Journal of Immunological Methods 438 (2016) 42–50

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Journal of Immunological Methods

journal homepage: www.elsevier.com/locate/jim

Research paper

A rapid alternative method to evaluate T-cell hybridoma activation using

an improved cytokine (IL-2) secretion assay
Gastelum-Aviña Paola a,c, Lares-Villa Fernando a, Espitia Clara b, Valenzuela Olivia c, Robles-Zepeda Ramon c,
Velazquez Carlos c,⁎, Garibay-Escobar Adriana c,⁎
a
Departamento de Ciencias Agronómicas y Veterinarias, Instituto Tecnológico de Sonora, 5 de febrero 818 sur., Cd. Obregón, Sonora 85000, Mexico
b
Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, Mexico D.F. 04510, Mexico
c
Departamento de Ciencias Químico-Biológicas, Universidad de Sonora, Blvd. Luis Encinas y Rosales s/n, Hermosillo, Sonora 83000, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: T-cell hybridoma assays have been widely used for the in vitro study of antigen processing and presentation be-
Received 7 July 2016 cause they represent an unlimited source of cells and they bypass the difficulty of maintaining T-cell clones in cul-
Received in revised form 26 August 2016 ture. One of the most widely used methods to assess hybridoma activation is measurement of CTLL-2 cell
Accepted 29 August 2016
proliferation, which is dependent on IL-2. However, continuous culture of this cell line results in a loss of sensi-
Available online 31 August 2016
tivity, and significant interassay variability can occur. Therefore, our goal was to develop a method to assess T-
Keywords:
cell hybridoma activation that was fast and sensitive with low variability based on the IL-2 secretion assay. The
T-cell hybridoma assay used flow cytometry detection and employed the hen egg lysozyme (HEL)-specific 3A9 hybridoma as a
IL-2 model. The original murine IL-2 secretion assay protocol from Miltenyi Biotec® was tested and modified; the
Cytokine secretion assay conjugated capture antibody (anti-CD45-anti-IL-2) was added together with the stimulus at the beginning of
CTLL-2 the antigen presentation assay instead of after antigenic stimulation. With this modification, the percentage of
FACS detectable CD4+ IL-2+ cells following HEL stimulation rose from 4.5% with the original protocol (0.8% without
HEL stimulus) to 94.1% (0.8% without stimulus) with the newly proposed method under the conditions evaluated
in this study. This modification allowed us to evaluate the activation of hybridomas directly and more rapidly
(~ 18 h) than the reference method that assayed CTLL-2 cell proliferation using the MTT reduction assay
(~48 h). In conclusion, the proposed method offered a rapid alternative for screening T-cell hybridomas and eval-
uating their antigen-specific activation.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction continuously maintain the cytokine-dependent cells in culture (Gieni

IL-2 is one of the most widely used cytokines to study the activity or
isolate antigen-specific T-cell lymphocytes (TcL) and T-cell hybridomas.
IL-2 bioassays involve two steps: in vitro cell activation and the direct or
indirect evaluation of the cytokine in the culture supernatant (Surman
and Hurwitz, 2002).
Among the currently available methods to measure cytokine pro-
duction, only biological assays provide a direct estimate of biologically
active cytokines in the culture supernatants. Bioassays that employ cy-
tokine-dependent cell lines are simple and provide accurate data [e.g.,
assays that measure the proliferation of IL-2-dependent cell lines
(CTLL-2) in the presence of cell culture supernatants] (Mire-Sluis et
al., 1995). However, there are inherent complications associated with
bioassays, such as problems with specificity and the need to
⁎ Corresponding authors.
E-mail addresses: velaz@guayacan.uson.mx (C. Velazquez),
agaribay@guayacan.uson.mx (A. Garibay-Escobar).
et al., 1995). Although proliferation assays with these cell lines are high-
ly sensitive, they show a high degree of inter- and intraassay variability
even when they are performed in the same laboratory by the same per-
son at different times. The origin of this variation can probably be traced
to differences in the initial cell count, cell culture media conditions or
cell culture itself (Saade et al., 2012).
Moreover, there are a variety of techniques for the direct measure-
ment of cytokines that involve either intracellular detection or quantifi-
cation of the total amount of cytokines present in the culture
supernatant. Although the conventional enzyme-linked immunosor-
bent assay (ELISA) is the standard method, only a single cytokine can
be evaluated per assay, and large sample volumes are required. Other
methods are available to measure several cytokines with smaller sam-
ple volumes using flow cytometry [e.g., antibody-conjugated beads (cy-
tometric bead array)]. However, similar to the ELISA these methods
cannot evaluate cytokine production per cell (Saade et al., 2012). In con-
trast, intracellular cytokine staining (ICS) is an alternative method that
has the advantage of simultaneously determining antigen-specific TcL

http://dx.doi.org/10.1016/j.jim.2016.08.011
0022-1759/© 2016 Elsevier B.V. All rights reserved.
 P. Gastelum-Aviña et al. / Journal of Immunological Methods 438 (2016) 42–50
functions and phenotypes; thus, this method is widely used to measure
antigenicity during the evaluation of the efficacy of vaccines and for TcL
epitope mapping (Saade et al., 2012; Brosterhus et al., 1999; Freer,
2014).
One recently developed technique for the individual identification of
antigen-specific TcLs is the cytokine secretion assay (CSA). In this test,
an artificial affinity matrix is created on the TcL surface with antibodies
specific for the cytokine being assessed; then, the cells are allowed to se-
crete the cytokine for a defined period of time under well-defined con-
ditions into a medium with low permeability for the secreted product.
The cytokine is captured on the cell surface and detected with specific
antibodies for cytometric analysis (Manz et al., 1995). This method
also allows the investigator to enrich the population or to sort the
cells of interest using magnetic beads that bind to the detection anti-
body. The great advantage of this technique is that it does not require
cell permeabilization or halting vesicular traffic as with the intracellular
staining techniques. Moreover, the detected cells remain viable for fur-
ther studies (Saade et al., 2012).
Therefore, in this study we focused on developing a method that
would allow us to evaluate the activation of antigen-specific T-cell hy-
bridomas through IL-2 secretion with low inter-assay variability and
faster cytokine detection. To accomplish this goal, we modified the orig-
inal IL-2 secretion and flow cytometry detection protocol from Miltenyi
Biotec® by adding the capture antibody at the beginning of the in vitro
antigen presentation assay. As a model, we used the 3A9 hybridoma
specific for hen egg lysozyme (HEL). The aim of this study was to com-
pare the sensitivity of the CTLL-2 cell proliferation assay (which we used
as reference method) with the modified flow cytometry protocol.
2. Materials and methods
2.1. Cell lines and antigens
The murine B-cell lymphoma line M12.C3.F6 (M12-Ak) expressing
MHC class II (I-Ak haplotype) (Velazquez et al., 2002), the 3A9 T-cell hy-
bridoma specific for the 48-61 epitope of HEL (Sigma-Aldrich, St. Louis,
MO, USA) and the murine cytotoxic T-cell line (IL-2-dependent) CTLL-2
were kindly provided by Dr. Emil R. Unanue (Department of Pathology
and Immunology, Washington University, St. Louis, MO, USA). The
3C2.D4 T-cell hybridoma specific for the PE_PGRS33 recombinant pro-
tein of Mycobacterium tuberculosis was generated by immunizing C3H/
HeJ mice with recombinant protein in the hind footpad; the hybridoma
was selected based on antigen sensitivity and subcloned by limiting di-
lution. The PE_PGRS33 recombinant protein was kindly provided by Dr.
Clara I. Espitia-Pinzón of the Department of Immunology, Biomedical
Research Institute of National Autonomous University of Mexico
(UNAM), and was used as an irrelevant antigen. The T-cell hybridomas
and M12.C3.F6 cells were cultured in Dulbecco's modified Eagle's medi-
um (DMEM) (Sigma, St. Louis, MO, USA) supplemented with 5% heat-
inactivated fetal bovine serum (FBS) (Gibco, Life Technologies, Carlsbad,
CA, USA) (D5F). The CTLL-2 cells were cultured in DMEM (Gibco, Life
Technologies, Carlsbad, CA, USA) supplemented with 10% heat-
inactivated FBS (D10F). All cell lines were incubated at 37 °C with 5%
CO2 and 95% humidity.
2.2. CTLL-2 culture
The CTLL-2 cell line was maintained with recombinant IL-2 (Sigma-
Aldrich, St. Louis, MO, USA) at a final concentration of 1 ng/mL or P815
cell culture supernatant (transfected with the murine IL-2 gene and a
G418 resistance marker) at a final concentration of 80 U/mL (kindly
provided by Dr. Emil R. Unanue). Every three days, the cells were
washed 5 times with DMEM and adjusted to a final concentration of
1.5 × 103 cells/mL in a total volume of 10 mL in a T25 flask or
8 × 105 cells/mL for the evaluation of T-cell hybridoma activation.
43
2.3. T-cell hybridoma assays
A total of 5 × 104 M12.C3.F6 cells (50 μL) as the antigen presenting
cell (APC), 105 T-cell hybridomas (100 μL) and a specific antigen [the
HEL (1, 0.1, 0.01, 0.001, 0 μM) or PE_PGRS33 protein (1 μM)] were incu-
bated in a 96-well tissue culture plate in a total volume of 200 μL/well.
After 16–20 h of incubation at 37 °C with 5% CO2 (triplicate wells)
(Astiazaran-Garcia et al., 2009), IL-2 production by the T-cell hybrid-
omas was evaluated by FACS and the CTLL-2 proliferation assay.
2.4. CTLL-2 proliferation assay
After 20 h of incubation in the T-cell hybridoma assay, 100 μL of cul-
ture supernatant was collected from each well of the 96-well tissue cul-
ture plate and transferred to another plate. The supernatants were
frozen at −80 °C and thawed at room temperature (RT). Fifty μL/well
of the CTLL-2 suspension (8 × 105 cells/mL) was added to the thawed
supernatant and incubated for 20 h. During the last 4 h of the cell cul-
ture, 15 μL of 5 mg/mL MTT solution (3-(4,5-dimethylthiazol-2-yl)-
2,5-dimethyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA)
was added to the wells. Viable cells reduce MTT to formazan; the crys-
tals formed were dissolved with 150 μL/well of acidic isopropanol and
the plates were read in an ELISA plate reader (iMark™ Microplate Read-
er, Bio-Rad) using a test wavelength of 570 nm and a reference wave-
length of 630 nm.
2.5. FACS
2.5.1. Surface staining
T-cell hybridomas were stained for cell surface markers using anti-
CD4-PE (eBioscience Cat# 12-0041-83, RRID:AB_465507), anti-CD3-
FITC (eBioscience Cat# 11-0031-85, RRID:AB_464883) or CD45-FITC
(BioLegend Cat# 103107, RRID:AB_312972). M12.C3.F6 cells were
stained with anti-CD4-PE, 40F-FITC (Dadaglio et al., 1997) or CD45-
FITC. The cells (0.5–1 × 106/tube) were incubated with each monoclonal
antibody (mAb) conjugate or isotype control at 1 μg/mL in 50 μL of
staining buffer (D5F-0.2% NaN3) for 30 min in the dark at 4 °C. After
two washes with cold PBS-0.1% bovine serum albumin (BSA), the cells
were fixed with 1% paraformaldehyde (Sigma, St. Louis, MO, USA). The
isotype control antibodies were FITC-conjugated anti-IgG1 (BD
Pharmingen, CA, USA) and PE-conjugated anti-IgG2b (eBioscience, San
Diego, CA, USA). Cell acquisition was performed on a fluorescence-acti-
vated cell sorter (Canto II FACS, Becton Dickinson, CA, USA). A total of
10,000 events were acquired. To exclude dead cells in the flow cytome-
try analysis, a simple staining with propidium iodide (0.5 μg/mL) was
done in an additional tube (without fixation). The exclusion of dead
cells was made according to the forward and side scatter properties in
a dot plot (the cells are distributed in two population based on cell
size) and we established a gate of alive cells. Therefore, FACS analysis
was carried out on cell populations that have a similar pattern to this
population in dot plot of FSC and SSC (all replicates were reproducible).
The data were analyzed with the BD FACSDiva software and Flowing
Software version 2.5.1 to overlay the histograms.
2.5.2. IL-2 secretion assay (original protocol)
For the flow cytometric analysis of activated T-cell hybridomas in
the antigen presentation assay described above, the cells were harvest-
ed and deposited in a 15 mL closable tube per sample (triplicate wells)
according to the manufacturer's instructions (original protocol) for
the mouse IL-2 secretion assay (130-090-492, Miltenyi Biotec®)
(Campbell et al., 2011). The cells were washed twice with 10 mL of
cold buffer (PBS with 0.5% BSA and 2 mM EDTA, pH 7.2) for 10 min at
4 °C (300 ×g) and the media was removed. The cell pellet was resus-
pended with cold D5F and the cells were labeled with the mouse anti-
IL-2 mAb (rat IgG2a) conjugated to the cell surface-specific mAb (rat
IgG2b) anti-CD45 (IL-2 capture antibody at final dilutions of 1:50–
44 P. Gastelum-Aviña et al. / Journal of Immunological Methods 438 (2016) 42–50

1:200). After 5 min of incubation on ice, 10 mL of warm D5F (37 °C) was
added and the cells were incubated for 45 min at 37 °C to allow IL-2 se-
cretion (turning the tube every 5 min). The cells were washed two
times as described above and resuspended in cold buffer. The cells
were stained for 10 min on ice with an anti-IL-2 mAb (rat IgG2b) conju-
gated to PE (1:50–1:200, IL-2 detection antibody) and anti-CD4-FITC
(1 μg/mL). Finally, the cells were washed, fixed and acquired as de-
scribed in Section 2.5.1.
2.5.3. IL-2 secretion assay (modified protocol)
To measure IL-2 secretion at the beginning of the assay, the capture
antibody was added at the same time as the HEL antigen in the antigen
presentation assay and then incubated for 16 h. The cells were harvest-
ed, washed and re-suspended in cold buffer and then incubated with
anti-IL-2 PE and anti-CD4-FITC for 10 min on ice. After washing, the
cells were fixed and acquired (triplicate wells of the antigen presenta-
tion assay were harvested together and stained in a single tube for
FACS). To evaluate the assay sensitivity, 0.5–1 × 106 T-cell hybridoma
3A9 cells were tested with different concentrations of rIL-2 (1, 0.5,
0.25, 0.125, 0.06, 0.03, 0.015, 0.007, 0.003, and 0 ng/mL). Staining with
the capture and detection antibodies was performed as described
above. The antigen presentation assay was evaluated at different time
points (0–16 h) using the modified protocol for comparison with the
CTLL-2 proliferation assay (see Section 2.4). Note: triplicate wells in an-
tigen presentation assay were harvested and stained together for the al-
ternative method.
2.6. Statistical analysis
The results were statistically assessed using the nonparametric test
Kruskal-Wallis test to compare the CTLL-2 proliferation assay and the al-
ternative method. The data were analyzed with the IBM SPSS Statistics
20 software.
3. Results
3.1. CD4 as a surface phenotypic marker of the T-cell hybridoma population
To evaluate the activation of T-cell hybridomas during the in vitro
antigen presentation assay, we measured IL-2 production by flow cy-
tometry with the commercially available kit from Miltenyi Biotec®.
This kit first employs a conjugated capture antibody (anti-CD45-anti-
IL-2) followed by a secondary anti-IL-2-PE detection antibody. Conse-
quently, it was first necessary to assess the expression of several cell
surface markers, such as CD3, CD4, CD45 and MHC-II, on the 3A9 and
M12.C3.F6 cell lines. We found that 65.3% of the population of 3A9 T hy-
bridoma cells expressed CD3 (mean fluorescence intensity (MFI) 476),
99.4% expressed CD4 (MFI 2741) and 99.9% expressed CD45 (MFI
13149). Similarly, 98.8% of the population of B lymphoma cells
expressed MHC-II (IAk haplotype) (MFI 6356), 0.3% expressed CD4
(MFI 147) and 100% expressed CD45 (4321) (Fig. 1). Therefore, we se-
lected CD4 as a hybridoma cell maker because there was a larger differ-
ence in the expression levels between the hybridoma cell population
(99.4%) and the M12.C3.F6 cell line (0.3%).
3.2. Addition of the IL-2 capture antibody at the beginning of the antigen
presentation assay improves the cytokine secretion assay protocol
Following the original protocol, the cells were labeled with anti-
CD45-anti-IL-2 after in vitro stimulation with the antigen (1 μM HEL
for 16 h), incubated for 45 min to allow binding of the secreted IL-2,
and then stained with anti-IL-2-PE and anti-CD4-FITC. The expression
of IL-2 by the hybridomas was compared between unstimulated (as a
negative control for activation) and stimulated cells.
Gating strategy for the analysis of activated T-cell hybridomas is
shown in Fig. 2. First, we established a region P1 in a dot plot of FSC
and SSC, and viable cells were selected based on PI staining (additional
tube, as explained in Section 2.5.1). Then, we selected a second region

Fig. 1. Expression of cell surface markers detected by flow cytometry on the 3A9 hybridoma and the murine B cell lymphoma M12.C3.F6 cells. Expression of the CD3, CD4 and CD45
molecules was assessed on the 3A9 hybridoma and the IAk haplotype MHC-II, CD4 and CD45 molecules were assessed on the M12.C3.F6 antigen presenting cells. A representative
experiment is shown with histograms comparing the mean fluorescence intensity of each molecule with the corresponding isotype control (IC). The histograms show the percentage
of the population expressing each marker.
 P. Gastelum-Aviña et al. / Journal of Immunological Methods 438 (2016) 42–50 45

(P2) from P1, corresponding to CD4-positive cells and finally from this
region we select the third region of IL-2-positive cells (P3). We com-
pared the percentage of positive cells and MFI of unstimulated and acti-
vated T-cell hybridomas.
The results showed that 0.8% of the unstimulated cells were CD4+
IL2+ (MFI 141) compared with just 4.5% of the HEL-stimulated cells
(MFI 293) (Fig. 3B1, B2). The supernatant collected following the HEL
antigen presentation assay (HEL SN) was also evaluated by adding it
during the period of IL-2 secretion; however, only 1.1% of the population
stained as CD4+ IL-2+ (Fig. 3B3). This result was considered inconsis-
tent with the CTLL-2 cell proliferation assay, where proliferation oc-
curred in the presence of the HEL SN collected from hybridomas
stimulated for 16 h (55.5% ± 1.5) and 20 h (100%) compared with the
supernatant from unstimulated cells (16.45% ± 3.7 for 16 h and
13.5% ± 0.8 for 20 h) (Fig. 4A).
Therefore, we modified the original protocol by adding the capture
antibody together with the stimulus at the beginning of the antigen pre-
sentation assay. The results showed that 94.1% of the cells stimulated
with HEL stained as CD4+ IL-2+ compared with only 0.8% of the
unstimulated controls (Fig. 3C2 and C1). The possibility that antibody
internalization was occurring during antigen processing was evaluated
by adding the antibody together with HEL from the beginning of the
test (16 h in total) and after 4 and 12 h of HEL stimulation; no differ-
ences were observed between these time points (data not shown).
Fig. 5 schematically illustrates both IL-2 secretion assays and compares
the original protocol, where the capture antibody was added after
antigenic stimulation (A. original protocol), with the modified protocol,
where the capture antibody was added at the same time (B. modified
protocol).
The antigen specificity of the experimental system was evaluated by
testing an irrelevant antigen (the PE_PGRS33 recombinant protein from
Mycobacterium tuberculosis) with the HEL-specific 3A9 hybridoma. The
system was found to be antigen-specific because only 1.3% of the cells
stimulated with the irrelevant antigen were CD4+ IL-2+ cells; this per-
centage was similar to the percentage in the unstimulated controls
(Fig. 3C3). We also assessed another antigen-specific system (the
PE_PGRS33-specific 3C2.D4 hybridoma and the PE_PGRS33 recombi-
nant protein). The method was found to be as sensitive as the 3A9-
HEL system, with 98.9% of the cells stimulated with PE_PGRS33
CD4+ IL-2+ (MFI 1627) compared with just 0.3% (MFI 99) of the
unstimulated control cells (Fig. 6).
3.3. The alternative method is sensitive and can detect different concentra-
tions of antigenic stimulation in the in vitro antigen presentation system
To assess the sensitivity of the modified IL-2 secretion assay, we de-
termined the minimum effective concentration of antigen that could
stimulate 50% of the CD4+ IL-2+ population (EC50) compared with the
CTLL-2 proliferation assay based on MTT reduction. With both tech-
niques, a significant difference (p b 0.05) was found between the
unstimulated controls and cells stimulated with the 0.1 and 1 μM HEL
concentrations. However, at the maximum concentration tested (1 μM

Fig. 2. Gating strategy for the analysis of activated T-cell hybridomas by flow cytometry. (A) Exclusion of dead cells with PI staining (T cell hybridoma cells 3A9 were stimulated with HEL
protein during 16 h and M12.C3.F6 cells were used as APCs). (B) Isotype control antibody (anti-IgG2b-PE), as a control of non-specific binding or the background fluorescence. (C)
Cytometric analysis of the percentage and mean fluorescence intensity of CD4+ IL-2+ cells (unstimulated 3A9 hybridomas) (D) HEL-stimulated 3A9 hybridomas. Data from one
representative experiment is shown. PI, propidium iodide.
46 P. Gastelum-Aviña et al. / Journal of Immunological Methods 438 (2016) 42–50

Fig. 3. Evaluation of 3A9 hybridoma activation by flow cytometry by comparing the original IL-2 secretion assay with the modified protocol. Cytometric analysis of the percentage of
CD4+ IL-2+ cells and the mean fluorescence intensity of unstimulated and HEL-stimulated (1 μM for 16 h) 3A9 hybridomas. (A) Controls: (1) isotype control (3A9 cells stained
with anti-IgG2b-PE), (2) isotype control added after first labeling the cells with the capture antibody (anti-CD45-anti-IL-2), (3) positive control (rIL-2 added at a final concentration of
1 ng/mL after labeling the 3A9 cells with the capture antibody and staining with the secondary anti-IL-2-PE), (4) MFI of the 3 controls. (B) Cells stained with the original protocol
wherein the first antibody was added after the presentation assay: (1) unstimulated cells, (2) cells stimulated with HEL, (3) the supernatant collected following the antigen
presentation assay was added to the 3A9 cells after labeling them with the capture antibody, (4) comparison of MFIs under the 3 conditions. (C) Cells stained with the modified
protocol wherein the first antibody was added together with the stimulus at the beginning of the antigen presentation assay: (1) cells without stimulation, (2) cells stimulated with
HEL, (3) cells stimulated with an irrelevant antigen (1 μM PE_PGRS33), (4) MFI under the 3 conditions. A representative experiment from at least 3 replicates is shown. IC, isotype
control; NS, non-stimulus; MFI, mean fluorescence intensity.

HEL), 100% proliferation was observed with the CTLL-2 assay compared
to 16% ± 3.2 for the unstimulated cells (Fig. 7A), whereas with the
newly proposed method the values were 94.1 ± 4.3% of the CD4+ IL-
2+ cells determined by FACS analysis and 2.3% ± 1.6 of the
unstimulated controls (mean of three independent experiments) (Fig.
7B). The EC50 was 0.01 to 0.04 μM HEL (x = 0.03 μM) for the CTLL-2
assay and 0.07 to 0.08 μM HEL (x = 0.08 μM) for the modified IL-2 secre-
tion assay (CD4+ IL-2+ activated 3A9 cells).
To compare the IL-2 detection limit of the CTLL-2 proliferation
assay with the newly proposed FACS method, the CTLL-2 cells
were incubated with various rIL-2 concentrations (Sigma-Aldrich,
St. Louis, MO, USA), and the percentage of proliferation of these
cells was evaluated (Fig. 8). rIL-2 EC50 values of 0.08 to 0.22 ng/mL
were obtained (x = 0.11 ng/mL) with the CTLL-2 technique and
0.26 to 0.28 ng/mL ( x = 0.27 ng/mL) with the newly proposed
FACS method.
A significant difference (p b 0.05) with the control without rIL-2
was found at a concentration of 0.125 ng/mL with the CTLL-2 prolif-
eration assay (43.8% ± 7.8 and 11.7% ± 0.4 for the control) (Fig. 8A),
whereas with the proposed FACS method a difference was found
from 0.25 ng/mL of rIL-2 (45.9% ± 2.6% and 8.4% ± 2.1 for the
control) (Fig. 8B). Therefore, the sensitivity of the IL-2 detection
limit was better with the CTLL-2 cell proliferation assay.
3.4. Detection of IL-2 as a hybridoma activation marker was faster with the
alternative method than with the proliferation assay using CTLL-2 cells
To determine the optimal stimulation time with the antigen for the
activation of the T-cell hybridomas using the proposed FACS method,
a kinetic activation assay was performed with 1 μM HEL and the results
were compared with the proliferation technique using CTLL-2 cells.
After 16 h of activation with HEL, the IL-2 secreted into the culture me-
dium induced the proliferation of CTLL-2 cells (55.5% ± 1.5) compared
with the unstimulated control (16.45% ± 3.7); indeed, 100% prolifera-
tion was observed after 20 h (13.5% ± 0.8, unstimulated control). The
HEL stimulation time that induced proliferation of 50% of the CTLL-2
population was 15.1 h. With the new method proposed here using cy-
tometry, 50% of the CD4+ IL-2+ cells were detected after 12.1 h of stim-
ulation with HEL during the presentation assay; after 16 h, the figure
rose to almost 100% (92.8% ± 2.3) compared with 11.4% ± 1.1 for the
corresponding unstimulated controls (Fig. 4). Therefore, considering
the total amount of time required for both antigenic stimulation and
 P. Gastelum-Aviña et al. / Journal of Immunological Methods 438 (2016) 42–50
Fig. 4. Activation kinetics of the 3A9 hybridomas evaluated by the CTLL-2 cell proliferation
method and by the modified cytokine secretion assay. (A) Percentage of proliferation of
CTLL-2 cells in the presence of supernatants collected at different time points after HEL
stimulation (1 μM). The O.D. values from the MTT reduction assay were converted into
proliferation percentages whereby the average O.D. obtained with the supernatant from
3A9 cells stimulated for 20 h was considered to be 100% proliferation (see Section 2.4.).
(B) Percentage of CD4+ IL-2+ hybridomas assessed through the modified cytokine
secretion assay (see Section 2.5.3). HEL-stimulated and unstimulated cells were
compared at 0, 4, 8, 12 and 16 h. Cells were cultured in triplicate wells and 1 μM HEL
was added every 4 h for each time point. The supernatants from all conditions were
collected at 20 h. A representative experiment from at least three independent
experiments is shown. NS, non-stimulus.
the activation measurement, hybridoma activation can be assessed in
less time (~18 h) with the modified FACS protocol than with the refer-
ence method (~48 h).
4. Discussion
Antigen-specific T-cell hybridomas play an important role in eluci-
dating the physiology and behavior of the cellular immune response.
Methods that assess the activation of these cells and TcLs are primarily
based on measuring the production or expression of cytokines with
techniques such as ELISA, flow cytometry and proliferation bioassays
using specific cytokine-dependent cell lines. IL-2 is one of the most com-
monly used activation markers, and bioassays with the CTLL-2 cell line
are widely used for this purpose (Surman and Hurwitz, 2002).
Radioactive, colorimetric or fluorometric markers are used to mea-
sure the proliferation and/or viability of these cell lines. However,
each marker has limitations in terms of sensitivity, cost or prolonged in-
cubation times (Soman et al., 2009). Because there is no standard test to
evaluate the activation of T-cell hybridomas and because each of the
tests offers advantages and disadvantages, we wanted to establish a
method that allowed us to reliably quantify IL-2. Although the bioassay
with CTLL-2 cells is frequently used to indirectly measure cell activation,
the continuous culture of these cells is associated with a loss of sensitiv-
ity, reliability and stability (Khatri et al., 2007; Weston et al., 1997, 1998;
Winandy et al., 1998). Therefore, we focused on developing an alterna-
tive method to assess hybridoma activation that was sensitive and
allowed earlier detection of IL-2 by flow cytometry.
Flow cytometry is a multi-parameter method to obtain information
from individual cells, the great advantages of flow cytometry are that
47
different cytokines can be detected per cell and that cell populations
can be differentiated based on the expression of a particular type of cy-
tokine in response to specific stimuli. The production and secretion of
cytokines can be assessed using beads (CBA) (Chen et al., 1999;
Morgan et al., 2004) or detected intracellularly using protein transport
inhibitors and permeabilization of the cell for antibody labeling (Jung
et al., 1993). In our case, we would have needed to add brefeldin
(BFA) as an inhibitor to assess IL-2 intracellularly during the hybridoma
assays; however, this compound is toxic for N4–6 h. Therefore, we have
needed to add BFA 4 h prior to finishing the antigen presentation assay.
However, at this time point (~12 h), IL-2 is likely to have already been
secreted and thus its detection by flow cytometry is low (Jung et al.,
1993; Nylander and Kalies, 1999).
Because the fixation, permeabilization and retention time of the cy-
tokine do not guarantee that the accumulation of IL-2 is sufficiently high
for detection with intracellular antibodies (Sargentini et al., 2009), we
decided to evaluate the method used by Miltenyi Biotec®, which has
been used for the characterization of cytokine-secreting TcLs. In this
method, cytokines or secreted products are retained on the surface of
the secreting cells, making them accessible for detection by techniques
such as flow cytometry (Manz et al., 1995; Campbell et al., 2011). This
method involves the use of an IL-2 capture antibody conjugated to a
cell binding antibody (anti-CD45), which allows the assessment of IL-
2 secreted during the antigen presentation assay and subsequent to T-
cell hybridoma activation. The original protocol recommends adding
the capture antibody after the period of stimulation with the specific an-
tigen and allowing an additional period of time (~45 min) for the secre-
tion of IL-2 at 37 °C and the binding of this cytokine to the antibody
bound to the cell surface. Then, the detection antibody (anti-IL-2-PE)
is added. Following the original protocol under the conditions evaluated
in this study, we found no differences in the percentage and MFI of IL-2+
cells between the unstimulated samples and the cells stimulated with a
specific antigen (Fig. 3B). However, we did observe a difference be-
tween cells incubated in the absence or presence of the HEL antigen
(data not shown) when evaluating the expression of the early activation
marker CD69 (Ziegler et al., 1994; Simms and Ellis, 1996) in the popula-
tion of 3A9 hybridoma cells.
Therefore, we evaluated different conditions and modified the IL-2
secretion assay protocol with the goal of capturing the cytokine from
the beginning of the antigen presentation assay. Thus, the capture anti-
body was added together with the stimulus. Based on the original pro-
tocol, IL-2 is secreted after 16 h of stimulation with antigen (Fig. 4A);
then, the cells are harvested, washed and labeled with the capture anti-
body. However, it is possible that some IL-2 has already been secreted
and is discarded with the cell washes prior to the antibody labeling
step. With the modified protocol proposed here, the capture of the cyto-
kine occurs as soon as it is produced, leading to faster and more efficient
results (Fig. 5).
Our findings indicated that both were sensitive methods for the
evaluation of T-cell hybridoma activation after stimulation with differ-
ent antigen (HEL) concentrations and for the detection of rIL-2.
Although the reference method proved to be twice as sensitive as the
modified protocol for the assessment of the activation of T-cell hybrid-
omas, the CTLL-2 proliferation assay required approximately 48 h to ob-
tain the result. In contrast, only ~18 h are required with the proposed
cytometry protocol. Therefore, the modified secretion protocol repre-
sents an alternative to evaluate the activation of antigen-specific T-cell
hybridomas, with fewer variables affecting the detection of IL-2 and re-
quiring only half the time. As mentioned above, continuous in vitro cul-
ture of the CTLL-2 cell line leads to problems that affect the sensitivity of
the assay. Furthermore, hybridoma cell activation is detected indirectly
with this method. In contrast, IL-2 detection with the proposed method
is direct, and the cytokine is captured as soon as it is secreted. IL-2 detec-
tion occurs extracellularly by flow cytometry, which avoids the need to
permeabilize the cells and use the toxic compound BFA for the intracel-
lular detection of the cytokine.
48 P. Gastelum-Aviña et al. / Journal of Immunological Methods 438 (2016) 42–50

Fig. 5. Schematic diagram of the IL-2 secretion assay comparing the original protocol with the proposed modified version. (A) Labeling with the capture antibody (anti-CD45-anti-IL-2) is
performed after 16 h of antigen stimulation; a period of time is allowed for cytokine secretion to proceed prior to staining with the detection antibody (anti-IL-2-PE). (B) Labeling with the
capture antibody is performed at the beginning of antigen presentation simultaneously with the addition of the antigenic stimulus. After 16 h of incubation, the cells were stained with the
detection antibody.

Fig. 6. Assessment of the specificity of the antigen presentation system using the modified cytokine secretion assay. The percentage of CD4+ IL-2+ hybridomas and the MFI for IL-2 of two T-
cell hybridomas after stimulation with their specific antigens were evaluated. (A) 3A9-HEL system (94.1%, MFI 1530) compared with the control without stimulus (0.8%, MFI 147). (B)
3C2.D4-PE_PGRS33 system (98.9%, MFI 1627) compared with the control without stimulus (0.3%, MFI 99). IC, isotype control, NS, non-stimulus.

In conclusion, the proposed protocol is a sensitive and rapid method
for the determination of IL-2 as an antigen-specific T-cell hybridoma ac-
tivation marker and allows capture of the cytokine as soon as it is secret-
ed. Additionally, it is possible to define the source of the cytokine in
combination with monoclonal antibodies using flow cytometry. This
method may be useful for assessments of cellular activation in primary
cultures, in human lymphocytes or during limiting dilution analysis for
hybridoma generation, where cytokine production can be very low.
However, additional studies are needed to evaluate the efficacy of this
method with these systems (Gieni et al., 1995).
Acknowledgements
The authors wish to thank the Mexican Council of Science and Tech-
nology (CONACYT) for supported the present work with the grant num-
ber 83224 and scholarship support number 305069.
 P. Gastelum-Aviña et al. / Journal of Immunological Methods 438 (2016) 42–50
Fig. 7. Sensitivity of the IL-2 secretion assay tested with the 3A9-HEL antigen presentation
system. We evaluated four HEL concentrations in the antigen presentation assay (0.001,
0.01, 0.1, and 1 μM) following incubation for 16 h with the capture antibody. The results
were compared with the CTLL-2 cell proliferation assay. A) Percentage of proliferation of
CTLL-2 cells stimulated with the supernatant collected after 20 h of activation with HEL
(0.001–1 μM). The percentage of proliferation was obtained from the O.D. values of the
MTT reduction assay (the average O.D. of cells proliferating in the presence of the 1 μM
HEL supernatant was considered 100%). A representative experiment of 3 independent
experiments is shown, each consisting of 3 replicates. The EC50 was 0.01 to 0.04 μM HEL
(x = 0.03 μM). B) Percent of CD4+ IL-2+ hybridomas evaluated using the alternative
method (see Section 2.5.). The results show the mean and standard deviation of 3
similar experiments. The EC50 was 0.07–0.08 μM HEL ( x = 0.08 μM). Significant
differences (p b 0.05) compared to the unstimulated control cells are indicated with an
asterisk.
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