Blocking NK Cell Inhibitory Self-Recognition Promotes
Antibody-Dependent Cellular Cytotoxicity in a Model of
Liat Binyamin,* R. Katherine Alpaugh,* Tracey L. Hughes,* Charles T. Lutz,†
Kerry S. Campbell,* and Louis M. Weiner2*
Human NK cells lyse Ab-coated target cells through the process of Ab-dependent cellular cytotoxicity (ADCC). Improving ADCC
responses is desirable because it is thought to be an important antitumor mechanism for some Abs. NK cell inhibitory receptors,
such as killer cell Ig-like receptors, engage with MHC class I molecules on self-cells to block NK cell activation. Accordingly, we
enhanced ADCC responses by blocking NK cell inhibitory receptors, thus perturbing induction of the self-recognition signal. In
a cell line model of anti-lymphoma therapy, the combination of rituximab with an Ab that blocks inhibitory self-recognition
yielded increased NK cell-mediated target cell lysis when compared with rituximab alone. To validate this proof-of-concept, we
then used a more representative approach in which an individual’s fresh primary NK cells encountered autologous, EBV-trans-
formed B cells. In this system, rituximab and a combination of Abs that block NK cell inhibitory receptors yielded improved NK
cell-mediated lysis over rituximab alone. The results show, for the first time, that disruption of inhibitory self-recognition can
efficiently promote ADCC in a human model, applying an autologous system in which physiologic checkpoints are in place. This
method provides an alternative approach to potentiate the therapeutic benefit of antitumor Abs that mediate ADCC. The Journal
of Immunology, 2008, 180: 6392–6401.
between the Ab Fc domains and Fc receptors expressed by acces-
sory cells (4). Several families of Fc receptors have been identi-
fied, and specific leukocyte populations characteristically express
defined Fc receptors (5). In particular, human NK cells express the
A (transmembrane) isoform of CD16 (Fc?RIIIA) (6). Fc-mediated
engagement of CD16A leads to phosphorylation of ITAMs on the
receptor-associated Fc?RI and TCR-? adaptor proteins. The phos-
phorylated ITAM then serves as a docking site for either the Syk
or ZAP70 tyrosine kinases, triggering a downstream cascade of
activation events that can lead to NK cell lysis of the Ab-coated
target (7). The mechanism of attack is analogous to that of CTLs,
involving the release of cytoplasmic granules containing perforin
and granzymes (8). Thus, ADCC uses the engagement of an Fc
he process of Ab-dependent cell-mediated cytotoxicity
(ADCC)3is considered to be a major antitumor mecha-
nism (1–3). This property is dependent upon interactions
receptor by Abs to direct an Ag-specific attack by NK cells that
otherwise lack specificity for a particular Ag. This mechanism of
NK cell lysis of Ab-coated cells has been shown to be a major
mechanism for direct antitumor effects in some settings, and also
for the regulation of innate and adaptive immune responses (1,
9–13). Indeed, several groups have provided a mechanistic basis
for the link between NK cells and Ag presentation, showing that
NK cells directly interact with and reciprocally activate dendritic
Rituximab is a chimeric anti-CD20 mAb that was the first un-
conjugated therapeutic anticancer mAb to be approved by the
FDA, and it is now a component of effective treatment for B cell
lymphomas that express CD20 (18–20). Rituximab efficiently ini-
tiates ADCC through CD16, thus its function is dependent on the
interaction between the Ab Fc domain and the Fc?RIIIA (21).
Valine (V) or phenylalanine (F) genetic polymorphisms of
Fc?RIIIA aa 158 alter affinity toward IgG Fc. Low-grade lym-
phoma patients with the homozygous higher affinity (V/V) poly-
morphism exhibit improved clinical response rates compared with
those possessing the lower affinity (V/F or F/F) polymorphism
after treatment with rituximab (1, 22, 23). Strategies to improve
mAb therapy are mainly focused on modifying mAb structure to
improve the affinity of Abs for Fc?RIII and other Fc?Rs to more
efficiently mediate ADCC (24).
NK cells are defined as a unique subset of lymphocytes that do
not express rearranged Ag recognition receptors (e.g., TCR or
BCR), but rather express numerous Ig-like receptors and C-type
lectin receptors that deliver a finely tuned balance of inhibitory and
activating signals. These receptors allow the NK cells to discrim-
inate self, healthy cells from transformed or pathogen-infected
cells, and regulate their effector function (“the missing self hy-
pothesis”) (25–28). It is now apparent that NK cells recognize and
kill target cells as a result of a balance of signaling by both inhib-
itory and activating NK cell receptors (29, 30). Most inhibitory
*Department of Medical Oncology and Division of Basic Science, Fox Chase Cancer
Center, Philadelphia, PA 19111; and†Department of Pathology and Laboratory Med-
icine, University of Kentucky, Lexington, KY 40536
Received for publication February 27, 2008. Accepted for publication February
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants CA50633, CA06927, AI050656, AI050656, and
CA083859 from the National Institutes of Health, by grants from the Frank Strick
Foundation and the Bernard A. and Rebecca S. Bernard Foundation, and by an ap-
propriation from the Commonwealth of Pennsylvania.
2Address correspondence and reprint requests to Dr. Louis M. Weiner, Lombardi
Comprehensive Cancer Center, Georgetown University Medical Center, Research
Building, Room E501, P. O. Box 571468, 3970 Reservoir Road, NW Washington,
DC 20057-1468. E-mail address: email@example.com
3Abbreviations used in this paper: ADCC, Ab-dependent cellular cytotoxicity; KIR,
killer cell Ig-like receptor; RISER, reversal of inhibitory self-recognition.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
receptors recognize MHC class I and class I-like molecules,
whereas the function and ligand specificity of some activating re-
ceptors still remains to be elucidated (11). The two major inhibi-
tory receptors in NK cells are the killer cell Ig-like receptors
(KIRs), which recognize HLA-A, HLA-B, or HLA-C and C-type
lectin CD94/NKG2A heterodimers, which recognize HLA-E
Fc?RIIIA engagement, as described, represents only one of
numerous mechanisms by which NK cells can be activated. The
triggering of natural cytotoxicity receptors (reviewed in Ref. 35),
activating CD94/NKG2 receptors, activating forms of KIRs, as
well as a lack of self-recognition by inhibitory KIRs or NKG2A/
CD94 could provide alternative activation signals (36).
KIRs constitute a polymorphic group of molecules that are en-
coded by multiple loci on chromosome 19 and vary in certain
structural features. Each member interacts with a different group of
closely related class I HLA-A, HLA-B, or HLA-C molecules.
Members of the KIR family have been shown to be highly poly-
morphic at the allelic and haplotypic level. To adapt to the rapid
evolution and divergence of MHC class I molecules, Khakoo et al.
(37) have shown that KIRs have also evolved very rapidly in pri-
mates. Significant diversity of KIRs occurs in human populations
because a given person inherits a defined repertoire of receptors
and these are differentially expressed on the individual NK cells
(38). Only the KIR family members with long cytoplasmic do-
mains (KIR2DL and KIR3DL) are associated with inhibitory sig-
naling. The inhibitory signal from the KIR is transduced via the
ITIMs located in the cytoplasmic domain of the receptor, whereas
the triggering signal from the activating KIRs is transduced via
association with DAP12 adaptor proteins bearing an ITAM. Given
that inhibitory and activating receptors are being expressed on the
same NK cell, a predominance of inhibitory signaling ensures tol-
erance to self HLA-expressing autologous cells (35). Although the
full complement of inhibitory receptors inherited by an individual
is not expressed on every NK cell of that individual, most, but not
all NK cells have at least one inhibitory receptor for a self MHC
class I molecule, which either can be a KIR or CD94/NKG2A
receptor (12, 39).
The role for KIR engagement in the process of negative control
of NK cell activation was described in several molecular-based
studies (40, 41). Also, the loss of MHC class I molecules from the
surfaces of virally infected cells or tumor cells was shown to be
associated with increased susceptibility to NK cell lysis (42). Basic
limitations in the ability to obtain in vivo mechanistic proof-of-
concept in rodent models are due to lack of KIR in mice. However,
several studies in syngeneic murine models have shown that the
manipulation of inhibitory NK cell signaling can prime NK cells to
exert in vivo antitumor activity and prevent allograft rejection (43–
45). These data also show that this blockade is frequently insuf-
ficient to mediate complete tumor eradication because the block-
ade is not associated with sufficiently potent concomitant
activation signals provided by the tumors.
Our approach differs from other approaches for the enhancement
of the ADCC properties of antitumor Abs in that we aim to down-
regulate the signaling mediated by NK cell inhibitory self-recog-
nition receptors to tip the balance toward ADCC-mediated tumor
attack. This approach can thus complement attempts to create more
powerful activating Abs that signal through CD16. In this study,
we define the potential clinical value of blocking inhibitory self-
recognition by human NK cells to promote mAb-mediated ADCC.
We show that lowering the threshold for NK cell activation
through the interruption of inhibitory self-recognition checkpoints
improves NK cell cytotoxicity toward Ab-coated transformed B
Materials and Methods
NK-92 and NK-92.26.5 cell lines (a subclone generated to express novel
genes, notably KIRs, by treatment with 5-aza-2?-deoxycytidine, as de-
scribed (46) were maintained in ?-MEM (Life Technologies) containing
10% FBS (HyClone Laboratories), 10% horse serum, 2 mM L-glutamate,
100 ?g/ml penicillin, 100 ?g/ml streptomycin, 1 mM sodium pyruvate (all
from Life Technologies), 100 ?M 2-ME (Fisher), 2 mM folic acid (Sigma-
Aldrich), 20 mM myoinositol (Sigma-Aldrich). Cell lines were supple-
mented with 2% culture supernatant of J558L cells transfected with the
human IL-2 gene provided by A. Lanzavecchia (Institute for Research in
Biomedicine, Bellinzona, Switzerland). Cells were passed with fresh IL-2
every 4 days.
Lymphoblast transfectant cell lines 721.221-B*5101 (B51) and
721.221-Cw4 (47) were maintained in RPMI 1640 containing 10% FBS, 2
mM L-glutamate, 100 ?g/ml penicillin, 100 ?g/ml streptomycin, 1 mM
sodium pyruvate, 1 mM HEPES buffer, and 50 ?M 2-ME.
SK-OV-3 ovarian carcinoma cells were maintained in DMEM contain-
ing 10% FBS, 2 mM L-glutamate, 100 ?g/ml penicillin, 100 ?g/ml strep-
tomycin, and 50 ?M 2-ME. NK cells were purified from donor whole
blood using the RossetteSep NK cell enrichment mixture (StemCell Tech-
nologies) according to the manufacturer protocol, and under Institutional
Review Board approval. EBV transformation of autologous B cells ob-
tained from donors was performed in the Fox Chase Cancer Center Cell
Culture Facility using the B95-8 strain of the EBV (48).
Generation of CD16-expressing NK-92 cells
CD16 cDNA (V158 polymorphic variant) provided by Dr. B. Perussia
(Thomas Jefferson University, Philadelphia, PA) was ligated into the bi-
cistronic retroviral expression vector, pBMN-IRES-EGFP, provided by Dr.
G. Nolan (Stanford University, Stanford, CA) using the BamHI/NotI re-
striction sites (49) to produce recombinant retrovirus for transduction of
NK cell lines with stably integrated cDNA. The oligonucleotides 5?-CTT
CTG CAG GGG GCT TTT TGG GAG TAA AAA TGT GTC T and
5?-AGA CAC ATT TTT ACT CCC AAA AAG CCC CCT GCA GAA G
were used to generate the CD16-F158 variant as well as primers overlap-
ping to the pBMN-IRES-EGFP vector 5?-GCA TCG CAG CTT GGA TAC
AC and 5?-GGC GGA ATT TAC GT AGC G and digestion with the
BamHI and NotI restriction sites. The integrity of all constructs was con-
firmed by sequencing in the Fox Chase Cancer Center Automated DNA
Sequencing Facility (Applied Biosystems). Transduction of the CD16 con-
struct was as previously described (49). Briefly, the packaging cell line,
Phoenix-Amphotropic was transfected with the pBMN-IRES-EGFP vector
etry was used to determine KIR expression on NK-92.26.5 cells. The Abs
HP3E4 (thin black histogram) binding KIR2DL1, KIR2DS1 and
KIR2DS4; DX9 (gray-shaded histogram) binding KIR3DL1; GL183 (thick
gray histogram) binding KIR2DL2, KIR2DL3, and KIR2DS2; and 5.133
(thick black histogram) binding KIR3DL1, KIR3DL2 and KIR2DS4 are
used. Thin gray histogram is isotype control stain. B, KIR3DL1 is a func-
tional inhibitory receptor on NK-92.26.5 cell line. NK-92.26.5 cells were
incubated with51Cr-labeled Fc?R-expressing P815 cells (redirected cyto-
toxicity assay) at various E:T ratios with no Ab (E), anti-CD56 (B159) (f),
or anti-KIR3DL1 (DX9) (F) Abs at 1 ?g/ml each.51Cr release was mea-
sured 4 h later. Results shown are representative of at least three indepen-
A, KIR expression NK-92.26.5 effector cells. Flow cytom-
6393 The Journal of Immunology
containing the CD16 gene using Lipofectamine Plus reagent (Life Tech-
nologies). Supernatants of these transfected cells grown in serum-free Opti-
MEM medium (Life Technologies) for 2 days were cocultured with NK-92
or NK-92.26.5 cell lines for 8 h in the presence of Lipofectamine Plus
reagent. Complete ?-MEM containing IL-2 was added for 3 days. At that
time 5–10% of the infected NK cells efficiently expressing enhanced GFP
and CD16 were sorted on a FACSVantage flow cytometer (BD Bio-
sciences) in the Fox Chase Cancer Center Cell Sorting Facility.
KIR and NK cell receptor-directed Abs used in these studies were: DX9
binding KIR3DL1 (mouse IgG1) produced from a hybridoma obtained
from Dr. L. Lanier (University of California, San Francisco, CA), HP3E4
binding KIR2DL1, KIR2DS1, and KIR2DS4 (mouse IgM; BD Pharmin-
gen), 143211 binding KIR2DL1 (mouse IgG1; R&D Systems), GL183
binding KIR2DL2, KIR2DL3, and KIR2DS2 (mouse IgG1; Immunotech),
5.133 binding KIR3DL1, KIR3DL2 and KIR2DS4 (mouse IgG1) produced
from a hybridoma obtained from Dr. M. Colonna (Washington University,
St. Louis, MO), Z199 binding NKG2A (mouse IgG2b; Beckman Coulter),
and HP3B1 binding CD94 (mouse IgG2a; Immunotech). B159 binding
CD56 (mouse IgG1) produced from a hybridoma obtained from Dr. B.
Perussia was used as a control Ab in ADCC assays. CD16 Ab, CLB-Fc
obtained from Dr. B. Perussia, and 3G8 (BD Pharmingen) were used to
detect CD16 expression of the two polymorphic variants at residue 158
c-erbB2) Abs were used to direct NK cell cytotoxicity (in ADCC assays).
CD56 R-PE (NCAM 16.2; BD Biosciences) and CD3-FITC (Leu-4; BD
Biosciences) conjugated Abs were used to identify NK cell populations.
DX17 is an Ab reactive with all HLA class I (HLA-A, -B, -C, -E, -G, and
-F) molecules provided by Dr. L. Lanier.
CD16 polymorphism analysis
Donors’ CD16 polymorphisms were determined in the Fox Chase Cancer
Center Cell Cancer Biomarker and Genotyping Facility using Sequence-
Specific Primer SSP kits (SSP UniTray; Pel-Freez) from Dynal Biotech.
ADCC studies were performed as previously described (50). Target cells
were labeled with Na2
1 h at 37°C in 500 ?l of FBS. The51Cr-labeled target cells were washed
twice and resuspended at the desired concentration in RPMI 1640. Ten
thousand cells were added to individual wells of 96-well flat-bottom plates
(Costar) containing NK cells (effector cells) at the indicated E:T ratio
and/or at indicated concentrations of Abs in supplemented RPMI 1640.
Each well contained a total volume of 200 ?l, and all assays were per-
formed in triplicate. The plates were centrifuged at 300 ? g for 3 min,
incubated for 4 h in a 5% (v/v) CO2incubator at 37°C, and then centrifuged
again at 300 ? g for 3 min. Supernatant (100 ?l) were removed from each
well for counting on a Packard Instruments Cobra Quantum, Series 5002
(PE Life Sciences). Cytotoxicity was estimated by measuring the quantity
of label released into culture supernatants using the formula: percentage of
lysis ? 100 ? (experimental release (cpm) ? spontaneous release (cpm))/
(total counts (cpm)/2 ? spontaneous release (cpm)), where the experimen-
tal release was defined as cpm released by target cells in the presence of
effector cells or Ab and the spontaneous release was defined as cpm re-
leased by target cells alone.
51CrO4(100 ?Ci/106targets; PE Life Sciences) for
The expression levels of NK cell receptors were determined by flow cy-
tometry with previously described techniques (51). Briefly, 1 ? 106cells
were incubated with the relevant Ab for 30 min at 4°C. The cells were
washed before the addition of fluorochrome-conjugated goat anti-mouse ?
Ab (Southern Biotechnology Associates). The degree of fluorescence was
determined using a FACScan flow cytometer (BD Biosciences), and data
were analyzed using FlowJo software (Tree Star).
Our goal in this study was to establish NK cell to target cell models
to study the impact of blocking self-inhibitory receptor interactions
or V158 variant as described in Materials and Methods. Cells were stained with anti-CD16 Abs to compare expression. Anti-CD16 Abs are CLB-Fc
(gray-filled histogram) and 3G8 (thick black histogram). Reactivity was detected with an R-PE-conjugated anti-mouse ? Ab. Thin black histogram is
secondary Ab alone. NK-92.26.5 cells (A), CD16-F158 transduced NK-92.26.5 cells (B), and CD16-V158 transduced NK-92.26.5 cells (C) are shown.
Flow cytometric analysis of CD16 transduced NK-92.26.5 cells. NK-92.26.5 cells were transduced with CD16 cDNA encoding either F158
CD16-F158.NK-92.26.5 (u), or CD16-V158.NK-92.26.5 (f) were incubated with51Cr-labeled 721.221-B*5101 (B51), CD20-expressing B cells (A) or
51Cr-labeled SK-OV-3, c-erbB2-expressing ovarian cancer cells (B) at a 10:1 ratio, in the presence of the indicated concentrations of rituximab or
trastuzumab, respectively.51Cr release was measured 4 h later. Results shown are mean ? SD of one representative experiment of at least three independent
ADCC is induced to different levels by CD16-F158 or CD16-V158 polymorphic variants of NK-92.26.5 cell line. NK-92.26.5 (?),
6394BLOCKING NK CELL SELF-RECOGNITION PROMOTES ADCC
with Abs to increase NK cell-mediated ADCC of Ab-coated lym-
KIR and CD16 expression on effector cells
To determine the impact of KIR blockade we first used the NK-92
subclone NK-92.26.5. These cells have been described elsewhere
(46) and we confirmed by flow cytometry that KIRs are expressed
on the surface (Fig. 1A). Abs that have been reported to function-
ally block KIR recognition were used for the flow cytometry anal-
ysis. These Abs are HP3E4 (52), DX9 (53), GL183 (54), and 5.133
(55). Of these Abs, only DX9 is monospecific for a single inhib-
itory receptor KIR3DL1. To study the ability of KIR3DL1 on the
NK-92.26.5 cells to deliver an inhibitory signal a “reverse ADCC”
assay (56, 57), using mouse Fc?R-positive P815 target cells, was
performed (Fig. 1B). Inhibition of P815 lysis by NK-92.26.5 was
observed when using the DX9 Ab, indicating that KIR3DL1 in-
hibitory signaling was triggered by the engagement with DX9 via
Fc receptors on the P815 cells. These results indicated that
KIR3DL1 is functional on NK-92.26.5 cells, as it can deliver a
dominant negative signal to those cells.
NK cell-mediated ADCC depends on Fc?RIIIA (CD16) inter-
actions (24). However, NK-92.26.5 does not naturally express
CD16 (Fig. 2A). Two genetic variants of the CD16 receptor have
different affinities for the Fc domain of the Ab and their presence
correlates with different clinical response rates when the ADCC-
mediating anti-CD20 Ab, rituximab, is used as a therapeutic agent
(1). To make NK-92.26.5 cells competent to mediate ADCC, these
cells were modified by retroviral transduction of CD16 cDNA to
stably express CD16. The two common polymorphic variants of
CD16-cDNA were separately transduced into NK-92.26.5 cells.
The variants contained the amino acid valine or phenylalanine at
position 158 of the protein sequence, with sequence assignment
based on numbering of the mature polypeptide (GenBank acces-
sion nos. BC017865.1 and NM_000569.6, respectively) (58). To
toxicity against 721.221-B*5101 (B51) cells by NK-
92.26.5 cells. A, NK-92.26.5 cells were incubated at an
E:T ratio of 5:1 with
(HLA-Bw4 transfected B cells) in the presence of var-
ious concentrations of B159 Ab (?) binding CD56 or
DX9 Ab (F) binding KIR3DL1. B, Cytotoxicity was
measured at different E:T ratios with no Ab (gray-filled
triangle), in the presence of B159 (?), DX9 (F), or
GL183 binding KIR2DL2, KIR2DL3, and KIR2DS2
(E) all at 1 ?g/ml.51Cr release was measured 4 h later.
Results shown are mean ? SD of one representative
experiment of at least three independent experiments.
KIR3DL1 blocking Ab induces cyto-
hibitory self-recognition receptor KIR3DL1. CD16-
V158 (A, C, and E) or CD16-F158 (B, D, and F) vari-
ants of the NK-92.26.5 cell line were incubated with
51Cr-labeled 721.221-B*5101 (B51) cells, at a 5:1 E:T
ratio and with various concentrations of DX9 (A and B)
and with (f) or without (?) 10 ng/ml rituximab, or with
various concentrations of rituximab (C and D) and with
(F) or without (E) 0.1 ?g/ml DX9. E and F, Cells were
incubated with no Ab (?), DX9 Ab alone (u) at 0.1
?g/ml, rituximab (gray-striped box) at 10 ng/ml, or with
rituximab plus DX9 Ab (f).51Cr release was measured
4 h later. Results shown are mean ? SD of one repre-
sentative experiment of at least three independent
ADCC is augmented by blocking the in-
6395The Journal of Immunology
determine CD16 expression levels on the cells, flow cytometry
assays were performed using two different anti-CD16 Abs,
CLB-Fc and 3G8 (Fig. 2). Using the CLB-Fc Ab we confirmed
that CD16 expression was at a comparable level in the two variant
cell lines. Fluorescence intensity staining with 3G8 was higher for
CD16-V158.NK-92, most likely because of higher affinity binding
compared with the CD16-F158.
To confirm that the CD16 NK-92.26.5 cells can mediate ADCC,
we used CD20?721.221-B*5101 (B51) or c-erbB2?SK-OV-3
target cells, in the presence of serial dilution of the Ag-reactive
mAbs rituximab or trastuzumab (59), respectively. ADCC was me-
diated only toward target cells that expressed the specific Ag (Fig.
3). CD16-V158 NK-92.26.5 cells mediated higher maximal cyto-
toxicity and were more sensitive to lower concentrations of Ab
compared with the corresponding CD16-F158 cells. This result is
in accordance with the flow data and previous reports regarding the
higher affinity of the CD16-V158 variants to the Fc domain of the
Ab (60). ADCC was not mediated by CD16?NK-92.26.5 cells or
by Ab that does not bind to the target cells (trastuzumab to B51 or
rituximab to SK-OV-3).
Increasing NK cell cytotoxicity by KIR blockade
To determine whether cytotoxicity of NK-92.26.5 against relevant
target cells could be increased by blocking inhibitory self-recog-
nition we performed cytotoxicity assays using B51 cells as targets.
B51 B cells were transfected to solely express HLA-B*5101,
containing the HLA-Bw4 epitope (47) for the engagement by
KIR3DL1 receptor. In this setting, blockade of the KIR3DL1 in-
teraction by the DX9 Ab promoted cytotoxicity in a concentration-
dependent manner (Fig. 4A). Attempted blockade of the other in-
hibitory receptor KIR2DL2 and KIR2DL3 by GL183 blocking Ab
failed to induce cytotoxicity against the B51 target cells (Fig. 4B).
Because these mechanisms are CD16-independent, results are
shown for the NK-92.26.5 cells but were equivalent to the results
with CD16-V158 or CD16-F158 transduced NK-92.26.5 cells
(data not shown).
To confirm that in the presence of the intact DX9 Ab CD16.NK-
92.26.5 cells are not cross-linked to each other to result in killing
of the NK cell population (i.e., by the Fc domain of DX9 that
potentially can bind CD16 on NK cells and the binding domain of
the DX9 Ab that binds KIR3DL1 on a second NK cell), a cyto-
toxicity assay was performed with only half of the CD16.NK-
92.26.5 cells labeled with51Cr (target cells), and in the presence of
intact DX9 Ab or F(ab?)2of the DX9 Ab. The results were con-
sistent with no detected cytotoxicity (data not shown).
Blocking inhibitory self-recognition improves ADCC
We then assessed the impact of combining the potentially com-
plementary NK cell-activating mechanisms of CD16 engagement
and blockade of inhibitory self-recognition.
Effector cells (NK-92.26.5 CD16-V158 or CD16-F158) were
incubated with B51 targets, at varied E:T ratios (5:1 E:T ratio
shown) using different concentrations of DX9 and in the presence
or absence of rituximab (10 ng/ml) (Fig. 5, A and B). The results
of the cytotoxicity assays for both CD16-V158 and CD16-F158
variants show that at any given DX9 concentration, adding ritux-
imab improved NK cell-mediated target cell lysis. We repeated
this experiment with both intact DX9 Ab and F(ab?)2fragments of
DX9 and found consistent results (data not shown). As expected,
at this low rituximab concentration, the amplitude of the effect was
greater with the higher affinity CD16 variant (V158). We then
incubated the effector and target cells in different concentrations of
rituximab and in the presence or absence of DX9 (0.1 ?g/ml) (Fig.
5, C and D). As shown, combining DX9 with any given rituximab
concentration yielded higher NK cell-mediated target cell lysis
receptor on fresh purified NK cells. NK cells were purified from a whole
blood sample obtained from a healthy donor as described in Materials and
Methods. A, Purity of NK cells was analyzed by flow cytometry staining
with CD3-FITC and CD56-PE Abs. B, Flow cytometric analysis was used
to determine KIR expression on purified NK cells. Staining with 143211
Ab (thick black histogram) binding KIR2DL1 and DX9 Ab (thick gray
histogram) binding KIR3DL1) is shown. Thin gray histogram is secondary Ab
alone. NK cells from this donor (expressing KIR2DL1 on all NK cells, but
KIR3DL1 on only a subpopulation, see Fig. 5B) were incubated with51Cr-
labeled 721.221-Cw4 cells (C) or with autologous EBV-transformed B cells
(D and E) at a 10:1 E:T ratio. Cytotoxicity was calculated using a 4-h51Cr
release assay in the presence of B159 binding CD56 (C and D), HP3E4 bind-
ing KIR2DL1, KIR2DS1, and KIR2DS4 (C and D), DX9 binding KIR3DL1
(E), and rituximab (C–E) in different combinations as indicated. All Abs were
used at 1 ?g/ml concentration. Results shown are mean ? SD of one repre-
sentative experiment of at least three independent experiments.
ADCC promotion by blocking inhibitory self-recognition
6396BLOCKING NK CELL SELF-RECOGNITION PROMOTES ADCC
than with rituximab alone. Cytotoxicity was also compared using
the different combinations of blocking inhibitory Abs at varied E:T
ratios (Fig. 5, E and F). A combination of rituximab plus DX9
yielded the highest NK cell-mediated target cell lysis, when either
CD16-V158.NK-92.26.5 or CD16-F158.NK-92.26.5 was the ef-
fector cell (Fig. 5, E and F, respectively). KIR3DL1 blockade
alone efficiently promoted B51 cell killing; in this E:T cell com-
bination, KIR3DL1 is the only receptor that contributes to inhib-
itory self-recognition. In the CD16 high affinity effector cell set-
ting, CD16-V158.NK-92.26.5, rituximab alone mediated a
significant level of killing and target cell lysis was only modestly
promoted when rituximab and DX9 were used together (Fig. 5E).
In the CD16-F158 effector cell setting, the level of cytotoxicity
significantly increased when combining DX9 and rituximab com-
pared with rituximab alone (Fig. 5F) and was equivalent to the
level of cytotoxicity achieved by the CD16-V158 cells when tested
with rituximab alone. This experiment provides a proof-of-concept
that blockade of inhibitory self-recognition can be applied to in-
crease the degree of ADCC by human NK cells.
A primary NK cell model with autologous target cells
Although our NK-92 cell line model system expressing KIR3DL1
provides a proof-of-concept, endogenous NK cells express multi-
ple inhibitory receptors to sustain MHC class I-mediated self-rec-
ognition. The therapeutic potential of KIR blockade in improving
antitumor ADCC responses is best modeled using NK cells and
autologous targets. In screening a number of volunteer donors, we
identified one unique individual who expressed KIR2DL1 on all
NK cells. As revealed by HLA phenotyping, this donor also ex-
presses the ligand for KIR2DL1 (HLA-C5, HLA-C6; American
Red Cross Laboratories). Flow cytometry analysis performed on
purified NK cells showed that an anti-KIR2DL1-specific Ab
(143211) stained all of this donor’s NK cells (Fig. 6B). In contrast
to a previous publication that described a severe immunodefi-
ciency syndrome in a patient with KIR2DL1 expressed on all NK
cells (61) our donor is healthy and exhibits entirely normal im-
mune function. Freshly isolated, highly purified NK cells (92%)
(Fig. 6A) were used as effector cells in ADCC experiments against
721.221-Cw4 B cells (expressing HLA-Cw4, a ligand for
KIR2DL1) or EBV-transformed B cells from the same individual.
Both target B cell populations were positively stained by rituximab
(data not shown). As shown in Fig. 6C, 721.221-Cw4 target cells
were not effectively killed by purified donor NK cells in the pres-
ence of either no Ab, inhibitory receptor blocking Ab HP3E4
(blocking KIR2DL1), or the B159 anti-CD56 Ab. Rituximab or a
combination of rituximab plus B159 control Ab achieved 40%
target cell lysis, while the combination of HP3E4 KIR2DL1 block-
ing Ab plus rituximab yielded a further 30% increase in cytotox-
icity over rituximab alone. In Fig. 6D, target cells were autologous
EBV-transformed B cells. Although the absolute percentage of ly-
sis varied between targets, the pattern was similar. The lower in-
tensity of cytotoxicity against autologous cells is presumably due
to additional protective interactions or lack of activating signals
(62). Importantly, however, exposure to only the KIR2DL1 block-
ing Ab (HP3E4) did not increase NK cell cytotoxicity against au-
tologous B cells (Fig. 6D), indicating that blocking KIR alone does
not break tolerance. In contrast, the combination of rituximab and
the KIR2DL1 blockade significantly improved the ability of the
NK cells to lyse these targets. In this donor, ADCC was promoted
by blocking the function of an inhibitory self-recognition receptor
that is ubiquitously expressed on all NK cells.
The expression of KIRs and other NK cell receptors was studied
in four other donors using specific Abs: Z199 (NKG2A), HP3B1
(CD94), DX9 (KIR3DL1), HP3E4 (KIR2DL1, 2DS1, 2DS4), and
GL183, which is an Ab with a broader KIR specificity (KIR2DL3,
KIR2DL2, KIR2DS2). Fig. 7 shows the diversity in expression
patterns as revealed by flow cytometry analysis. As shown, con-
siderable interdonor variability was observed in the expression of
the various NK cell receptors. CD16 polymorphisms were also
determined. We then performed ADCC assays using freshly iso-
lated NK cells (effector cells) and autologous EBV-transformed B
cells (target cells). Effector and target cells were incubated with
NK cell Abs, with or without rituximab. Table I shows the lysis of
EBV-transformed B cells when incubated with different Ab com-
binations. When each of the NK cell receptors Abs was individu-
ally used to block inhibitory self-recognition in rituximab-based
ADCC assays, we determined that the impact of interference with
self-recognition has variable and generally modest effects on
ADCC promotion as compared with rituximab alone.
versity of NK cell receptors and
CD16 polymorphisms among donors.
NK cells were purified from whole
blood samples obtained from healthy
donors as described in Materials and
Methods. Flow cytometry analysis
was used to determine NK cell recep-
tor expression on purified NK cells of
each individual using available Abs
described in Materials and Methods.
CD16 polymorphism status was de-
termined by the Fox Chase Cancer
Center Biomarker and Genotyping
Facility as described in Materials and
Expression of the di-
6397 The Journal of Immunology
We then tested the hypothesis that interfering with multiple in-
hibitory receptors would effectively tip the balance in favor of
Ab-promoted cytotoxicity. We used a combination of Abs, termed
reversal of inhibitory self-recognition (RISER) combination, to
block inhibitory self-recognition. The RISER combination consists
of the Abs that were used in the experiments described in this
study. We found that a mixture of Abs with broad coverage of a
large number of NK cell receptors, most of which are inhibitory,
had a dramatic influence on the capacity of donor NK cells to lyse
autologous B cell targets in the presence of rituximab, but not in its
absence (Table I). This effect was equivalent or more pronounced
than that obtained when autologous self-recognition was blocked
through incubation with DX17, a pan anti-MHC class I Ab (63).
As re-demonstrated in Fig. 8A, applying rituximab and the RISER
combination of Abs was superior in achieving Ab-mediated NK
cell cytotoxicity over rituximab alone. The improvement in cyto-
toxicity was independent of CD16 polymorphism status.
We also tested an anti-CD19 Ab (Xencor) in this system (Fig.
8B). The Fc domain of this Ab was manipulated to improve affinity
to Fc receptor and thus to more efficiently promote ADCC com-
pared with an Ab of identical specificity and affinity, but with a
wild-type IgG1 Fc domain (data not shown). When freshly isolated
NK cells and autologous EBV-transformed B cells were incubated
with the manipulated CD19 Ab in the absence or presence of the
NK cell RISER combination of Abs, NK cell-mediated cytotoxic-
ity was augmented in all donors, suggesting that the blockade of
inhibitory self-recognition provides a benefit that is additive to the
effects of optimizing Ab structure to promote ADCC.
In this study, we show for the first time that ADCC can be pro-
moted by blocking inhibitory self-recognition receptors on human
NK cells. This mechanism was shown in an appropriate autologous
human system, providing physiologic “checkpoints”. This ap-
proach offers a new direction in the development and improvement
of new and existing therapeutic Ab treatments of cancer. A broad
assortment of NK cell inhibitory and activating receptors create
important immune response checkpoints by surveying self-cells
that express different ligands (39). These checkpoints are exquis-
itely regulated in part by the defined inherited repertoire of inhib-
itory KIRs distributed on different NK cell subpopulations within
NK cells from five healthy donors (n ? 5) were incubated with51Cr-
labeled autologous EBV-transformed B cells at a 10:1 E:T ratio in the
presence of rituximab (A) or mutant anti-CD19 Ab (Xencor) (B) or in
the absence (?) or presence (f) of NK cell RISER Abs combination
binding NKG2A, CD94, KIR3DL1, KIR2DL1, KIR2DL3, KIR2DL2,
and KIR2DS2. Cytotoxicity was calculated using a 4-h
assay. All Abs were used at 1 ?g/ml concentration. Results shown are
mean ? SD of one representative experiment of at least three indepen-
RISER augments Ab-induced NK cell-mediated ADCC.
Table I. Reversal of inhibitory self-recognition (RISER) enhances rituximab-promoted ADCCa
Percentage of Lysis of EBV-Transformed
B Cells by Autologous NK Cells
Pan anti-MHC I Ab (DX17)
Rituximab ? CD56
Rituximab ? NKG2A (Z199)
Rituximab ? CD94 (HP3B1)
Rituximab ? KIR3DL1 (DX9)
Rituximab ? KIR2DL1 (HP3E4)
Rituximab ? KIR3DL1, 3DL2, 2DS4 (5.133)
Rituximab ? KIR2DL3, 2DL2, 2DS2 (GL183)
Rituximab ? Pan anti MHC I antibody (DX17)
Rituximab ? RISER combination*
1 ? 0.2
0.4 ? 0.1
1.3 ? 0.1
6.4 ? 0.1
4 ? 0.8
11.6 ? 0.6
9.6 ? 0.6
6 ? 0.85
21.2 ? 0
4.4 ? 0.3
6 ? 0.6
24.4 ? 0.8
31.2 ? 0.2
0.2 ? 0.1
0.5 ? 0.1
0 ? 0.1
14.9 ? 1.5
14.5 ? 0.5
30.3 ? 0.6
20.2 ? 1.3
22 ? 0.3
13.2 ? 1.3
15.5 ? 0.9
13.1 ? 0.6
15.3 ? 0.3
43.4 ? 1.4
1.2 ? 0.2
10.6 ? 0.1
8.2 ? 0.3
50.9 ? 3.4
43.4 ? 1.4
58.8 ? 3.9
53.3 ? 4.4
55.8 ? 3.0
51.5 ? 1.1
47.3 ? 0.8
59.8 ? 1.2
60.3 ? 2.2
76.3 ? 1.6
3.4 ? 1.7
5.9 ? 2.7
2.7 ? 0.5
25.8 ? 5.8
27.5 ? 5.5
41.6 ? 0.3
29.3 ? 0.5
20.1 ? 2.1
29.6 ? 5.2
29.2 ? 1.9
41.1 ? 0.1
25.2 ? 4.4
53.4 ? 3.9
3.2 ? 2.2
11.5 ? 0.1
13.2 ? 0.8
79.1 ? 1.5
81.5 ? 1.6
92.6 ? 1.5
88.9 ? 1.8
81.2 ? 4.5
78.7 ? 7.5
73.2 ? 0.7
81.9 ? 1.8
82.2 ? 1.7
96.9 ? 1.4
aNK cells were freshly purified from healthy donors whole blood samples (effector cells). EBV-transformed B cells were labeled with51Cr
(target cells). Cells were incubated at effector to target cells ratio of 10:1 in the presence of Ab combination as specified. All Abs were used at
1 ?g/ml concentration. Cytotoxicity was calculated using a 4 h51Cr -release assay. Results shown are representative of at least three independent
bRISER combination, a combination of NK cell receptor antibodies (Z199, HP3B1, DX9, HP3E4, 5.133, GL183, 1 ? g/ml each).
6398BLOCKING NK CELL SELF-RECOGNITION PROMOTES ADCC
an individual; this basis provides a mechanism by which NK cells
sense changes in self-molecule expression or expression of stress-
activating ligands while protecting healthy cells. Based on our re-
sults, disrupting interactions that lead to inhibitory self-recognition
can efficiently improve ADCC for the elimination of Ab-coated
CD16 is the predominant Fc receptor on NK cells and has been
shown to be involved in the capacity of NK cells to mediate ADCC
(2). In our cell line model, NK-92.26.5 cells were transduced with
one of the two variants of CD16 (determined by the aa 158 poly-
morphism valine (V) or phenylalanine (F)) (Fig. 2). CD16.NK-
92.26.5 effector cells were treated with DX9 to block KIR3DL1
inhibitory receptor and used to mediate ADCC against rituximab-
coated 721.221-B*5101 (B51) target cells. Lysis of the target cells
was improved when DX9 was used in combination with rituximab,
compared with rituximab alone, indicating that our approach can
improve ADCC (Fig. 5).
The results show that NK cells that do not express CD16 pre-
dictably do not mediate Ab-dependent cytotoxicity. However,
CD16-expressing NK cells efficiently mediate ADCC against cells
bearing relevant target Ags, CD20?or c-erbB2?, using rituximab
or trastuzumab, respectively (Fig. 3). Moreover, in determining
how the impact of CD16 polymorphisms on the degree of response
correlates with Fc binding affinity (1), an enhanced cytotoxicity
was seen with the V158 variant compared with F158 at any given
Ab concentration (Fig. 3).
To validate our hypothesis that RISER will augment Ab-depen-
dent NK cell-mediated cytotoxicity we combined the two comple-
mentary NK cell-activating mechanisms of CD16 engagement and
blockade of inhibitory self-recognition (Fig. 5). The Ab isotypes of
mouse origin that we used in our study show low affinity binding
to human CD16 (64). We initially used F(ab?)2fragments of the
DX9 Ab in our cytotoxicity experiments. This process was per-
formed in anticipation of the potential problem of cross-linking
two NK cells and a possible competition with the Ab that mediates
ADCC. In our CD16.NK-92 cells system, an intact mouse IgG1
Ab (DX9) was used with no such effect, as described in Results. In
our model, the blockade of KIR3DL1 on NK-92.26.5 cells was
sufficient to significantly activate lysis toward 721.221-B*5101
(B51) target cells (on which the KIR3DL1 cognate ligand HLA-B
B*5101 is the only expressed class I MHC molecule). We found
that although blocking of KIR3DL1-HLA-Bw4 interaction by the
DX9 Ab or mediating ADCC by rituximab were separately effec-
tive in triggering NK cell cytotoxicity alone, the combination of
these two approaches yielded the most efficient cytotoxicity level.
Our results indicate that blocking KIR-HLA interactions could
overcome the effects of CD16 Fc receptor low affinity in
An individual NK cell population contains a diverse repertoire
of NK cells, each expressing one of a varied spectrum of inhibitory
receptors that recognize class I MHC self-molecules and thereby
achieve tolerance. Therefore, it is predictable, that in a biologic
setting, the blockade of any single receptor is unlikely to have a
substantial overall impact on inhibitory self-recognition. Thus,
broad targeting of inhibitory NK cell receptors might be required
to effectively block NK cell recognition, allowing a nonrestricted
enhanced antitumor response in patients. The NK-92.26.5 studies
provided a useful starting point for obtaining proof-of-concept and
elucidating key principles that must be fulfilled in order for inhib-
itory self-recognition to cooperate with CD16 signaling to effi-
ciently promote ADCC. Nonetheless, an autologous setting using
primary NK cells and controlled by realistic checkpoints was
A unique donor gave us the opportunity to test how KIR block-
ade affected NK-mediated lysis in a human autologous in vitro
system (Fig. 6). Ab blockage of KIR2DL1, which was expressed
on virtually all NK cells from this subject, did not allow cytotox-
icity of either 721.221 or autologous target cells that expressed
HLA-C ligands for KIR2DL1. However, Ab blockage of
KIR2DL1 did augment rituximab-mediated ADCC of these target
cells. This property offers potential advantages for clinical trans-
lation because cytotoxicity will not be directed to healthy cells, but
more specifically toward Ab-coated tumor cells. When we tested
DX9 to block KIR3DL1 (expressed on 13% of the donor NK cells)
in Fig. 6B, neither basal NK activity nor ADCC were augmented
(Fig. 6E), suggesting that the inhibitory receptors on a more sub-
stantial proportion of the NK cell population must be blocked to
effectively improve NK cell cytotoxicity.
More donors were characterized for varying expression patterns
of inhibitory receptors and CD16 polymorphism status (Table I),
and these cells were tested for the impact of blocking inhibitory
receptors on ADCC. Because the interference with self-recognition
through a single inhibitory receptor had variable and generally
modest effects on ADCC promotion, we applied a combination of
NK cell-binding Abs to interfere with multiple inhibitory recep-
tors. One challenge for this set of experiments was that most anti-
KIR Abs bind to polymorphic extracellular domain determinants
that are shared among several activating and inhibitory receptors
of a given KIR family (e.g., KIR2D). However, we found that a
mixture of mAb molecules with broad coverage of a large number
of NK cell receptors, most of which are inhibitory, had a dramatic
influence on the capacity of donor PBMC or NK cells to lyse
autologous B cell targets in the presence of rituximab, but not in its
absence (Table I). The results of blocking autologous recognition
by pan anti-MHC class I Ab were similar to those obtained when
autologous self-inhibition was blocked with the NK cell receptor
combination of Abs, suggesting that we successfully achieved
maximal inhibition of MHC class I-regulated self-recognition,
even though some activating signals were targeted (Table I). Due
to the variability among our donors and among NK cell subpopu-
lations within each donor, attempts to identify a critical subset of
KIR receptors required to achieve these effects have not yielded
consistent results, suggesting that optimal ADCC promotion by
interfering with inhibitory self-recognition requires the blocking of
multiple inhibitory receptors. A combination of Abs targeting
NKG2A/CD94 and multiple KIRs generally yielded the most con-
sistent promotion of cytotoxicity.
Other laboratories have used strategies to modify mAb structure
to better interact with the immune system and more efficiently
mediate ADCC (65). These approaches have included manipula-
tion of the mAb Fc region. Examples include introducing muta-
tions within the Fc domain of mAb to selectively tune the affinity
for Fc?RIII and other Fc?Rs (66) and modifying Fc glycosylation
by the Ab-producing cell line (67). Importantly, the improvement
in rituximab-mediated ADCC when combined with the RISER
combination of Abs (Fig. 8A) was also seen using an anti-CD19
Ab with a mutated Fc domain that mediates enhanced ADCC
(CD19 Ab) obtained from Xencor (Fig. 8B). Accordingly, the
blockade of inhibitory self-recognition further augments ADCC
promoted by optimized CD16 signaling. Thus, if the current gen-
eration of high affinity ADCC promoting anti-CD20 Abs possesses
improved antitumor activity in lymphoma, additional gains may be
anticipated by combining our approach.
Interestingly, in some of our donors, the relatively low in vitro
rituximab-promoted cytotoxicity of autologous cells was not con-
sistent with the ubiquitous and rapid systemic B cell depletion that
follows rituximab therapy. It must be considered that we have used
6399 The Journal of Immunology
short-term assay with purified NK cells. However, additional
mechanisms such as macrophage- and neutrophil-mediated dam-
age of Ab-coated cells, complement fixation, and signaling pertur-
bation of the B cells probably contributed to rituximab-initiated B
cell depletion in vivo. We believe, nonetheless, that more efficient
ADCC promotion is still a valuable benefit, particularly because
CD16 polymorphism status can dictate clinical response to ritux-
imab therapy in lymphoma. Increased ADCC thus offers the pos-
sibility of improving treatment outcomes, particularly in individ-
uals who carry the low response polymorphism forms of CD16
(F/F and V/F). This accomplishment can be furthered by altering
target cell sensitivity to effector cell lysis, by modulating the af-
finity of the Ag-binding domains for the tumor target, and by im-
proving the affinity of the Ab Fc domain for activating cellular Fc
receptors (65). Our results suggest that lowering the threshold for
NK cell activation through the blockade of inhibitory cellular re-
ceptors can complement attempts to create more powerful activat-
ing Abs that signal through CD16. This novel approach offers a
clear pathway for clinical development of a new way to improve
We thank Christine Quigley and Sharon Howard in the Fox Chase Cancer
Center Cell Culture Facility for establishing EBV-transformed cell lines
from donor blood samples.
K.S.C. has submitted a patent application for NK-92 cells transduced to
express CD16 variants.
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6401The Journal of Immunology