Journal of Immunological Methods 294 (2004) 15 – 22

www.elsevier.com/locate/jim

Research paper

CD107a as a functional marker for the identification of natural

killer cell activity
Galit Alter, Jessica M. Malenfant, Marcus Altfeld*
Partners AIDS Research Center, Massachusetts General Hospital and Division of AIDS, Harvard Medical School, Massachusetts, USA
Received 17 June 2004; received in revised form 5 August 2004; accepted 10 August 2004
Available online 25 September 2004

Abstract

Natural killer (NK) cells are a subset of lymphocytes that play a central role in the innate immune response to tumors and
infections. An important limitation in the field of NK research is attributable to the deficit of assays available for the detection of
the functional activity of NK cells. Recently, lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) has been
described as a marker of CD8+ T-cell degranulation following stimulation. Here we describe CD107a as a marker of NK cell
functional activity using multi-parameter flow cytometry. CD107a is significantly upregulated on the surface of NK cells
following stimulation with MHC devoid targets. Additionally, CD107a expression correlates with both cytokine secretion and
NK cell-mediated lysis of target cells. However, as well as being coordinately expressed on nearly all cytokine secreting cells,
CD107a was also expressed on a large subset of NK cells that did not secrete cytokine following stimulation. These data
suggest that employing CD107a as a marker of NK cell functional activity may allow for the identification of a large fraction of
activated NK cells that may degranulate in the absence of cytokine secretion. Cumulatively, the data presented here demonstrate
that CD107a is a sensitive marker of NK cell activity.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Natural Killer Cells; IFN-g; TNF-a; CD107a; Cytotoxicity; Degranulation

1. Introduction CD56 (Cooper et al., 2001). This subset of lympho-

Natural killer (NK) cells are a subset of large
granular lymphocytes defined by a lack of T-cell
receptor (CD3) and by the surface expression of
* Corresponding author. Tel.: +1 617 724 2461; fax: +1 617
724 8586.
E-mail address: maltfeld@partners.org (M. Altfeld).
0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jim.2004.08.008
cytes plays an integral role in the control of a number
of viral infections and in tumor cell clearance
(Yokoyama and Scalzo, 2002). NK cells are an
important component of the innate immune response
as they are able to lyse tumor cells or virally infected
cells without prior antigen sensitization. NK cells are
also a critical bridge between the innate and adaptive
immune response as they secrete large amounts of
cytokines and chemokines that can shape and drive
16 G. Alter et al. / Journal of Immunological Methods 294 (2004) 15–22
the ensuing adaptive immune response (Moretta et al.,
2002). While NK cells are present predominantly in
the peripheral circulation, they home to tissues
following chemoattractant release by infected or
malignant cells (Moretta et al., 2002).
NK cells contain high concentrations of preformed
cytolytic granules in their cytoplasm as they circulate
in the periphery (Cooper et al., 2001). These lytic
vesicles contain a number of cytolytic proteins such as
perforin and granzyme uniquely designed to induce
death in target cells upon release (Cooper et al., 2001;
Burkhardt et al., 1989; Tschopp and Nabholz, 1990).
Subsequent to activation, following the integration of
complex signals from both activating and inhibitory
receptors on the surface of these cells, NK cells
rapidly release these granules at the immunological
synapse inducing death of the target cell (Cooper et
al., 2001; Moretta et al., 2002; Cerwenka and Lanier,
2001). Lining the membrane of these cytolytic
granules is the lysosomal-associated membrane pro-
tein-1 (LAMP-1 or CD107a) (Winchester, 2001;
Peters et al., 1991). This and other LAMP family
member proteins are highly glycosylated membrane
proteins that represent approximately 50% of the
proteins in the lysosomal membrane (Fukuda, 1991;
Kannan et al., 1996). Members of the LAMP family
have short cytosolic tails, which are thought to
interact with trans Golgi mediators that are involved
in sorting and targeting proteins to the lysosomal
pathway (Winchester, 2001). The highly glycosylated
portion of the molecule on the luminal side of the
vesicle has also been proposed to be involved in
protecting the cellular membrane from attack by the
lytic enzymes contained in the granules, and thus may
subsequently protect the extracellular membrane of
the effector cell following degranulation (Fukuda,
1991). Yet their precise function is still largely
unclear. Recently, CD107a expression on the cell
surface has been described as a marker of cytotoxic
CD8+ T-cell degranulation and was shown to be
strongly upregulated on the cell surface following
stimulation in concordance with a loss of perforin
(Betts et al., 2003).
Given the strong cytotoxic capacity of NK cells,
we chose to investigate the potential role of
CD107a as a marker of NK cell activation and
function. We demonstrate that CD107a is signifi-
cantly upregulated on the surface of NK cells
following stimulation with MHC devoid target cells
and following phorbol-12-myristate-13-acetate/iono-
mycin stimulation. This marker is expressed within
2 h of stimulation, and correlates strongly with both
cytokine secretion and NK cell-mediated lysis of
target cells.
2. Materials and methods
2.1. Study subjects
Peripheral blood mononuclear cells were isolated
from whole blood by Ficoll-Hypaque density gradient
centrifugation from 12 healthy volunteers. Each
subject gave informed consent for participation in
the study.
2.2. Cell lines
The K562 cell line, a highly undifferentiated
human erythroleukemic cell line which does not
express MHC class 1 molecules, was provided by
the American Type Culture Collection (ATCC;
Manassas, VA) and maintained in RPMI 1640 (Sigma,
St. Louis, MO) containing 10% FBS (Atlanta Bio-
logicals, Norcross, GA), 2 mM -glutamine (Cellgro,
Herndon, VA), 50 IU/ml penicillin (Cellgro) at
0.25 106 cells/ml.
2.3. Multi-parameter flow cytometry
The frequency of degranulating and cytokine
secreting NK cells was quantitated by multi-param-
eter intracellular cytokine staining. PBMCs were
resuspended at 106 cells/ml in RPMI 1640 (Sigma)
containing 10% FBS (Atlanta Biologicals), 2 mM -
glutamine (Cellgro), 50 IU/ml penicillin (Cellgro).
Cells were then stimulated with MHC devoid, K562
cells (ATCC), at an effector to target ratio of 10:1,
medium alone served as the negative control. Cells
were stimulated with phorbol-12-myristate-13-acetate
(PMA) (2.5 g/ml) and ionomycin (0.5 g/ml)
(Sigma) as a positive control. CD107a-PeCy5 anti-
body (BD Biosciences, San Jose, CA) was added
directly to the tubes at 20 l/ml. Cells were
incubated for 1 h at 37 C in 5% CO2 after which
brefeldin A (Sigma) was added at a final concen-
 G. Alter et al. / Journal of Immunological Methods 294 (2004) 15–22
tration of 10 g/mL as well as 6 l of monensin
(Golgi-Stop, BD Biosciences) at a final concentration
of 6 g/mL and incubated for an additional 5 h at 37
C in 5% CO2. While brefeldin A prevents the
exocytosis of cytokine containing vesicles allowing
for the visualization of cytokine production follow-
ing stimulation, monensin prevents the acidification
of endocytic vesicles avoiding the degradation of
reinternalized CD107a proteins from the surface and
allowing for the visualization of this marker
following stimulation. PBMCs were stained for
surface NK cell markers CD56-PECy7 (BD Bio-
sciences) and CD3-PECy5.5 (Caltag, Burlingame,
CA) for 30 min. Samples were then fixed and
permeabilized according to manufacturer’s directions
(Caltag), and stained for intracellular interferon-g
(IFN-g)-APC and tumor necrosis factor-a (TNF-a)-
PE (BD Biosciences) for an additional 30 min.
After washing, cells were resuspended in 1%
paraformaldehyde (Sigma) until five-color flow
cytometric analysis was performed on a LSRII
instrument (BD Biosciences). A total of 50,000 to
250,000 events were acquired and analyzed using
FlowJo software. The analysis was performed on
gated cells that fell within the lymphocyte popula-
tion. We then gated on the CD3 /CD56+ popula-
tion. Within this population of cells, we noted the
independent expression of CD107a, IFN-g, or TNF-
a for each sample following stimulation. A response
was considered positive if the frequency of CD107a
expressing or cytokine secreting CD3 /CD56+ cells
following stimulation with MHC devoid targets was
at least three-fold greater than in unstimulated
controls.
2.4. Chromium release cytotoxicity assay
The ability of NK cells to lyse MHC devoid
target cells was examined using a standard chromium
release cytotoxicity assay (Kiessling et al., 1975).
Two million K562 cells were labeled with 50 Ci
Na2(51Cr04) (1 Ci=37 GBq; New England Nuclear)
for 1 h at 37 C, 5% CO2. PBMCs were added as
effectors at E/T ratios of 10:1 and 50:1. Supernatant
was harvested after a 4-h incubation at 37 C and
5% CO2. For E/T titration assays, effectors were
plated at 1:1, 5:1, 10:1, 25:1, 50:1, and 100:1 for the
4-h incubation. For the time course experiment,
17
PBMCs and 51Cr labeled K562 cells were plated at
10:1 and 50:1 and were incubated for variable
lengths of time. The percent lysis was calculated as
[((sample count spontaneous release)/(maximal
release spontaneous release)) 100].
2.5. Statistical analysis
Unpaired T-tests were employed to assess differ-
ences between the level of NK activity at different
time points for each functional marker. Spearman
correlation was used to examine the relation between
CD107a expression and a number of NK cell func-
tional responses following stimulation with MHC
devoid targets. P-values of less then 0.05 were
considered significant.
3. Results
3.1. CD107a is expressed at high levels on NK cells
following stimulation
Recently, CD107a has been shown to be a marker
for cytotoxic CD8+ T-cell activity as its expression
was associated with a loss of perforin following
antigen stimulation (Betts et al., 2003). Although NK
cells are also cytolytic effector cells, the role of this
marker has not been addressed for this subset of
lymphocytes. Here, we assessed whether CD107a
was expressed on the surface of NK cells following
stimulation. Freshly isolated PBMCs were incubated
with MHC devoid target cells or PMA/ionomycin.
Following a 6-h incubation in the presence of
monensin and CD107a antibody, NK cells were
stained for CD3 and CD56. Representative data from
one subject is shown in Fig. 1A–C. Surface
expression of CD107a was low in unstimulated NK
cells (0.33%) (Fig. 1A). Following stimulation with
MHC devoid target cells, CD107a expression on the
surface of NK cells increased 52-fold, resulting in
17.4% of CD3 CD56+ NK cells expressing CD107a
(Fig. 1B). Following maximal stimulation of NK
cells with PMA/ionomycin, CD107a expression on
the surface of NK cells was up-regulated 88-fold,
reaching frequencies of 29.17% of NK cells (Fig.
1C). Overall results for CD107a expression in the 12
subjects tested are shown in Fig. 1D. There was an
18 G. Alter et al. / Journal of Immunological Methods 294 (2004) 15–22

Fig. 1. CD107a is expressed at high levels on the surface of NK cells following stimulation. Flow cytometry figures represent the percent
positive CD3 /CD56+ cells that express CD107a following no stimulation (A), stimulation with K562 cells (B) or with PMA/ionomycin (C) for
a single representative subject. The dot plot represents the percent CD3 /CD56+ NK cells that express CD107a following no stimulation ( ),
stimulation with K562 cells ( ) or PMA/ionomycin ( ) for all subjects tested in this study (D).

average 25-fold increase in CD107a expression
compared to baseline activity following co-incuba-
tion of NK cells with MHC devoid K562 cells for all
subjects tested ( p 0.001, Student’s T-test as com-
pared to baseline CD107a expression). Following
PMA/ionomycin stimulation, we observed an average
32-fold increase in CD107a expression, reaching an
average frequency of 21.5% ( p 0.001, Student’s T-
test as compared to baseline CD107a expression)
(Fig. 1D). Taken together, these data demonstrate that
CD107a is significantly upregulated on the surface of
NK cells following activation.
Traditionally, one means by which NK cell
functional activity can be quantitated is via intra-
cellular cytokine secretion profiles following stim-
ulation. Thus we next characterized the distribution
of NK cells that expressed CD107a with respect to
those that secreted IFN-g and TNF-a following
stimulation with K562 cells. As few as 0.66% of
IFN-g positive NK cells were not also expressing
CD107a following stimulation, and the number of
TNF-a positive NK cells not expressing CD107a was
negligible (0.08%). In contrast, significantly more
NK cells expressed CD107a following stimulation
than either IFN-g or TNF-a (Fig. 2A). Of the
CD107a+CD3-CD56+ NK cells, an average of 66%
expressed CD107a alone, 11% expressed both TNF-
a and CD107a, 13% expressed CD107a and IFN-g,
and 10% of NK cells expressed all three functional
markers (Fig. 2B). Taken together, these data
demonstrate that CD107a represents a more compre-
hensive marker of NK activity as it is expressed on a
larger proportion of functionally active cells that are
not detected using simple assessment of cytokine
secretion.
3.2. Kinetics of CD107a expression on NK cells
following stimulation
Following the demonstration that CD107a stain-
ing allows for the identification of a significantly
larger fraction of activated NK cells than intra-
cellular cytokine staining, we studied the kinetics by
which this marker is expressed on activated NK