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 developed a custom assay using recall antigens to understand the in vitro activity of our cells. Access the full case study here to learn about our process: https://astartebio.com/resources/case-studies/using-recall-antigen-assay-tool-understanding-immunity/

1. CASE STUDY
ASTARTE IN ACTION
Using a Recall Antigen
Assay as a Tool for
Understanding Immunity

2. 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.
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.
(See Figures 1 and 2)
6. 150 uL of medium was removed from each
well and used for cytokine analysis.
900
800
700
600
500
400
300
200
100
0
Kinetics of IFNγ Production
Tetanus Toxoid LPS
pg/mLIFNγ
0 1 2 3 4 5
Days in Culture
Figure 1. IFNγ concentrations for samples containing
tetanus toxoid and CMV were measured on culture day
one, two, three, and four. Day four may or may not have
been peak concentration.
7000
6000
5000
4000
3000
2000
1000
0
Kinetics of TNFα Production
PHA LPS
pg/mLTNFα
0 1 2 3 4 5
Days in Culture
Figure 2. Peak cytokine concentration for samples
containing PHA was observed on culture day three
using TNFα. Peak cytokine concentration for samples
containing LPS was observed on culture day one
using TNFα.

3. 3
Results
Recall Response is Highly
Reproducible, But Some
Variation is Expected
Repeated testing of a reference lot of PBMC using
the recall antigen assay resulted in varying levels
of cytokine production, even when the lot of
antigen, culture medium, and other conditions
were kept constant. (See Figure 3)
Due to this variability, the recall antigen assay
should not be used to compare separate runs or
experiments. Although recall patterns seem to be
stable across experiments, the variation makes it
difficult to compare results quantitatively.
However, comparisons of results within the same
run or experiment are acceptable. We have used
this recall antigen assay to evaluate different lots
of antigen with high reproducibility. (See Figure 4)
800
700
600
500
400
300
200
100
0
Vial-to-Vial Reproducibility
pg/mLIFNγ
3
Experiment Number
4 521
Figure 3. The response to tetanus toxoid was measured
using the same lot of PBMC and the same lot of antigen
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.
4000
3500
3000
2500
2000
1500
1000
500
0
Comparison of CMV Antigen Lots
pg/mLTNFα
3
µg/mL Antigen
10.3
Lot #2Lot #1
Figure 4. 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
shows lot-to-lot variability for CMV.
Donor Response to Recall
Antigens Varies Over Time
The degree of recall response can vary over time as
measured by our recall antigen assay, even for the
same donor. Be cautious of using the same donor
for multiple experiments, as even samples from the
same donor can vary depending on factors such as
time of collection. Our samples were all collected at
approximately the same hour of the day, but other
variables can impact the immune response. More
research is needed to understand these variables.
As shown in Figure 5, samples from donor 287
collected at different times over a 20-month period
had a positive response to both tetanus toxoid and
CMV, but the degree of response varied over time
even as all other variables were kept constant.

4. 4
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
Donor 287pg/mLIFNγ
Tetanus Toxoid CMV
Jan-16 Aug-16 Dec-16 Jun-17
Figure 5. The response to tetanus toxoid and CMV
antigens were measured using the PBMC samples taken
from the same donor at different times over a 20-month
period. Except for a lower response in January 2016,
donor 287 maintained a robust response to tetanus
toxoid and CMV.
Our experiments have shown that tetanus
immunizations have a lasting impact. Most of
our donors have a positive response to tetanus
toxoid (defined as 100 pg/mL or more), although
the degree of response has a wide range of up to
10,000 pg/mL. This range could be attributed to
the recency of a booster vaccination.
Compared to a one-time tetanus immunization,
the responses to CMV, a chronic infection, were
much less variable for donor 287. Because CMV-
positive donors have the virus for life, their T cells
are more likely to be exhausted by the constant
battle to keep the virus in check. This may explain
the relative ease of demonstrating the effect of a
PD-1 antibody on the in vitro immune response
to CMV, and is also a model for response to
cancer antigens, which are chronically present
and likely to drive T cells to exhaustion.
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 6 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.
µg/mL Antigen
IgG4Nivolumab
4500
4000
3500
3000
2500
2000
1500
1000
500
0
Donor 349
pg/mLIFNγ
0.3 1 100.10
Figure 6. 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.

5. 5
Conclusion
The assay we developed is a reliable
measure of immune responses, but
some variation is expected. When using
our recall antigen assay to test for
functionality and immunity, always use a
variety of donors and test multiple times
to ensure your results are dependable.
Our recall antigen assay has been useful in
demonstrating the functionality of PBMC and
has also been used as a model for monitoring
the effects of test compounds on immune
cell activation.
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