimization of therapeutics involving
TILs or CAR-T cells that are engineered to recognize specific
tumor proteins have gained much attention.3
Thus, there exists a need for development of robust and
relevant strategies to monitor novel therapeutics for
sufficient activation and antitumor efficacy while minimizing
toxicity concerns. This includes assessment of cell health,
viability, phenotype, and function.
To address the need for rapid monitoring of T cell function,
we developed an optimized, high-content, multiplexed
assay using high-throughput cytometry to measure T cell
activation. The iQue® Human T Cell Activation Kit collapses
the traditional workflow by evaluating cell phenotype,
T cell activation markers, cell proliferation, cell viability, and
secreted cytokines in a single well assay using
a 96 or 384-well plate format.
Figure 1: Workflow Figure
“+” denotes highly expressed | secreted, “+|-“denotes partially expressed/ secreted, “-“ denotes low or no expression/secretion (table insert).
3
Assay Principles
The iQue® Human T Cell Activation Kit has been designed
to quantify T cell activation by measuring phenotype and
function. Assay plates can be set up to look at signals from
various T cell sources (e.g., PBMCs or CAR-T cells) using
various methods of cell activation (e.g., Dynabeads™,
effector and target cell co-culture). At the assay endpoint,
an aliquot of the sample from each well is transferred into an
assay plate. Cytokine analyses can be performed separately
or multiplexed with the phenotyping components for added
flexibility depending on research needs. To perform the assay,
10 µL of sample containing cells and supernatant together
or separately were transferred to an assay plate and analyzed
utilizing the iQue® Human T Cell Activation Kit protocol
illustrated in Figure 1. Data was acquired on the iQue®
High-Throughput Screening (HTS) Cytometry Platform
using the violet, blue, and red (VBR) laser configuration and
analyzed using the provided Forecyt® software template to
quantify cytokines TNFα and IFNγ, T cell subsets (via CD3,
CD4, CD8) and surface activation markers (CD69, CD25,
HLA-DR), and cell viability via the iQue® Cell Membrane
Integrity Dye provided in the kit. The kit also contains the
optional iQue® Cell Proliferation and Encoding (B/Green)
Dye that can be used to assess proliferation or to stain cells
samples of interest (e.g., target cells). In these experiments,
the encoder function of this dye was replaced with the use
of stably transfected green-labeled target cells (Incucyte®
Nuclight Green Lentivirus).
Methods
Antibody-Mediated T Cell Activation
Ramos cells modified to express a nuclear restricted green
fluorescent protein (Incucyte® Nuclight Green Lentivirus)
were seeded (15K/well) with PBMCs (5:1 effector-to-target;
E:T) and either activated with increasing concentrations of
an anti-hCD3xCD19 BiTE (10 ng/mL; 5-fold serial dilution),
or an anti-hCD3xβGAL control antibody (10 ng/mL), or
CD3/CD28 Dynabeads™ (75K beads/well; 3-fold serial
dilution). Every 24 hours, 10 µL of supernatant was removed
and cytokine analysis (IFNγ and TNFα) was performed using
iQue Qbeads® found in the iQue® Human T Cell Activation
Kit. At 72 hours, cells were dissociated and the numbers of
live, green-labeled target cells were counted. T cell subsets
and activation markers were also evaluated using the iQue®
Human T Cell Activation Kit antibody panel.
TILs Assay Model
BT474, SKOV-3 or A549 cells were seeded in ultra-low
attachment (ULA) plates and incubated for 72 hours to
promote spheroid formation. Pre-activated (1:1 Dynabeads™
to cell ratio) PBMCs (5:1 E:T) were added for 24 hours.
After 24 hours in co-culture, the non-infiltrated tumor
cells were washed off and transferred into a separate plate,
leaving only the spheroids and TILs in the assay plate.
Once the non-infiltrated cells had been separated, the
spheroids and TILs were dissociated to create a single cell
suspension. To examine the effect of fibroblasts in different
donors, spheroids were formed with either BT474 cells
alone or with 50% BT474s and 50% CCD106SK fibroblasts
(NHDF). PBMCs from two different donors and CD3/CD28
Dynabeads™ were added for 40 hours. Samples were
assessed for T cell subset and activation marker expression
using the iQue® Human T Cell Activation Kit.
CAR-T Cell Activation Assay
CD19, second-generation CAR-T cells (~50% transduction
efficiency) or control matched-donor, mock-transduced T
cells (Creative BioLabs) were placed into culture with CD19
antigen positive Ramos or CD19 antigen negative Jurkat
cells at various E:T ratios. Samples were analyzed on Day 2, 4,
and 7 using the iQue® Human T Cell Activation Kit and the
iQue® Human T Cell Companion Kit (IL-2).
“On Target Off Tumor” CAR-T Profiling
HER2 high (AU565), HER2 low (MDA-MB-231) or HER2
negative (MDA-MB-468) cell lines were modified to express
a nuclear restricted green fluorescent protein (Incucyte®
Nuclight Green Lentivirus), seeded into ULA plates and
allowed to form single spheroids over 3 days in the presence
of Matrigel® (1.25%). Once formed, anti-HER2 CAR-T cells
or mock transduced control T cells were added to the wells at
various E:T ratios.
On Day 2, 4, and 7, samples were analyzed on the iQue®
HTS platform. Supernatants were collected for secreted
protein analysis before cultures were gently dissociated
to remove Matrigel® and break up the spheroids. Samples
were assessed for phenotype and function using the iQue®
Human T Cell Activation Kit.
4
Results and Discussion
Antibody-Mediated T Cell Activation
BiTE antibodies, such as Blinatumomab, are being used
clinically for treatment of B-lymphocytic leukemia alongside other late-stage cancers.4
The CD3xCD19 construct,
simultaneously engages the CD3 on T cells and the tumor
associated antigen, CD19, present on B cells. This interaction
causes the clonal expansion and activation of T cells as well as
direct contact between CD3+ T cells and CD19+ tumor cells,
resulting in tumor specific cell lysis. To model the therapeutic
effects on B cell leukemia in vitro, an anti-hCD3xCD19 BiTE
antibody was evaluated alongside a control antibody targeting CD3 or Dynabeads™.
Cytokine production was evaluated from the supernatant
samples taken daily. The CD3xCD19 BiTE induced production
of TNFα and IFNγ in a concentration dependent manner,
reaching maximal concentrations of 1.5±0.3 ng/mL and
1.3±0.3 ng/mL by day 3, respectively (Figure 2A and B).
Interestingly, CD3/CD28 Dynabead™ stimulation evoked
vastly increased production of both cytokines, with TNFα
reaching 3.8±0.2 ng/mL and IFNγ 14.4±2.5 ng/mL.
Comparison to the levels of cytotoxicity as measured by target
cell count (Figure 2C) highlight that despite the low level of
cytokine release, the BiTE antibody induces relatively high
levels of target cell death compared to CD3/CD28 stimulation.
On day 3 post-treatment, cells were lifted from the
assay plate and labeled with the iQue® Human T Cell
Activation Kit antibody panel. As expected, the CD3/CD28
Dynabeads™ induced concentration-dependent increases
in the proportions of CD69, CD25 and HLA-DR positive
populations, yielding comparable EC₅₀ values for each
marker of 4690, 16958 and 13865 beads/well, respectively
(Figure 3A). The maximal population percentages were 29%
for CD69, 69% for CD25 and 32% for HLA-DR.
Figure 2: Temporal Cytokine Production and Immune Cell Killing in Response to CD3xCD19 BiTE Antibody
Daily supernatant samples (10 μL) taken for analysis of cytokine (TNFα and IFNγ) concentrations by the iQue® HTS platform (A and B). Temporal
cytokine concentrations were compared to the level of immune cell killing as measured by the live green-labeled target cell count data in (C), with
bars for CD3/CD28 Dynabeads™ in teal, BiTE in grey and CD3xβGAL antibody control in black.
Days
TNFα [ng/mL]
0
5
4
3
2
1
Control
0.02 ng/mL
0.4 ng/mL
10 ng/mL BiTE Antibody
75,000 Beads
1 2 3
Live Green Cell Count [x10³]
0
50
40
30
20
10
309
926
2.8 K
8.3K
25K
75K
0.0032 ng/mL
0.016 ng/mL
0.08 ng/mL
0.04 ng/mL
2 ng/mL
10 ng/mL
Control
A
Days
IFNγ [ng/mL]
0.0
20.0
15.0
10.0
5.0
2.0
1.0
0.5
1 2 3
1.5
B
C
Dynabead™ Density
SSC
CD3
A
5
Figure 3: Immune Cell Activation in Response to CD3xCD9 BiTE Antibody
Concentration response curves to activation induced by (A) CD3/CD28 Dynabeads (100 to 75K beads/well) or (B) CD3xCD19 BiTE antibody
(0.6 pg/mL to 10 ng/mL).
In contrast, inclusion of the BiTE antibody displays a clear left
shift in the CD69 expression pattern, with low concentrations
(20 pg/mL) capable of inducing almost exclusive expression
of this early activation marker (EC₅₀ value of 5.5 pg/mL,
Figure 3B). CD25 (mid activation) and HLA-DR (late
activation) are induced but at much higher concentrations
of BiTE, yielding EC₅₀ values of 87 pg/mL and 72 pg/mL,
respectively. Maximal subset percentages were 45% for
CD69, 92% for CD25 and 46% for HLA-DR.
These data suggest different mechanisms of activation
may yield altered proportions of CD8 positive subsets.
The findings presented require further investigation to
fully understand the implications of this observation.
Activation of Tumor Infiltrating Lymphocytes (TILs)
It is widely accepted that increased numbers of TILs
within a tumor are often associated with improved clinical
prognosis.5
Evidence is now growing which suggests that
the composition of the TILs, in terms of the cell subsets
present and their activation status, is also highly important
in denoting the quality of the anti-tumor response the TILs
can exert.5-7 To investigate the phenotypic profile of these
cells in vitro, a TILs assay model with wild type BT474 (breast
cancer) spheroids and PBMCs, activated with a range of
concentrations of CD3/CD28 Dynabeads™ was employed.
After 24 hours in co-culture, single cell suspensions of
non-infiltrated and infiltrated T cells from dissociated
tumor spheroids were analyzed.
Figure 4: Comparison of the Activation Status of Infiltrated or Non-Infiltrated T Cells from Dissociated BT474 Spheroids
(A) Spheroids and TILs were dissociated to a single cell suspension and analyzed using the iQue® Human T Cell Activation Kit. Plate view shows individual
well plots of side scatter (SSC) vs. CD3 with gates highlighting the number of CD3 positive TILs per iQue® sip from each well. Each column represents
a different Dynabead™ density (n = 4). An outlier (highlighted in red) was excluded from subsequent analyses. (B) and (C) Activation marker expression
comparison between the non-infiltrated and infiltrated T cells.
Log (bead number)
Expression [%]
0
100
80
60
40
20
2 3 4 5
CD8/CD69
CD8/CD25
CD8/HLA-DR
A
Log (CD3xCD19) [g/L]
Expression [%]
0
100
80
60
40
20
-13 -12 -11 -10 -19 -8 -7
B
CD3/CD28 Dynabead Density
Expression [%]
0
100
80
60
40
20
CD69
CD25
HLA-DR
247 740 2.2K 6.6K 20K
B Non-Infiltrated
CD3/CD28 Dynabead Density
Expression [%]
0
100
80
60
40
20
CD69
CD25
HLA-DR
247 740 2.2K 6.6K 20K
C Infiltrated
6
A Dynabead™ concentration-dependent increase in
infiltration of T cells into the spheroid was evidenced by
the relative number of CD3+ TILs per well, as shown in the
Forecyt® plate view diagram (Figure 4A). Figures 4B and 4C
compare the expression of early (CD69), mid (CD25) and
late (HLA-DR) stage activation markers between the noninfiltrated and infiltrated T cells. At all but the topmost
concentration of Dynabeads™, the expression of the three
activation markers was higher on the infiltrated T cells than
on the non-infiltrated. On the infiltrated cells, elevated
expression of CD69 was maintained, regardless of the density
of Dynabeads™ present, with an average of 67.1±0.6%.
Non-infiltrated cells displayed increased sensitivity to
changes in external stimuli with CD69 expression increasing
in a Dynabead™ concentration dependent manner, from
2.3±0.2% to 86.9±0.7% when the Dynabead™ density was
increased from 247 to 20K per well. These data show that
activation marker expression on TILs is greater than on
non-infiltrated T cells.
Studies that have measured rates of TILs across multiple
tumor types have found differences between cancers in
the likelihood of the presence of high-density TILs and the
significance of these in terms of prognostic outcomes.8-10 To
verify whether our model could be used to reveal differences
in infiltration between different tumor types, we formed
spheroids from breast (BT474), ovarian (SKOV-3) and lung
(A549) cancer cell lines and analyzed the rate of infiltration
and the phenotype of pre-activated PBMCs after 24 hours of
co-culture using iQue® Human T Cell Activation Kit.
Both the BT474 and SKOV-3 spheroids displayed very
similar levels of infiltration with an average of 617± 97 and
567±105 CD3+ cells sampled per well, respectively, while
average infiltration into the A549 cells was much lower at
282±41 (Figure 5A). This indicated there may be a tumorspecific element to the degree of infiltration. The TILs from
each of the different spheroid types were also analyzed for
the ratio of CD8 to CD4 cells. A high proportion of cytotoxic
CD8+ TILs is generally associated with more successful
pathological complete response in cancer.9
In the spheroid
types we tested, both the SKOV-3 and A549 spheroids
contained a high proportion of CD8+ TILs with average ratios
of 1.65 and 1.35 CD8:CD4 cells, respectively (Figure 5B).
Comparatively, the TILs in the BT474 spheroids had a much
lower average CD8:CD4 ratio of 0.60. Further work is needed
to investigate how this translates to the clinical scenario,
but the ability to determine the CD8:CD4 ratio in vitro has
potential to be a highly useful predictive tool.
The next experiments were designed to investigate the
effect fibroblasts have on the degree of infiltration into a
tumor. It has been shown previously that fibroblasts can lead
to the formation of treatment-resistant tumors, which in
turn results in poor clinical outcomes.10 Across both PBMC
donors tested, there was a large reduction in infiltration when
fibroblasts were included in the spheroid compared to tumor
cells alone, with a 52% reduction in infiltration of Donor 1
and 55% of Donor 2 T cells with 25K Dynabeads™ per well
(Figure 5C and D). Although the effects of fibroblasts were
similar, the overall infiltration levels were strikingly different
between the two donors, suggesting that immune cell
donor-specific factors are impactful. It was noted that with
both donors, at the top concentration of Dynabeads™,
there was a decrease in the level of infiltration relative to
the second highest density, possibly related to spheroid
disruption during washing. Thus, these data demonstrate
that tumor type and the addition of fibroblasts impacts the
number and profile of TILs.
CAR-T Activation
CAR-T cells are designed to selectively target and kill tumor
cells through interaction with a specific surface antigen while
limiting off-target side effects. To demonstrate this specificity
in vitro, anti-CD19 CAR transduced T cells or donor matched
mock transduced T cells were used in an immune cell killing
assay and functionally profiled at different time points using
the iQue® HTS platform.
On Day 2, 4, and 7, samples were analyzed on the iQue®
platform to assess phenotype and function using the iQue®
Human T Cell Activation Kit, and quantification of IL-2 via
the iQue® Human T Cell Companion Kit. Results show that,
when combined with antigen positive Ramos cells, there was
a rapid upregulation of T cell activation markers CD69 and
CD25 (Figure 6A and B, respectively) on the CD8+ cells.
This upregulation demonstrated some time dependence,
with the highest levels observed on Day 7, but there was
little difference between CAR-T cell densities. Expression
of activation markers was low in co-cultures with antigen
negative Jurkat cells or in the presence of mock transduced
T cells (< 7%). In the presence of Ramos cells, concentrations
of secreted cytokines IFNγ and IL-2 (indicators of activation)
increased at early time points, but then dropped by Day 7,
indicating a transient response. Overall, this quantification
demonstrates a clear antigen specific activation of antiCD19 CAR-T cells as measured by both surface markers and
secreted proteins.
7
Figure 5: BT474 Spheroid Infiltration by CD3+ TILs is Affected by Tumor Type, the Presence of Fibroblasts (NHDF), and Immune Cell Donors
TILs subsets were analyzed using the iQue® Human T Cell Activation Kit. (A) and (B) Graphs show the average number of TILs (CD3+) per iQue® sip per
spheroid type and the associated ratio of CD8:CD4 cells within the TILs population. (C) and (D) Spheroids were formed with either BT474 cells alone or
with 50% BT474s and 50% CCD106SK fibroblasts (NHDF). PBMCs from two different donors and CD3/CD28 Dynabeads™ were added for 40 hours.
Graphs show the relative number of CD3+ TILs at each Dynabead™ density.
Post clinical success of anti-CD19 CAR-T therapies for liquid
tumors, there has been increased interest in applying similar
therapies to solid tumors, for example, in the fight against
breast cancer. An obvious target of interest in this area is
the HER2 (ERBB2) receptor which has been identified to
be over-expressed in many breast cancers. Unfortunately,
in early trials, there were serious adverse events in the clinic
linked to “on target off tumor” effects and further testing was
stopped.11,12 It was indicated that the CAR cells had attacked
other “off tumor” cells throughout the body that expressed
low levels of HER2 epitope and were, therefore, defined as
“on target.” There is additional evidence in the literature that
the affinity of the CAR-T interaction with the HER2 antigen
can also contribute to this effect.13 To model potential “on
target off tumor” effects in vitro, a spheroid co-culture with
anti-HER2 CAR-T cells was used to mimic the immune killing
of a solid tumor.
Infiltrated CD3 Count
800
600
400
200
0
BT474 SKOV-3 A549
A T Cell Infiltration by Tumor Type
CD8:CD4 Ratio
2.0
1.5
1.0
0.5
0.0
BT474 SKOV-3 A549
B Infiltrated CD8:CD4 Ratios
Infiltrated CD3 Count
1000
600
400
200
0
2 3 4
800
5
Log (Dynabead™) Density
BT474
50% BT474 +50% NHDF
C PBMC Donor 1 +/- Fibroblasts
Infiltrated CD3 Count
1000
600
400
200
0
2 3 4
800
5
Log (Dynabead™) Density
D PBMC Donor 2 +/- Fibroblasts
8
Figure 6: Antigen Specific Activation of Anti-CD19 CAR-T
Samples were quantified on Day 2, 4, and 7 for surface marker expression and secreted protein using the iQue® Human T Cell Activation Kit with iQue®
Human T Cell Companion Kit (for IL-2). Graphs (A, B) show expression levels in CD8+ T cells of CD69 or CD25, and graphs (C, D) show levels of IFNγ or
IL-2. Grey bars represent Ramos with mock transduced T cells, black bars are CD19 CAR-T with Ramos cells, and teal bars are CAR-Ts in combination with
Jurkat cells. The 3 bars represent Day 2, 4, and 7, all data shown as mean ± SEM of 3 wells.
The results show an increase in CD69 and CD25 activation
markers on the CD8+ population for both HER2 high
expressing AU565 and HER2 low expressing MDA-MB-231
cell co-cultures with anti-HER2 CAR-T cells (Figure 7A
and B). This effect was absent in the presence of mock
transduced T cell. The HER2 negative MDA-MB-468 cells
showed no change compared to control T cells for CD69 and
low levels for CD25 which decreased by Day 4. Supernatants
were assessed for IFNγ using iQue Qbeads® detection as
part of the kits. Once again both AU565 and MDA-MB-231
CAR-T co-cultures demonstrated high levels while nothing
was detected in the MDA-MB-468 co-culture wells.
These data indicate anti-HER2 CAR-T driven activation of
T cells in co-cultures with high expressing AU565 and low
expressing MDA-MB-231 cells, indicating the potential for
“on target off tumor” effects with these cells. The lack of any
activity in the presence of MDA-MB-468 cells demonstrates
the expected specificity of the anti-HER2 CAR-T cells.
CD69+ CD8+ [%]
25
20
10
5
0
Ctrl T 3:1 CAR-T 3:1 CAR-T 3:1
15
Ramos Jurkat
A
CD25+ CD8+ [%]
80
40
20
0
Ctrl T 3:1 CAR-T 3:1 CAR-T 3:1
60
Ramos Jurkat
B
IFNγ [ng/mL]
3
2
1
0
Ctrl T 3:1 CAR-T 3:1 CAR-T 3:1
Ramos Jurkat
C
IL-2 [ng/mL]
0.6
0.4
0.2
0.0
Ctrl T 3:1 CAR-T 3:1 CAR-T 3:1
Ramos Jurkat
D
9
Figure 7: “On Target Off Tumor” Activation of T Cells in a Solid Tumor Co-Culture Model
Samples were quantified on Day 2, 4, and 7 for surface marker expression and secreted protein using the iQue® Human T Cell Activation Kit. Graphs
(A and B) show expression levels in CD8+ T cells of CD69 or CD25, and graph (C) shows levels of IFNγ for each target cell co-culture with either
non-activated mock transduced T cell or anti-HER2 CAR-T cells. The 3 bars represent Day 2, 4, and 7, all data shown as mean ± SEM of 4 wells.
Conclusion
This application note demonstrates the value of using the
iQue® High-Throughput Screening cytometry platform
in conjunction with the validated iQue® Human T Cell
Activation Kit to assess immune cell phenotype and function
during the development of antibody-mediated and cellbased cancer therapies using minimal sample volume.
The high-throughput iQue® HTS platform combined with
the built-in, visual-based iQue Forecyt® software allows for
assessment of multiparametric data of cell health, viability,
phenotype, and effector function coupled with cytokine
analysis from the same well, using simple workflows and
minimal sample volumes. These functions enable broad
in vitro characterization of therapeutic effects on immune
cell activation and function including the following features.
• Concentration response curves give insight into
concentration-dependent effects of BiTE therapy on
T cell activation and secreted cytokines. In conjunction
with target cell counts, this gives insight into effector
functions against tumor targets. Development of novel
therapeutic antibodies relies on rapid identification and
characterization of candidate molecules as early in the
development process as possible.
• Infiltrated immune cell populations can be analyzed and
compared to their non-infiltrated counterparts. Both
the numbers and phenotypes of TILs can be important
indicators of clinical outcomes and are impacted by many
environmental and physiological factors. The ability to
analyze these in vitro could aid the development of novel
therapeutics.
• Antigen-specific activation by characterization of T cell
surface markers and quantification of secreted proteins
enable insights into tumor antigen specificity of CAR-T
cells. Importantly, potential on-target off-tumor toxic
effects can be exposed in vitro which may bear important
consequences in a clinical setting.
Together, this method offers a rapid, robust, and convenient
solution for characterization of cellular responses, and is
ideal for assisting in the development of new immunological
therapies
0
60
50
40
30
20
10
Control T
CAR-T 2:1
Control T
CAR-T 2:1
Control T
CAR-T 2:1
CD69+ as a % of CD8+
AU565 MDA-MB-231 MDA-MB-468
0
60
50
40
30
20
10
Control T
CAR-T 2:1
Control T
CAR-T 2:1
Control T
CAR-T 2:1
CD25+ as a % of CD8+
AU565 MDA-MB-231 MDA-MB-468
0.0
2.5
2.0
1.5
1.0
0.5
Control T
CAR-T 2:1
Control T
CAR-T 2:1
Control T
CAR-T 2:1
IFNγ [ng/mL]
AU565 MDA-MB-231 MDA-MB-468
A B C
10
References
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doi:10.1038/s41577-020-0306-5
2. Zhang Y, Zhang Z. The history and advances in cancer
immunotherapy: understanding the characteristics of
tumor-infiltrating immune cells and their therapeutic
implications. Cell Mol Immunol. 2020;17(8):807-821.
Doi:10.1038/s41423-020-0488-6
3. Schoenfeld AJ, O'Cearbhaill RE. How Do We Meet
the Challenge of Chimeric Antigen Receptor T-Cell
Therapy for Solid Tumors? Cancer J. 2021;27(2):134-142.
doi:10.1097/PPO.0000000000000516
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Acknowledgement
The authors would like to acknowledge Clare Szybut and
Lauren Kelsey for contributing to this work while employed at
Sartorius.
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