3260 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
Unlicensed NK cells target neuroblastoma
following anti-GD2 antibody treatment
Nidale Tarek,1 Jean-Benoit Le Luduec,2 Meighan M. Gallagher,2 Junting Zheng,3
Jeffrey M. Venstrom,4 Elizabeth Chamberlain,1 Shakeel Modak,1 Glenn Heller,3
Bo Dupont,2 Nai-Kong V. Cheung,1 and Katharine C. Hsu4,5
1Department of Pediatrics, 2Immunology Program, Sloan-Kettering Institute, 3Department of Epidemiology-Biostatistics, and 4Department of Medicine,
Memorial Sloan-Kettering Cancer Center, New York, New York, USA. 5Department of Medicine, Weill Cornell Medical College, New York, New York, USA.
Survival outcomes for patients with high-risk neuroblastoma (NB) have significantly improved with anti-disi-
aloganglioside GD2 mAb therapy, which promotes NK cell activation through antibody-dependent cell-medi-
ated cytotoxicity. NK cell activation requires an interaction between inhibitory killer cell immunoglobulin-like
receptors (KIRs) and HLA class I ligands. NK cells lacking KIRs that are specific for self HLA are therefore
“unlicensed” and hyporesponsive. mAb-treated NB patients lacking HLA class I ligands for their inhibitory
KIRs have significantly higher survival rates, suggesting that NK cells expressing KIRs for non-self HLA are
mediating tumor control in these individuals. We found that, in the presence of mAb, both licensed and unli-
censed NK cells are highly activated in vitro. However, HLA class I expression on NB cell lines selectively
inhibited licensed NK cell activity, permitting primarily unlicensed NK cells to mediate antibody-dependent
cell-mediated cytotoxicity. These results indicate that unlicensed NK cells play a key antitumor role in patients
undergoing mAb therapy via antibody-dependent cell-mediated cytotoxicity, thus explaining the potent “miss-
ing KIR ligand” benefit in patients with NB.
Neuroblastoma (NB), an embryonal malignancy of neuroectoder-
mal origin, is the most common extracranial solid tumor of child-
hood. Nearly two-thirds of patients present at diagnosis with evi-
dence of metastatic disease and have poor long-term survival due
to residual disease, despite aggressive approaches, including high-
dose multiagent chemotherapy, surgery, radiation therapy, and
autologous stem cell transplantation (ASCT) (1, 2). Treatment of
patients with high-risk NB with monoclonal antibodies (mAb) tar-
geting the disialoganglioside surface antigen GD2 has resulted in
lower recurrence rates and improved overall survival (OS) (3–5). In
addition to complement-dependent cytotoxicity, the anti-GD2 mAb
3F8 achieves NB killing through antibody-dependent cell-mediated
cytotoxicity (ADCC) mediated by myeloid and NK cells (4).
NK activity is regulated by inhibitory and activating signals
following engagement of cell membrane receptors with their
cognate ligands on target cells (6). Different mechanisms of
NK activation and inhibition have been described upon NK
interaction with NB. Untreated NB tumors and cell lines are
widely reported to have reduced to no HLA class I expression,
rendering them potentially susceptible to NK killing due to
lack of engagement of HLA class I–specific inhibitory killer
cell immuno globulin-like receptors (KIRs) (7, 8). In addition to
CD16-mediated activation by mAb, direct activation of NK cells
by NB through NKp30, NKp44, NKp46, and the DNAM-1 recep-
tor has also been described (9–11). To evade NK surveillance,
NB cells exhibit poor cell surface expression of the activating
ligands MICA, MICB, and ULBPs; and high serum concentra-
tions of soluble MICA in patients with NB results in depressed
NK function (8). Furthermore, the 4Ig-B7-H3 molecule, widely
expressed among solid tumors, including NB (12, 13), is a potent
inhibitor of NK function (14).
NK cells spare autologous cells from killing through interaction of
inhibitory receptors with self-HLA class I antigens on the autologous
cell. KIR2DL2 and KIR2DL3 recognize HLA-C allotypes characterized
by Asn80 (grouped as HLA-C1); KIR2DL1, and to a weaker extent some
KIR2DL2/3 allotypes, recognize HLA-C allotypes characterized by
Lys80 (grouped as HLA-C2); KIR3DL1 recognizes HLA-A and HLA-B
allotypes with the Bw4 epitope; and the heterodimeric CD94/NKG2A
receptor recognizes complexes of HLA-E bound to peptides from
the leader sequences of HLA class I molecules (15, 16). Interaction
between self-specific inhibitory KIRs and their cognate HLA ligands
is fundamental to a process referred to as licensing (17), in which NK
cells expressing inhibitory KIRs for self HLA (S-KIRs) are “licensed”
and have higher resting capacity for response (IFN-γ production,
cytotoxicity, and ADCC) (18, 19). Lacking S-KIRs, “unlicensed” NK
cells are significantly less responsive at rest and include cells express-
ing inhibitory KIRs for non-self HLA (NS-KIR). Under inflammatory
conditions, however, unlicensed NK cells can exhibit higher response
(20–22). Independent of KIRs, CD94/NKG2A expression is associated
with modest response capacity (19). Because the KIR and HLA genes
are located on different chromosomes and segregate independently,
approximately 60% of individuals have inhibitory KIRs for which they
lack the cognate HLA class I ligands (a condition referred to herein as
missing KIR ligand) (23, 24) and therefore potentially possess sub-
stantial numbers of unlicensed NK cells expressing NS-KIRs.
We previously reported a significant association between KIR/
HLA genotypes predictive of missing KIR ligand and survival in 169
patients with NB treated with the anti-GD2 mAb 3F8 following
ASCT (25). A smaller study of 38 patients treated with Hu14.18-IL2,
an anti-GD2 mAb fused to IL-2, also suggested improved response
among patients missing KIR ligand (26). The improved clinical out-
comes suggest that unlicensed NK cells expressing NS-KIRs may play
Authorship note: Nidale Tarek and Jean-Benoit Le Luduec contributed equally to
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(9):3260–3270. doi:10.1172/JCI62749.
Related Commentary, page 3053
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
an important role in patients treated with mAb. In this study, we con-
firm the impact of missing KIR ligand in patients with NB receiving
3F8 and demonstrate that ASCT is not necessary for this associa-
tion. We demonstrate that 3F8 in vitro activates NK cells expressing
S-KIRs, NS-KIRs, and NKG2A for ADCC, but cytokine-induced
upregulation of HLA class I expression on NB targets selectively
abrogates activation of S-KIR–positive and NKG2A-positive NK cells.
These results not only demonstrate the dominance of the unlicensed
NS-KIR–positive NK cell in ADCC and antitumor cytotoxicity but
also confirm that missing KIR ligand KIR/HLA genotype combina-
tions represent an important prognostic marker in patients with NB
treated with mAb. KIR/HLA genotypes indicative of missing KIR
ligand may therefore be a positive prognostic indicator for other
patient populations with malignancies treated with mAb.
Missing KIR ligand is associated with improved survival in patients with
high-risk NB receiving anti-GD2 mAb. We previously reported that
patients receiving ASCT with 3F8 had superior OS and progres-
sion-free survival (PFS) if they lacked HLA class I ligands for
autologous inhibitory KIRs (25). To test whether ASCT is neces-
sary for this benefit, we evaluated updat-
ed outcomes of 166 previously reported
patients who received 3F8 following ASCT
(25) and 76 new patients who received 3F8
following chemotherapy without ASCT.
Patient characteristics for both cohorts
are listed in Table 1. The median follow-
up was 74 months from the institution of
3F8 immunotherapy, and the OS and PFS
were similar in both patient groups (data
Patients were considered missing KIR
ligand if they lacked one or more HLA
class I ligand for their inhibitory KIRs. In
contrast, patients with “all ligands pres-
ent” possessed all HLA class I ligands
for their inhibitory KIRs. 153 patients
(63%) were missing KIR ligand, and the
proportions of missing KIR ligand were
comparable among patients who received
chemotherapy without ASCT (62%) and
those who received ASCT (64%). In both
the non-ASCT and ASCT settings, missing
KIR ligand was associated with increased
OS and PFS (Figure 1, A and B), indicating
that ASCT is not necessary for the “miss-
ing ligand” effect and suggesting that the
effect may be more related to the mAb
3F8 or to NK-NB interaction in general.
Among patients receiving 3F8, the medi-
an OS and PFS were 114 and 50 months,
respectively, for patients missing KIR
ligand compared with a median OS and
PFS of 51 and 18 months, respectively, for
patients with all ligands present (Figure
1C). Thus, compared with that of patients
with all KIR ligands, patients lacking one
or more class I ligands for autologous
inhibitory KIRs had significantly longer
OSs (hazard ratio [HR] = 0.57 [95% CI, 0.39–0.83], P = 0.003) and
PFSs (HR = 0.58 [95% CI, 0.42–0.81], P = 0.001). In the multivari-
ate analysis, which controlled for age, MYCN amplification, and
bone metastases, the missing ligand effect was maintained for OS
(HR = 0.48 [95% CI, 0.32–0.71], P < 0.001) and PFS (HR = 0.52
(95% CI, 0.36–0.74), P < 0.001) (Table 2), and there were no signifi-
cant differences in disease status between the missing KIR ligand
and all ligands present groups (P = 0.66; Supplemental Table 1; sup-
plemental material available online with this article; doi:10.1172/
JCI62749DS1). Although lack of any one class I ligand was associat-
ed with improved survival over that of those who possessed all KIR
ligands, the highest OSs (HR = 0.38 [95% CI, 0.18–0.79], P = 0.01)
and PFSs (HR = 0.28 [95% CI, 0.13–0.58], P = 0.001) were evident in
patients who were HLA-Bw6/Bw6 and lacking the HLA-Bw4 ligand
for autologous KIR3DL1 (Supplemental Figure 1).
Licensed and unlicensed NK cells are activated in the presence of mAb
3F8. The improved outcome in patients with high-risk NB missing
KIR ligand implies that unlicensed NK cells expressing NS-KIRs in
these individuals become activated and contribute significantly to
tumor control. To determine whether unlicensed NK cells express-
ing NS-KIRs can be mobilized by 3F8 for ADCC and tumor toxic-
Patients treated with chemotherapy
plus 3F8 (n = 76)
Patients treated with ASCT
plus 3F8 (n = 166)
166 (100%) Stage IV cancer
Disease status at 3F8 therapy
All ligands present
Missing ≥1 ligand
APatients who received 3F8 as part of induction therapy in combination with chemotherapy. CR/
VGPR, complete response/very good partial response.
3262 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
ity in vitro, we evaluated unlicensed and licensed NK cell activ-
ity from normal individuals in response to NB cell lines in the
presence or absence of 3F8. Donors with KIR and HLA genotypes
predictive of missing KIR ligand (n = 16) were selected for evalu-
ation, allowing functional comparisons between NK populations
expressing S-KIRs or NS-KIRs within each individual. Because
donors were of a variety of KIR and HLA ligand backgrounds,
licensed and unlicensed NK populations were defined by different
KIR/self-HLA combinations (Supplemental Table 2). Four donors
with all ligands present were also evaluated.
Multicolor flow cytometry provided single-cell assessment of
NK cells exclusively expressing a single inhibitory KIR, permitting
evaluation of unlicensed NK cells expressing NS-KIRs distinct
from licensed NK cells expressing S-KIRs. Among individuals
with missing KIR ligand, unlicensed NK cells exclusively express-
ing a single NS-KIR represented 3%–35% of the total NK repertoire
(Supplemental Table 3). Among these same individuals, licensed
NK cells exclusively expressing a single S-KIR represented 8%–31%
of the total NK repertoire. In individuals with all ligands present,
licensed NK cells exclusively expressing 1 S-KIR represented 19%–
35% of the total NK repertoire. Thus, unlicensed and licensed NK
cells expressing a single inhibitory KIR can represent substantial
segments of the total NK repertoire.
In each individual, we compared CD107 mobilization as a
marker of cytotoxic response among NS-KIR–positive and
S-KIR–positive NK cells following stimulation with the HLA
class I–negative K562 target cell line or with HLA-genotyped NB
cell lines (Supplemental Table 4) in the presence or absence of
mAb 3F8. Among all individuals, both NS-KIR and S-KIR NK cell
populations had dim CD56 expression, indicating their equiva-
Missing KIR ligand is associated with improved OS and PFS in patients with high-risk NB treated with 3F8. Among patients receiving 3F8, patients
lacking class I ligands for autologous inhibitory KIRs (dotted lines) have higher OS and PFS compared with those of patients with all KIR ligands
present (solid lines) following (A) chemotherapy alone (OS, HR = 0.53 [95% CI, 0.26–1.08], P = 0.077; PFS, HR = 0.49 [95% CI 0.28–0.88],
P = 0.014) or (B) ASCT (OS, HR = 0.57 [95% CI, 0.37–0.88], P = 0.011; PFS, HR = 0.62 [95% CI, 0.41–0.94], P = 0.022), confirming that ASCT is
not necessary for the missing ligand effect. (C) Among all patients receiving 3F8, patients missing KIR ligands have higher OS (HR = 0.57 [95%
CI, 0.39–0.83], P = 0.003) and PFS (HR = 0.58 [95% CI, 0.42–0.81], P = 0.001) compared with those of patients with all KIR ligands.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
lent terminal differentiation and potential for cytotoxic response
(27, 28). Furthermore, there was no difference in CD16 expres-
sion among the NK populations (data not shown).
As expected, in an HLA-C1/C1, HLA-Bw4/Bw6 individual,
licensed NK cells exclusively expressing the self-specific KIR2DL3
or KIR3DL1 receptor were responsive to K562 cells, while unli-
censed NK cells expressing the non-self-specific KIR2DL1 receptor
were hyporesponsive (Figure 2A). Although NB cell lines have low
to no cell surface expression of HLA class I molecules (7, 8) (Figure
3A), rendering them potentially susceptible to NK cytotoxicity, all
NK cells, regardless of their licensed status, responded poorly to
stimulation with the NB cell line LAN-1 (Figure 2A). In the pres-
ence of the anti-GD2 mAb 3F8, however, licensed single-positive
NK (spNK) cells for S-KIRs were highly activated when stimulated
(Figure 2A). Unexpectedly, the unlicensed NS-KIR spNK cells also
demonstrated higher activation in the presence of 3F8, compared
with that of the K562 control. KIR-negative NK cells were also
activated but to a lesser degree. These results were confirmed with
the IMR-32 cell line and 4 other NB cell lines (data not shown),
demonstrating that multiple NK subsets are recruited by 3F8 to
mediate ADCC against NB target cells. Cytotoxicity assays con-
firmed that addition of 3F8 was critical for NK-mediated NB lysis
(Supplemental Figure 2).
Aggregate analysis of NK response to the NB cell line, LAN-1,
from 16 individuals demonstrated that NK cells expressing S-KIRs
or NS-KIRs showed strong CD107 mobilization mediated by 3F8
(P < 0.0001) (Figure 2B). A higher percentage of S-KIR spNK cells
compared with NS-KIR spNK cells exhibited CD107 mobilization
(P = 0.004), confirming that licensed NK cells are more activated
for ADCC (18, 19). The aggregate analysis also showed response
among KIR-negative NK cells, albeit to a lower level than that in
the NS-KIR–positive population (P = 0.006).
Based on the results in the KIR-negative NK cells, we surmised
that CD94/NKG2A may contribute to the NK cell response to
LAN-1 cells in the presence of 3F8. Indeed, NKG2A expression
contributed to response among all NK cell populations, including
the KIR-negative cells (Figure 2C). Among individuals with miss-
ing KIR ligand, NKG2A-positive KIR-negative NK cells represent-
ed 13%–51% of the total NK repertoire, and there was no preferen-
tial coexpression of NKG2A among NS-KIR or S-KIR populations
(data not shown).
HLA class I expression on tumor cells selectively inhibits licensed NK cells,
sparing ADCC by unlicensed NK cells. In vitro, NK cells exclusively
expressing S-KIRs or NS-KIRs are activated by 3F8 for ADCC against
NB, although comparatively S-KIR–positive NK cells have higher
response. In vivo, however, higher survival among patients missing
KIR ligands for autologous inhibitory KIRs suggests not only that
unlicensed NK cells expressing NS-KIRs are major contributors to
tumor control but also that licensed NK cells expressing S-KIRs in
patients with all ligands present are inhibited or silenced. Others
have shown that HLA class I molecules, including the nonclassical
HLA-E, are upregulated on NB cells following exposure to chemo-
therapy, isotretinoin, and anti-GD2 antibodies (29–32), therapeutic
agents that the patients with high-risk NB in this study received. We
therefore hypothesized that upregulation of self-HLA class I ligands
on NB cells specifically inhibits licensed NK cells expressing inhibi-
tory receptors for self-HLA ligands while sparing unlicensed NK
cells lacking self-specific inhibitory receptors.
To isolate the role of KIR and HLA in NK-NB interactions, we
evaluated NKG2A-negative NK cells for CD107 mobilization and
IFN-γ production in response to NB cell lines and 3F8 with induced
self-HLA expression. HLA class I expression on NB cells was induced
following culture for 72 hours with IFN-γ (33). Reproducing autolo-
gous NK-NB interactions in a patient missing KIR ligand, NK cells
from an HLA-C1/C1/Bw6/Bw6 individual were incubated with the
target LAN-1 cells. Although the HLA genotype of LAN-1 cells is
HLA-C1/C1/Bw4/Bw6, IFN-γ–induced HLA class I expression did
not include HLA-Bw4 (Figure 3A), therefore mimicking an HLA-
C1/C1/Bw6/Bw6 tumor target. While both S-KIR–positive and
NS-KIR–positive NK cells were activated by LAN-1 cells with 3F8,
upregulation of HLA expression on the target cell led to selective
inhibition of the self-KIR2DL3 spNK cells (Figure 3B). In contrast,
the non-self-specific KIR2DL1 and KIR3DL1 spNK cells main-
tained their level of activation in the presence of the class I–express-
ing tumor target. Reproducing autologous NK-NB interactions in
an individual with all ligands present, NK cells from an HLA-C1/
C2/Bw4/Bw4 individual were incubated with the HLA-C1/C2/Bw4/
Bw4 cell line BE(2)N. IFN-γ induced HLA class I expression, includ-
ing HLA-Bw4, on the BE(2)N target cell line (Figure 3A). Licensed
NK cells exclusively expressing KIR2DL1, KIR2DL3, or KIR3DL1
were activated by BE(2)N cells in the presence of 3F8, and all were
inhibited by induced HLA class I expression on the target cell (Fig-
ure 3B). Confirming that the inhibition was due to interaction of
cognate HLA class I molecules on the target cells with self-specific
KIRs on the licensed NK cells, addition of anti–HLA class I blocking
antibodies to the effector/target mix fully restored activity among
the S-KIR NK cells, while leaving activity among the NS-KIR NK
cells unchanged (Figure 3B). Selective inhibition of licensed NK
cells by induced cognate HLA class I ligands was verified with the
NB-1691 and IMR-32 cell lines (data not shown).
In the aggregate analysis of unlicensed and licensed NK response
to LAN-1 cells from 9 individuals homozygous for HLA-C1 and
therefore lacking KIR ligand HLA-C2, HLA class I upregulation on
the NB target cell led to selective inhibition of CD107 mobilization
among self-specific KIR2DL2/3+ cells and, consequently, a stronger
relative response in the non-self-specific KIR2DL1+ cells (P = 0.003,
Factors associated with survival
Univariate analysis of OS Multivariate analysis of OS Univariate analysis of PFS Multivariate analysis of PFS
Missing any ligand
Age ≥1.5 years
HR (95% CI)
HR (95% CI)
0.48 (0.32–0.71) <0.001
P value HR (95% CI)
HR (95% CI)
3264 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
Figure 3C). Among NKG2A-negative cells, NS-KIR–positive NK
cells were more responsive than KIR-negative NK cells (P = 0.001).
Similar results were obtained with intracellular IFN-γ response
(data not shown). Aggregate analysis of NK activity among 3 indi-
viduals with all ligands present was consistent, with inhibition of
all S-KIR–bearing NK subsets by NB target cells expressing the cog-
nate HLA class I ligands (P < 0.0001, Figure 3C).
When NKG2A-positive cells were evaluated, NKG2A coexpres-
sion enhanced ADCC response of all NK cell subsets, but it did
not prevent HLA class I–mediated inhibition of licensed cells
(Supplemental Figure 3). Moreover, NKG2A-positive cells were
also inhibited by HLA-E expression on the BE(2)N target cell, as
evidenced by suppression of activity among the NKG2A-positive
KIR-negative subset (Supplemental Figure 3).
Cytokines released by activated NK cells induce HLA class I expres-
sion on NB cell lines. Induced HLA expression has been described
in NB tumors isolated from patients receiving treatment (29).
To determine whether upregulation of HLA class I molecules
on NB target cell lines is directly induced by agents commonly
used in the treatment of NB, the NB cell lines LAN-1 and BE(2)
N were incubated with GM-CSF, isotretinoin, or with different
chemotherapy agents. No increase in HLA class I expression on
the NB cell lines was detected following treatment with these
agents (data not shown). We then investigated whether cytokines
released from NK cells activated by 3F8-dependent ADCC could
induce HLA class I expression on NB cells. PBMCs and NB tar-
gets were coincubated in the presence of 3F8, with or without
GM-CSF, which enhances 3F8-dependent ADCC (34). Superna-
tant obtained following 24 hours of coculture was collected and
used as a culture medium for the NB cell lines LAN-1 and BE(2)
N (Figure 4). Supernatant from PBMCs alone did not affect HLA
class I expression on NB cell lines, and the addition of GM-CSF
to PBMCs resulted in supernatant that minimally increased HLA
class I expression on the NB target. Coincubation of 3F8 with
PBMCs and NB cells, however, produced a supernatant capable
of inducing HLA class I expression on LAN-1 and BE(2)N cells
3F8 activates NK cells expressing S-KIRs, NS-
KIRs, and NKG2A against NB targets. In healthy
individuals, CD107 degranulation was analyzed
in the presence of NB target cells (LAN-1 cells),
with and without mAb, among subsets of NK cells.
(A) In individual no. 8, S-KIR spNK cells dem-
onstrate strong activation in response to K562
compared with that of NS-KIR spNK cells. All NK
populations show minimal activation in response to
LAN-1 cells alone but are activated by the addi-
tion of 3F8 in response to LAN-1 cells. Data rep-
resent the average of 3 separate experiments. (B)
Aggregate function of S-KIR and NS-KIR spNK
cells from 16 healthy individuals. Addition of 3F8
to LAN-1 cells results in activation of S-KIR–posi-
tive, NS-KIR–positive, and KIR-negative NK cells
(P < 0.0001), with S-KIR spNK cells more respon-
sive (P = 0.004) and KIR-negative NK cells less
responsive (P = 0.006) than NS-KIR spNK cells. (C)
NKG2A expression contributes to NK response to
LAN-1 cells in the presence of 3F8 among NK cells
expressing S-KIR, and NS-KIR (P < 0.0001), and
KIR-negative NK cells (P = 0.0004). NS-KIR–posi-
tive, NKG2A-negative NK cells are more respon-
sive than KIR-negative, NKG2A-negative NK cells
(P = 0.0008) and are equally responsive to KIR-
negative, NKG2A-positive cells (P = 0.13). Sym-
bols represent individual samples (mean ± SEM).
*P < 0.05, **P < 0.01, ***P < 0.001.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
Licensed NK cells are selectively inhibited by HLA class I ligands induced on NB targets. (A) Incubation with IFN-γ results in increased HLA class
I antigen expression on LAN-1 and BE(2)N cells. HLA-Bw4 and HLA-E antigens are readily induced on BE(2)N but not LAN-1 cells. (B) CD107
degranulation and IFN-γ production in NKG2A-negative, KIR-positive NK cells in response to IFN-γ–treated NB targets in the presence of 3F8.
When NK cells from individual no. 17 (HLA-C1/C1/Bw6/Bw6) are incubated with LAN-1 cells, mimicking effectors and NB cells in a patient lacking
HLA-C2 and HLA-Bw4 ligands, both NS-KIR–positive (KIR2DL1 single positive [KIR2DL1sp] and KIR3DL1sp) and S-KIR–positive (KIR2DL3sp)
NK cells are activated in the presence of 3F8, but only KIR2DL3 spNK cells are inhibited by IFN-γ–induced expression of self HLA on tumor target.
When NK cells from individual no. 20 (HLA-C1/C2/Bw4/Bw4) are incubated with BE(2)N cells, mimicking effectors and NB cells in a patient with
all ligands present, S-KIR–positive NK subsets (KIR2DL1sp, KIR2DL3sp, and KIR3DL1sp) are inhibited by IFN-γ–induced expression of cognate
ligands on the tumor target. In both individuals, blocking antibodies to HLA class I fully restores response among S-KIR–positive NK cells. (C)
Aggregate CD107 response among NK populations from 9 HLA-C1/C1 individuals to LAN-1 cells. HLA class I upregulation on the target inhibits
S-KIR spNK cells, resulting in a comparatively stronger response from NS-KIR spNK cells (P = 0.003). Aggregate CD107 response among spNK
cells from 3 HLA-C1/C2/Bw4 individuals to BE(2)N demonstrates that HLA class I upregulation on the target inhibits all self-specific NK subsets.
Symbols represent individual samples (mean ± SEM). **P < 0.01,***P < 0.001.
3266 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
(Figure 4A) to levels nearly equivalent to the level attained fol-
lowing the direct addition of IFN-γ (Figure 3A). HLA-Bw4 expres-
sion was also specifically induced, albeit to a lower level than that
induced in the other class I molecules.
We surmised that release of cytokines by tumor-activated PBMCs
in the presence of mAb was responsible for the supernatant-medi-
ated upregulation of HLA class I on the tumor target. Using ELISA,
we determined the concentrations of IFN-γ released in the differ-
ent supernatants. PBMCs alone did not produce detectable IFN-γ,
and the addition of GM-CSF resulted in the release of a minimal
amount of IFN-γ at 24 hours (Figure 4B). However, PBMCs activat-
ed by NB target cells in the presence of 3F8 released a substantial
amount of IFN-γ, peaking at 12 hours, and the addition of GM-
CSF to 3F8 further enhanced IFN-γ production, reaching 60 pg/ml
by 24 hours (Figure 4B). Using titration assays, we determined that
HLA expression on LAN-1 and BE(2)N cells could be induced with
as little as 10 pg/ml IFN-γ (Figure 4C). We therefore conclude that
concentrations of IFN-γ released by tumor-activated PBMCs in the
presence of 3F8 and GM-CSF are adequate to induce HLA expres-
sion on the tumor.
When treated with anti-GD2 mAb, patients with NB who lack
one or more HLA ligand for their inhibitory KIRs respond bet-
ter to treatment, have lower rates of relapse, and survive longer
compared with patients who possess all HLA KIR ligands. These
results are independent of stem cell transplantation and suggest
that the missing ligand effect is a result of either the 3F8 mAb
therapy and/or the interaction between NK cells and the NB tar-
get. The response and survival advantages associated with missing
KIR ligand genotypes, denoting individuals who lack HLA ligands
for his/her autologous inhibitory KIR, suggested to us that the
IFN-γ released by activated NK cells induces
upregulation of HLA class I on NB cells in vitro. (A)
HLA-Bw4 and HLA-A, HLA-B, and HLA-C expres-
sion on the LAN-1 and BE(2)N NB cell lines is
shown following 72 hours in different culture condi-
tions. Supernatants collected from PBMCs coincu-
bated with LAN-1 and BE(2)N cells and 3F8, with
or without GM-CSF, induced HLA class I expression
on LAN-1 and BE(2)N cells, respectively; in compari-
son, supernatant collected from PBMCs incubated
with or without GM-CSF did not induce HLA expres-
sion. (B) By ELISA, PBMCs alone or PBMCs with
GM-CSF produced no or minimal IFN-γ. PBMCs
activated by LAN-1 cells in the presence of 3F8 and
GM-CSF released a substantial amount of IFN-γ
compared with baseline and reached 60 pg/ml at
24 hours. (C) Titration assays demonstrate that HLA
class I expression can be induced on LAN-1 and
BE(2)N cells with 10 pg/ml IFN-γ and that expres-
sion increased in a dose-dependent manner.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
unlicensed NK cells expressing KIRs for non–self-HLA ligands in
these individuals may be more effective in tumor eradication than
the licensed NK cells expressing KIRs for self HLA. We considered
2 possible mechanisms underlying these clinical findings: acti-
vation among the NS-KIR–specific NK population by 3F8 and/
or selective inhibition of the S-KIR–specific population. For NB
treated with 3F8, both mechanisms appear to be important.
In vitro, licensed NK cells expressing S-KIRs and unlicensed
NK cells expressing NS-KIRs are both activated by NB targets in
the presence of 3F8, but only the licensed cells are inhibited by
cytokine-induced expression of self-HLA molecules on tumor tar-
gets. While NKG2A expression contributes to the ADCC response,
NKG2A-bearing NK cells are also silenced by HLA-E expression on
tumor cells (27, 35). Taken together, the data suggest that upon
encountering target cells with HLA class I expression, unlicensed
NK cells bearing receptors for non-self HLA are the primary medi-
ators of ADCC. Several studies examining tumor samples from
patients with NB and mouse models indicate that upregulation
of HLA class I occurs in vivo. All patients in this study received
chemotherapy, GM-CSF, and anti-GD2 mAb therapy, and most
patients received isotretinoin. It has been reported that tumor
specimens from patients with NB treated with chemotherapy
upregulate class I expression as a result of partial differentiation
(29); retinoid derivatives induce cellular differentiation and HLA
class I expression on NB cells (30); and, in NB-bearing mice treated
with anti-GD2 mAb, recurrent tumors exhibit greater than 5-fold
higher MHC class I expression (31). We could not demonstrate in
vitro that agents used to treat NB could directly upregulate HLA
class I expression on certain NB cell lines, suggesting that in vivo
conditions may have contributed to previous findings or that the
cell lines we used may not respond with HLA class I upregulation
to these agents (30). We show clearly, however, that PBMCs
responding to NB in the presence of mAb produce cytokines, in
particular IFN-γ, at concentrations sufficient to induce HLA class
I expression on NB cells. Other cytokines, such as tumor necrosis
factor, can further enhance HLA class I expression, including HLA-
Bw4 (data not shown), and it is possible that these cytokines are
also released in ADCC-dependent lymphocyte activation. These in
vitro findings offer insight to in vivo conditions, in which recruit-
ment of NK cells to the tumor microenvironment may result in
effector/target interactions, leading to significant local cytokine
concentrations. Taken together, the data indicate that it is highly
likely that NK activation in vivo by 3F8-dependent ADCC leads to
cytokine release, inducing HLA class I expression on the NB target,
inhibiting licensed NK cells, and facilitating the striking missing
KIR ligand effect (Figure 5).
The in vitro studies presented in this report represented activity
among NK cells expressing a single inhibitory KIR. Examination
of the NK repertoire among 20 individuals revealed that the single-
positive unlicensed and licensed NK populations, while varying in
size from individual to individual, can each represent a significant
proportion of the total NK repertoire. The repertoire also includes
smaller populations of NK cells that express two or more inhibi-
tory receptors, often resulting in higher levels of responsiveness
(19, 36). However, interaction between just one receptor and its
HLA class I ligand is sufficient to inhibit these highly responsive
NK cells (19). Thus, licensed NK cells expressing S-KIRs, whether
expressing one or more than one inhibitory KIR, will be inacti-
vated by the presence of self-HLA class I on the tumor target.
These findings are highly relevant to patients with other malig-
nancies treated with mAbs, such as rituximab, whose mechanisms
of action involve NK-mediated ADCC (37). Higher HLA class I
expression on CD20-positive lymphoma cells is associated with
attenuation of rituximab-induced NK-mediated ADCC (38). How-
ever, correlation of KIR/HLA genotyping among patients receiving
rituximab and other mAb therapies has not been systematically
explored for the missing ligand effect.
Our data support the incorporation of KIR and HLA genotypes
as a prognostic marker in patients with high-risk NB receiving
antibody immunotherapy. Indeed, KIR/HLA genotypes are as
Unlicensed NK cells dominate NB killing in the presence of 3F8. 3F8 activates both licensed and unlicensed NK cells through CD16. Engagement
of inhibitory KIR with its cognate HLA class I ligand on NB cells inhibits licensed NK cell-mediated killing, leaving activation of unlicensed NK cells
expressing KIR for non-self or missing ligands intact.
3268 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
by Peter Houghton, Nationwide Children’s Research Institute Center for
Childhood Cancer, Columbus, Ohio, USA) and 3 cell lines, BE(1)N, BE(2)
N, and SKNLP, derived from patients with NB treated at Memorial Sloan-
Kettering Cancer Center were cultured in RPMI-1640 medium supple-
mented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin,
100 μg/ml streptomycin, 1% sodium pyruvate, and 1% 2-mercaptoethanol
and incubated at 37°C with 5% CO2. To upregulate HLA class I expression,
recombinant human IFN-γ (PeproTech) was added at 1,000 units/ml for
72 hours (33). For supernatant experiments, PBMCs (1 × 105 cells per well)
were placed in culture alone or with NB cell lines at a ratio of 1:1, with or
without 3F8 and GM-CSF at 2 ng/ml (34) for 24 hours, in 96-well U-bottom
plates with 200 μl of medium, described above, per well. The supernatant
was collected, centrifuged, filtered through a 22-μm filter (Millipore), and
then used as culture media for the same NB cell lines for 72 hours prior
to assaying HLA expression. To test induction of HLA class I expression
by therapeutic agents, NB cell lines were cultured in complete medium in
the presence or absence of vincristine (50 ng/ml and 200 ng/ml), thiotepa
(20 μg/ml and 200 ng/ml), and doxorubicin (1 μg/ml) for 48 hours; isotret-
inoin (5 μM) for 72 hours; or GM-CSF (2 ng/ml) for 72 hours prior to
assaying HLA expression. Both PBMCs and purified NK cells from healthy
individuals were used for analysis of NK function. PBMCs were isolated
by Ficoll density gradient centrifugation. NK cells were isolated following
CD3+ depletion and CD56+ enrichment using the CliniMACS system (Milt-
enyi). Effector cells were cultured in medium, described above, supplement-
ed with human IL-2 (Proleukin, Prometheus) at 1,000 U/ml and incubated
at 37°C with 5% CO2 for 12 to 16 hours prior to assaying function.
NK activation and flow cytometric analysis. CD107 mobilization and
intracellular IFN-γ production were used as indicators of effector cell acti-
vation (48–50). PBMCs (5 × 105 cells per well) or NK cells (2 × 105 cells per
well) were incubated with NB target cells at ratios of 1:3 and 1:1, respec-
tively, with or without 0.8 μg/ml 3F8 for 4 hours in 96-well U-bottom
plates with 200 μl medium, described above, per well. The 3F8 concen-
tration was selected after titration assays demonstrated no difference in
in vitro NK activation at higher concentrations (Supplemental Figure
4) and is less than the serum concentration achieved in patients treated
with 3F8 (1–4 μg/ml) (51). For HLA class I blocking, NB cells were prein-
cubated for 30 minutes at 37°C with an antibody cocktail composed of
DX17 (BD Biosciences), and 6A4 and A6.136 (both provided by Alessan-
dro Moretta, Università di Genova, Genoa, Italy) (52) prior to the addition
of PBMCs or purified NK cells and 3F8. Isotype controls were used for
each sample. PerCPCy5.5-conjugated anti-CD107a or Pacific Blue–conju-
gated anti-CD107a (H4A3 clone, Biolegend) was added to each well prior
to incubation. Cells were stained with anti-CD3-APC-Cy7 (clone SK7, BD
Biosciences), anti-CD56-PE-Cy7 (clone B159, BD Biosciences), or anti-
CD56-ECD (clone N901, Beckman Coulter); anti-KIR2DL1/2DS1-APC
(clone HP-MA4, eBioscience), anti-KIR2DL2/2DL3/2DS2-FITC (clone
CH-L, BD Biosciences), anti-KIR3DL1-PE (clone DX9, BD Biosciences), or
anti-KIR3DL1–Alexa Fluor 700 (clone DX9, Biolegend); anti-NKG2A-PE
(clone Z199, Beckman Coulter); or Pacific Blue–conjugated anti-NKG2A
(clone Z199, Beckman Coulter). For IFN-γ evaluation, brefeldin-A (10
μg/ml, Sigma-Aldrich) and GolgiStop (6 μg/ml, BD Biosciences) were
added to the mixture 1 hour following incubation, and FIX & PERM
(Invitrogen) was used for staining. LIVE/DEAD Aqua Fixable Dead Cell
Stains (Invitrogen) were used to exclude dead cells from analysis. Cells
were analyzed using multicolor flow cytometry on a LSRII instrument
with FACS Diva software (BD Biosciences). Results were interpreted using
FlowJo software (Tree Star). For multicolor compensation and gating,
unstained, single-color, and FMO controls were used. NK cells exclusively
expressing a single inhibitory KIR were evaluated as previously described
(19, 22). HLA class I and GD2 expression on NB cells was evaluated with
strong a predictor of response and survival, if not better than sev-
eral conventional NB biomarkers (25). An analogous study includ-
ing only patients with NB, particularly those who do not receive
antibody treatment, should be performed to confirm whether
mAb is necessary for this effect.
Since licensed NK cells expressing S-KIRs have higher ADCC
capacity in general, rescuing licensed NK activity from class I
inhibition is desirable and could increase response in all patients,
including those with all KIR ligands present. Use of newly devel-
oped clinical antibodies directed to the inhibitory KIRs would pre-
vent their engagement with HLA cognate ligands and potentially
restore licensed NK function (39). In addition, the use of exog-
enous NK cells in the treatment of patients with NB may be poten-
tially useful if the patient lacks class I ligands for the donor inhibi-
tory KIRs. Augmentation of innate immunity through adoptive
transfer of allogeneic NK cells or the use of agents that increase
endogenous NK cell number and activity, such as IL-2, lenalido-
mide, and anti-CD137 antibody, may all improve NB control, par-
ticularly in the presence of 3F8 (40–42).
Unlicensed NK cells expressing NS-KIRs have a clinically sig-
nificant impact in the control of acute myelogenous leukemia in
allogeneic hematopoietic stem cell transplantation (23, 43, 44),
and it is clear from this study that their impact extends to other
tumor targets. While transplantation and mAb therapy are hardly
normal physiologic conditions, they both take advantage of the
important pool of unlicensed NK cells, previously thought to be
hyporesponsive and therefore potentially less clinically relevant. In
mice, unlicensed NK cells play a dominant role in clearing murine
cytomegalovirus (45, 46), in which the proinflammatory cytokine
environment of viral infection leads to higher function among
unlicensed cells. These cells can be recalled upon viral rechal-
lenge to eradicate infected cells more efficiently than licensed NK
cells, whose cytotoxic and proliferative function is diminished
in the presence of MHC class I expression (45, 46). It is possible
that activity among unlicensed NK cells in patients with NB may
also be augmented by the proinflammatory state associated with
tumor burden. The existence of unlicensed NK cells that have been
presumed to be hyporesponsive in an otherwise parsimoniously
constructed human immune system has been a paradox. These
findings contribute to our understanding of the critical role of
the unlicensed NK population in tumor control.
Patients. This is a retrospective analysis of 242 patients with high-risk NB
treated at Memorial Sloan-Kettering Cancer Center between 1994 and
2007 with anti-GD2 mAb 3F8. All but 1 patient received GM-CSF, 76%
of patients received oral isotretinoin, and 1 patient received oral β-glucan
(http://clinicaltrial.gov/; study nos. NCT00072358, NCT00002560, and
KIR and HLA genotyping. Genomic DNA, extracted from PBMCs or mar-
row mononuclear cells after remission using the QIAamp DNA Blood Mini
Kit (Qiagen), was KIR genotyped using previously published methods (47).
Patient HLA alleles were identified by a combination of HLA serology,
sequence-based amplification (PCR sequence-specific primer), and oligo-
nucleotide probing of genomic DNA (PCR-specific oligonucleotide probe).
HLA genotyping for healthy donors was performed at HistoGenetics.
Target cells, effector cells, and culture conditions. The human erythroleukemia
cell line K562 (ATCC) and human NB cell lines IMR-32 (ATCC) and LAN-1
(provided by Robert Seeger, Children’s Hospital Los Angeles, Los Angeles,
California, USA) as well as the human NB cell line NB-1691 (13) (provided
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 9 September 2012
Study approval. Informed consent for specimen collection was obtained
from patients or legal guardians in accordance with institutional review
board guidelines at Memorial Sloan-Kettering Cancer Center. These stud-
ies were approved by the institutional review board of Memorial Sloan-
Kettering Cancer Center.
We acknowledge the neuroblastoma team at Memorial Sloan-
Kettering Cancer Center and Marcel Van Den Brink for use of
the LSRII flow cytometer. This study was supported in part
by Alex’s Lemonade Stand Foundation, the NIH (HL088134,
CA083766, and CA106450), the CALGB Clinical Scholars
Award, the Robert Steel Foundation, and Katie Find a Cure. We
acknowledge the support Novartis Oncology, provided through
the CALGB Foundation.
Received for publication January 9, 2012, and accepted in revised
form June 28, 2012.
Address correspondence to: Katharine C. Hsu, Department of
Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York
Ave., New York, New York 10065, USA. Phone: 646.888.2667; Fax:
646.422.0298; E-mail: email@example.com.
Nidale Tarek’s present address is: Department of Pediatrics, The Uni-
versity of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Jeffrey M. Venstrom’s present address is: Department of Medicine,
UCSF, San Francisco, California, USA.
anti-HLA-ABC-FITC (clone G46-2.6, BD Biosciences), anti-HLA-Bw4-FITC
(clone FH007, One Lambda), anti-HLA-E-PE (clone 3D12, Biolegend), and
anti-GD2–Alexa Fluor 488 (clone 2Q549, Abcam).
NK cytotoxicity. Cell-mediated cytotoxicity was analyzed by flow cytome-
try with the PKH26 membrane dye and TO-PRO-3 DNA label as previously
described (19), with and without 3F8.
Cytokine assay. IFN-γ production was measured by ELISA (R&D Sys-
tems) according to the manufacturer’s instructions and analyzed using
an MRX plate reader (Dynex Technology) and Biolinx 2.22 software
Statistics. OS was defined as the time from 3F8 therapy to the date of
death or last follow-up. PFS was defined as the time from 3F8 therapy to
the date of relapse, death, or last follow up. Survival probabilities were
estimated using the Kaplan-Meier method. The log-rank test was used to
compare survival probabilities among patients missing any or possessing
all KIR ligands. HRs of missing KIR ligand indicators were estimated by
multivariable Cox regression, controlling for age, MYCN amplification,
and bone disease. Fisher’s exact test was used to determine whether the
disease status distribution differed between the missing ligand and all
ligands present patient groups. To investigate activation of licensed and
unlicensed NK cells, CD107 mobilization and intracellular IFN-γ were
measured for each population following incubation with NB cell lines in
the presence or absence of 3F8. Paired 2-tailed Student’s t test was used to
compute the comparisons within the licensed or unlicensed populations,
and the unpaired 2-tailed Student’s t test was used to compute compari-
sons between the unlicensed and licensed populations. For relative fold
increase calculations, the unpaired Student’s t test was used to compare the
relative activity of licensed and unlicensed NK cells. A test with a P value of
less than 0.05 was considered statistically significant.
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