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IMMUNOBIOLOGY

Regulation of human NK-cell cytokine and chemokine production by target cell

recognition
Cyril Fauriat,1 Eric O. Long,2 Hans-Gustaf Ljunggren,1 and Yenan T. Bryceson1
1Center
for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; and 2Laboratory of
Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD

Natural killer (NK)–cell recognition of in-
fected or neoplastic cells can induce cyto-
toxicity and cytokine secretion. So far, it
has been difficult to assess the relative
contribution of multiple NK-cell activa-
tion receptors to cytokine and chemokine
production upon target cell recognition.
Using Drosophila cells expressing li-
gands for the NK-cell receptors LFA-1,
NKG2D, DNAM-1, 2B4, and CD16, we stud-
ied the minimal requirements for secre-
tion by freshly isolated, human NK cells.
Target cell stimulation induced secretion
of predominately proinflammatory cyto-
kines and chemokines. Release of chemo-
kines MIP-1␣, MIP-1␤, and RANTES was
induced within 1 hour of stimula-
tion, whereas release of TNF-␣ and IFN-␥
occurred later. Engagement of CD16, 2B4,
or NKG2D sufficed for chemokine re-
lease, whereas induction of TNF-␣ and
IFN-␥ required engagement of additional
receptors. Remarkably, our results re-
vealed that, upon target cell recognition,
CD56dim NK cells were more prominent
cytokine and chemokine producers than
CD56bright NK cells. The present data dem-
onstrate how specific target cell ligands
dictate qualitative and temporal aspects
of NK-cell cytokine and chemokine re-
sponses. Conceptually, the results point
to CD56dim NK cells as an important
source of cytokines and chemokines upon
recognition of aberrant cells, producing
graded responses depending on the mul-
tiplicity of activating receptors engaged.
(Blood. 2010;115:2167-2176)

Introduction
Natural killer (NK) cells respond directly to infected or neoplastic
cells through engagement of a multitude of germline-encoded
receptors by ligands on target cells.1-3 Beside their ability to kill
aberrant cells, NK cells are also critical components of the innate
immune response by virtue of their capacity to produce a variety of
cytokines and chemokines.4-6 Murine models have demonstrated a
dependence on NK cell–derived cytokines in early responses to
obligate intracellular parasites such as Listeria, Toxoplasma, and
Leishmania and in resistance to cytomegalovirus infection.7-10 In
many of these systems, NK cells respond to cues from sentinel
immune cells, including dendritic cells, macrophages, and pathogen-
infected tissue cells.11-13 These cues are communicated by release
of cytokines, including interleukin-1 (IL-1), IL-10, IL-12, IL-15,
and IL-18.14 Thus, secondary to triggering of innate immune cells
by pattern recognition receptors, NK cells can relay and amplify
cytokine signals. Among the most prominent cytokines produced
by NK cells are tumor necrosis factor-␣ (TNF-␣) and interferon ␥
(IFN-␥). Moreover, NK cells have been reported to secrete several
other factors, including immunoregulatory cytokines such as IL-5,
IL-10, IL-13, the growth factor GM-CSF, and the chemokines
MIP-1␣, MIP-1␤, IL-8, and RANTES.15-22
In humans, NK cells are usually defined as CD3–CD56⫹ cells,23
and can be further subdivided based on CD56 expression. Typi-
cally, CD56dim NK cells constitute the majority (90%) of peripheral
blood NK cells, whereas CD56bright NK cells are more abundant in
secondary lymphoid tissues.14,24 CD56dim NK cells express high
levels of the low-affinity Fc receptor CD16, display variegated
expression of several types of inhibitory receptors for MHC class I,
and express high levels of perforin. In contrast, CD56bright NK cells
express no or low levels of CD16, exclusively express the
inhibitory receptor CD94/NKG2A, and have 10-fold lower per-
forin expression than CD56dim NK cells.25-27 Because of these and
other findings, CD56dim and CD56bright NK-cell subsets are consid-
ered to be developmentally distinct and to occupy different
functional niches.12,28-30
Human NK-cell responses to exogenous cytokines have been
extensively studied.21 In contrast, relatively less is known with
respect to NK-cell cytokine and chemokine production upon target
cell recognition. For example, the full spectrum of cytokines
released by freshly isolated, resting NK cells upon target cell
recognition has not been fully characterized. Furthermore, the
minimal requirements for induction of cytokine secretion upon
engagement of specific ligands on target cells are not known. To
understand how NK cells may contribute to, and maybe even act as
primary initiators of, immune responses upon target cell recogni-
tion, studies on how receptor-ligand interactions dictate qualitative
and temporal aspects of cytokine and chemokine secretion are
important. Here, we have set out to study in detail cytokine and
chemokine production by human peripheral blood NK cells upon
target cell recognition.
To overcome the complexity in receptor-ligand interactions
between NK cells and target cells, we have developed a reconstitu-
tion system using Drosophila cells as targets.31 A notable advantage
of such a system is that cytokine and chemokine secretion by
primary, unmanipulated NK cells can be studied in the context of
specific receptor-ligand interactions. This system has recently

Submitted August 13, 2009; accepted November 3, 2009. Prepublished online
as Blood First Edition paper, December 1, 2009; DOI 10.1182/blood-2009-08-
238469.
A Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.

BLOOD, 18 MARCH 2010 䡠 VOLUME 115, NUMBER 11 2167

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2168 FAURIAT et al BLOOD, 18 MARCH 2010 䡠 VOLUME 115, NUMBER 11

revealed cooperation among NK-cell receptors for discrete events A NK cells

in cytotoxicity, including NK-cell cytolytic granule polarization 5000
NK cells + K562 cells
4000
and exocytosis.32,33

Secretion (pg/mL)
3000
Here, we addressed how specific engagement of the receptors 2000
1000
NKG2D (CD314), DNAM-1 (CD226), 2B4 (CD244), LFA-1 250
(CD11a/CD18), and CD16, or combinations thereof, regulate 200

cytokine and chemokine production by freshly isolated, resting 150

100
human NK cells. The data provide insight into the regulation of
50
cytokine and chemokine secretion by different NK-cell subsets 0
upon target cell recognition.

C CL /M P-1

S
C XC 8/ in
L1 9/M -8
G /IP G
-C 0
IL SF

-2
-4
-5
-6
IL IL- 7
2p 0
40
IL 3
IL 5
IF 7
C L2/ NF γ
C M -α

C L 5 / /M 1 α
6 N β

IL 2Rα
IL α

-α
β
C T N-

M -1

-
-1 1

-1
-1
-1
L2 A 1

R
-1
C /Eo TE
C CL tax

0 I
XC L IL

IL
IL
IL
IL
IL

N
C R IP
C 4 IP

-1

IL
IF

C L3 C

-
X
C
B 10000
Methods
Cells
1000

Secretion (pg/mL)
Human NK cells were isolated from peripheral blood by negative selection
(NK-cell isolation kit; Miltenyi Biotec). Approval was obtained from the
100
Regional Ethics Review Board for the use of peripheral blood mononuclear
IFN-γ
cells from healthy donors. CD56dim and CD56bright NK-cell subsets were
TNF-α
sorted based on CD56 expression by flow cytometry (FACSAria; BD
10 MIP-1α
Biosciences). Freshly isolated NK cells were maintained in complete
MIP-1β
medium (RPMI 1640 supplemented with 10% fetal bovine serum and 2mM
RANTES
L-glutamine; all from Invitrogen). NK-cell populations contained at least 1
99% CD3⫺CD56⫹ cells and were used within 2 days of isolation. The cell 0 1 2 3 4 5 6 7 8 9 10 11 12
line K562 (ATCC) was maintained in complete medium. The transfection Time (hrs)
and maintenance of Drosophila S2 cells has been previously described and Figure 1. Profile and kinetics of NK-cell secretion upon interaction with K562
expression of the ligands for NK receptors was monitored prior to cells. (A) Resting NK cells were incubated alone or with K562 cells for 6 hours at
experiments.31,33 A rabbit serum raised against S2 cells and used to coat 37°C. Supernatants were harvested, and the concentrations of indicated cytokines
S2 cells with IgG has been described.32 and chemokines were determined by a multiplex immunoassay. Values represent
mean ⫾ SD of 8 different donors. (B) NK cells were mixed with K562 cells and
incubated at 37°C. Supernatants were harvested at different time points, as
Antibodies and fluorescent reagents indicated, and the concentrations of IFN-␥, TNF-␣, MIP-1␤, MIP-1␤, and RANTES
were determined by a multiplex immunoassay. Values represent mean ⫾ SD of
Fluorochrome-conjugated monoclonal antibodies (mAbs) used for flow 5 different donors.
cytometry were anti-CD3 (clone UCHT1; Dako), anti-CD56 (clone NCAM
16.2; BD Biosciences), anti-CD107a (clone H4A3; BD Biosciences),
anti–MIP-1␣ (clone 93342; R&D Systems), anti–MIP-1␤ (clone D21- paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline, permeabil-
1351; BD Biosciences), anti–TNF-␣ (clone MP6-XT22; eBioscience), and ized, and stained intracellularly with fluorochrome-conjugated mAbs to
anti–IFN-␥ (clone 25723.11; BD Biosciences). Streptavidin-conjugated cytokines and chemokines. Finally, cells were washed and analyzed on a
Qdot 605 was used to detect biotinylated anti-CD107a and a fixable cell CyAn ADP LX 9-color flow cytometer (Dako).34 Data were analyzed with
viability dye (LIVE/DEAD 405 nm) was used to exclude dead cells from FlowJo 8.6 software (TreeStar). Pie charts were generated with SPICE
the analysis (both from Invitrogen). software, Version 4.1 (M. Roederer, Vaccine Research Center, National
Institute of Allergy and Infectious Diseases, National Institutes of Health).
Cytokine secretion measurements
Statistical analysis
Resting NK cells (2 ⫻ 105) were washed twice in and mixed with 4 ⫻ 105
S2 cells or K562 cells in 200 ␮L of complete medium. In experiments with All analyses were performed with GraphPad software (GraphPad Soft-
sorted cells, 1 ⫻ 105 CD56dim or CD56bright NK cells were mixed with ware). Statistical analyses of minimal requirements and comparisons
1 ⫻ 105 S2 cells. Cells were incubated for the indicated time at 37°C in between CD56dim and CD56bright responsiveness were performed using a
5% CO2. Thereafter, supernatants were collected and stored at ⫺20°C 2-way analysis of variance (nonparametric) with Bonferroni posttest.
pending measurement. The concentrations of cytokines were quantified by
a multiplex immunoassay (Luminex 100 IS; Invitrogen) using a kit that
detected 25 different cytokines (Biosource; listed in Figure 1A). To address Results
the kinetics of secretion of select cytokines, 5 simplexes (for IFN-␥, TNF-␣,
RANTES, MIP-1␣, and MIP-1␤) were mixed according to the manufactur- Secretion profile of resting NK cells upon interaction with K562
er’s instructions (Biosource). cells

Intracellular staining of cytokines
Resting NK cells (2 ⫻ 105) were added to 4 ⫻ 105 S2 cells in 200 ␮L of
complete medium. IL-12, IL-15, and IL-18 (all from Peprotech) were added
in some experiments. Cells were incubated for 1 hour at 37°C in 5% CO2.
Thereafter, Brefeldin A (GolgiPlug; Becton Dickinson) was added to the
cultures, which were incubated for 5 more hours. After 6 hours of incuba-
tion, the cells were spun down and resuspended in 50 ␮L of staining buffer
(phosphate-buffered saline supplemented with 2% fetal bovine serum and
2mM ethylenediaminetetraacetic acid) added fluorochrome-conjugated
mAbs for surface staining. Thereafter, cells were washed, fixed with 2%
We first set out to determine the factors released by freshly isolated
peripheral blood NK cells upon short incubations with susceptible
K562 target cells. Resting NK cells were mixed with K562 cells for
6 hours. Supernatants were harvested and the concentration of
25 different soluble factors was determined by a multiplex
immunoassay (Figure 1A). As anticipated, stimulation of NK cells
by K562 cells induced IFN-␥ and TNF-␣ secretion. In addition,
stimulation induced ample secretion of several chemokines, includ-
ing MCP-1 (CCL2), MIP-1␣ (CCL3), MIP-1␤ (CCL4), RANTES
(CCL5), IL-8 (CXCL8), and IP-10 (CXCL10). Low levels of
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BLOOD, 18 MARCH 2010 䡠 VOLUME 115, NUMBER 11 NK-CELL RESPONSES TO TARGET CELL RECOGNITION 2169

Figure 2. Minimal requirements for receptor-ligand 300

interactions for secretion of cytokines and chemo- IFN-γ
kines by NK cells. Resting NK cells were mixed with *
S2 cells expressing ligands for NK-cell receptors, as 200
indicated, and incubated for 6 hours at 37°C. For some
stimulations, S2 cells were preincubated with diluted
*
100 *
anti–S2 cell serum (⫹ IgG). Supernatants were har-
vested, and the concentrations of cytokines and chemo-
kines were determined by a multiplex immunoassay. 0
Values represent mean ⫾ SD of 5 different donors. Data 800
are representative of 3 independent experiments. For TNF-α
clarity, selected statistical analyses are indicated. 600 *
*P ⬍ .05, ***P ⬍ .001.
400 *
200

3500
Secretion (pg/mL)
3000 MIP-1α
2500
2000 ***
*
1500
1000
*
500
0
12500
MIP-1β
10000 ***
***
7500

5000
***
2500 ***
0
2500
RANTES
2000

1500 ***
1000
***

500

0 – S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2
ICAM1 CD48 ULBP1 CD155 ICAM1 ICAM1 ICAM1 CD48 ICAM1 ICAM1 CD48 ULBP1 CD155 ICAM1 ICAM1 ICAM1 CD48 ICAM1
CD48 ULBP1 CD155 ULBP1 CD48 CD48 ULBP1 CD155 ULBP1 CD48
ULBP1 ULBP1
+ + + + + + + + + +
IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG

chemokines MIP-1␣, MIP-1␤, and RANTES were also detected in
supernatants from NK cells incubated without target cells, suggest-
ing a degree of constitutive secretion. However, incubation with
K562 cells resulted in a 44-, 181-, and 6.5-fold increase in secretion
of MIP-1␣, MIP-1␤, and RANTES, respectively, compared with
supernatants from NK cells alone. Notably, stimulation of NK cells
by K562 cells also induced release of soluble IL-2R␣ (CD25).
GM-CSF, IL-5, and IL-13 were weakly or not secreted after
interaction of NK cells with K562 cells, contrasting previous
studies using cytokine-cultured NK cells.15,17 Low levels of IL-1␤,
IL-6, IL-7, IL-10, IL-12p40, IFN-␣, and MIG (CXCL9) secretion
were measured after target cell stimulation. Secretion of IL-2, IL-4,
IL-5, IL-13, IL-15, IL-17, and eotaxin (CCL26) was not detected.
Next, the temporal profile of cytokine and chemokine secretion
was analyzed (Figure 1B). Chemokine secretion could be detected
within 1 hour of mixing NK cells with K562 cells. In contrast,
TNF-␣ and, in particular, IFN-␥ secretion occurred later. After
6 hours of incubation, the highest levels of secreted cytokines were
measured. Thereafter the concentrations of chemokines remained
stable throughout the time course, whereas IFN-␥ and TNF-␣
concentrations, if anything, decreased.
Thus, interactions with a susceptible target cell line can induce a
profoundly proinflammatory profile of chemokine and cytokine
secretion. Moreover, kinetic studies revealed a rapid release of
chemokines, relative to cytokine secretion.
Minimal receptor engagement requirements for secretion of
proinflammatory cytokines and chemokines by resting NK cells
Having identified soluble factors that were secreted by resting NK
cells upon interaction with a susceptible target cell, the contribution
of individual receptor-ligand interactions to cytokine and chemo-
kine secretion was assessed. Resting NK cells were mixed with
Drosophila Schneider 2 (S2) cells expressing ICAM-1 (CD54),
CD48, ULBP1, and CD155 (which bind receptors LFA-1, 2B4,
NKG2D, and DNAM-1, respectively), or combinations thereof,
and incubated for 6 hours. In these experiments, the concentrations
of IFN-␥, TNF-␣, MIP-1␣, MIP-1␤, and RANTES were deter-
mined using a multiplex immunoassay (Figure 2). Secretion of
TNF-␣ required coexpression of CD48 and ULBP1 on S2 cells,
whereas IFN-␥ required combined expression of ICAM-1, CD48,
and ULBP1. In contrast, expression of CD48 was sufficient to
induce secretion of chemokines MIP-1␣, MIP-1␤, and RANTES.
 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2170 FAURIAT et al
Coexpression of CD48 and ULBP1 on S2 cells did not enhance
secretion of chemokines compared with S2 cells expressing CD48
alone. However, expression of ICAM-1, in addition to CD48 and
ULBP1, on S2 cells could augment chemokine secretion, as
observed for IFN-␥. To engage CD16 on NK cells, S2 cells were
preincubated with an anti–S2 cell polyclonal serum.32 IgG coating
of S2 cells was sufficient to induce significant TNF-␣ secretion, but
much less IFN-␥ secretion (Figure 2). In regard to IFN-␥ secretion,
expression of ICAM-1 or CD48 on S2 cells augmented IgG-
mediated secretion. Chemokine secretion was readily induced by
IgG-coated S2 cells. Expression of ICAM-1 or CD48 on S2 cells
could augment IgG-induced chemokine secretion.
Thus, in the absence of IgG, engagement of 2B4 was sufficient
to induce chemokine secretion, whereas stronger signals evoked by
2B4 and NKG2D were required for secretion of TNF-␣ or IFN-␥.
Coengagement of LFA-1 augmented cytokine and chemokine
secretion induced by 2B4 and NKG2D. CD16 engagement could
induce secretion of chemokines and cytokines. CD16-mediated
secretion of IFN-␥ and chemokine secretion, but not TNF-␣, was
augmented by LFA-1 coengagement.
Longer time points were also assessed and secretion patterns
resembled that induced by K562 cell stimulation (supplemental
Figure 1, available on the Blood website; see the Supplemental
Materials link at the top of the online article). Notable features were
a slight induction of chemokines by S2 cells expressing ULBP1, as
previously described with cytokine-cultured NK cells,35 and that
costimulation could accelerate chemokine secretion.
A range of S2 cell combinations engaging several activating
receptors was assessed for the 25 factors listed in Figure 1A.
Corroborating findings with NK-cell stimulation by K562 cells,
profiles of secreted cytokines and chemokines were proinflamma-
tory (data not shown). Thus, NK-cell activation by CD16, 2B4, and
NKG2D, in addition to costimulation by DNAM-1 and LFA-1,
induced secretion of proinflammatory, but not immunoregulatory,
cytokines.
CD56dim NK cells are major producers of cytokines and
chemokines upon target cell recognition
Whereas analysis of supernatants from cell-mixing experiments
identified the predominant chemokines and cytokines secreted by
freshly isolated NK cells in response to target cell recognition, a
different approach was required to identify the phenotypic features
of the cytokine- and chemokine-producing cells, to quantify the
frequency of responding cells, and to examine to what extent
individual cells could produce multiple soluble factors. To this end,
multicolor flow cytometry was performed. Production of IFN-␥,
TNF-␣, MIP-1␣, and MIP-1␤ was compared in CD56dim and
CD56bright NK-cell subsets after 6 hours of incubation with differ-
ent S2 cells (Figure 3A). Coexpression of ICAM-1, CD48, and
ULBP1 on S2 cells was required to induce IFN-␥ production by
CD56dim NK cells. Expression of CD48 on S2 cells was sufficient
to induce TNF-␣ production. Interestingly, despite LFA-1, 2B4,
and NKG2D being expressed at similar levels on different NK-cell
subsets (Bryceson et al36; data not shown), very few CD56bright NK
cells expressed IFN-␥ or TNF-␣ after incubation with target cells
expressing ligands for these receptors. Expression of CD48 or
ULBP1 on S2 cells was sufficient to induce MIP-1␣ and MIP-1␤
production by CD56dim NK cells (Figure 3A and supplemental
Figure 2), corroborating results quantifying chemokine secretion.
CD56bright NK cells did produce some MIP-1␣ and MIP-1␤ upon
stimulation by S2 cells coexpressing CD48 and ULBP1. However,
a consistently higher frequency of CD56dim NK cells produced
BLOOD, 18 MARCH 2010 䡠 VOLUME 115, NUMBER 11
chemokines upon such stimulations. For example, CD48 expres-
sion on S2 cells induced MIP-1␣ production in 9% (⫾ 6%) of
CD56bright NK cells, as opposed to 43% (⫾ 7%) of CD56dim NK
cells. Thus, strikingly, upon target cell recognition CD56dim NK
cells were more prominent chemokine and cytokine producers than
CD56bright NK cells. Strengthening this notion, greater proportions
of CD56dim NK cells than CD56bright NK cells produced IFN-␥,
TNF-␣, MIP-1␣, and MIP-1␤ upon K562 cell stimulation (Figure
3B). Moreover, experiments comparing cytokine and chemokine
secretion from equal numbers of sorted CD56dim and CD56bright NK
cells separately incubated with K562 cells or various S2 cell
transfectants also showed greater secretion by CD56dim NK cells
(Figure 3C and data not shown).
Ig-coated S2 cells were sufficient to induce TNF-␣ and IFN-␥
production by CD56dim, but not CD56bright, NK cells (supplemental
Figures 2-3). However, Ig-coated S2 cells induced 29% (⫾ 11%)
MIP-1b⫹ CD56bright NK cells, demonstrating that engagement of
CD16 can elicit responses by a subset of CD56bright NK cells
(supplemental Figure 3).
Reports defining CD56bright NK cells as the major source of NK
cell–derived cytokines have relied on exogenous cytokines for
stimulation.26 To ensure that the low cytokine response of CD56bright
NK cells upon target cell recognition was not an artifact of our
experimental procedure, we evaluated the response of freshly
isolated NK cells to 24 hours of stimulation with cytokines IL-12,
IL-15, or IL-18, or combinations thereof (Figure 3D). These
cytokines induce strong IFN-␥ secretion by CD56bright NK cells in
72-hour assays.26 In agreement with other reports, a greater
frequency of CD56bright NK cells, relative to CD56dim NK cells,
produced IFN-␥ and TNF-␣ in response to stimulation by exog-
enous cytokines. In comparison, production of MIP-1␣ and MIP-1␤
was equal or greater in CD56dim NK cells. Next, we tested whether
the greater frequency of cytokine-producing CD56bright NK cells
after 24 hours of stimulation could reflect temporal differences in
responsiveness between the 2 subsets. After 6 hours of stimulation,
somewhat higher frequencies of CD56bright NK cells, relative to
CD56dim NK cells, produced IFN-␥ and TNF-␣ in response to
IL-12 plus IL-18 (Figure 4). Remarkably, as with 24 hours of
stimulation, production of MIP-1␣ and MIP-1␤ was, if anything,
more pronounced in CD56dim NK cells relative to CD56bright NK
cells. Moreover, after 24 hours of stimulation with K562 cells,
the frequency of cytokine- and chemokine-producing CD56bright
NK cells was still lower than that of CD56dim NK cells (data not
shown). Together, these results suggest that CD56bright NK cells
are not inherently slower to respond to target cell recognition.
Rather, CD56dim NK cells excel in cytokine and chemokine
production upon recognition of target cells. Furthermore, a
greater proportion of the CD56dim than the CD56bright subset of
NK cells produced chemokines in response to exogenous
cytokine stimulation.
IL-12 plus IL-18 potentiate cytokine secretion upon target cell
recognition by CD56dim NK cells
Studies have shown that cytokines, such as IL-2, IL-12, IL-15, and
IL-21, can enhance IFN-␥ and chemokine secretion induced by
target cell recognition.20,22,37-39 Therefore, we examined how exog-
enous cytokine stimulation by IL-12 plus IL-18 influenced NK-cell
production of IFN-␥, TNF-␣, MIP-1␣, and MIP-1␤ upon interac-
tion with K562 cells (Figure 4). Addition of IL-12 and IL-18
augmented the frequency of IFN-␥–producing CD56dim NK cells
after 6 hours of incubation with K562 cells. Interestingly, IL-12
and IL-18 did not significantly increase MIP-1␣ and MIP-1␤
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BLOOD, 18 MARCH 2010 䡠 VOLUME 115, NUMBER 11 NK-CELL RESPONSES TO TARGET CELL RECOGNITION 2171

A
30
CD56dim NK cells
IFN-γ TNF-α ***
20 CD56bright NK cells
***
Positive cells (%) ***
10 ** *
0

100 MIP-1α MIP-1β

80 *** ***
60 *** ***
40 **
20 *
0 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2
CD48 CD48 ICAM1 CD48 CD48 ICAM1 CD48 CD48 ICAM1 CD48 CD48 ICAM1
ULBP1 CD48 ULBP1 CD48 ULBP1 CD48 ULBP1 CD48
ULBP1 ULBP1 ULBP1 ULBP1

B 100
MIP-1β
Positive cells (%)

IFN-γ TNF-α MIP-1α ***

80 ***
***
60 ***
40 **
*
20

0
– K562 – K562 – K562 – K562 – K562 – K562 – K562 – K562
C
500 500 1500 5000
Secretion (pg/mL)

IFN-γ TNF-α MIP-1α MIP-1β

400 400 4000
1000
300 300 3000

200 200 2000

500
100 100 1000

0
– K562 – K562 – K562 – K562 – K562 – K562 – K562 – K562

D
100
IFN-γ TNF-α MIP-1α MIP-1β
Positive cells (%)

80

60

40

20

0 – – – – – – – –
IL-12
IL-15
IL-18
IL-12+IL-15
IL-15+IL-18
IL-12+IL-18

IL-12
IL-15
IL-18
IL-12+IL-15
IL-15+IL-18
IL-12+IL-18

IL-12
IL-15
IL-18
IL-12+IL-15
IL-15+IL-18
IL-12+IL-18

IL-12
IL-15
IL-18
IL-12+IL-15
IL-15+IL-18
IL-12+IL-18

IL-12
IL-15
IL-18
IL-12+IL-15
IL-15+IL-18
IL-12+IL-18

IL-12
IL-15
IL-18
IL-12+IL-15
IL-15+IL-18
IL-12+IL-18

IL-12
IL-15
IL-18
IL-12+IL-15
IL-15+IL-18
IL-12+IL-18

IL-12
IL-15
IL-18
IL-12+IL-15
IL-15+IL-18
IL-12+IL-18

Figure 3. CD56dim NK cells produce cytokines and chemokines upon target cell recognition. Resting NK cells were mixed with S2 cells expressing ligands for NK-cell
receptors (A) or K562 cells (B), as indicated, and incubated for 6 hours at 37°C. After stimulation, the cells were surface stained with fluorochrome-conjugated anti-CD56 mAb,
fixed, permeabilized, and stained intracellularly with fluorochrome-conjugated mAbs to cytokines and chemokines. The percentage of CD56dim or CD56bright NK cells producing
IFN-␥, TNF-␣, MIP-1␣, and MIP-1␤, as indicated, was determined by flow cytometry. Values represent mean ⫾ SD of at least 6 different donors. (C) Sorted CD56dim or
CD56bright NK cells were incubated alone or with K562 cells for 6 hours at 37°C. Supernatants were harvested, and the concentrations of cytokines and chemokines were
determined by a multiplex immunoassay. Values represent mean ⫾ SD of 5 different donors. (D) Resting NK cells were incubated alone or stimulated with 10 ng/mL IL-12,
100 ng/mL IL-15, or 100 ng/mL IL-18, or combinations thereof, for 24 hours at 37°C. After stimulation, the cells were surface stained with fluorochrome-conjugated anti-CD56
mAb, fixed, permeabilized, and stained intracellularly with fluorochrome-conjugated mAbs to cytokines and chemokines. The percentage of CD56dim or CD56bright NK cells
producing IFN-␥, TNF-␣, MIP-1␣, and MIP-1␤, as indicated, was determined by flow cytometry. Values represent mean ⫾ SD of 5 different donors. For clarity, selected
statistical analyses are indicated. *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001.

production by CD56dim NK cells in response to K562 cells. K562 pathways for cytokine and chemokine production in CD56bright and
cells alone did not induce cytokine and chemokine production by CD56dim NK-cell subsets.
CD56bright NK cells, and addition of IL-12 and IL-18 did not
increase the frequencies of cytokine- and chemokine-producing Costimulation signals lower the threshold for CD16-induced
CD56bright NK cells relative to IL-12 plus IL-18 alone. In conclu- NK-cell cytokine and chemokine production
sion, exogenous cytokines potentiated cytokine production by
CD56dim NK cells in response to K562 cells. In such settings, Next, we analyzed how the strength of stimulation through
chemokine production by CD56dim NK cells could be ascribed increased CD16 engagement affected the frequency of cytokine-
mainly to target cell recognition, whereas cytokine and chemokine and chemokine-producing CD56dim NK cells (Figure 5). By
production by CD56bright NK cells could be ascribed to exogenous varying the concentration of anti-S2 serum with which S2 cells
cytokines. These data suggest different requirements and signaling were coated, the density of ligands for CD16 could be modulated
 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

2172 FAURIAT et al BLOOD, 18 MARCH 2010 䡠 VOLUME 115, NUMBER 11

CD56dim NK cells Figure 4. Costimulation of NK cells by exogenous

CD56bright NK cells cytokines enhances cytokine production by CD56dim
100 NK cells upon interaction with K562 cells. Resting NK
IFN-γ TNF-α MIP-1α MIP-1β cells were incubated alone or stimulated with cytokines
***
Positive cells (%)

80 IL-12 and IL-18 (10 ng/mL and 100 ng/mL, respectively)

for 6 hours at 37°C with or without K562 cells. After stimula-
*** ***
60 tion, the cells were surface stained with fluorochrome-
* conjugated anti-CD56 mAb, fixed, permeabilized, and
***
40 stained intracellularly with fluorochrome-conjugated mAbs
*** * **
* * to cytokines and chemokines. The percentage of CD56dim
20 or CD56bright NK cells producing IFN-␥, TNF-␣, MIP-1␣,
and MIP-1␤, as indicated, was determined by flow cytom-
0 etry. Values represent mean ⫾ SD of 7 different donors.
IL-12+IL-18 – + – + – + – + – + – + – + – + – + – + – + – + – + – + – + – +
For clarity, selected statistical analyses are indicated.
K562 – – + + – – + + – – + + – – + + – – + + – – + + – – + + – – + +
*P ⬍ .05, **P ⬍ .01, ***P ⬍ .001.

on target cells.32 Experiments assessing degranulation of CD56dim
NK cells upon modulation of IgG density on target cells produced
similar results as have been reported previously (supplemental
Figure 4; Bryceson et al32). Increasing the concentration of IgG
augmented cytokine and chemokine responses in a nonlinear
fashion. Expression of CD48 or ICAM-1 on S2 cells lowered the
concentrations of IgG required to augment the frequency of
cytokine- and chemokine-producing CD56dim NK cells, with
coexpression of CD48 and ICAM-1 on S2 cells providing the
greatest response to low concentrations of IgG. NK-cell cytokine
and chemokine responses reached a plateau at the highest concen-
trations of IgG. At a 10⫺3 dilution of anti-S2 serum, coexpression
of ICAM-1 and CD48 increased the frequency of IFN-␥– and
TNF-␣–producing CD56dim NK cells 2.8- and 2.3-fold, respec-
tively. Nonlinear regression analysis revealed that the half-
maximal percentage of IFN-␥–producing CD56dim NK cells in-
duced by CD16 engagement alone could be achieved with 10- and
63-fold lower IgG concentration when S2 cells expressed CD48 or
ICAM-1 and CD48, respectively (data not shown). S2 cells
expressing ICAM-1 achieved the half-maximal level of response of
untransfected S2 cells with a 3-fold lower concentration of IgG.
Similar observations were made for TNF-␣ secretion. In addition,
the half-maximal MIP-1␣ or MIP-1␤ responses could be reached
with 7- and 10-fold lower concentrations of IgG upon S2 cell
expression of ICAM-1 and CD48, respectively. Thus, coengage-
ment of different activating receptors can lower the activation
threshold for CD56dim NK-cell cytokine and chemokine produc-
tion, increasing the frequency of responding cells even in the
context of high ligand densities for powerful NK cell–activating
receptors such as CD16.
Interrelationships between different NK-cell responses induced
by target cell recognition
In addition to determining the frequency and phenotype of
responding cells, multicolor flow cytometric analysis can also
provide information on how many different responses an individual
cell can mediate. To gain insight into the relationships between
different NK-cell responses and to what extent individual cells can
perform multiple responses, intracellular expression of TNF-␣,
IFN-␥, MIP-1␣, and MIP-1␤ and surface expression of CD107a
were simultaneously assessed on CD56dim NK cells (Figure 6).
Surface expression of CD107a (lysosome-associated membrane
protein-1) is a marker of lytic granule exocytosis.32 In line with
previous findings, untransfected S2 cells did not induce cytokine or
chemokine expression, and CD107a was not expressed on the cell
surface (Figure 6A). Stimulation of resting NK cells by S2 cells
expressing ICAM-1, CD48, and ULBP1 induced intracellular
expression of TNF-␣, IFN-␥, MIP-1␣, and MIP-1␤ and surface
expression of CD107a on subsets of CD56dim NK cells (Figure 6A).
The greatest frequency of responding cells was consistently
observed upon staining with MIP-1␤, closely followed by that of
MIP-1␣. Expression of MIP-1␤ correlated strongly with expres-
sion of MIP-1␣. Expression of TNF-␣, IFN-␥, and CD107a was
confined to a MIP-1␤⫹ NK-cell subset. CD107a expression did not
necessarily correlate with expression of TNF-␣ or IFN-␥. How-
ever, IFN-␥ expression was contained mostly within a TNF-␣⫹
NK-cell subset. Thus, the data demonstrate specific interrelation-
ships and a considerable degree of cellular overlap among different
responses enacted by CD56dim NK cells. For example, IFN-␥

30 40
IFN-γ TNF-α
30
20
20
10
Positive cells (%)

10

Figure 5. Increasing activating ligand density on 0 0

target cells augments the frequency of cytokine- and 10–9 10–8 10–7 10–6 10–5 10–4 10–3 10–2 10–9 10–8 10–7 10–6 10–5 10–4 10–3 10–2
chemokine-producing CD56dim NK cells. Before mix- 100 100
ing with NK cells, S2 cells expressing ligands for NK-cell MIP-1α MIP-1β
receptors, as indicated, were preincubated with serial 80 80
dilutions of anti–S2 cell serum. Resting NK cells were
incubated with S2 cells for 6 hours at 37°C. After stimula- 60 60 S2
tion, the cells were surface stained with fluorochrome- S2–ICAM1
conjugated anti-CD56 mAb, fixed, permeabilized, and 40 40
stained intracellularly with fluorochrome-conjugated mAbs S2–CD48
20 20
to cytokines and chemokines. The percentage of CD56dim S2–ICAM1–CD48
NK cells producing IFN-␥, TNF-␣, MIP-1␣, and MIP-1␤,
0 0
as indicated, was determined by flow cytometry. Values
10–9 10–8 10–7 10–6 10–5 10–4 10–3 10–2 10–9 10–8 10–7 10–6 10–5 10–4 10–3 10–2
represent mean ⫾ SD of 3 different donors.
[IgG]
 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 18 MARCH 2010 䡠 VOLUME 115, NUMBER 11 NK-CELL RESPONSES TO TARGET CELL RECOGNITION 2173

A
30K 30K
3
10

20K 20K
SSC-H

FSC-A
2
10

CD56
1
10K 10K 10

0
Lymphocytes Singlets CD56dim NK cells
0 0
1 2 3
0 10K 20K 30K 0 10K 20K 30K 0 10 10 10
FSC-H FSC-H DCM Stimulation

1.7 4.7 5.8 0.7 6.3 0.2 5.79 0.7

3 3 3 3
10 10 10 10
MIP-1β

2 2 2 2
10 10 10 10
S2
1 1 1 1
10 10 10 10

0 2.2 0 0.3 0 0.2 0 0.3

5.9 49.9 41 14.8 47.9 7.9 43 12.8

103 103 103 103
S2
ICAM1
MIP-1β

102 102 102 102

CD48
101 101 101 101 ULBP1

0 3.9 0 0.6 0 0.2 0 1.3

1 2 3 1 2 3 1 2 3 1 2 3
0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10
MIP-1α TNF-α IFN-γ CD107a
1.3 12.8 2 14 7.3 6.8 9.5 3.8 8.9 6.2
10
3
10
3
10
3
10
3
103 S2
ICAM1
CD107a

TNF-α
10
2
10
2
10
2
10
2
102
CD48
1 1 1 1 10
1 ULBP1
10 10 10 10

0 0 0 0
43 39.8 9 4.9 0 1.9

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10

MIP-1β MIP-1α TNF-α IFN-γ IFN-γ

B
MIP-1β TNF-α IFN-γ CD107a
4 responses

3 responses

2 responses

1 response

No response
S2 S2 S2 S2 S2 S2 S2
ICAM-1 CD48 ULBP1 CD48 ICAM1 CD48
ULBP1 CD48 ULBP1
ULBP1 +
IgG
Figure 6. Interrelationships between different NK-cell responses induced by target cell recognition. Resting NK cells were mixed with S2 cells expressing ligands for
NK-cell receptors, as indicated, and incubated for 6 hours at 37°C. For some stimulations, S2 cells were preincubated with diluted anti–S2 cell serum (⫹ IgG). After stimulation,
the cells were surface stained with fluorochrome-conjugated anti-CD56 and anti-CD107a mAbs, fixed, permeabilized, and stained intracellularly with fluorochrome-conjugated
mAbs to cytokines and chemokines. (A) Lymphocytes were gated on forward scatter height (FSC-H) versus side scatter height plots (SSC-H). Single-cell events were gated on
forward scatter height (FSC-H) versus forward scatter area plots (FSC-A). CD56dim NK cells were gated on CD56 versus dead cell marker (DCM) plots. The second and third
rows show MIP-1␣, TNF-␣, IFN-␥, and CD107a staining in relation to MIP-1␤ staining after stimulation with S2 cells, as indicated. The bottom row shows MIP-1␤, MIP-1␣,
TNF-␣, and IFN-␥ staining in relation to CD107a staining, and IFN-␥ staining in relation to TNF-␣ staining (right panel). Gates were set using fluorochrome-conjugated isotype
control mAbs. The plots are derived from one representative donor. (B) CD56dim NK cells were gated as described in panel A, and a Boolean gating strategy was used for
analysis. Pie charts represent the frequency of cells positive for the given number of measured responses (MIP-1␤, TNF-␣, IFN-␥, and CD107a). Thus, cells can be categorized
into the number of responses they display. Arcs depict the relative frequency of cells specifically positive for MIP-1␤, TNF-␣, IFN-␥, and/or CD107a staining, as indicated.
Values represent the mean of 6 different donors.

production by individual CD56dim NK cells is likely to be nor TNF-␣ was increased relative to S2 cells only expressing
accompanied by MIP-1␤ and TNF-␣ production by the same cell. CD48. A small fraction of cells coexpressed MIP-1␤, TNF-␣, and
Further analysis was performed to enumerate responses by surface CD107a. Coexpression of ICAM-1, CD48, and ULBP1
individual CD56dim NK cells upon recognition of target cells further increased the frequency of cells producing MIP-1␤ and
expressing increasing numbers of ligands for activating receptors TNF-␣. Moreover, IFN-␥ was produced by a sizable fraction of
(Figure 6B). Given the high correlation between expression of CD56dim NK cells. If anything, surface expression of CD107a
MIP-1␣ and MIP-1␤, MIP-1␣ was omitted from this analysis. decreased, compared with stimulation with S2 cells coexpressing
Incubation of resting NK cells alone, with untransfected S2 cells, or CD48 and ULBP1. Upon stimulation with S2 cells coexpressing
S2 cells expressing ICAM-1 did not induce responses by CD56dim ICAM-1, CD48, and ULBP1, a small fraction of cells responded
NK cells. Expression of CD48 or ULBP1 on S2 cells induced with all 4 responses examined, whereas a greater fraction of cells
MIP-1␤ and weak degranulation by CD56dim NK cells. Coexpres- responded with 3 responses. Last, when NK cells were stimulated
sion of CD48 and ULBP1 on S2 cells specifically synergized for with IgG-coated S2 cells expressing CD48 and ULBP1, more than
degranulation, as neither the frequency of cells producing MIP-1␤ 90% of the CD56dim NK cells responded in terms of at least one
 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2174 FAURIAT et al
A A current view of NK-cell biology regards CD56bright and
M 7a
IP α
β
α
M P-1
-1
10
-γ
F-
N
CD
TN
I
IF
0 1 2 3 4 5 6 NK cells are regarded as specialized for cytotoxic function.12,30,43
Stimulation time (hours)
B IFN-γ
TNF-α
CD107a
MIP-1α
MIP-1β
Strength of stimulation
Figure 7. Schematic representation of activation thresholds and kinetics of
resting CD56dim NK-cell responses. (A) Approximate times required for induction of
different NK-cell responses such as degranulation (surface expression of CD107a),
chemokine secretion (MIP-1␣ and MIP-1␤), and cytokine secretion (IFN-␥ and
TNF-␣) are indicated on the time scale. (B) The figure depicts the relative signal
strength required for induction of different NK-cell responses such as degranulation
(surface expression of CD107a), chemokine secretion (MIP-1␣ and MIP-1␤), and
cytokine secretion (IFN-␥ and TNF-␣).
parameter and 16% of the cells responded in terms of all
parameters examined. Analogously, increasing densities of IgG on
target cells augmented the frequencies of CD56dim NK cells
displaying multiple different responses, and costimulation reduced
the density of IgG required for NK cells to display multiple
responses (supplemental Figure 5).
Together, these data provide insights into the heterogeneous
response of NK cells to target cell recognition, revealing specific
interrelationships and a hierarchy among the NK-cell responses for
degranulation of lytic granules and production of chemokines and
cytokines. Moreover, the data illustrate how increasing signal
strength can induce multiple different effector responses from
individual NK cells.
Discussion
With a multiplicity of receptors evolved to sense cellular homeosta-
sis and distress, NK cells are well equipped to act as primary
initiators of immune responses upon recognition of infected or
neoplastic cells.40 Such responses are not confined to cytotoxic
effector mechanisms, but also involve the secretion of cytokines
and chemokines. Here, we define the cytokines and chemokines
that are secreted by normal, freshly isolated human peripheral
blood NK cells upon interaction with target cells expressing
complex or defined sets of ligands for specific activating receptors.
We demonstrate that engagement of the activating receptors CD16,
2B4, or NKG2D sufficed for rapid secretion of chemokines, and
that coengagement of additional activating receptors could acceler-
ate and increase chemokine secretion. In contrast, secretion of
IFN-␥ required engagement of multiple different receptors and
occurred later, revealing a tighter control and a higher activation
threshold for induction of cytokine secretion (Figure 7). Unexpect-
edly, the results revealed CD56dim, rather than CD56bright, NK cells
to be more prominent producers of cytokines upon target cell
recognition. Furthermore, upon both target cell recognition and
exogenous cytokine stimulation, CD56dim NK cells excelled in
production of the chemokine MIP-1␤. These experiments highlight
CD56dim NK cells as an important proinflammatory cytokine
source during early immune responses to aberrant cells.
BLOOD, 18 MARCH 2010 䡠 VOLUME 115, NUMBER 11
CD56dim NK cells as developmentally distinct subsets occupying
different functional niches.41,42 In general, CD56bright NK cells are
considered to be the major source of cytokines, whereas CD56dim
The anatomic distribution of the 2 subsets underlies this perceived
dichotomy. Enriched in secondary lymphoid tissues, CD56bright NK
cells can intimately interact with other immune cells, influencing
ongoing adaptive immune responses through secretion of soluble
factors. Conversely, in the periphery, CD56dim NK-cell cytotoxic
activity can provide immunosurveillance of infected or neoplastic
cells. Notably, CD56bright NK cells produce more cytokines in
response to phorbol myristate acetate and ionomycin stimulation
than CD56dim NK cells.26,27 This fact has been ascribed to the
relatively low expression of the phosphatase SHIP-1 and the high
expression of the phosphatase inhibitor SET in CD56bright NK cells,
facilitating a lower activation threshold for cytokine secretion.44,45
Although we also found that exogenous cytokines induced a
greater frequency of IFN-␥– and TNF-␣–producing CD56bright NK
cells in 6- and 24-hour assays, our data highlight a prominent role
for CD56dim NK cells in producing cytokines and chemokines upon
target cell recognition. In fact, CD56dim NK cells responded more
vigorously in terms of cytokine and chemokine production relative
to CD56bright NK cells. These data substantiate and extend previous
findings demonstrating that CD56dim NK cells can produce cyto-
kines in response to a tumor cell line.46 Here, cytokines and
chemokines were induced by 2B4 and NKG2D synergy or K562
cells that express ligands for the natural cytotoxicity receptor
NKp30, NKG2D, and DNAM-1.47,48 Comparatively, CD56bright and
CD56dim NK-cell subsets express similar levels of activating
receptors.36 Therefore, differential expression of molecules convey-
ing signals from receptors such as 2B4, NKG2D, and NKp30 might
underlie the relatively more potent response of CD56dim NK cells to
target cell recognition. Division of NK-cell subsets on the basis of
variegated inhibitory receptor expression or maturation markers
such as CD27 may further distinguish CD56dim NK-cell subsets in
terms of cytokine production and will be addressed in further
experiments.46,49,50
NK cells have previously been reported to secrete a plethora of
proinflammatory and immunoregulatory cytokines. In our experi-
mental setting, using resting human NK cells freshly isolated from
peripheral blood, K562 target cell recognition consistently induced
secretion of a proinflammatory cytokine profile characterized by
MIP-1␣, MIP-1␤, RANTES, TNF-␣, and IFN-␥. This profile was
observed also with S2 cells expressing ligands for NKG2D and
2B4, IgG-coated S2 cells triggering CD16, and K562 cells
triggering NKp30. NKG2D and 2B4 synergy is immunoreceptor
tyrosine-based activation motif independent, whereas CD16 and
NKp30 are associated with immunoreceptor tyrosine-based activa-
tion motif–containing adaptor proteins.51-53 Thus, diverse signals
induce proinflammatory cytokine and chemokine secretion by NK
cells. Strikingly, upon NK-cell recognition of target cells, we did
not detect secretion of immunoregulatory cytokines, such as IL-5,
IL-10, IL-13, or GM-CSF. Such immunoregulatory cytokines are
more often reported to be secreted by NK cells upon stimulation
with exogenous cytokines. For example, IL-10 is secreted predomi-
nately by CD56bright NK cells upon stimulation by IL-12 with IL-2
or IL-15 (Fehniger et al21; Wolk et al54; C.F. and Y.T.B. unpublished
observations, March 2009). In mice, such NK-cell secretion of
IL-10 requires STAT4,55 which signals downstream of the IL-12
and IL-23 receptors.56 Our data suggest that NK cells are initially
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BLOOD, 18 MARCH 2010 䡠 VOLUME 115, NUMBER 11 NK-CELL RESPONSES TO TARGET CELL RECOGNITION 2175

wired to promote immune responses by secreting proinflammatory
cytokines upon target cell recognition.
With Drosophila S2 cells expressing ligands for NK-cell
receptors, the minimal requirements for cytokine and chemokine
production were dissected. Interestingly, IFN-␥ secretion required
the coengagement by LFA-1, 2B4, and NKG2D, reflecting a
minimal requirement previously identified for natural cytotoxic-
ity.33 Likewise TNF-␣ secretion also required receptor coengage-
ment, although here 2B4 and NKG2D synergy sufficed. Data
corroborate an earlier study using antibody-coated beads, where
mAbs for different NK cell activating receptors could not induce
secretion of TNF-␣ and IFN-␥ on their own by freshly isolated NK
cells.36 However, S2 cells revealed that secretion of IFN-␥ is
particularly dependent on LFA-1 engagement, as opposed to
secretion of TNF-␣. This might reflect not only LFA-1–mediated
adhesion, but also signaling.57 Remarkably, chemokines such as
MIP-1␣, MIP-1␤, and RANTES could be induced by engagement
of 2B4 or NKG2D. In contrast to other activating receptors,
engagement of CD16 was sufficient to induce some IFN-␥ and
TNF-␣ secretion, in addition to chemokine secretion. The strong
propensity of CD16 to induce NK-cell activation reflects the role of
antibody-dependent cell-mediated cytotoxicity as an effector arm
of the adaptive immune system. Thus, in vivo, selection of B cells
can safeguard robust activation mediated by CD16 engagement.
Notably, upon stimulation of receptors such as 2B4 and NKG2D,
intracellular cytokine and chemokine expression was apparent,
sometimes without much secretion detected. The divergence in
intracellular expression versus secretion after stimulation might
reflect regulation of cytokine and chemokine exocytosis. This facet
of regulation will be interesting to explore in more detail.
Temporally, the secretion of chemokines always preceded that
of TNF-␣ and IFN-␥. Comparing the frequency of cytokine- and
chemokine-producing cells revealed that a higher proportion of NK
cells produced chemokines relative to cytokines after 6 hours of
stimulation. Costimulation of receptors by engagement of LFA-1 or
other coactivation receptors could, in many instances, accelerate
and increase cytokine and chemokine secretion, and increase the
frequency of cells producing these factors. As we could simulta-
neously monitor production of MIP-1␣, MIP-1␤, TNF-␣, and
IFN-␥, as well as CD107a, in individual cells, the relationships
between production of specific chemokines, cytokines, and degranu-
lation could be assessed. These experiments revealed that produc-
tion of TNF-␣ and IFN-␥ and degranulation were contained within
the chemokine-producing NK-cell subset. Moreover, most IFN-␥–
producing cells also expressed TNF-␣, but the inverse relationship
did not hold. Production of TNF-␣ and IFN-␥ did not necessarily
correlate with degranulation, but this could in part reflect differ-
ences in kinetics of these responses (Figure 7A). Taken together,
these results reveal different activation thresholds for distinct
responses. Chemokine production is an early feature of NK-cell
responses and is triggered with relatively weak activating signals,
requiring less stimulation than that necessary for degranulation or
cytokine production. Induction of TNF-␣ and IFN-␥ is more
stringently controlled, with a greater requirement for receptor
cooperation and release at later time points after stimulation.
Thus, the data reveal a hierarchy among factors released upon
NK-cell interactions with target cells, with graded responses
according to the degree of ligand expression for activating
receptors (Figure 7B).
Altogether, this study provides detailed insight into regulation
of NK-cell cytokine secretion upon target cell recognition. Engage-
ment of individual activating receptors on resting NK cells suffices
for chemokine secretion, to alert and recruit other immune cells.
More complex interactions, upon which multiple activating NK-
cell receptors are engaged, can induce production of TNF-␣ and
IFN-␥, to promote cellular resistance to infections and shape
adaptive immune responses. These chemokines and cytokines are
readily produced by the CD56dim NK-cell subset. Chemokine and
cytokine production by CD56dim NK cells may thus be an important
component of immunosurveillance.
Acknowledgments
We thank S. Wood for critical reading of the paper and
J. Michaëlsson for helpful discussions.
This work was supported by grants from the Swedish Founda-
tion for Strategic Research, Research Council, and Cancer Society
(C.F., H.-G.L., and Y.T.B.), by the Intramural Research Program at
National Institutes of Health, National Institute of Allergy and
Infectious Diseases (E.O.L.), and Mary Beve’s Foundation, David
& Astrid Hagelen’s Foundation, the Karolinska Institute Research
Foundation, and Jonas Söderquist’s Stipend (Y.T.B.).
Authorship
Contribution: C.F. designed research, performed experimental
work, analyzed and interpreted data, and drafted the paper; E.O.L
and H.-G.L. designed research and contributed to drafting the
paper; and Y.T.B. designed research, analyzed and interpreted data,
and drafted the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Cyril Fauriat or Yenan T. Bryceson, Center for
Infectious Medicine, Department of Medicine, Karolinska Institute,
Karolinska University Hospital Huddinge, S-14186 Stockholm, Swe-
den; e-mail: cfauriat@wanadoo.fr or yenan.bryceson@ki.se.

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 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

2010 115: 2167-2176

doi:10.1182/blood-2009-08-238469 originally published
online December 1, 2009

Regulation of human NK-cell cytokine and chemokine production by

target cell recognition
Cyril Fauriat, Eric O. Long, Hans-Gustaf Ljunggren and Yenan T. Bryceson

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