ASTARTE IN ACTION

Using a Recall Antigen

Assay as a Tool for
Understanding Immunity

CASE STUDY

Getting on with Discovery

Table of Contents
3 Introduction

3 Methods

3 Results: The Rate of Cytokine Production

Varies Based on Antigen and Time
Several cytokines were measured using the recall antigen assay and optimal days
in culture were analyzed for each.

4 Results: Limitations in Cell Proliferation

Measurement
Cell proliferation was measured using both the ATP and BrdU uptake methods,
which yielded different results.

5 Results: Recall Response is Highly

Reproducible, But Some Variation is Expected
Ge
Cytokine production varied, even when the lot of antigen, culture medium, and
other conditions were kept constant.

6 Results: Recall Antigen Assay Results Are

Consistent Over Time
Longitudinal data from the recall antigen assay yielded fairly consistent results
over time.

7 Results: The Recall Antigen Assay Can Be

Manipulated to Increase or Decrease the
Immune Response
The assay can be used as a model for designing cancer immunotherapies and
vaccines or testing autoimmune disease and allergy drug candidates.

7 Conclusion

2
Introduction
While there is no industry-accepted
protocol for measuring the functionality
of peripheral blood mononuclear cells
(PBMC), it’s an important test that should
be conducted for quality control.
We needed a reliable, reproducible way to measure
the functionality of our cryopreserved PBMC, so
we developed a custom assay using recall antigens
to understand the in vitro activity of our cells. By
testing PBMC for immune response to several
different antigens over time, we intended to better
understand an acceptable range of variation in
recall response from PBMC samples from the same
donor over time.
The recall antigen testing assay needed to
effectively evaluate the quality of important
immune cell types, including T cells, B cells, and
monocytes. We strategically designed the assay to
use several antigens:
• Phytohaemagglutinin (PHA): As a positive
control
• Lipopolysaccharide (LPS): To stimulate B cells
and monocytes
• Cytomegalovirus (CMV): As a natural, chronic
infection for a subset of the CMV-infected
population
• Tetanus Toxoid: Due to its widespread use in
vaccinations
Methods
1. Cryopreserved PBMC were thawed in a 37°C
water bath and the concentration adjusted to
2.5 x 106 per mL in X-VIVO™ 15 medium.
2. Cells were added to a round bottom, 96 well
plate at 100 uL per well.
3. Dilutions of antigens and mitogens were
prepared in X-VIVO 15 medium. Tetanus toxoid
and Cytomegalovirus (CMV) antigens were
diluted to 2 ug/mL. Lipopolysaccharide (LPS)
was diluted to 200 ng/mL. Phytohemaglutinin
(PHA) was titrated to determine the optimal
concentration, which we found to be 1 ug/mL.
4. Antigens were added to the well plate at
100 uL per well in triplicate. 100 uL of medium
was added to three wells as a control.
5. Samples were incubated at 37°C, 6% CO2 for
four days. Note: Day four is not the optimal time
to measure cytokine concentrations for all of the
antigens and mitogens in this assay. Day four
was chosen to maximize the chance of seeing a
response to all four antigens.
6. 150 uL of medium was removed from each
well and used for cytokine analysis.
Results
The Rate of Cytokine Production
Varies Based on Antigen and Time
Cytokine analysis can show us how immune cells
react to a stimulus or change in the immune
system and is a great measure of cell quality. To
assess the overall quality of PBMC samples using
the antigen recall assay, we measured production
of 10 different cytokines over time in response to
CMV and tetanus toxoid.
We found that IFNγ had the most significant
increase over background production in response
to both antigens used. The other cytokine that was
increased for both recall antigens was TNFα.
We chose to track these two cytokines routinely.
While IFNγ is sufficient to detect responses to
CMV and tetanus, TNFα was a logical choice for
monitoring the stimulation of monocytes by LPS.
TNF is detectable within 4 hours of LPS stimulation,
but with antigen stimulation, TNF did not increase
until day 3.
3
 This difference between the rate of cytokine to collect media for cytokine analysis after 4 days,
production means it is not possible to measure which gave some margin for detection of positive
cytokine at the optimal time for T cell antigens responses to the antigens.
and the LPS and PHA mitogens. Instead, we chose

Cytokine Production Over Time

IL-
IFNg IL-10 IL-13 IL-1b IL-2 IL-4 IL-6 IL-8 TNFa
12p70
Day 1 399.5 5.2 ND 20.9 3.9 33.0 ND 16.6 41,606.8 70.4
CMV Day 2 2,515.3 4.6 ND 47.7 2.5 40.3 ND 31.2 20,221.3 52.6
Day 3 6,624.3 5.4 ND 23.9 4.2 6.4 ND 8.9 50,166.9 168.7

Day 1 34.4 24.5 1.0 26.3 7.3 81.8 ND 156.5 73,880.4 65.0
Tetanus Day 2 518.8 7.9 ND 118.7 12.6 78.5 ND 291.3 131,109.0 50.1
Day 3 6,273.8 10.4 ND 126.0 9.1 126.6 ND 159.2 142,611.6 133.6

Figure 1. Note: ND = Not Detectable

Limitations in Cell Proliferation We measured cell proliferation using both the ATP
Measurement and BrdU uptake methods, which yielded different
results. Figure 2 shows that cell proliferation was
When using CellTiter-Glo®, a reagent based on higher for both donor samples used in the recall
adenosine triphosphate (ATP) bioluminescence assay when stimulated with PHA than for any other
content, increased cell metabolism and higher ATP antigen. Figure 3 tells a different story with more
per cell is interpreted as proliferation. However, variable proliferation measurements depending
increased cell metabolism can also increase ATP on donor and antigen. Lot number 1406 had
per cell, which may complicate interpretation. proliferation above control for all 4 stimuli (tetanus,
PHA, LPS and CMV) with the highest proliferation
Another method for measuring proliferation is the observed in response to CMV.
uptake of bromodeoxyuridine (BrdU), which labels
cells replicating their DNA.

Proliferation Measured by ATP Proliferation Measured by BrdU Uptake

40,000 1.600
Relative Luminescence Units

35,000 1.400
Absorbance at 450 nm

30,000 1.200
25,000 1.000
20,000 0.800
15,000 0.600
10,000 0.400
5,000 0.200
0 0.000
Control Tetanus PHA LPS CMV Control Tetanus PHA LPS CMV
1406JN12 1559JY12 1406JN12 1559JY12

Figure 2. PBMC proliferation measured by ATP content Figure 3. PBMC proliferation measured by uptake of
using CellTiter-Glo®. bromodeoxyuridine.

4
When measuring proliferation, note that both Vial-to-Vial Reproducibility
methods have their limitations. We found the ATP
800
method had a poor signal to noise ratio — the
700
increase in ATP following antigen stimulation was
not significantly higher than the relatively large 600

amount of ATP in the absence of stimulation — 500

pg/mL IFNγ
and the BrdU uptake incorporation assay to be 400
cumbersome. 300
200
These two methods can produce vastly different 100
results, especially with immune cell populations 0
1 2 3 4 5
in which a small subset may proliferate with the
Experiment Number
majority simply maintaining their metabolism. With
these limitations in mind, we decided to use the Figure 4. The response to tetanus toxoid was measured
cytokine production (see Figure 1) as the optimal using the same lot of PBMC and the same lot of antigen
measure of primary immune cell response. on five separate occasions. Each experiment reported an
immune response to tetanus toxoid, but IFNγ production
varied from less than 100 pg/mL to 700 pg/mL.

Recall Response is Highly Comparison of CMV Antigen Lots

Reproducible, But Some Variation
is Expected
4000
3500
3000
Repeated testing of a reference lot of PBMC using
pg/mL TNFα

2500
the recall antigen assay resulted in varying levels of
cytokine production, even when the lot of antigen, 2000

culture medium, and other conditions were kept 1500

constant. 1000
500
Due to this variability, the recall antigen assay 0
0.3 1 3
should not be used to compare separate runs or
µg/mL Antigen
experiments. Although recall patterns seem to be
Lot #1 Lot #2
stable across experiments, the variation makes it
difficult to compare results quantitatively. Figure 5. The recall antigen assay was used to measure
cytokine production of the PBMC samples using two
different lots of CMV at varying concentrations. This graph
However, comparisons of results within the same
shows lot-to-lot variability for CMV.
run or experiment are acceptable. We have used
this recall antigen assay to evaluate different lots of
antigen with high reproducibility.

See Figures 4 and 5 for results from vial-to-vial

reproducibility testing using tetanus toxoid and a
comparison of CMV antigen lots.

5
 Recall Antigen Assay Results Are produced little to no response to TNFα when
Consistent Over Time stimulated with tetanus toxoid. The low response
does not indicate that donor 290 is unresponsive
An examination of the longitudinal data from to tetanus toxoid because there was IFNγ found in
running the recall antigen assay consistently on the sample.
multiple donors revealed that the results of the
assay are fairly consistent over time. Figures 6 and
7 are just two examples of our longitudinal data, Recall Antigen Testing: Donor 290
although we have examined the assay on other 3,000
donors with similar results and continue to run 2,500

pg/mL TNFα
these longitudinal studies. 2,000
1,500

PBMC from donor 369 produced IFNγ when 1,000

stimulated with either CMV or tetanus toxoid. 500

0
The responses have been relatively steady since Feb 2018 Sep 2017 May 2017 Jan 2017
May 2017. A pipetting error may have caused the Sample Collection Date
large standard deviation observed in July 2017.
CMV Tetanus Toxoid
PBMC collected in February 2017 had much lower
cytokine output, which raises questions as to what Figure 7. PBMC from donor 290 were tested in the recall
antigen assay over a year. This graph depicts the TNFα
may have affected the cells at that time.
production observed. IFNγ production in response to CMV
Recall Antigen Testing: Donor 369 was off scale in every batch.
25,000

20,000 We have noticed that some donors produce TNFα

pg/mL IFNγ

15,000 in response to tetanus toxoid or CMV while others

10,000
do not. There is also a tendency for donors to
5,000
produce higher (>4,000 pg/mL) or lower (<2,000
0
May 2018 Mar 2018 Jan 2018 Nov 2017 Jul 2017 May 2017 Feb 2017
pg/mL) TNFα following stimulation with LPS, a
Sample Collection Date substance known to activate monocytes.
CMV Tetanus Toxoid
TNFα levels do not necessarily relate to the
Figure 6. PBMC from donor 369 were evaluated in the
recall antigen assay over a year. The response to tetanus percentage of monocytes present; it seems to be a
toxoid had greater fluctuation than the response to CMV donor-specific characteristic. We are continuing to
antigen. monitor this characteristic to determine if we can
relate it to other donor characteristics.
Looking at the recall responses over time for
another donor, we found that PBMC from donor
290 were positive for CMV response as measured
by TNFα production. However, donor 290

6
 The Recall Antigen Assay Can
Be Manipulated to Increase or
Decrease the Immune Response
There is great excitement surrounding the progress
of targeted therapies that use the immune system
to attack cancer cells. The activity of one such drug,
Nivolumab, can be demonstrated in our recall
antigen assay by measuring the immune response
to CMV antigen.
Nivolumab works by intensifying the immune
response to cancer. Figure 8 shows that the
cultures in our assay containing Nivolumab had a
greater response to CMV antigen than the control
cultures.
Similar approaches can also be tested using this
assay to monitor the impact of drug candidates on
the in vitro immune response. Whether you want to
boost the response as in cancer or vaccine design
or blunt the immune attacks as in autoimmune
disease or allergy drug candidates, this assay can
be used as a model.
Conclusion
The assay we developed is a useful
measure of immune cell quality. Our
customers have found the data from the
assay helpful in reviewing cell quality prior
to purchase to select lots that will work
well in their assays.
While we did not set out to measure donors
longitudinally or compare cytokine profiles
between donors, this data has been beneficial
in understanding the variability between
donors and in a single donor over time. Our
recall antigen assay is also used as a model for
monitoring the effects of test compounds on
immune cell activation.

Donor 349

4500
4000
3500
3000
pg/mL IFNγ

2500
2000
1500
1000
500
0
0 0.1 0.3 1 10
µg/mL Antibody
Nivolumab IgG4
Figure 8. The response of donor 349 to CMV antigens was
measured when Nivolumab (Opdivo®) was added to the
culture. IgG4 was used as a negative control for Nivolumab.
The secretion of IFNγ was increased 100% or more by this
checkpoint inhibitor.

7
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