Cytometry Part B (Clinical Cytometry) 00:00–00 (2014)
Original Article
Establishment of a Healthy Human Range for
the Whole Blood “OX40” Assay for the
Detection of Antigen-Specific CD41 T Cells
by Flow Cytometry
Ross Sadler,1 Elizabeth A. L. Bateman,1 Victoria Heath,1 Smita Y. Patel,2
Phillip P. Schwingshackl,1 Alice C. Cullinane,1 Lisa Ayers,1 and Berne L. Ferry1*
1
Department of Immunology, Churchill Hospital, Oxford University Hospitals NHS Trust, Oxford, United Kingdom
2
Department of Clinical Immunology, John Radcliffe Hospital, Oxford University Hospitals NHS Trust,
Oxford, United Kingdom
Background: Clinical investigation of antigen-specific T cells in potentially immunodeficient patients
is an important and often challenging aspect of patient diagnostic work up. Methods for detection of
microbial exposure to the T-cell compartment exist but are laborious and time consuming. Recently, a
whole blood technique involving flow cytometry and detection of CD25 and OX40 (CD134) expression on
the surface of activated CD41 T cells was shown to be accurate and concordant when compared with
more traditional methods of antigen-specific T-cell detection.
Methods: Whole heparinized blood was collected from healthy donors and set up using the “OX40”
assay to detect antigen-specific CD41 T-cell responses to Varicella Zoster Virus, Epstein-Barr Virus
(EBV), Cytomegalovirus, Candida albicans, and Streptococcus pneumoniae.
Results: The “OX40” assay technique was clinically validated for routine use in an NHS clinical immunology laboratory by analysis of incubation length (40–50 h), sample transport time (up to 24 h at room
temperature), concordance with serology testing, proliferation and interferon-gamma production. In addition, 63 healthy controls (age range 21–78) were tested for responses to generate a healthy control reference range.
Conclusions: The OX40 assay, as presented in this report, represents an economical, rapid, robust
whole blood technique to detect antigen-specific T cells, which is suitable for clinical immunology diagC 2014 International Clinical Cytometry Society
nostic laboratory use. V
Key words: flow cytometry; costimulation/costimulatory; diagnostics; CD41 T cells
How to cite this article: Sadler R, Bateman EAL, Heath V, Patel SY, Schwingshackl PP, Cullinane AC, Ayers L,
Ferry BL Establishment of a Healthy Human Range for the Whole Blood “OX40” Assay for the Detection of Antigen-Specific CD41 T Cells by Flow Cytometry. Cytometry Part B 2014;00B: 000–000.
Additional Supporting Information may be found in the online version of this article.
This report is independent research arising from a Healthcare Scientist Research Fellowship. The views expressed in this publication are
those of the authors and not necessarily those of the NHS, the
National Institute of Health Research or the Department of Health.
Ross Sadler contributed to study design, data acquisition (all), and
article writing/editing. Elizabeth Bateman contributed to study design,
data acquisition (OX40 optimization), and article editing. Victoria
Heath contributed to data acquisition (CFSE comparison). Smita Y.
Patel contributed to clinical input regarding XIAP patients and article
editing. Phillip Schwingshackl contributed to data acquisition (OX40
optimization). Alice Cullinane contributed to data acqusition (serologi-
C 2014 International Clinical Cytometry Society
V
cal comparison). Lisa Ayers contributed to article writing/editing.
Berne Ferry contributed to study design and article writing/editing.
Correspondence to: Berne Ferry, Department of Immunology,
Churchill Hospital, OUH Trust, Oxford, OX3 7LJ, United Kingdom.
E-mail: Berne.Ferry@ouh.nhs.uk
Grant sponsor: National Institute for Health Research and the Chief
Scientific Officer.
Received 12 August 2013; Revised 17 December 2013; Accepted
29 January 2014
Published online 00 Month 2014 in Wiley Online Library
(wileyonlinelibrary.com).
DOI: 10.1002/ccc.21165
2
SADLER ET AL.
Detection of previous exposure to a pathogen is an
important aspect of clinical investigation (1). Traditionally
this has been accomplished via two strategies. First, by
detection of the pathogen in the subject, this can be carried out by culturing of patient fluid, or more recently by
molecular techniques such as quantitative polymerase chain
reaction (2). The second approach has been to analyze
components of the immune system for their ability to specifically recognize the pathogen in question, this has most
commonly been achieved in the clinical environment by
serology testing (3) but now includes limited T-cell function
detection methods, for example ELISpot assays, for pathogens such as tuberculosis (4). This second approach has
the advantage of not only informing the clinician whether
the patient has previously been exposed to the pathogen
but also can indicate the degree of specific immune
response the patient is capable of mounting.
Serology has been and will continue to be useful in
measuring antibody responses to pathogens (3). However,
there is an urgent and increasing need for rapid, robust
and accurate clincally validated assays capable of measuring T-cell antigen-specific responses, especially in
patients unable to produce antibody or who are on immunoglobulin replacement therapy. Previous work by Zaunders et al. (5) showed a flow cytometric technique for
detecting antigen-specific CD41 T cells via upregulation of
CD25 and OX40 (CD134) on the surface of the cell after
approximately 48 h incubation with antigen. The “OX40”
assay exhibited concordance with research methods of
antigen-specific T-cell detection such as carboxyfluorescein
succinimidyl ester (CFSE) proliferation studies and intracellular cytokine production (5). Further work showed the
potential of this assay for the detection of latent TB infection in healthy control cohorts and HIV-infected cohorts
and also in detection of Hepatitis C infection (6,7).
The potential use for this assay in assessing T-cell functional responses in routine clinical laboratories is wideranging and significant. Currently, no healthy control ranges
exist for pathogen-specific responses using this assay. In
this study, we have optimized and validated the “OX40”
assay for use in a routine clinical laboratory. This optimized
assay was tested for its robustness and the effect of sample
processing and incubation times with antigen and then
used to generate healthy control ranges for pathogenspecific responses to an array of clinically relevant pathogens. This normal range was clinically validated against two
patients with a characterized cellular immunodeficiency.
METHODS AND MATERIALS
Healthy Controls Subjects and X-Linked Inhibitor
of Apoptosis Patients
For all experiments, including the establishment of normal ranges, healthy controls (n 5 63, median age 5 40
years, range 5 21–78 years, M:F 5 27:36) were consented
according to accepted ethics code 11/SC/0184 awarded
by the South Berkshire research ethics committee. Heparinized blood of 7 ml and 3 ml of blood for serum collection was taken from each healthy control. Two male
patients with X-linked inhibitor of apoptosis (XIAP)
immunodeficiency, leading to recurrent severe EpsteinBarr Virus (EBV) infection were also consented according
to the ethics code previously. Patients 1 and 2 have been
described previously (8). At the time of the study,
Patients 1 and 2 were 34 and 31 years of age respectably.
Both patients were in good health at the time of sampling
and were not suffering from symptomatic signs of EBV
infection. All samples were processed within 6 h of venepuncture for all experiments except for the those used to
investigation the effect of duration of blood storage.
Reagents
Phytohemagluttinin (PHA) was obtained from SigmaAldrich (Dorset, UK) and used at 15 mg/ml, Cytomegalovirus (CMV), Varicella Zoster Virus (VZV), EBV, Vero
lysate, and human foreskin fibroblast (HFF) lysate were
all supplied by Source Bioscience (Nottingham, UK) and
used at 2 mg/ml. Candida albicans (Candida) lysate was
supplied by Oxford Biosystems (Oxford, UK) and used
at 2 mg/ml. Whole ethanol killed Streptococcus pneumoniae used at 1 3 105 cfu/tube was a kind gift from Richard Malley (Boston, Massachusetts).
OX40 Assay
Heparin blood from each subject was diluted 1:1 with
Iscoves modified medium (Sigma-Aldrich) and aliquoted
into 500 ml volumes in sterile capped 5 ml polystyrene
flow cytometry tubes (BD Bioscience, Oxford, UK). To
each relevant sample, 2 mg/ml of antigen or 15 mg/ml of
PHA was added. Each assay was run with two negative
control tubes which were media only and a cell lysate control (either Vero or HFF; Source Bioscience), run at 2 mg/
ml. After incubation at 37 C 5% CO2 for 42–44 h, samples
were vortexed and 100 ml was aliquotted to a new tube.
Blood was then stained with the following monoclonal fluorochromes; CD25–FITC, CD134-PE, CD14-PerCP, Viaprobe
(7-AAD), CD4-APC, and CD3-PE-Cy7 (BD Bioscience). Samples were incubated for 15 minutes at room temperature
before having 1 ml of FACSLyse (BD Bioscience) solution
added for 5 minutes. Samples were washed twice with PBS
before being fixed with 1% paraformaldehyde and run on a
BD FACSCanto II (BD Bioscience). Analysis was carried out
using BD FACSDiva software (version 6.1; BD Biosciences).
Samples were gated to acquire 30,000 CD3 positive
events, which were then negatively gated for CD14 and
7AAD. Quantification of CD31CD41CD251CD1341
events were calculated by setting gate coordinates on
the
media
only
sample
to
equal
0.1%
CD31CD41CD251CD1341 events as a percentage of
all CD31CD41 events. These coordinates were applied
to subsequent antigen tubes to produce a percentage
value of CD251CD1341 double positive events.
CFSE Proliferation Assay
Peripheral blood mononuclear cells (PBMC)s were
purified using a lymphoprep gradient (Axis-Shield, Oslo,
Norway). PBMCs were counted and diluted to 1 3 106/
ml in RPMI (Sigma-Aldrich) before being stained with
Cytometry Part B: Clinical Cytometry
CLINICAL VALIDATION OF THE “OX40” ASSAY FOR ANTIGEN-SPECIFC CD41 T CELLS
3
1 mM/ml of CFSE for 15 minutes at 37 C. After incubation, cells were quenched with 10 ml of ice cold RPMI
(Sigma-Aldrich) before being spun at 350g for 5 min.
This wash step was repeated before adding 200 ml of
cells/well. Various antigens were added to wells at concentrations ranging from 0.2 to 1 mg/ml, cells were then
incubated for 5 days at 37 C 5% CO2. After this time,
cells were washed in 1 ml of PBS at 350g for 5 min
before being incubated for 15 minutes at room temperature with CD3-PE-CY7 and CD4-APC (BD Bioscience).
After a final wash, cells were acquired on a BD FACScanto II and analyzed. Cells were first gated using CD3
and then by forward/side scatter to capture T cells that
showed “blast-like” appearance. Proliferating cells were
measured by a decrease in CFSE fluorescence intensity
compared to unstimulated control.
Concordance was calculated as the percentage of the
true positives and true negatives generated by the assay
compared to the total number of results generated (true
positives 1 true negatives 1 false positives 1 false negatives).
Sensitivity values were calculated as the percentage of the
true positive results generated by the assay when compared
to the total number of true positives 1 false negatives. Specificity values were calculated as the percentage of the true
negatives generated by the assay when compared to the
total number of true negatives 1 false positives.
Serological ELISAs for CMV/EBV/VZV
Initial development of the “OX40” assay (5) used a fourcolor system that gated on lymphocytes using forward and
side scatter, followed by identification of CD31CD41 cells
before measuring CD25 and CD134 expression (Fig. 1Ai).
A concern with this original strategy was that using an initial forward/side scatter gate when examining activated
cells could lead to some cells that showed increased granularity from their activation being overlooked from analysis. This is highlighted in Figure 1Aii with analysis of a
PHA stimulated sample that has been shown with an
overly expanded initial gate to show potentially missed
events. The importance of using an expanded gate is further shown in Figure 1B where the same sample stimulated with CMV lysate was analyzed with a “compact” lymphocyte gate and an “expanded” lymphocyte gate. Figure
1Bii demonstrates CD251CD1341 CMV-specific T cells
were present in the forward/side scatter plot outside the
“compact” lymphocyte gate (Fig. 1Bi), leading to different
CD251CD1341 T-cell percentages (4.6 vs 5.3%).
A concern with using an “expanded” gating strategy
was the potential inclusion of either dying cells that
would be aberrantly upregulating cell markers or CD14
monocytes that possess large amounts of FC receptors
on their surface, leading to nonspecific monoclonal antibody binding. To confront these two potential issues, it
was decided that initial gating with a comprehensive
CD31 gate should be sufficiently inclusive to include all
true T cells followed by an additional negative selection
gate using CD14 and the viability marker 7-AAD to help
minimize nonspecific cell contamination (Fig. 2A).
Detection and quantification of antigen-specific T cells
was measured against viral (VZV, EBV, and CMV), fungal
(Candida), and bacterial (S. pneumoniae) antigens.
These encompassed both pathogens that inhabit perenially and those that are associated with an isolated infection (Fig. 2B). As a further control measure to test the
specificity of the response, individuals were tested
against lysates of two established cell lines used to propagate the VZV and CMV antigen used in the experiments
(Fig. 2B). No individual tested showed a response to
either cell line that was >0.1% above the unstimulated
control, in addition, increasing concentrations of cell
Patient serology to EBV, CMV, VZV, and Candida
[immunuglobulin G (IgG)] were measured according to
manufacturers instructions (Diasorin, Vercelli, Italy).
S. pneumoniae Serotype Testing
The quantification of specific IgG to 12 S. pneumoniae serotypes was performed using a multiplex assay.
Conjugation of pneumococcal polysaccharide to beads
and performing the multiplex assay follows techniques
described previously (9–11). Briefly, serotypes 1, 3, 4, 5,
7F, 9V, 18C, 19A, 6B, 14, 19F, and 23F (Statens Serum
Institut, Copenhagen, Denmark) were conjugated and
coupled onto activated carboxylated beads (Bio-Rad,
Hemel Hempstead, UK). Samples were acquired and analyzed on a Bio-Plex-200 machine using Bio-Plex Manager
6.0 software (Bio-Rad) and the quantity of serotypespecific IgG in lg/ml was determined by comparing the
mean fluorescent intensity (FI) to a Log-5PL standard
curve generated from FI against expected IgG concentration for 89-sf (Food and Drug Administration, MD).
Interferon-Gamma Production From Whole Blood Cultures
Whole blood stimulated by the OX40 assay (as
described previsouly) was removed from the incubator
after 42–44 hrs incubation. 200 ml of cell free supernatant was removed from the cultures and immediately
stored at 280 C until required. Interferon-gamma (IFNg) concentrations were measured by Luminex assay
using a Bio-Plex-200 machine and a human IFN-g kit
supplied by Bio-Rad. Analysis was carried out using BioPlex Manager 6.0 software (Bio-Rad).
Statistics
Statistical analysis was performed using Microsoft Excel
and Prism GraphPad version 5 Software (GraphPad Prism,
San Diego, CA). Reference ranges were generated by calculating the median, 5th and 95th percentile for each
pathogen. For 2 group comparisons, nonparametric
Mann-Whitney tests were carried out. Correlation analysis
was made using a Spearman Rank test. For all analyses,
P < 005 was considered to be statistically significant.
Cytometry Part B: Clinical Cytometry
RESULTS
A CD31 Gate Followed by a Negative Gate to Remove
Dying/Dead Cells and CD141 Cells Is Essential to Capture
All Relevant Cells While Eliminating Contaminants
4
SADLER ET AL.
FIG. 1. (A) Original gating strategy from Zaunder et al. (5) on healthy control blood that has been unstimulated or stimulated with PHA. Initial gating of cells made by gating lymphocyte population using forward and side scatter, PHA stimulated lymphocytes show increased granularity and size
requiring a bigger gate to capture lymphocyte population. Black events within gates are CD31CD41CD251OX401 cells. (B) Comparison of different
sized initial lymphocyte gates using forward/side scatter on collection of CD31CD41CD251OX401 cells. The gating strategy on a healthy control
sample stimulated with CMV was analyzed, first using a compact lymphocyte gate (i) and second using an expanded lymphocyte gate (ii). Black
events within gates are CD31CD41CD251OX401 cells. Percentage CD251OX401 cells as a proportion of all CD31 CD41 cells is marked in the
quadrant. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Cytometry Part B: Clinical Cytometry
CLINICAL VALIDATION OF THE “OX40” ASSAY FOR ANTIGEN-SPECIFC CD41 T CELLS
5
FIG. 2. New proposed gating strategy for the detection of CD14-7AAD-CD41CD251OX401 T cells. (A) A healthy control sample was left unstimulated or stimulated with PHA. Cells were first gated as CD31 before being gated as CD142 and 7AAD2. These cells were then selected according
to CD4 expression and then analyzed for CD25 and OX40 expression. Black cells within gates are CD31CD14-7AAD-CD41CD251OX401 cells. (B)
Representative plots of healthy control whole blood stimulated with various pathogens using the strategy outlined in (A). Percentage CD251OX401
cells as a proportion of all CD31 CD41 cells is marked in the quadrant. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Cytometry Part B: Clinical Cytometry
6
SADLER ET AL.
FIG. 3. Concordance of OX40 assay results with CD41 T-cell proliferation by CFSE staining. Representative plots of both OX40 assay responses
and CFSE proliferation assay responses in the same individual after stimulation with PHA, VZV, EBV, CMV, or Candida. Values given as percentage
CD251OX401 cells in stimulated culture minus value obtained from the same quadrant in the relevant unstimulated control. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
line lysate did not increase the level of CD251CD1341
T cells (n 5 63, data not shown).
OX40 Assay Results Are Concordant With Antigen-Specific
T-Cell Proliferation Assays
To determine the accuracy of the “OX40” assay, parallel assays were setup on the same healthy controls to
measure VZV, EBV, CMV, and Candida status (n 5 15) by
both the OX40 assay and an antigen-specific CD41 Tcell proliferation assay using CFSE. Although this assay is
measuring proliferation rather than activation of CD41
T cells in response to the antigen ie like the OX40 assay,
it was preferred to other techniques such as intracellular
cytokine staining or ELispot since it captures the entire
antigen-specific population rather than one particular
cohort such as IFN-g 1 CD41 T cells for example. There
was 100% concordance between results generated by
the two different assays with all antigens measured, in
all individuals both in either detecting the serologically
confirmed presence or absence of a particular pathogen.
Correlative analysis of levels of proliferating antigenspecific CD41 T cells vs OX401CD251 CD41 T cells
for each individual against the selected pathogens
showed variable results with significant correlations
seen in CMV, EBV, and VZV responses (r 5 0.93, 0.82,
and 0.68, respectively, P < 0.001 for all results, data not
shown) but not candida (r 5 0.04, data not shown). Figures 3A–3E show the representative OX40 and CFSE
proliferation plots of a healthy control that had confirmed VZV1EBV1 CMV2 serology.
The 40–50 h Incubation Is Suitable for Consistant
Measurements
For use in a clinical environment, incubation times
must be pragmatic and reproducible with regard to
results. To assess incubation timing on individual
response variation, time course experiments were performed, measuring PHA and CMV responses in five CMV
positive individuals with stimulation times between 40
and 50 h (Fig. 4). The highest variance seen between a
40-h and a 50-h incubation sample in any one individual
was 8.5 and 0.9% for PHA and CMV, respectively (Figs.
4A and 4B). Variance for all samples between 40 and 50
h incubation in each individual gave a mean coefficient
of variation (CV%) of 9.1 and 12.9% for PHA and CMV,
respectively. These CV% values are within an acceptable
Cytometry Part B: Clinical Cytometry
CLINICAL VALIDATION OF THE “OX40” ASSAY FOR ANTIGEN-SPECIFC CD41 T CELLS
7
FIG. 4. Differences in antigen-specific CD41 T-cell responses depending on incubation time with antigen. Five healthy control whole blood samples were either stimulated with PHA (A) or CMV (B). Samples were stained and processed for detection of CD251OX401 CD41 T cells at 2-h intervals from 40 to 50 h poststimulation. (C) IFN-gamma production from whole blood samples after stimulation with either EBV or CMV. Whole blood
from healthy controls was incubated for 42–44 h with either CMV or EBV lysate. Samples were grouped as either both serologically and OX40 assay
negative or both positive. Correlations between the amount of IFN-gamma released after each stimulation and OX40 assay result generated in that
same individual were also carried out. Identical results (individuals producing 0% OX40 result and 0 pg/ml) are overlaid on the graph.
Cytometry Part B: Clinical Cytometry
8
SADLER ET AL.
Table 1
Concordance, Sensitivity, and Specificity of OX40 Assay Antigen-Specific CD41 T-Cell Responses With Serology in Healthy
Controls
Pathogen
tested
VZV
CMV
EBV
Number
of samples
False Positives/
true Positives
False negatives/
true negatives
Concordance
(%)
Sensitivity (%)/
specificity (%)
OX40 assay cutoff
determined for positivity
51
55
53
0/48
0/24
1/43
1/2
0/31
2/7
98.0
100.0
94.3
98/100
100/100
96/88
0.4%
0.5%
0.2%
CV% for flow cytometric assays on live blood cells indicating that clinical testing using this assay could allow
for some flexibility in the incubation times of samples
without fear of producing significantly different results.
For the remainder of the work in this study, all samples
were incubated for between 42 and 44 h as this suited
the work flow of the clinical laboratory.
Concordance of OX40 Assay Response in Healthy
Individuals With Serological Analysis
To further confirm the validity of the “OX40” assay
for detecting previous antigen exposure, serological
analysis in a group of healthy controls was compared
with their response against the same pathogens on the
“OX40” assay. A cohort of healthy controls was used to
test antigen-specific T-cell responses to VZV, EBV, and
CMV. Table 1 shows the amount of true positives, false
positives, true negatives, false negatives, overall concordance and sensitivity and specificity in the OX40 assay
compared with serology using set cutoff percentage values for positivity within each antigen test, using the
serological result as the gold standard. CMV was most
concordant with 100% concordance to serology, this
included both seronegative and seropositive individuals.
VZV testing produced 1 discordant result, which was
found in a 22 year old female who had contracted
chicken pox at the age of 18 months. While serology for
VZV was negative, the “OX40” assay response was 0.4%.
The median level of VZV response for all healthy controls tested below the age of 25 was 1.3% (n 5 6, data
not shown), indicating that this individual produced a
relatively low VZV-specific response. EBV concordance
was 94.3% with 1 false positive and 2 false negatives.
No specific data regarding the infection of these patients
could give a reason for these discrepancies. Using these
results, OX40 assay cutoffs for positivity could be determined using the lowest recorded responses seen for
each antigen (Table 1). As expected, sensitivity and
specificity values for each antigen test gave similar
results to the concordance values within each antigen,
CMV producing the highest values (100% sensitivity and
specificity) and EBV the lowest (96% sensitivity and 88%
specificity) (Table 1). Serology testing against Candida
and S. pneumoniae was also carried out. Since these
are organisms that inhabit the human body consistently
from a young age, every individual tested produced a
serological response. These data were used for all subsequent OX40 assays in new individuals to determine
pathogen exposure positivity without carrying out
serology.
OX40 Assay Responses to CMV and EBV Correlate With
Production of IFN-c in Whole Blood Samples
To further validate the accuracy of the OX40 assay, a
comparison of IFN-g production was made in CMV1 vs
CMV2 individuals and EBV1 vs EBV2 individuals as
determined by both the OX40 assay and serology. A significant difference in IFN-g production was seen
between both CMV1 vs CMV2 and EBV1 vs EBV2
individuals (Fig. 4C). Correlation analysis of the relative
frequency of OX401CD251 T cells and the amount of
IFN-g produced for each individual showed significant
correlation for both EBV and CMV (CMV r 5 0.97,
P < 0.0001, EBV r 5 0.8, P < 0.0001 Fig. 4C). These data
confirm that OX401 CD251 CD41 T cells can be considered to be activated antigen-specific T cells responding to the specific antigen stimulus.
Establishment of a Healthy Human Range for OX40
Responses to PHA, VZV, EBV, CMV, Candida, and S.
pneumoniae
A total of 63 healthy controls with an age range of
21–78 (median 40 years) were recruited and T-cell antigen specificity to a range of commonly encountered
pathogens determined. Data for each respective pathogen response were only included for individuals that
showed an OX40 response that was higher than the cutoffs as determined by serological analysis and stated in
Table 1. Reference ranges were calculated as the 5th
and 95th percentiles (Fig. 5). The data showed that PHA
stimulation produced the widest normal range (3.8–
54.1%) and EBV the narrowest (0.2–3.9%). Gender difference analysis of all antigen reference ranges showed no
significant differences (data not shown). Correlation
analysis of age with specific antigen responses showed a
weak positive trend for increased CMV responses with
increasing age (r2 5 0.29, P 5 0.006). No other antigen
showed a correlation with age. For these reasons, agespecific reference ranges were not calculated at this
point in time.
OX40 Assay EBV-Specific Responses in Patients With an
XIAP Mutation Are Elevated Compared to the Reference
Range
In order to test the potential clinical utility of having
a healthy control reference range for specific pathogens,
a pathogen-specific response was measured in patients
Cytometry Part B: Clinical Cytometry
CLINICAL VALIDATION OF THE “OX40” ASSAY FOR ANTIGEN-SPECIFC CD41 T CELLS
9
FIG. 5. Establishment of healthy control normal ranges for antigen-specific CD41 T cell responses to common pathogens using the OX40 assay. A
cohort of 63 health controls was used to generate a normal response range. Samples were all incubated for 42–44 h and data was only used for individuals that showed a positive response to the pathogen as determined by serology (Table 1). Median values and normal range high and low cut-off
values, defined as the 5th and 95th percentiles are shown for each plot.
with a diagnosed immunodeficiency. Mutations in XIAP
result in affected individuals suffering from recurrent
EBV infection (12). EBV-specific responses were investigated in two siblings (Patients 1 and 2) with a characterized XIAP mutation (8). The amount of OX401CD251
CD41 T cells generated in both siblings in response to
EBV was dramatically raised (23.3 and 20.6% Fig. 6A)
compared with the reference range (95th percentile for
EBV 5 3.9%) whilst PHA responses and candida
responses were within the reference range generated
(95th percentile for PHA 5 54.1%, 95th percentile for
Candida 5 5.0%; Fig. 6B). Specific comparisons of the
patient data with age/sex-matched individuals data from
the reference range cohort also showed no distinct differences in PHA and candida responses (Fig. 6B). Further confirmation of a large EBV-specific response in
Patient 1 was carried out using a CFSE proliferation
assay again showing a raised response of 38.2% [mean
EBV response by CFSE proliferation in healthy
cohort 5 6.3% (data not shown); Fig. 6A].
Effects of Length of Blood Storage Prior to Assay Setup
To determine the optimum time between venepuncture and “setting up” of the OX40 assay, five CMV and
EBV seropositive healthy controls were analyzed for
their antigen-specific T-cell responses to PHA, CMV and
EBV. Blood was used at 6 h of venepuncture, 24 h postvenepuncture or 48 h postvenepuncture. In all experiments blood was incubated with antigen for 42–44 h.
Analysis of antigen-specific responses showed that all
responses against all antigens (except for one control
against EBV) were above/on the lower limit of the reference range after 24 h (Supporting Information Fig. 1).
Cytometry Part B: Clinical Cytometry
However, the mean CV% produced over the three time
points measured for each individual, in all 3 antigen
stimulations used, was increased compared to typical
interassay variation for flow cytometric data, with EBV
responses showing the highest variance (PHA 5 33.5
CV%, CMV 5 34.3 CV%, and EBV 5 84.2 CV%; Supporting
Information Fig. 1). This could also in part be due to
EBV responses showing the smallest numerical values
which can skew CVs to higher values. One control who
showed a consistently low PHA response during the
generation of the PHA normal range was also tested and
this proved to be consistently low, staying below the
normal range cutoff (Supporting Information Fig. 1).
These results suggest that samples can be left for up to
24 h at room temperature before assay set up if assessing antigen specificity only but not for accurate quantification of antigen-specific response.
DISCUSSION
Clinical investigations into the presence and extent of
antigen-specific T-cell immunity are becoming more
important and the need for antigen-specific functional
CD4 T-cell assays that can be performed in routine diagnostic clinical laboratories is clear (1). In this report, the
OX40 antigen-specific functional CD41 T-cell assay was
clinically validated for use in a routine clinical immunology laboratory and the validated assay was used to establish a healthy control normal response range for antigenspecific T-cell responses to five common pathogens.
OX40 is a member of the TNF superfamily and is considered a later costimulatory marker, important in the
activation and survival of T cells as they under go differentiation from na€ıve to effector memory status from 1
FIG. 6. Investigations into EBV-specific CD41 T cell responses in patients with XIAP defects. (A) Whole blood from two patients with a characterized XIAP defect was stimulated with either PHA, EBV, or Candida for 42–44 h before being run on the OX40 assay. Patient 1 was also tested for
CD41 proliferation in response to PHA or EBV. (B) Comparison of % CD251CD1341 CD41T cells generated in response to PHA, EBV or Candida
in XIAP patients and in age/sex-matched healthy controls. Dotted line indicates the 95th percentile value from the reference range generated. [Color
figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
CLINICAL VALIDATION OF THE “OX40” ASSAY FOR ANTIGEN-SPECIFC CD41 T CELLS
to 2 days after antigen recognition (13). There is a concern that this assay could be detecting na€ıve CD41 T
cells in individuals that are specific for the stimulating
antigen rather than pre-existing CD41 T-cell memory.
However, this is unlikely for two reasons. First, strong
concordance data with serology from this report support the idea that only memory cells are recognising
antigen and reacting with upregulation of OX40. Second, incubation of CMV seronegative individuals blood
with CMV antigen does not induce OX40 upregulation
for at least 8 days (R. Sadler, unpublished observations),
suggesting that T-cell antigen-specific responses
described in this report were due to pre-existing effector memory T cells. Another potential issue is that the
assay quantifies proliferated antigen-specific T cells—
exaggerating the true memory compartment of the individual to that antigen. Previous work reported that proliferation of memory T cells began 48 h post second
antigen exposure (5), indicating that incubations of
approximately 48 h or below were suitable for detecting
baseline CD41 T-cell memory levels.
Additional optimization and clinical validation of the
OX40 assay in the study has shown that a different gating strategy increases confidence in the results generated by the assay. This is particularly important since
the level of detection of this assay appeared to be
60.5% in some cases ((5) and this study). Comparisons
with a CFSE proliferation assay, an already established Tcell function assay used in clinical immunology laboratories, showed 100% qualitative concordance between the
assays in all antigens measured but did not show quantitative correlation when candida responses were analyzed. This was the only antigen measured that did not
quantitatively correlate between the two assays and
interestingly it was also the only pathogen that can be
considered a commensal of humans. This universal
exposure of the body to candida could explain the differences seen in proliferation and activation of exposed
CD41 T cells. Crucially, for the purposes of logistics of
sample processing in the laboratory, studies show that
an antigen incubation window can be used between 40
and 50 h, allowing some flexibility when performing the
assay. Sample transportation analysis revealed that by
delaying assay setup, an accurate qualitative result of
exposure to pathogen could still be obtained as long as
samples were processed within 24 h. However, quantifiable levels of antigen-specific response were not concordant between stored blood and blood that was <6 h
old, indicating that comparison with this healthy control
range can only be made when blood is processed within
6 h of venepuncture. In addition, only CMV and EBV
responses were analyzed and investigations into the
other pathogens chosen here should be carried out.
Antigen-specific T-cell responses are currently investigated in a number of ways. MHC-tetramer staining is
considered the gold standard for detecting antigenspecific T cells. This technique is predominantly used in
CD81 T-cell work and while highly specific and informative, paradoxically the MHC restrictive nature of this
Cytometry Part B: Clinical Cytometry
11
technique severely limits who can be measured. In addition, the complex labor required and high consumable
costs, together with the paucity of available MHC tetramers makes it difficult to use the technique in routine
NHS laboratories. Measurement by antigen-specific CFSE
or ELIspot are both possible but require PBMC sorting
and extensive optimization to become interpretable.
The OX40 assay is a whole blood assay with a minimal
setup time before incubation, it allows for both qualitative and quantitative analysis of antigen-specific T cells.
Qualitative analysis can be useful in patients with dysgammaglobulinemia or who are on IVIG and therefore
are not able to be measured by more established serological methods. Investigations into EBV-specific CD41
T-cell responses in two patients with a mutation in XIAP
revealed grossly abnormal levels of antigen-specific
OX401CD251CD41 T cells. These raised levels could
be a result of the T-cell compartment having to dedicate
greater levels of resource to a particular pathogen to
compensate for a functional failing in another component of the immune system such as the NK cells or
CD81 T cells. These data support the notion that quantifying levels of antigen-specific CD41 T cells using the
OX40 assay against particular pathogens can be useful in
investigating patients with suspected immunodeficiency.
The OX40 assay can only be used to detect antigenspecific CD41 T-cell responses, not CD81 T cells. Analysis of CD81 T cells does not show the same kinetics
or accuracy with regards to CD25 and CD134 expression as CD41 T cells (R. Sadler unpublished observations). Further work needs to be carried out to identify
a similar technique to the OX40 assay that can work for
CD81 T cells. Previous studies phenotypically analysing
vaccinia tetramer positive CD8 T cells in vaccinated individuals reveal many candidate markers such as CD38
and HLA-DR (14,15).
In summary, this study shows clinical validation and
establishment of a healthy human range for the detection of antigen-specific CD41 T-cell responses to a number of common pathogens using the OX40 assay. This
allows quick, cheap, whole blood analysis of patient Tcell immunity to aid in immune profiling of patients.
ACKNOWLEDGMENTS
The authors would like to thank Richard Malley,
Department of Epidemiology and Department of Immunology & Infectious Diseases, Harvard School of Public
Health, Boston, Massachusetts, for the gift of whole
ethanol killed Streptococcus pneumoniae. The authors
would also like to acknowledge the staff at the Department of Immunology, Churchill Hospital, Oxford, UK.
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