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IMMUNOBIOLOGY
Brief report
2B4 inhibits NK-cell fratricide
Ruth T. Taniguchi,1 Dustin Guzior,1 and Vinay Kumar1
1University
of Chicago Department of Pathology, Committee on Immunology, Chicago, IL
2B4 (CD244) and its ligand, CD48, are
expressed on all natural killer (NK) cells.
In studies using 2B4-deficient, CD48deficient, or wild-type NK cells with blocking antibodies, we found that in the absence of 2B4-CD48 interactions, activated
murine NK cells kill each other. We also
show that NK-NK fratricide in the absence
of 2B4-CD48 interaction is dependent on
perforin both in vitro and in vivo. 2B4 has
been reported to have activating, costimulatory, and inhibitory functions on murine
NK cells. 2B4-mediated inhibition of NKcell fratricide explains some of the paradoxes of 2B4 function reported in studies
of murine NK cells. We show that in the
absence of 2B4 signaling, activated NK
cells have defective cytotoxicity and proliferation because of fratricide and not
due to the absence of a 2B4-dependent
activation signal. (Blood. 2007;110:
2020-2023)
© 2007 by The American Society of Hematology
Introduction
2B4 is expressed by all natural killer (NK) cells as well as a subset
of memory CD8⫹ ␣␤ T cells, ␥␦ T cells, basophils, and monocytes.1 The ligand to 2B4, CD48, is a glycophosphatidylinositollinked molecule expressed on all nucleated hematopoetic cells,
including NK cells themselves.2 Murine 2B4 has been reported
to have activating and inhibitory activities on NK cells.3-8 These
studies raise questions of how triggering the same 2B4 receptor
on NK cells can lead to variable functional outcomes. Here we
show that 2B4 can inhibit NK-NK fratricide and that fratricide
can explain some of the apparent dual functions of 2B4 on
murine NK cells.
Materials and methods
Mice
Wild-type (WT) C57BL/6 (B6), rag knockout (KO), ␤2m KO, and perforin
KO mice were purchased from Jackson Laboratories (Bar Harbor, ME).
2B4 KO mice were generated in B6-derived embryonic stem (ES) cells as
previously described.8 CD48-/- cells were generously provided by Dr Arlene
Sharpe (Harvard University, Boston, MA).9 The mice were maintained at
the University of Chicago in a pathogen-free animal housing facility. The
mouse protocols were reviewed and approved by the Institutional Animal
Care and Use Committee (IACUC) at the University of Chicago. All KO
mice were derived or crossed onto the B6 background and were used at 5 to
10 weeks of age for experiments.
Fluorescently labeled monoclonal antibodies (mAbs) purchased from BD
Biosciences (San Jose, CA) are the following: anti-2B4 (2B4), anti-CD48
(HM48–1), anti-CD3 (145–2C11), and anti-DX5 (DX5). Fluorescently
labeled anti-NK1.1 (PK136) mAb was purchased from eBioscience (San
Diego, CA). Apoptosis was detected using BD Pharmingen (San Jose, CA)
Annexin V-FITC Apoptosis Kit I.
In vitro cytotoxicity assay and spontaneous release assay
Target LAK cells were labeled with 100 ␮L of sodium chromate (51Cr) for
1 hour at 37°C, washed, and then plated at 2000 cells per well. Effector
LAK cells were added at the indicated ratios in triplicates. After 6 hours of
incubation at 37°C, supernatants were collected for analysis, and percent
lysis was calculated using standard methods. For fratricide assays using
blocking mAb, LAK cells were labeled with 51Cr for 1 hour at 37°C. LAK
cells were incubated alone at 5E4 cells per well in the presence of
10 ␮g/mL 2.4G2 plus indicated blocking mAb and incubated at 37°C for
6 hours. Percent specific lysis was calculated using the following
equation: % specific lysis ⫽ ([cpm in the presence of blocking mAb] ⫺
[spontaneous release without ab])/([CPM with 0.5% Triton X] ⫺
[spontaneous release without ab]).
Proliferation assay
LAK proliferation was measured by 3H-thymidine incorporation as described previously.10
BLT ester assay
NK LAK preparation
NK lymphokine-activated killer (LAK) cells were prepared as described
previously.10
Antibodies and fluorescence-activated cell sorter
(FACS) analysis
Anti-2B4, anti-CD48, and anti-CD16/32 blocking antibodies were produced by 2B4, HM48–1, and 2.4G2 hybridoma cell lines, respectively.
Plates were coated overnight with 15␮g/mL ␣NK1.1 mAb. Coated
plates were used to stimulate 3 ⫻ 105 LAK cells per well in the presence
of 10 ␮g/mL 2.4G2 mAb with or without 10 ␮g/mL ␣CD48 mAb or
␣2B4 mAb. After 6 hours of incubation at 37°, 50 ␮L supernatant
was analyzed from triplicate samples for N-␣-benzyloxycarbonyl-Llysine thiobenzyl (BLT) esterase activity as previously described by Cho
et al.11 The % specific esterase release ⫽ (experimental esterase
release ⫺ spontaneous release)/(maximum release with Triton X ⫺
spontaneous release).
Submitted February 27, 2007; accepted May 28, 2007. Prepublished online as
Blood First Edition paper, May 30, 2007; DOI 10.1182/blood-2007-02-076927.
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.
The online version of this article contains a data supplement.
© 2007 by The American Society of Hematology
2020
BLOOD, 15 SEPTEMBER 2007 䡠 VOLUME 110, NUMBER 6
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BLOOD, 15 SEPTEMBER 2007 䡠 VOLUME 110, NUMBER 6
2B4 INHIBITS NK CELL FRATRICIDE
In vivo NK stimulation and analysis
cells can kill each other. To confirm that 2B4-CD48 interactions are
required for the inhibition of fratricide, WT LAK were used to kill
CD48 KO and WT LAK targets in chromium release assays. WT
LAK cells kill CD48 KO LAK cells but not WT LAK cells (Figure
1B), showing that 2B4-CD48 interactions are required for the
inhibition of fratricide. We next compared the role of major
histocompatibility complex (MHC)–dependent and 2B4-dependent
inhibition in regulating fratricide. To test this, we used WT LAK
cells to kill ␤2m KO and WT LAK targets with and without
anti-CD48 blocking antibodies. ␤2m KO LAK cells, which have
low MHC class I expression, are killed slightly more than WT LAK
cells, indicating that the presence of self class I MHC can inhibit
fratricide (Figure 1C). However, if CD48 on the ␤2m KO or WT
LAK targets are blocked with anti-CD48 antibody, a much higher
level of fratricide occurs, indicating that class I–dependent inhibition is not as potent as CD48-dependent inhibition in controlling
LAK fratricide (Figure 1C).
NK-mediated lysis of most tumor targets requires perforin.14 To
determine whether NK fratricide in the absence of 2B4-CD48
interactions is also dependent on perforin, fratricide among perforin KO and WT LAK cells was compared. WT and perforin KO
LAK cells were labeled with 51Cr and incubated in the presence of
anti-2B4, anti-CD48, or control blocking antibodies. An increase of
chromium release from WT LAK cells occurs in the presence of
anti-2B4 or anti-CD48 blocking antibodies in a dose-dependent
Mice were injected with 100 ␮g CpG 1826 (Coley Pharmaceutical,
Wellesley, MA) in 100 ␮L PBS intraperitoneally. Five days after injection,
NK cells from the blood, liver, and spleen were enumerated using Sphero
AccuCount Blank Particles, 10.2 ␮m (Spherotech, Lake Forrest, IL) and
NK1.1⫹, CD3- fluorescent antibody staining. The fold expansion of blood
NK cells was calculated by dividing the number of NK per milliliter in
CpG-injected mice by that of noninjected mice. The fold expansions of
spleen- and liver-derived NK cells were calculated by dividing the total
number of NK cells per spleen or liver of CpG-injected mice by that of
noninjected mice.
Results and discussion
2B4 inhibits NK cytotoxicity against CD48-expressing tumor cells
and T-cell blasts.8,12,13 Because all nucleated hematopoetic cells
express CD48, we hypothesized that 2B4 can also inhibit NKmediated lysis of other nontransformed leukocyte subsets, including NK cells themselves. To determine if 2B4 inhibits NK-NK
fratricide, killing assays were performed using LAK cells as both
targets and effectors. WT and 2B4 KO LAK cells were used as
effectors against WT LAK targets in chromium release assays. As
expected, WT LAK cells failed to kill other WT cells. However,
LAK cells from 2B4 KO mice killed WT LAK cells effectively
(Figure 1A). Thus, in the absence of 2B4-mediated inhibition, NK
30%
WT Effector
20%
2B4 KO
Effector
10%
WT LAK Effectors
WT LAK Effectors
C
30%
30%
WT Target
CD48 KO
Target
20%
10%
Specific Lysis (%)
B
WT LAK Targets
Specific Lysis (%)
Specific Lysis (%)
A
WT
20%
WT +
α D48
β m KO
10%
β
α
0%
100:1
50:1
25:1
12:1
E:T Ratio
D
m KO +
D48
0%
0%
100:1
-10%
2021
50:1
25:1
E:T Ratio
12:1
100:1
50:1
25:1
E:T Ratio
E
Figure 1. In the absence of 2B4-CD48 interactions, activated NK cells undergo perforin-dependent fratricide. LAK cells activated with IL-2 in vitro were used as effectors
and targets in cytotoxicity assays. (A) WT and 2B4 KO LAK cells were used as effectors against WT LAK cells. (B) WT LAK cells were used as effectors against WT and CD48
KO LAK cells. (C) WT LAK cells were used as effectors against untreated, or anti-CD48 antibody–coated WT, or ␤2m KO LAK cells. To coat LAK targets, cells were incubated
for 15 minutes in 10 ␮g/mL anti-CD48 antibody at room temperature. Coated cells were then washed and used in killing assays. (D) WT and perforin KO LAK cells were loaded
with chromium and incubated in the presence of anti-CD16/CD32 blocking antibody plus anti-2B4, anti-CD48, anti-NK1.1, or anti-H-2Kb antibody. Chromium release, which
indicates lysis due to fratricide, was measured after 6 hours of incubation. (E) WT and perforin KO LAK cells were incubated in the presence of anti-CD16/CD32 blocking
antibody plus anti-2B4, anti-CD48, or anti-NK1.1 antibody. Annexin V and PI staining was measured after 6 to 7 hours of incubation with blocking antibody. The percents of
Annexin V⫹, PI⫹ apoptotic cells are noted in the upper right quadrants of the FACS dot plots. Results are representative of 4 independent experiments. Error bars are standard
deviation (SD).
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2022
BLOOD, 15 SEPTEMBER 2007 䡠 VOLUME 110, NUMBER 6
TANIGUCHI et al
manner, whereas control antibodies against H-2Kb and NK1.1
failed to increase the lysis of NK cells (Figure 1D). No chromium
release could be detected from perforin KO LAK cells with any
blocking antibody, indicating that NK-NK cell lysis is perforin
dependent (Figure 1D).
Perforin-dependent NK fratricide in the presence of anti-2B4 or
anti-CD48 blocking antibodies was also confirmed via annexin V
and PI staining. The percentage of apoptotic WT, but not perforin
KO NK cells, is higher in the presence of anti-2B4 and anti-CD48
blocking antibodies compared with control (anti-NK1.1) antibody
(Figure 1E), confirming that NK fratricide and apoptosis occurs in
the absence of 2B4-CD48 interactions. To rule out the possibility of
NK cell death due to antibody-dependent cellular cytotoxicity
(ADCC), FcR blockage using anti-CD16/32 antibody was performed in all experiments using blocking antibodies. That such
blockade was effective in preventing ADCC is supported by the
data showing that no cell death could be detected with anti-NK1.1
control antibodies (Figure 1D,E).
We and others have previously reported that 2B4-CD48 interactions among NK cells are required for optimal NK functions.5,10
There are 2 distinct mechanisms that can explain these results:
(1) 2B4-CD48 signaling may activate optimal NK functions, or
(2) 2B4-CD48 signaling may inhibit NK fratricide to allow for
optimal NK function. To determine if 2B4-mediated activation or
inhibition is required for optimal NK functions, the cytotoxicities
of WT and perforin KO NK cells in the absence of 2B4-CD48
interactions were compared. Because perforin KO LAK cells have
impaired cytolytic function,14 the cytolytic ability of LAK cells
stimulated by anti-NK1.1 antibody–coated plates was measured
indirectly by using the BLT esterase assay to detect granzyme
secretion.11 Consistent with previous reports,5 WT LAK cells have
decreased esterase release in the presence of anti-2B4 or anti-CD48
blocking antibodies (Figure 2A). In contrast, perforin KO LAK
cells do not exhibit a decrease in esterase activity in the presence of
anti-2B4 or anti-CD48 blocking antibodies (Figure 2A).
In the absence of 2B4-CD48 interactions, NK cells have also
been found to have defective proliferation.5 To determine if
defective proliferation in the absence of 2B4-CD48 interactions
was due to a lack of an activation signal or a lack of inhibition
signal, the proliferation of NK cells from WT and perforin KO
mice in the presence of anti-2B4, anti-CD48, or control antibody
was measured by 3H-thymidine following culture with IL-2.
Blocking 2B4-CD48 interactions among WT but not perforin KO
NK cells decreased proliferation in IL-2 (Figure 2B). These data
indicate that the 2B4-CD48 interactions among LAK cells do not
activate optimal NK proliferation but inhibit perforin-mediated
fratricide. Together these data show that in the absence of 2B4
signaling, activated NK cells have defective function in vitro due to
a lack of inhibition leading to fratricide.
To determine if perforin-dependent NK fratricide in the absence of
2B4-CD48 interactions affects NK proliferation in vivo, WT, 2B4 KO,
perforin/2B4 (perf/2B4) double knockout (DKO), and perforin (perf)
KO mice were injected with CpG intraperitoneally. NK proliferation in
the blood, liver, and spleen of these mice was compared 5 days after
CpG injection. The numbers of NK cells in untreated WT, 2B4 KO,
perf/2B4 DKO, and perf KO mice are similar8 (Figure S1, available on
the Blood website; see the Supplemental Materials link at the top of the
online article). The degree of NK expansion in the blood and livers of
2B4 KO mice was less than that of the WT mice (Figure 2C). This defect
in expansion of NK cells was not seen in perf KO mice or perf/2B4
DKO mice (Figure 2C). These data indicate that the decrease in
expansion of NK cells in the blood and livers of 2B4 KO mice compared
with the WT mice is perforin dependent. NK proliferation in the spleen
and migration out of the spleen can be detected during infection15 or
under lymphopenic conditions16 with different kinetics. However,
5 days after injection with CpG, expansion of activated NK cells in the
spleens of WT and 2B4 KO could not be detected, possibly due to NK
migration into other tissues at this time point. Therefore, 2B4-dependent
inhibition of fratricide could not be detected in the spleen 5 days after
injection with CpG (Figure S1).
Unlike in NK cells from B6 mice, 2B4-CD48 interactions
predominantly activate human NK cells.17 The molecular bases for
the different functions of 2B4 across different species are unclear
and are currently under investigation in our laboratory. Here we
restricted our examination of 2B4 function to B6 murine NK cells.
Some studies of B6 mouse models indicate that 2B4-CD48
interactions augment NK-cell functions,5-7,10 while other studies
indicate that 2B4 inhibits functions.3,4,8,12 The data here show that
2B4 can inhibit NK-cell fratricide among activated NK cells and
that fratricide provides an explanation for some of these conflicting
findings. We demonstrate that in the absence of 2B4, activated NK
cells have defective proliferation and cytotoxicity due to fratricide
and not the absence of an activation signal. In addition to blocking
fratricide, 2B4-CD48 interactions among NK cells can also inhibit
IFN-␥ production (Figure S2). Together these data suggest that
there are activation signals between NK cells that can stimulate
A
no block
αCD48
α 2B4
B 12
20%
WT
* *
10%
5%
CPM (*10^4)
10
15%
8
6
4
2
0
0%
WT
Perforin KO
Day 3
Day 4
Day 5
16
14
12
10
8
6
4
2
0
Perforin KO
α 2B4
αCD48
IgG
no block
Day 3
Day 4
Day 5
C
fold expansion of NK
25%
CPM (*10^4)
Specific BLT Esterase Release (%)
30%
WT
2B4 KO
perf KO
12
10
25
8
6
4
2
0
15
perf/2B4 DKO
20
10
5
0
blood
liver
Figure 2. Perforin-dependent fratricide causes defective NK function in the absence of 2B4-CD48 interactions. (A) WT and perforin KO LAK cells were stimulated with
anti-NK1.1 mAb–coated plates in the presence of anti-2B4 or anti-CD48 blocking antibodies. After 6 hours of stimulation, culture supernatants were measured for granzyme
secretion via the BLT esterase assay. *P ⬍ .05. Results are representative of 3 independent experiments. (B) WT and perforin KO NK cells were cultured in complete media
supplemented with IL-2 in the presence of anti-2B4 or anti-CD48 blocking antibodies. Thymidine incorporation was measured at different times during culture. Results are
representative of 3 independent experiments. (C) Five days after injection with CpG, NK cells from WT, 2B4 KO, perf KO, and perf/2B4 DKO mice were counted. Fold
expansions were calculated by dividing NK numbers of mice injected with CpG by NK numbers in noninjected control mice; n ⫽ 3 mice per group, and data are representative
of 2 independent experiments. Error bars are SD.
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BLOOD, 15 SEPTEMBER 2007 䡠 VOLUME 110, NUMBER 6
cytotoxicity and cytokine production, which can be inhibited by
2B4. Because 2B4 inhibition of fratricide is only apparent on NK
cells activated with IL-2 in vitro or CpG in vivo, another possibility
is that activated NK cells constitutively degranulate, and adhesion
among NK cells is sufficient to induce granule polarization18
leading to fratricide. In any case, the specific receptor and/or
adhesion molecule interactions that 2B4 inhibits among NK cells
are unknown and warrant more studies.
In addition to providing an explanation to some of the conflicting
findings in studies of 2B4, this work provides evidence for a non-MHC—
related mechanism of self-tolerance required for activated NK cells.
Because CD48 is expressed on all nucleated hematopoetic cells, we
hypothesize that 2B4-mediated inhibition may play an important role in
activated NK-cell tolerance of other leukocyte subsets as well.
Acknowledgments
This work was supported by the Molecular and Cellular Biology
Training Grant (3T32GM007183–31S1).
2B4 INHIBITS NK CELL FRATRICIDE
2023
We thank the National Institutes of Health (NIH) AIDS
Research and Reagent Program, Division of AIDS, National
Institute of Allergy and Infectious Diseases (NIAID), NIH, for the
generous gift of h-rIL-2. We also thank Megan McNerney for
helpful comments on this work and manuscript.
Authorship
Contribution: R.T.T. designed and executed experiments, analyzed
and interpreted data, and wrote the paper; D.G. designed and
executed the thymidine proliferation experiments and analyzed and
interpreted these data; V.K. provided guidance for the progress of
the project and wrote the paper; and all authors checked the final
version of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Ruth T. Taniguchi, University of Chicago
Department of Pathology, Committee on Immunology; 5812 S
Ellis Ave, Rm S-315, MC 3083, Chicago, IL 60637; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2007 110: 2020-2023
doi:10.1182/blood-2007-02-076927 originally published
online May 30, 2007
2B4 inhibits NK-cell fratricide
Ruth T. Taniguchi, Dustin Guzior and Vinay Kumar
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