Chapter 7

CD107a Degranulation Assay to Evaluate Immune Cell

Antitumor Activity
Seila Lorenzo-Herrero, Christian Sordo-Bahamonde, Segundo Gonzalez,
and Alejandro López-Soto

Abstract
Cancer development is under surveillance by the immune system of the host. Tumor cells can be recog-
nized and killed by cytotoxic lymphocytes— such as CD8+ T lymphocytes and natural killer (NK) cells—
mainly through the immune secretion of lytic granules that kill target cells. This process involves the fusion
of the granule membrane with the cytoplasmic membrane of the immune effector cell, resulting in surface
exposure of lysosomal-associated proteins that are typically present on the lipid bilayer surrounding lytic
granules, such as CD107a. Therefore, membrane expression of CD107a constitutes a marker of immune
cell activation and cytotoxic degranulation. In this chapter, we detail the steps required to isolate peripheral
blood mononuclear cells (PBMCs), coculture them with target tumor cell lines, and evaluate the cytotoxic
immune function by means of flow cytometry evaluation of CD107a expression on the surface of NK cells.

Key words NK cells, Antitumor immunity, CD107a

1  Introduction

Natural killer (NK) cells are a subset of immune cells that take a
central part in the innate immune response. They are able to elimi-
nate normal stressed cells such as virus-infected as well as malig-
nant transformed cells [1].
NK cell activity is regulated by a tight balance of signals trans-
duced through activating and inhibitory receptors expressed on
their surface, although, in contrast to T cells, prior antigen priming
is not required for their activation. Among this array of regulatory
receptors, natural killer group 2, member D (NKG2D) stands out
as a key receptor in regulating NK cell function. In humans,
NKG2D ligands include stress-regulated molecules such as MHC
class I polypeptide-related sequence A/B (MICA/B) and UL16
binding protein (ULBP) molecules, which are over-expressed on
the surface of tumor cells [2–4].

Alejandro López-Soto and Alicia R. Folgueras (eds.), Cancer Immunosurveillance: Methods and Protocols, Methods in Molecular Biology,
vol. 1884, https://doi.org/10.1007/978-1-4939-8885-3_7, © Springer Science+Business Media, LLC, part of Springer Nature 2019

119
120 Seila Lorenzo-Herrero et al.

NK cells are predominantly cytolytic immune cells and their

main mechanism of action relies on a process called degranulation.
Thus, NK cells hold preformed cytotoxic granules bearing perforin
and granzymes in their cytoplasm, which, upon target cell recogni-
tion, release their content into the immunological synapse, result-
ing in the lysis of the target cell without affecting the immune
effector cell [5, 6].
Lysosome-associated membrane protein-1 (CD107a or
LAMP-1) is a highly glycosylated transmembrane protein present
in the lysosomes. In NK cells and cytotoxic T cells, CD107a is one
of the most abundant proteins present in the lytic granules. This
molecule is found lining the membrane of the vesicles, hiding its
highly glycosylated portion in the luminal side and exposing a
short tail to the cytoplasm [7]. As a consequence of the degranula-
tion process, the outer membrane of the granules merges with the
NK cell plasma membrane, leading to surface exposure of CD107a
molecules, which has been proposed as a mechanism to protect the
effector cell from degranulation-associated suicide [8].
Moreover, CD107a membrane expression has been widely
described as a marker for activated cytotoxic lymphocytes, both
CD8+ T cells [9] and NK cells [10]. Indeed, a direct correlation
between CD107a surface expression evaluated by flow cytometry
analysis and NK cell cytotoxic activity and cytokine secretion has
been established [11, 12], making it a reliable assay for evaluation
of activation of immune cell cytotoxic responses against a variety of
stimuli, including tumor cells [13–15]. This method has also been
proposed as an assay to diagnose genetic disorders of cytotoxicity,
describing defective degranulation of NK cells as a prognostic fac-
tor in some diseases [16–18].
CD107b/LAMP-2 and CD63/LAMP-3, two proteins from
the same family as CD107a, are also present in the membrane of
the lytic vesicles in NK and cytotoxic T cells. In contrast to
CD107a, flow cytometry detection of CD107b surface exposure
due to NK cell activation is weak and CD63 membrane expression
does not clearly correlate to activation in NK cells [8], making
these molecules a poor marker for NK cell degranulation.
Measuring the activation of NK cells by detection of surface
CD107a has several advantages compared to other assays. Above
all, this method provides direct information about the effector
population, whereas cytotoxicity assays only detect the final lysis of
target cells. Furthermore, labeling of CD107a allows the discrimi-
nation of multiple NK cell subsets based on their level of activation
[11]. Nevertheless, this assay should be complemented with NK
cell cytotoxicity and/or detection of cytokine production assays in
order to achieve a more profound knowledge of the immune
mechanisms that are taking place against a given stimulus.
 In vitro NK Cell Degranulation Assay 121

Herein, we describe a detailed protocol of an in vitro degranu-

lation assay based on the detection of CD107a surface expression
in NK cells by flow cytometry, a sensitive method to measure the
level of activation of these immune cells to cancer cells. More spe-
cifically, we evaluated the NK cell response to the tumor cell line
K-562, which is a “gold-standard” cell line to evaluate anticancer
immune responses. Furthermore, the contribution of NKG2D to
the degranulation against a given target is also assessed by using a
blocking antibody against this immunoreceptor. Likewise, this
protocol can also be used for the detection of CD107a in CD8+ T
cells.

2  Materials

2.1  Cell Culture 1. K-562: human lymphoblastoid cell line established from a
chronic myelogenous leukemia patient and obtained from the
2.1.1  Cell Line Culture
American Type Culture Collection (ATCC). These are human
leukocyte antigen (HLA) class-I and -II negative cells.
2. Complete growth medium: Roswell Park Memorial Institute
(RPMI) 1640 medium supplemented with 10% heat-inacti-
vated fetal bovine serum (FBS), 1  mM sodium pyruvate,
2  mM  L-­glutamine, 100  U/mL penicillin, and 10  μg/mL
streptomycin.
3. 25 and 75 cm2 flasks for cell culture.
4. 5 and 10 mL disposable serological pipettes.
5. 0.4% Trypan Blue solution.
6. Neubauer chamber for cell counting.

2.1.2  PBMC culture 1. Complete growth medium (see Subheading 2.1.1).

2. Recombinant human interleukin-2 (IL-2) (ORF Genetics):
100  U/μL stock solution in deionized water (dH2O), ali-
quoted and stored at −80 °C.
3. 25 and 75 cm2 flasks for cell culture.
4. 5 and 10 mL disposable serological pipettes.
5. 0.4% Trypan Blue solution.
6. Neubauer chamber for cell counting.

2.2  Isolation 1. Buffy-coats from healthy donors.

of Peripheral Blood 2. 1× Phosphate-buffered saline (PBS): 2.7  mM KCl, 4.3  mM
Mononuclear Cells Na2HPO4, 1.4  mM KH2PO4, and 137  mM NaCl in
(PBMCs) dH2O. Adjust pH to 7.4.
3. Histopaque®-1077 (Sigma Aldrich) or any other commer-
cially available solution of Ficoll density gradient medium.
122 Seila Lorenzo-Herrero et al.

4. 15 and 50 mL conical centrifuge tubes.

5. Plastic 1.5 mL Pasteur pipettes.
6. 5 and 10 mL disposable serological pipettes.

2.3  PBMC/Tumor Cell 1.

Protein transport inhibitor containing monensin BD
Coculture and Staining GolgiStop™ (BD Biosciences): stored undiluted at 4 °C.
2. Ionomycin: 1  mg/mL stock solution in dimethyl sulfoxide
(DMSO), aliquoted and stored at −80 °C.
3. Phorbol 12-myristate 13-acetate (PMA): 50  μg/mL stock
solution in DMSO, aliquoted and stored at −80 °C.
4. U-bottom 96-well plates for cell culture.
5. 5  mL round-bottom fluorescence-activated cell sorting
(FACS) tubes (12 × 75 mm).

2.3.1  Antibodies 1. Normal mouse control IgG (Santa Cruz Biotechnology).

2. Purified mouse antihuman NKG2D (Clone 1D11, IgG1)
(Santa Cruz Biotechnology).
3. Phycoerythrin (PE)-conjugated mouse antihuman CD107a
(Clone H4A3, IgG1) (BD Biosciences).
4. Fluorescein isothiocyanate (FITC)-conjugated mouse antihu-
man CD3 (Clone UCHT1, IgG1) and Allophycocyanin
(APC)-conjugated mouse antihuman CD56 (Clone C5.9,
IgG2b) (Cytognos).

2.4  Flow Cytometry 1. Flow cytometer FACSCanto II (BD Biosciences), controlled by

Acquisition BD FACSDiva software for acquisition and data analysis, or
and Analysis equivalent.

3  Methods

3.1  Cell Culture 1. K-562 cells are cultured as a suspension in 25 cm2 or 75 cm2
flasks at 37 °C in 5% CO2. Maintain cell cultures at 106 cells/
mL by adding fresh complete growth medium every 2–3 days.
2. Once a week, transfer the cell suspension to a 15 mL conical
tube and centrifuge cells at 250 × g for 5 min at room tem-
perature (RT). Aspirate supernatant and resuspend the cell
pellet in fresh pre-warmed (37  °C) complete medium at an
adequate density.

3.2  Isolation 1. The day before setting the CD107a assay, collect buffy-coats
of Peripheral Blood from healthy donors. Blood is always manipulated under ster-
Mononuclear Cells ile conditions and processed within a few hours of collec-
(PBMCs) tion (see Note 1).
 In vitro NK Cell Degranulation Assay 123

2. To dilute the concentrated blood, prepare one 50 mL conical

tube per healthy donor by adding 25 mL of 1× PBS. Cut the
cannula of the blood bag and pour another 25 mL of blood in
each tube. Mix gently by inverting the tube (see Note 2).
3. For PBMC isolation by density gradient, prepare one 50 mL
conical tube per healthy donor by adding 15  mL of
Histopaque-­1077® pre-warmed at RT (see Note 3).
4. Carefully pour 35 mL of diluted blood over the Histopaque so
they do not mix and remain as two separated phases.
5. Immediately afterward, centrifuge the tubes (one per healthy
donor) at 900 × g for 22 min and RT, without brake to avoid
disruption of the gradient layers.
6. After centrifugation, handle the tubes carefully (see Note 4).
Transfer the mononuclear cell layer to a new 15 mL conical
tube with a plastic Pasteur pipette and add 10 mL of 1× PBS
to the tube. Mix gently by inversion.
7. Centrifuge the tubes at 250 × g for 5 min at RT and aspirate
the supernatant with a glass Pasteur pipette connected to a
vacuum system or an alternative method. Wash the pellet with
1× PBS once more and centrifuge again. If the supernatant
remains cloudy, add a third wash with 1× PBS (see Note 5).
8. Once washed, resuspend the cell pellet in 10  mL of pre-­
warmed (37 °C) complete growth medium avoiding the for-
mation of cell clumps (see Note 6).
9. To determine cell density, count the cell suspension with a
Neubauer chamber. Briefly, prepare a 1:10 dilution of the cells
in growth medium and mix 1:1 with Trypan Blue solution.
Incubate the mixture for 2–3 min at RT and count the non-­
dyed (alive) cells.
10. Maintain PBMCs overnight in cell culture at a density of

2 × 106 cells/mL in complete growth medium supplemented
with 100 U/mL IL-2 if necessary (see Notes 7 and 8).

3.3  PBMC/Tumor Cell 1. Determine cell density of K-562 and PBMC cultures with a
Coculture and Staining Neubauer chamber as indicated in Subheading 3.2, step 9 (see
Note 9).
2. Centrifuge PBMCs at 250 × g for 5 min at RT and resuspend
the cell pellet in fresh pre-warmed (37 °C) complete growth
medium at 2 × 106 cells/mL. Split the cell culture in two dif-
ferent flasks and add 10  μg/mL of antihuman NKG2D or
10 μg/mL of control IgG. Incubate for 1 h at 37 °C and 5%
CO2. Subsequently, wash PBMCs with 1× PBS and centrifuge
to eliminate the excess of antibody.
124 Seila Lorenzo-Herrero et al.

3. Prepare growth medium for the positive controls of the assay

by supplementing 100 ng/mL PMA and 2 μg/mL ionomycin
to complete medium (see Notes 10 and 11).
4. After washing PBMCs, centrifuge both K-562  cells and
PBMCs at 250 × g for 5 min at RT. Aspirate the supernatant
and resuspend the cell pellet in fresh complete growth medium.
Adjust K-562 concentration to 106 cells/mL and PBMCs
concentration to 107 cells/mL for a 10:1 effector:target (E:T)
ratio (see Note 12).
5. In a U-bottom 96-well culture plate, add 100 μL of each cell
suspension to set the following conditions at least in duplicates
(Table 1) (see Note 13). The final volume per well is 200 μL.
6. Right afterward, add 10 μL of antihuman CD107a antibody
to each well and incubate for 4 h at 37 °C and 5% CO2.
7. After 1 h of incubation, prepare a monensin solution by add-
ing 2 μL of GolgiStop to 75 μL of complete growth medium
(see Note 14). Add 5  μL/well of the solution and mix by
pipetting up and down.
8. Following the 4  h incubation, transfer the whole volume of
each well to a 5 mL FACS tube, wash the cells twice with 1×
PBS and centrifuge them at 400 × g for 5 min at RT.
9. Prepare a master mix solution of the surface antibodies by
mixing 5 μL of antihuman CD3 antibody, 5 μL of antihuman
CD56 antibody, and 90 μL of 1× PBS per tube (see Note 15).
10. Resuspend cells in 100 μL/tube of antibody solution and mix
them carefully by brief vortexing. Then incubate for 25 min at
RT in the dark.
11. After the incubation period, wash cells with 1× PBS and cen-
trifuge them at 400 × g for 5 min at RT. Discard the superna-
tant and resuspend the cell pellet in 300–400 μL/tube of 1×
PBS. Maintain cells on ice and protected from light until flow
cytometry acquisition.

Table 1
Experimental conditions for a conventional CD107a assay (96-well plate)

Condition PBMCs K-562 cells Complete growth medium

Basal 100 μL/well – 100 μL/well
degranulation
Coculture 100 μL/well 100 μL/well –
Positive control 100 μL/well – 100 μL/well supplemented with PMA and
ionomycin
 In vitro NK Cell Degranulation Assay 125

P2 events

100
All events P1 events

Cell count
75
250

50
P2

5
10
P3: 6,6%

25
200

4
10

0
CD56-APC
2 3 4 5
0 10 10 10 10
150
SSC-A

CD107a-PE

3
10
100

P2 events

2
50

0 10
P1

5
10
CD56-APC
4
50 100 150 200 250

10
2 3 4 5
0 10 10 10 10

FSC-A CD3-FITC

3
10
P3: 6,6%

2
10
2 3 4 5
0 10 10 10 10

CD107a-PE

Fig. 1 Analysis of PBMC and NK cell staining by flow cytometry. First, create a FSC/SSC dot plot to locate alive
PBMCs within the whole sample (P1). In a second dot plot, separate lymphocyte subsets based on their
expression of CD3 and CD56. In humans, NK cells are defined as CD3−CD56+ (P2). To determine the percent-
age of CD107a+ cells, create a third gate to enclose the PE-positive subgroup (P3). CD107a data is routinely
represented as a percentage using density plots

3.4  Flow Cytometry 1. Acquire samples in a conventional flow cytometer that, at least,
Acquisition detects 488 nm (blue) and 638 nm (red) channels.
and Analysis 2. First, determine the position of alive PBMCs based on forward
(FSC) and side (SSC) scatter parameters (Fig. 1) (see Note 16).
3. In humans, NK cells are defined as CD3−CD56+ cells. Thus, in
the present protocol, this subset is gated as APC-positive FITC-­
negative cells (see Note 17). To determine the percentage of
CD107a+ NK cells, gate the subgroup of PE-positive NK cells
(Fig. 1).
4. Blocking of NKG2D activating receptor in NK cells signifi-
cantly reduces the degranulation of NK cells against K-562 can-
cer cells (Fig. 2).

4  Notes

1. CD107a assays are usually performed with at least two differ-

ent buffy-coats at a time to ensure the reproducibility of the
results.
2. Buffy-coats have a high cell density and erythrocytes can
aggregate in major clumps, trapping mononuclear cells. As a
consequence, those mononuclear cells sediment, diminishing
lymphocyte recovery. To reduce cell aggregates, it is impor-
tant to dilute whole blood with 1× PBS.
126 Seila Lorenzo-Herrero et al.

a) Basal Co-culture
degranulation with K-562

Control
IgG
2,4% 74,2%
CD56-APC

α-NKG2D

4,3% 56,5%

CD107a-PE

b) + K-562
80
CD107a+CD3-CD56+ cells (%)

60

40

20

0
Control IgG + - + -
α-NKG2D - + - +
Fig. 2 Functional relevance of NKG2D activating receptor in NK cell degranulation against K-562 leukemia cell
line. IL-2 stimulated PBMCs were pretreated with 10 μg/mL control IgG or 10 μg/mL antihuman NKG2D for 1 h,
as detailed in Subheading 3.3. Afterward, PBMCs were co-incubated with K-562 cancer cells at a 10:1 (E:T)
ratio for 4 h. Finally, cells were stained with specific antibodies and analyzed by flow cytometry. In panel (a),
representative dot plots are displayed. In panel (b), mean data of independent experiments are represented
(mean ± standard deviation, n = 2). As shown in the results, incubation with an NKG2D-blocking antibody
significantly reduces the degranulation of NK cells against K-562 cancer cells, supporting the relevance of
such receptor in NK cell activation
 In vitro NK Cell Degranulation Assay 127

3. The density of Ficoll changes with temperature, affecting both

the time and effectivity of the gradient separation. Optimal
results can be achieved at temperatures of 18–20 °C.
4. After gradient centrifugation, four differentiated layers appear
in the conical tubes, being the following: a top layer, which is
mainly plasma; a white cloudy layer, where the mononuclear
cells remain; the Ficoll layer and, at the bottom, a cell pellet
with erythrocytes and granulocytes (Fig. 3).
5. Prolonged contact with Ficoll solution can be toxic to cells, so
it is recommended to wash the cell pellet immediately after
recovery of the mononuclear cell layer to eliminate remaining
Ficoll traces, avoiding cell damage.
6. In some cases, erythrocytes can be present in the mononuclear
cell layer, contaminating the sample recovered. This can be
avoided by using pre-warmed Ficoll, as low temperatures pre-
vent erythrocytes to sediment through the high density Ficoll.
Alternatively, erythrocytes can be partially eliminated from the
cell pellet after centrifugation and washing based on their
higher adherence to the plastic tube walls by carefully resus-
pending the cell pellet and transferring the cell solution into a
new 15 mL tube, leaving the red blood cell layer attached to
the tube. This method can reduce the amount of mononuclear
cells recovered.
7. According to the manufacturer, reconstituted IL-2 is best
stored in working aliquots at −20  °C, avoiding repeating

Fig. 3 Isolation of PBMCs by Ficoll density gradient. Once the gradient is settled,
four different layers can be identified in the conical tube. Mononuclear cells
appear as a white cloudy layer
128 Seila Lorenzo-Herrero et al.

freeze-thaw cycles. Once an aliquot is thawed, it is recom-

mended to keep it at 4 °C for no longer than 2 weeks.
8. The level of NK cell degranulation highly depends on the tar-
get cancer cell line, with no detectable CD107a staining
observed in NK cells cocultured with certain poorly immuno-
genic tumor cell lines. Addition of IL-2 to the culture medium
overcomes this problem by activating NK cells, hence increas-
ing their responsiveness against a given cell target (Fig. 4).
9. CD107a assays can also be performed using IL-2-stimulated
purified NK cells as the effector population. To this end, a
wide variety of human NK cell isolation kits, based on mag-
netic cell labeling and separation, are commercially available.
10. PMA and ionomycin are supplied as a powder. Once resus-
pended, store them in single-use aliquots (5–10 μL) to avoid
repeated freeze-thawing. These reagents should be stored at
−20 °C, protected from light.
11. The working concentrations for PMA and ionomycin are

50 ng/mL and 1 μg/mL, respectively. In this case, the growth
medium for positive controls is supplemented with 2× PMA
and 2× ionomycin, as it will be diluted 1:1 with PBMC sus-
pension (Table 1).

Basal Co-culture
degranulation with K-562

6,6% 42,4%
CD56-APC

+ IL-2

5,7% 73,9%

CD107a-PE

Fig. 4 Comparative CD107a assay of IL-2 pretreated effector cells against K-562
leukemia cells. PBMCs were cultured overnight with or without IL-2 (100 U/mL).
Next day, PBMCs were co-incubated with K-562 cells at a 10:1 (E:T) ratio for 4 h,
as detailed in Subheading 3.3. Afterward, cells were stained and samples were
analyzed by flow cytometry. As shown in the figure, pretreatment with IL-2
increases the degranulation of NK cells against a given target
 In vitro NK Cell Degranulation Assay 129

12. In this protocol, calculations are made to set a co-incubation

with 105 target cells and 106 effector cells per well in a final
volume of 200 μL, which corresponds to a 10:1 (E:T) ratio. In
case the experimental settings differ from those described here,
appropriate densities to resuspend both cell types need to be
recalculated accordingly.
13. It is necessary to determine basal degranulation and stimula-
tion by target cells for all the conditions (e.g., control and
NKG2D-blocked PBMCs). Additionally, a positive control
should also be included to check the ability of PBMCs to
degranulate. Phorbol esters, such as PMA, are commonly used
to this end.
14. The adequate concentration of BD GolgiStop™ is achieved by
adding 4 μL of the reagent to every 6 mL of cell culture. In
the current protocol, GolgiStop is added to the cell culture
after 1 h of co-incubation in a more concentrated dilution to
obtain the final optimal concentration.
15. When preparing a master mix of antibodies for staining, it is
advisable to add 10% more volume of each component to the
mix to avoid falling short of volume.
16. Sometimes, target and effector cells have similar FSC and SSC
values, so that they appear in the same position in the dot plot.
This should not be a problem as the effector cells are labeled
with specific antibodies that differentiate them from the rest.
However, it is recommended to previously check whether
your working target cells have autofluorescence that could
interfere with the identification of the NK cell population.
17. A minimum of 5 × 103 events of your population of interest
should be acquired in order to obtain representative and reli-
able data. Acquiring a lower number of events can lead to
misguided data, as outliers represent a higher percentage of
the population.

Acknowledgments

This work was supported by a Spanish grant of Instituto de Salud

Carlos III (PI16/01485). S.L.H. holds a predoctoral Severo
Ochoa Fellowship (BP14–150) from FICYT of Principado de
Asturias, Spain.
130 Seila Lorenzo-Herrero et al.

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