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Prepublished online May 30, 2007;
doi:10.1182/blood-2007-02-076927
2B4 inhibits NK cell fratricide
Ruth T Taniguchi, Dustin Guzior and Vinay Kumar
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Blood First Edition Paper, prepublished online May 30, 2007; DOI 10.1182/blood-2007-02-076927
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2B4 inhibits NK cell fratricide
Ruth T. Taniguchi, Dustin Guzior, and Vinay Kumar
From the University of Chicago Department of Pathology, Committee on Immunology
Chicago, IL 60637, USA
Corresponding author and reprints: Ruth T. Taniguchi; University of Chicago Department
of Pathology, Committee on Immunology; 5812 S. Ellis Ave., Room S-315, MC 3083,
Chicago, IL 60637; email: [email protected]; phone: (773)834-7921; fax: (773)702-9379
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 this 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.
Copyright © 2007 American Society of Hematology
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Abstract
2B4 (CD244) and its ligand, CD48, are expressed on all NK cells. In studies using 2B4
deficient, CD48 deficient, 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 NK cell 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 the absence of a 2B4dependent activation signal.
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Introduction
2B4 is expressed by all NK cells, as well as a subset of memory CD8+ αβ T cells, γδ T
cells, basophils and monocytes1. The ligand to 2B4, CD48, is a GPI- linked molecule
expressed on all nucleated hematopoetic cells, including NK cells themselves2. 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.
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Materials and methods
Mice
Wild type C57BL/6 (B6), Rag KO, β2M KO and Perforin KO mice were purchased from
Jackson Laboratories. 2B4 KO mice were generated in B6-derived ES cells as previously
described 8. CD48 -/- 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 have been reviewed and
approved by the Institutional Animal Care and Use Committee (IACUC) at the University
of Chicago. All knockout mice are derived or crossed onto the B6 background and were
used at 5-10 weeks of age for experiments.
NK LAK preparation
NK LAK were prepared as described previously 10.
Antibodies and 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. Fluorescently labeled mAbs
purchased from BD biosciences 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. Apoptosis was detected using BD Pharmingen™
Annexin V-FITC Apoptosis Kit I.
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In vitro cytotoxicity assay and spontaneous release assay
Target NK LAK cells were labeled with 100µl of sodium chromate (51Cr) for 1 hour at
37˚C, washed, then plated at 2000 cells/well. Effecter NK 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, NK LAK cells were labeled with 51Cr as described
above. LAK cells were incubated alone at 5E4 cells/well in the presence of 10µ/mL 2.4G2
+ indicated blocking mAb, and incubated at 37˚C for 6 hours. %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% tritonX)-(spontaneous
release without ab)].
Proliferation assay
NK LAK proliferation was measured by 3H-thymidine incorporation as described
previously 10.
N-α-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) assay
Plates were coated overnight with 15µg/mL αNK1.1 mAb. Coated plates were used to
stimulate 3 x 10^5 LAK cells per well in the presence of 10µg/mL 2.4G2 mAb +/10µg/mL αCD48 mAb or α2B4 mAb. After 6 hours of incubation at 37˚, 50µl supernatant
was analyzed from triplicate samples for BLT esterase activity as previously described by
Cho, D.H. et al.11. % specific esterase release= (experimental esterase release- spontaneous
release)/ (maximum release with triton x - spontaneous release).
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In vivo NK stimulation and analysis
Mice were injected with 100µg CpG 1826 (Coley Pharmaceutical) in 100µL PBS i.p. 5
days after injection, NK cells from the blood, liver and spleen were enumerated using
Sphero™ AccuCount Blank Particles, 10.2 µm (Spherotech Inc.) and NK1.1+, CD3fluorescent antibody staining. The fold expansion of blood NK cells was calculated by
dividing the number of NK per ml in CpG-injected mice to that of non-injected mice. The
fold expansions of spleen and liver derived NK cells were calculated by dividing the total
number of NK per spleen or liver of CpG-injected mice to that of age and sex matched,
non-injected mice.
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Results and discussion
2B4 inhibits NK cytotoxicity against CD48-expressing tumor cells and T cell blasts
8,12,13
. Since all nucleated hematopoetic cells express CD48, we hypothesized that 2B4 can
also inhibit NK mediated lysis of other non-transformed leukocyte subsets, including NK
cells themselves. To determine if 2B4 inhibits NK-NK fratricide, killing assays were
performed using lymphokine activated NK cells (LAK) 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 cells can kill each other. To confirm that 2B4CD48 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 kill CD48 KO
LAK, but not WT LAK (Figure 1B), showing that 2B4-CD48 interactions are required for
the inhibition of fratricide. We next compared the role of MHC-dependent and 2B4dependent inhibition in regulating fratricide. To test this, we used WT LAK to kill β2m
KO and WT LAK targets, with and without anti-CD48 blocking antibodies. β2m KO
LAK, which have low MHC class I expression, are killed slightly more than WT LAK,
indicating that the presence of self class I MHC can inhibit fratricide (Figure 1C).
However, if CD48 on the β2m 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 NK LAK fratricide
(Figure 1C).
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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 occurs in the presence of anti-2B4 or anti-CD48 blocking antibodies in a dose
dependent manner, while 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 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 to 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 published that 2B4-CD48 interactions among NK
cells are required for optimal NK functions 5,10. There are two distinct mechanisms that
can explain these results: 1) 2B4-CD48 signaling may activate optimal NK functions, or 2)
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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 cytotoxicity of WT and perforin KO NK cells in the absence of 2B4-CD48 interactions
were compared. Since perforin KO NK LAK 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 secretion11.
Consistent with previous reports5, WT LAK have decreased esterase release in the presence
of anti-2B4 or anti-CD48 blocking antibodies (Figure 2A). In contrast, perforin KO LAK
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 found to have
defective proliferation5. To determine if defective proliferation in the absence of 2B4CD48 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). This data indicates that the 2B4-CD48 interactions
among NK LAK 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 affect NK proliferation in vivo, WT, 2B4 KO, perforin/2B4 (perf/2B4) DKO
and perforin (perf) KO mice were injected with CpG i.p. NK proliferation the blood, liver
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and spleen of these mice were compared 5 days after CpG injection. The numbers of NK
cells in untreated WT, 2B4 KO, perf/2B4 DKO and perf KO are similar8 (supplementary
Figure 1). 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). This data indicates that the decrease
in expansion of NK cells in the blood and livers of 2B4 KO mice compared to 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 (supplementary Figure 1).
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 functions5-7,10, while
other studies indicate that 2B4 inhibit functions 3,4,8,12. The data here shows 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-γ production (supplementary Figure 2).
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Together these data suggests that there are activation signals between NK cells that can
stimulate cytotoxicity and cytokine production, which can be inhibited by 2B4. Since 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 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. Since 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.
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Acknowledgements
This work was supported by the Molecular and Cellular Biology Training Grant
(3T32GM007183-31S1). We thank the NIH AIDS Research and Reagent Program,
Division of AIDS, NIAID, NIH for the generous gift of h-rIL-2. We also thank Megan
McNerney for helpful comments on this work and manuscript. The authors have no
financial conflict of interest.
References
1. McNerney ME, Lee KM, Kumar V. 2B4 (CD244) is a non-MHC binding
receptor with multiple functions on natural killer cells and CD8(+) T cells. Mol Immunol.
2005;42:489-494
2. Kumar V, McNerney ME. A new self: MHC-class-I-independent natural-killercell self-tolerance. Nat Rev Immunol. 2005;5:363-374
3. McNerney ME, Guzior D, Kumar V. 2B4 (CD244) - CD48 interactions provide
a novel MHC class I-independent system for NK cell self-tolerance in mice. Blood. 2005
4. Lee KM, McNerney ME, Stepp SE, Mathew PA, Schatzle JD, Bennett M,
Kumar V. 2B4 acts as a non-Major Histocompatibility Complex binding inhibitory receptor
on mouse natural killer cells. J Exp Med. 2004;199:1245-1254
5. Lee KM, Forman JP, McNerney ME, Stepp S, Kuppireddi S, Guzior D,
Latchman YE, Sayegh MH, Yagita H, Park CK, Oh SB, Wulfing C, Schatzle J, Mathew
PA, Sharpe AH, Kumar V. Requirement of homotypic NK cell interactions through
2B4(CD244)/CD48 in the generation of NK effector functions. Blood. 2005
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Fischer A, Latour S. Regulation of natural cytotoxicity by the adaptor SAP and the Srcrelated kinase Fyn. J Exp Med. 2005;202:181-192
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Latour S, Veillette A. Negative regulation of natural killer cell function by EAT-2, a SAPrelated adaptor. Nat Immunol. 2005;6:1002-1010
8. Vaidya SV, Stepp SE, McNerney ME, Lee JK, Bennett M, Lee KM, Stewart CL,
Kumar V, Mathew PA. Targeted disruption of the 2B4 gene in mice reveals an in vivo role
of 2B4 (CD244) in the rejection of B16 melanoma cells. J Immunol. 2005;174:800-807
9. Gonzalez-Cabrero J, Wise CJ, Latchman Y, Freeman GJ, Sharpe AH, Reiser H.
CD48-deficient mice have a pronounced defect in CD4(+) T cell activation. Proc Natl Acad
Sci U S A. 1999;96:1019-1023
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10. Assarsson E, Kambayashi T, Schatzle JD, Cramer SO, von Bonin A, Jensen
PE, Ljunggren HG, Chambers BJ. NK cells stimulate proliferation of T and NK cells
through 2B4/CD48 interactions. J Immunol. 2004;173:174-180
11. Cho DH, Song HK, Kang HS, Yoon SR, Lee HG, Pyun KH, Lee WJ, Kim YB,
Choi I. Ligation of ICAM-1 molecules inhibits target cell-induced granule exocytosis of
IL-12-activated natural killer cells. Cell Immunol. 2000;199:1-7
12. Mooney JM, Klem J, Wulfing C, Mijares LA, Schwartzberg PL, Bennett M,
Schatzle JD. The murine NK receptor 2B4 (CD244) exhibits inhibitory function
independent of signaling lymphocytic activation molecule-associated protein expression. J
Immunol. 2004;173:3953-3961
13. Lee KM, Bhawan S, Majima T, Wei H, Nishimura MI, Yagita H, Kumar V.
Cutting edge: the NK cell receptor 2B4 augments antigen-specific T cell cytotoxicity
through CD48 ligation on neighboring T cells. J Immunol. 2003;170:4881-4885
14. Kagi D, Ledermann B, Burki K, Seiler P, Odermatt B, Olsen KJ, Podack ER,
Zinkernagel RM, Hengartner H. Cytotoxicity mediated by T cells and natural killer cells is
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15. Prlic M, Gibbs J, Jameson SC. Characteristics of NK cell migration early after
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16. Jamieson AM, Isnard P, Dorfman JR, Coles MC, Raulet DH. Turnover and
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17. Valiante NM, Trinchieri G. Identification of a novel signal transduction surface
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granule polarization and degranulation controlled by different receptors in resting NK cells.
J Exp Med. 2005;202:1001-1012
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Figure 1. In the absence of 2B4-CD48 interactions, activated NK cells undergo
perforin-dependent fratricide. NK LAK cells activated with IL-2 in vitro were used as
effectors and targets in cytotoxicity assays. (A) WT and 2B4 KO NK LAK were used as
effectors against WT NK LAK. (B) WT NK LAK cells were used as effectors against WT
and CD48 KO NK LAK. (C) WT LAK cells were used as effectors against untreated, or
anti-CD48 antibody coated WT, or β2m KO NK LAK. To coat NK LAK targets, cells
were incubated for 15 minutes in 10µg/mL anti-CD48 antibody at room temperature.
Coated cells were then washed and used in killing assays. (D) WT and perforin KO LAK
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 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 6-7 hours of incubation with blocking antibody.
Results are representative of four independent experiments.
Figure 2. Perforin-dependent fratricide causes defective NK function in the absence
of 2B4-CD48 interactions. (A) WT and perforin KO NK LAK cells were stimulated with
anti-NK1.1 mAb coated plates, 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. *, indicates P<0.05. Results are representative of three
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
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antibodies. Thymidine incorporation was measured at different times during culture.
Results are representative of three independent experiments. (C) 5 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 non-injected control mice. N= 3 mice per group and data is representative of 2
independent experiments.
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Figure 1
A
B
WT LAK Effectors
WT LAK Targets
WT Effector
20%
2B4 KO
Effector
Specific Lysis (%)
C
10%
30%
Specific Lysis (%)
30%
WT Target
CD48 KO
Target
20%
10%
0%
100:1
50:1
25:1
12:1
0%
100:1
50:1
25:1
-10%
E:T Ratio
E:T Ratio
C
WT LAK Effectors
Specific Lysis (%)
30%
WT
20%
WT +
αCD48
β2m KO
10%
β2m KO +
αCD48
0%
100:1
50:1
E:T Ratio
25:1
12:1
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Figure 1
D
WT LAK
Perforin KO LAK
Specific Lysis (%)
Specific Lysis (%)
25%
20%
15%
10%
5%
0%
-5%
15
5 1.7 0.6 0.2 0.1
ug/mL antibody
E
25%
α2B4
20%
αCD48
15%
αNK1.1
10%
αH-2Kb
5%
0%
-5%
15
5
1.7 0.6 0.2 0.1
ug/mL antibody
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only.
Figure 2
A
Specific BLT Esterase Release (%)
30%
25%
20%
15%
no block
* *
α2B4
αCD48
10%
5%
0%
WT
Perforin KO
B
WT
12
CPM (*10^4)
CPM (*10^4)
10
8
6
4
2
0
Day 3
Day 4
Day 5
16
14
12
10
8
6
4
2
0
Perforin KO
α2B4
αCD48
IgG
no block
Day 3
Day 4
Day 5
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Figure 2
fold expansion of NK
C
12
10
25
8
6
4
2
0
15
WT
20
2B4 KO
perf KO
10
5
perf/2B4
DKO
0
blood
liver