Impaired Natural Killing of MHC Class
I-Deficient Targets by NK Cells Expressing a
Catalytically Inactive Form of SHP-1
This information is current as
of June 17, 2017.
Bente Lowin-Kropf, Béatrice Kunz, Friedrich Beermann and
Werner Held
J Immunol 2000; 165:1314-1321; ;
doi: 10.4049/jimmunol.165.3.1314
http://www.jimmunol.org/content/165/3/1314
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References
Impaired Natural Killing of MHC Class I-Deficient Targets by
NK Cells Expressing a Catalytically Inactive Form of SHP-11
Bente Lowin-Kropf,* Béatrice Kunz,* Friedrich Beermann,† and Werner Held2*
N
ATURAL killer cells are activated to kill when encountering target cells. However, the engagement of NK cell
inhibitory receptors with their MHC class I ligand aborts
this activation process. MHC class I-deficient target cells thus fail
to deliver an inhibitory signal and, as a consequence, become susceptible to NK cell-mediated lysis. This mode of reactivity has
been termed “missing self recognition.” Consequently, the decision whether a target cell is killed depends on the balance between
opposing activating and inhibitory signals (1).
In the mouse, two types of MHC class I-specific receptors have
been identified. These belong to the Ly49 and CD94/NKG2 receptor families, members of which recognize distinct MHC class I
molecules and sometimes discriminate alleles thereof (2–5). While
the majority of MHC class I receptors inhibit, some also activate
NK cell function. Both types of receptors are expressed by partially overlapping subpopulations of NK cells and thus generate a
rather complex MHC receptor repertoire.
Inhibitory MHC receptors are able to block signals from triggering receptors in trans by recruiting effector molecules to their
cytoplasmic immunoreceptor tyrosine-based inhibition motif
(ITIM).3 So far, two phosphatases, namely SHP-1 and SHP-2,
*Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne,
Epalinges, Switzerland; and †Swiss Institute for Experimental Cancer Research
(ISREC), Epalinges, Switzerland
Received for publication December 22, 1999. Accepted for publication May 17, 2000.
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.
1
W. H. is the recipient of a START fellowship and supported in part by a grant from
the Swiss National Science Foundation.
2
Address correspondence and reprint requests to Dr. Werner Held, Ludwig Institute
for Cancer Research, Lausanne Branch, Ch. de Boveresses 155, 1066 Epalinges,
Switzerland. E-mail address: [email protected]
3
Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibition
motif; ADCC, Ab-dependent cell-mediated cytotoxicity; B6, C57BL/6; ␤2m, ␤2-microglobulin; dn, dominant-negative; LCR, locus control region; me, motheaten; mev,
Copyright © 2000 by The American Association of Immunologists
have been shown to interact with tyrosine-phosphorylated ITIMs
of Ly49 and NKG2A receptors (6, 7). An important role for SHP-1
in the inhibitory pathway of mature NK cells is evident from several studies. The overexpression of a catalytically inactive SHP-1
mutant in human NK cell clones prevents MHC class I-mediated
inhibition of natural killing and Ab-dependent cell-mediated cytotoxicity (8, 9). Furthermore, NK cells from motheaten (me) and
viable motheaten (mev) mice that show complete and partial loss of
SHP-1 enzymatic activity, respectively (10), are partially impaired
in Ly49A-mediated inhibition of natural cytotoxicity (11).
Signaling through MHC-specific inhibitory receptors may be
one mechanism by which mature NK cells remain self-tolerant
(12–14). A reduced capacity to transduce inhibitory signals due to
the lack of active SHP-1 may thus interfere with the maintenance
of self-tolerance in mature NK cells and/or its induction during
development. Conclusions regarding the role of SHP-1 in these
processes based on the analysis of me or mev-mice suffer from a
caveat as effects on NK cells may be secondary to the chronic
activation of macrophage/myeloid populations in these mice (15,
16). Moreover, both me and mev-mice have multiple hematopoietic
and immunological disorders and die by 1–3 mo of age from progressive inflammatory disease.
In this paper we have evaluated the role of SHP-1 in NK cell function and development. To control for possible side effects observed in
mev-mice, we have generated transgenic mice that express a catalytically inactive, dominant-negative form of SHP-1 (dnSHP-1) only in
lymphoid cells. We show that, although transgenic dnSHP-1 expression partially blocks Ly49-mediated inhibition, the generation of NK
cells is not impaired. However, transgenic NK cells show reduced
natural cytotoxicity toward MHC-deficient target cells, suggesting
that non-MHC-specific NK cell activation is significantly impaired.
Therefore, SHP-1 is required for the development of functional NK
viable motheaten; p-NPP, para-nitrophenyl phosphate; PTP, protein tyrosine phosphatase; SHP, SH2-containing protein tyrosine phosphatase.
0022-1767/00/$02.00
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NK cell function is negatively regulated by MHC class I-specific inhibitory receptors. Transduction of the inhibitory signal involves
protein tyrosine phosphatases such as SHP-1 (SH2-containing protein tyrosine phosphatase-1). To investigate the role of SHP-1
for NK cell development and function, we generated mice expressing a catalytically inactive, dominant-negative mutant of SHP-1
(dnSHP-1). In this paper we show that expression of dnSHP-1 does not affect the generation of NK cells even though MHC
receptor-mediated inhibition is partially impaired. Despite this defect, these NK cells do not kill syngeneic, normal target cells. In
fact dnSHP-1-expressing NK cells are hyporesponsive toward MHC-deficient target cells, suggesting that non-MHC-specific NK
cell activation is significantly reduced. In contrast, these NK cells mediate Ab-dependent cell-mediated cytotoxicity and prevent
the engraftment with ␤2-microglobulin-deficient bone marrow cells. A similar NK cell phenotype is observed in viable motheaten
(mev) mice, which show reduced SHP-1 activity due to a mutation in the Shp-1 gene. In addition, NK cells in both mouse strains
show a tendency to express more inhibitory MHC-specific Ly49 receptors. Our results demonstrate the importance of SHP-1 for
the generation of functional NK cells, which are able to react efficiently to the absence of MHC class I molecules from normal
target cells. Therefore, SHP-1 may play an as-yet-unrecognized role in some NK cell activation pathways. Alternatively, a reduced
capacity to transduce SHP-1-dependent inhibitory signals during NK cell development may be compensated by the down-modulation of NK cell triggering pathways. The Journal of Immunology, 2000, 165: 1314 –1321.
The Journal of Immunology
cells that are able to efficiently react to the absence of MHC class I
from normal cells.
Materials and Methods
Mice
Cell lines and cell culture
The moloney murine leukemia virus-induced lymphoma cell line YAC-1,
the SV40-transformed peritoneal macrophage cell line IC-21 (19), the xenogeneic hamster cell line CHO, the lymphoma cell line RMA, and the
MHC class I-deficient variant RMA-S were used as target cells. The murine monocyte cell line C1498 and the Dd-transfectant C1498.Dd were a
gift from W. Seamann (University of California, San Francisco, CA). Con
A-activated T cell blasts were prepared as described previously (20).
Briefly, erythrocyte-depleted spleen cells were cultured at 2 ⫻ 106 cells/ml
for 48 h in DMEM supplemented with 10% FCS and 2.5 ␮g/ml Con A
(Sigma, Buchs, Switzerland). Before use as targets in a standard 4-h 51Crrelease assay, dead cells were removed by centrifugation over a Ficoll
gradient (Pharmacia, Uppsala, Sweden). To generate IL-2-activated NK
cells, spleen cells were depleted of erythrocytes and passed over a nylon
wool column. Nonadherent cells were cultured for 3 days in DMEM supplemented with 10% FCS and 500 ng/ml recombinant human IL-2 (a gift
from Glaxo IMB, Geneva, Switzerland). Cultures of mev-derived cells
were depleted of macrophages by discarding plastic-adherent cells at
day 2. Adherent and nonadherent cells were harvested at day 3 and used
as effector cells.
Abs and reagents
Anti-Ly49A (JR9-318 and A1), anti-Ly49C/I (SW5E6), and anti-Ly49G2
(4D11) have been described (21–24). Abs against NK1.1 (PK136), CD3
(145-2C11), CD45.2 (104), and Thy-1 (30-H12) were purchased from
PharMingen (San Diego, CA). For immunoblots, immunoprecipitations,
and intracellular FACS staining, a monoclonal and a polyclonal anti-FLAG
Abs were purchased from Kodak (INTEGRA Biosciences, Wallisellen,
Switzerland) and Zymed (San Francisco, CA), respectively. Monoclonal
and polyclonal (C-19) anti-SHP-1 Abs were obtained from Transduction
Laboratories (Lexington, KY) and Santa Cruz Biotechnology (Santa Cruz,
CA), respectively.
Flow cytometry
Spleen and bone marrow cell suspensions were depleted of erythrocytes
and thereafter passed over nylon wool columns. Nonadherent cells were
collected and 1.5 ⫻ 106 cells were incubated with 2.4G2 hybridoma supernatant (anti-CD16/32) for 20 min on ice to block nonspecific Ab binding via Fc␥R. Cells were then stained with the appropriate Abs described
above. For intracellular staining, surface-labeled cells were fixed for 10
min in PBS/1% paraformaldehyde at room temperature. After one wash in
PBS, cells were incubated for 1 h with a rabbit anti-FLAG Ab diluted in
PBS/3% FCS/0.5% saponin (Sigma). Cells were washed once in PBS/3%
FCS/0.5% saponin and incubated for 30 min with CyChrome 3-conjugated
donkey anti-rabbit IgG (Jackson ImmunoResearch; Dianova, Hamburg,
Germany). After one wash in PBS/3% FCS/0.5% saponin, cells were resuspended in PBS/3% FCS and analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA).
Immunoprecipitation and immunoblot
Thymocytes and IL-2-activated NK cells were washed once in PBS. Then
108 cells per ml were solubilized in RIPA lysis buffer (50 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 10% glycerol, and protease inhibitors (Complete,
Boehringer Mannheim, Mannheim, Germany)) for 30 min on ice. Postnuclear lysates were either directly separated by SDS-PAGE or incubated
for 4 h with the appropriate Ab bound to Protein G-Sepharose (Pharmacia).
After four washes with RIPA lysis buffer, precipitated proteins were separated by SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (Amersham, Little Chalfont, U.K.). Membranes were incubated
with the appropriate Abs and revealed with the ECL system (Amersham).
The phosphatase activity of the immunoprecipitated proteins was determined as described (45). Briefly, the immune complex was washed once
with a buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and
0.1% Triton X-100; twice with a buffer containing 50 mM Hepes (pH 7.6),
150 mM NaCl, and 0.1% Triton X-100; and twice with assay buffer containing 40 mM MES (pH 5.0) and 1.6 mM DTT. The washed immune
complex pellet was incubated in 200 ␮l of assay buffer containing 25 mM
para-nitrophenyl phosphate (p-NPP) at 30°C. The reaction was terminated
by the addition of 200 ␮l of 1 N NaOH, and the absorbance at 405 nm was
determined.
Cytotoxicity assays
To determine cytotoxic activities, a conventional 51Cr-release assay was
performed (25). Briefly, 106 target cells were labeled with 50 ␮Ci of 51Cr
for 1 h at 37°C. After three washes, 5 ⫻ 103 labeled target cells were mixed
with IL-2-activated NK cells in duplicate at various E:T ratios in 96-well
U-bottom plates. For Ab inhibition studies, effector cells were preincubated
for 15 min at room temperature with the Ly49A-specific mAb A1 or an
isotype-matched control (mAb F23.1, anti-TCR V␤8) at a concentration of
20 ␮g/106 cells. For Ab-dependent cell-mediated cytotoxicity (ADCC) assays (20), target cells, after 51Cr labeling, were incubated with 10 ␮g/ml of
anti-Thy-1.2 mAb (clone 30-H12, PharMingen) for 30 min on ice. Target
cells were washed twice before addition to the effector cells. After 4 h of
incubation at 37°C, supernatants were harvested and radioactivity was
measured in a gamma-counter. The percentages of NK cells in the effector
cell cultures were determined using flow cytometry. The lysis curves were
moved relative to the content of NK cells in the B6 effector cell population.
Bone marrow graft rejection
One day after lethal irradiation with a 137Cs source (950 rad), groups of
four recipient mice were injected i.v. with 5 ⫻ 106 bone marrow cells from
␤2m-deficient mice. Five days later, the proliferation of donor cells was
assessed by measuring the splenic incorporation of 125I-labeled 5-iodo-2⬘deoxyuridine (125I-UdR). Recipient mice were injected i.p. with 3 ␮Ci of
125
I-UdR, and 1 day later the spleens were removed. After rinsing with
PBS, whole spleen radioactivity was measured in a gamma-counter.
Results
Generation of transgenic mice expressing dnSHP-1
To generate mice expressing dnSHP-1, a point mutation (C453S)
was introduced into the catalytic site of the phosphatase (8, 9). A
FLAG epitope was added at the C terminus of dnSHP-1 to allow
discrimination from endogenous SHP-1. These modifications did
not interfere with the capacity of SHP-1 to bind to the phosphorylated Ly49A receptor (data not shown). To obtain lymphocyterestricted transgene expression, the dnSHP-1 construct was inserted into an expression cassette which is driven by the human
␤-globin promoter and a CD2 locus control region (17) (Fig. 1A).
Following injection of the transgene construct into fertilized B6
oocytes we obtained three transgenic founder lines. Only one of
these, line no. 6, expressed appreciable levels of dnSHP-1 (data not
shown). To further increase the levels of dnSHP-1, line no. 6 was
bred to homozygosity. Unless stated otherwise, homozygous line
no. 6 dnSHP-1 transgenic mice were used for the experiments
shown hereafter. Immunoprecipitations revealed the presence of
dnSHP-1 in IL-2-activated NK cells and thymocytes of transgenic
mice (Fig. 1B). Single cell analysis of intracellular dnSHP-1 using
an anti-FLAG antiserum and flow cytometry detected dnSHP-1 in
freshly isolated splenic NK cells and T cells (Fig. 1C). However,
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To generate dnSHP-1 transgenic mice, a C453S point mutation was introduced into a cDNA encoding murine SHP-1 by PCR-based mutagenesis. In
addition, a FLAG-tag was added to the C terminus. The dnSHP-1-encoding
cDNA was then inserted into a cassette in which expression is controlled
by the ␤-globin promoter and a CD2 downstream locus control element
(LCR) (17). Transgenic mice were generated by standard methods in the
C57BL/6 (B6) background. Founder mice were screened by PCR using the
following primers: 5⬘ SHP-1, 5⬘-CATGCAGGGCCCATCATTGTGCATTC
CTGCGCTGGC-3⬘; and 3⬘ FLAG, 5⬘-CTTGTCATCGTCGTCCTTG
TAGTC-3⬘.
Ly49A transgenic mice (line no. 2) were described before (18).
Ly49A ⫻ dnSHP-1 double-transgenic mice were generated by crossing
homozygous dnSHP-1 transgenic mice (H-2b) with Ly49A transgenic mice
(H-2b). Double-transgenic offspring was back-crossed to homozygous dnSHP-1 mice. Appropriate offspring was identified by FACS analysis using
a FLAG- or Ly49A-specific Ab. B6 mice were purchased from Harlan
(Zeist, The Netherlands). ␤2-microglobulin (␤2m)-deficient mice and homozygous mev mice were obtained from The Jackson Laboratory (Bar
Harbor, ME). All mice were used at 6 – 8 wk of age.
1315
1316
ROLE OF SHP-1 IN NK CELL DEVELOPMENT AND FUNCTION
NK cells expressed the transgene at significantly lower levels than
T cells (⬃4-fold) (see Discussion). No expression was detected in
B cells and macrophages, confirming that dnSHP-1 expression is
confined to T and NK cells (Fig. 1C and data not shown).
T cell and NK cell development in dnSHP-1 transgenic mice
dnSHP-1 transgenic mice appear healthy and show no overt symptoms of inflammatory disease as do mev-mice. In accordance with
recent reports (26, 27), the thymus of B6 dnSHP-1 mice showed a
normal size and subset distribution (Table I and data not shown).
Splenic CD4 and CD8 T cells included equal populations of
CD62Llow and CD44high (memory) cells in B6 and B6 dnSHP-1
transgenic mice (data not shown). Notably, the ratio of splenic
CD4/CD8 cells was slightly lower among B6 (1.3 ⫾ 0.2) as compared to B6 dnSHP-1 (1.7 ⫾ 0.2) T cells. Spleens and bone marrow of transgenic and nontransgenic mice contained comparable
FIGURE 2. SHP-1 phosphatase activity is reduced in transgenic NK
cells. SHP-1 was immunoprecipitated from the lysates of IL-2 activated
NK cells from B6 and dnSHP-1 transgenic mice. A, SHP-1 and dnSHP-1
in the immunocomplex were visualized using a monoclonal anti-SHP-1
and anti-FLAG Ab, respectively. B, SHP-1 phosphatase activity was then
measured using p-NPP as a substrate. Background values (OD405 ⬍ 0.1 in
each case) are subtracted.
numbers of NK cells (Table I), and these expressed a normal set of
cell surface markers such as NK1.1, DX5, CD2, and 2B4 (data not
shown). Thus, expression of dnSHP-1 does not overtly interfere
with the generation of T and NK cell compartments.
Reduced SHP-1 phosphatase activity in NK cells expressing
dnSHP-1
To evaluate the effect of transgene expression on SHP-1 phosphatase activity, total SHP-1 protein was immunoprecipitated from
IL-2-activated transgenic and nontransgenic NK cells. The protein
tyrosine phosphatase (PTP) activity in the immune complex was
then assayed using p-NPP as a substrate. Based on immunoblotting
with a monoclonal anti-SHP-1 Ab, the amounts of SHP-1 immunoprecipitated from transgenic and control NK cells are comparable. However, the PTP activity in immunoprecipitates from transgenic NK cells was reduced as compared to control NK cells (Fig.
2). Therefore, transgenic NK cells contain sufficient amounts of
inactive dnSHP-1 to compete with endogenous SHP-1 for substrate binding.
Inefficient Ly49-mediated inhibition by dnSHP-1 transgenic NK
cells
Table I. NK and T cell development in dnSHP-1-transgenic micea
B6
B6 dnSHP-1
Bone marrow
Spleen
NK cell number (⫻105)
2.2 ⫾ 0.1
1.8 ⫾ 0.4
8.3 ⫾ 3.8
10.3 ⫾ 3.1
Thymus
Spleen
T cell number (⫻106)
157 ⫾ 45
163 ⫾ 6
14 ⫾ 4
21 ⫾ 4
a
Data are derived from five independent experiments (⫾SD) using freshly isolated nylon wool nonadherent spleen or bone marrow cells and total thymocytes.
Numbers for bone marrow represent NK cells residing in femur and tibia of the two
hind legs.
To assess whether dnSHP-1 expression affects NK cell function,
we first assessed Ly49A-mediated inhibition of natural killing. To
this end we generated mice double transgenic for dnSHP-1 and the
Dd-specific inhibitory receptor Ly49A. Short-term activated bulk
NK cells from Ly49A transgenic mice are unable to lyse Dd-transfected C1498 target cells, but readily kill untransfected C1498
cells (Fig. 3) (18). In contrast, NK cells derived from dnSHP-1 ⫻
Ly49A double-transgenic mice lysed C1498.Dd cells quite efficiently, suggesting that Ly49A-mediated inhibition is defective
(Fig. 3). The reversal of inhibition was however only partial, since
blocking of the Ly49A-Dd interaction with the Ly49A-specific
mAb A1 further enhanced the lysis of C1498.Dd cells. Killing of
the parental C1498 target by single- and double-transgenic NK
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FIGURE 1. Generation of dnSHP-1 transgenic mice. A, A dnSHP-1 mutant (C453S) was expressed under the control of the ␤-globin promoter and
a CD2 downstream LCR. B, Transgene expression in IL-2 activated NK
cells and thymus from a B6 dnSHP-1 transgenic mouse (⫹) or a nontransgenic littermate (⫺) was determined by immunoprecipitation using a rabbit
anti-SHP-1 polyclonal Ab. The transgenic dnSHP-1 in the immunocomplex was revealed with an anti-FLAG mAb. C, Transgene expression was
assessed among nylon wool nonadherent spleen cells using intracellular
flow cytometry among NK cells (NK1.1⫹CD3⫺), T cells (CD3⫹), or B
cells (NK1.1⫺CD3⫺; ⬎90% sIg⫹). Intracellular dnSHP-1 transgene expression (thick line) was determined using polyclonal anti-FLAG Abs.
Nontransgenic littermates served as a negative control (thin line).
The Journal of Immunology
1317
(11), which have ⬃10 –20% of normal SHP-1 enzymatic activity
(10). Therefore, the expression of dnSHP-1 or reduced activity of
endogenous SHP-1 reduces the capacity to mediate MHC class
I-dependent inhibitory signals.
NK cells from dnSHP-1 transgenic and mev mice have an
altered Ly49 receptor repertoire
cells was comparable, both in the absence or presence of the blocking (A1) or an isotype-matched control antibody (Fig. 3). These
results suggest an effect of dnSHP-1 on the class I-specific inhibitory pathway and establish that dnSHP-1 transgene levels are sufficient to interfere with the transduction of Ly49-mediated inhibitory signals. Similar to our findings, Ly49A-mediated inhibition is
partially impaired in NK cells derived from homozygous mev-mice
Defective natural killing of class I-deficient target cells by NK
cells from dnSHP-1 transgenic and mev mice
NK cell self-tolerance may also be ensured by the modulation of
triggering pathways (25, 28 –31). Therefore, we compared the activity of B6, B6 dnSHP-1, and mev-derived NK cells in cytotoxicity assays using various target cells. NK cell-sensitive tumor targets such as YAC-1 (H-2a) and CHO were killed equally well by
all three effector populations (Fig. 4). The normal killing of xenogeneic CHO cells suggests that Ly49D-mediated NK cell activation (32) is functional in dnSHP-1 transgenic and mev NK cells. In
Table II. Ly49 receptor usage by dnSHP-1-transgenic and mev/mev NK cellsa
Percentage of NK1.1⫹ CD3⫺ Cells
Self-MHC
(H-2b) Specific
Function
B6
B6 dnSHP-1
B6 mev/mev
Bone marrow
Ly49A
Ly49C/I
Ly49G2
Ly49D
⫺
⫹
⫺
⫺
Inh.
Inh.
Inh.
Act.
24.7 ⫾ 3.0
25.5 ⫾ 4.7
47.3 ⫾ 7.0
46.5 ⫾ 6.8
26.1 ⫾ 5.0
44.8 ⫾ 2.2*
41.0 ⫾ 6.2
39.4 ⫾ 3.5
40.6 ⫾ 1.7**
41.1 ⫾ 3.3*
60.7 ⫾ 5.7
29.3 ⫾ 2.3*
Spleen
Ly49A
Ly49C/I
Ly49G2
Ly49D
⫺
⫹
⫺
⫺
Inh.
Inh.
Inh.
Act.
18.7 ⫾ 1.3
47.3 ⫾ 5.8
49.6 ⫾ 5.9
56.6 ⫾ 1.1
16.8 ⫾ 3.3
60.4 ⫾ 3.0*
43.7 ⫾ 1.4
50.6 ⫾ 3.9
30.8 ⫾ 3.5*
41.2 ⫾ 2.4
54.7 ⫾ 7.2
27.9 ⫾ 2.3**
a
Data are derived from three or more independent experiments (⫾SD) using freshly isolated nylon wool nonadherent spleen
or bone marrow cells. Statistically significant differences as compared to B6 were determined using the two-tailed Student’s t
test. Inh, inhibitory receptor; Act, activating receptor.
ⴱ, p ⬍ 0.02.
ⴱⴱ, p ⬍ 0.001.
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FIGURE 3. Ly49A function is partially impaired in dnSHP-1-expressing NK cells. IL-2-activated NK cells from Ly49A transgenic and
Ly49A ⫻ dnSHP-1 double-transgenic mice were tested for the lysis of
C1498 and C1498.Dd target cells. Tests were performed in the absence of
Ab (f) or in the presence of Ly49A-specific mAb A1 (䡺) and isotypematched control antibody (F23.1) (µ). The figure is representative of the
results obtained in two of three experiments. The observed variability may
be due to the up-regulation of endogenous SHP-1 expression upon culturing NK cells in IL-2 (data not shown). As determined by flow cytometry,
the effector cell populations contained 8% (Ly49A transgenic) and 16%
(Ly49A ⫻ dnSHP-1 double transgenic) of NK1.1⫹CD3⫺ NK cells,
respectively.
As shown above, NK cells in dnSHP-1 transgenic mice develop
despite reduced MHC-specific inhibition. The question thus arises
whether and how these cells remain self-tolerant. One mechanism
to avoid the emergence of auto-aggressive NK cells may involve
an adaptation of the inhibitory MHC receptor repertoire. Indeed,
the analysis of transgenic NK cells revealed changes in the repertoire of Ly49 receptors. Significantly more NK cells expressed the
self-MHC (H-2b)-specific inhibitory Ly49C/I receptors. In contrast, NK cell subsets expressing the non-self-MHC (H-2d)-specific
inhibitory receptors Ly49A and Ly49G2 or the activating receptor
Ly49D were present at normal frequencies (Table II). NK cells
derived from homozygous mev-mice showed even more profound,
yet partially distinct alterations in their Ly49 receptor repertoire
compared to dnSHP-1 transgenic mice. In the bone marrow, significantly more NK cells expressed the inhibitory receptors Ly49A
and Ly49C/I, whereas cells positive for the activating receptor
Ly49D were under-represented. Similar repertoire changes were
observed in the spleen, except that Ly49C/I-positive NK cells were
present at normal frequencies (Table II). Cell surface levels of
Ly49 receptors on NK cells from transgenic and B6 mice were not
notably different, except for Ly49C/I, which was marginally increased. In contrast, NK cells from mev-mice displayed significantly reduced Ly49C/I and Ly49D cell surface levels (Table III).
Therefore, reduced SHP-1 activity tends to expand the usage of
some inhibitory Ly49 receptors, while their cell surface levels may
be unaffected or lower.
1318
ROLE OF SHP-1 IN NK CELL DEVELOPMENT AND FUNCTION
Table III. Ly49 receptor expression levels in dnSHP-1-transgenic and mev/mev NK cellsa
Relative Expression Level
Spleen
Self-MHC
(H-2b) Specific
Function
B6
B6 dnSHP-1
B6 mev/mev
Ly49A
Ly49C/I
Ly49G2
Ly49D
⫺
⫹
⫺
⫺
Inh.
Inh.
Inh.
Act.
100
100
100
100
94 ⫾ 12
119 ⫾ 13
106 ⫾ 7
98 ⫾ 12
94 ⫾ 18
62 ⫾ 5*
110 ⫾ 33
70 ⫾ 4*
a
Data are derived from three independent experiments using freshly isolated nylon wool nonadherent spleen cells. Numbers
represent average mean fluorescent intensity of Ly49 expression relative to B6 values (⫽100) (⫾SD). Statistically significant
differences as compared to B6 were determined using the two-tailed Student’s t test. Inh, inhibitory receptor; Act, activating
receptor.
ⴱ, p ⬍ 0.01.
FIGURE 4. Normal lysis of NK cell-sensitive tumor targets by IL-2activated NK cells from dnSHP-1 transgenic mice. IL-2-activated NK cells
from B6 (f), dnSHP-1 transgenic (䡺), and mev (µ) mice were tested for
the lysis of YAC-1, IC-21, and CHO target cells. The percentages of NK
cells in the respective effector cell cultures were 16% for B6, 9% for B6
dnSHP-1 Tg, and 9% for mev. Data show a representative experiment of
two or three with similar results. The lysis curves were shifted relative to
the content of NK cells present in the B6 effector cell population.
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contrast, IC-21 cells were reproducibly somewhat more resistant to
lysis by mev-derived NK cells (Fig. 4).
Syngeneic (B6-derived) T cell blasts and RMA cells (H-2b)
were resistant to the lysis by all three effector cell types (Fig. 5).
These data suggest that NK cells with low SHP-1 activity are selftolerant. Interestingly, dnSHP-1 transgenic and mev-derived NK
cells were very inefficiently lysing ␤2m-deficient T cell blasts or
the MHC class I-deficient RMA variant RMA-S. Specific lysis was
reduced 10- to 30-fold as compared to lysis by B6-derived NK
cells (Fig. 5). Therefore, in the absence of MHC class I ligands
(and thus MHC class I-mediated inhibition and activation), dnSHP-1 transgenic- and mev-derived NK cells are hyporesponsive,
suggesting that the function of non-MHC-specific activation receptors is impaired. The effect was reproducibly more pronounced
in mev compared to dnSHP-1 transgenic NK cells, possibly reflecting lower levels of functional SHP-1 in the former mouse strain.
FIGURE 5. Impaired lysis of MHC class I-deficient tumor targets and
Con A blasts by IL-2-activated NK cells from dnSHP-1 transgenic (Tg)
and mev mice. IL-2-activated NK cells from B6 (f), dnSHP-1 transgenic
(䡺), and mev (µ) mice were tested for the lysis of B6- and ␤2m⫺/⫺-derived
Con A blasts or RMA and RMA-S tumor targets. For ADCC, prior to the
assay RMA target cells were coated with anti-Thy-1 Ab and washed. The
percentages of NK cells in the respective effector cell cultures were 12%
for B6, 24% for B6 dnSHP-1 transgenic and 7% for mev. The lysis curves
were shifted relative to the content of NK cells present in the B6 effector
cell population. Data show a representative experiment of two or three with
similar results.
The Journal of Immunology
To further assess whether specific NK cell activation pathways
function normally in mev or B6 dnSHP-1 NK cells, we tested activation via CD16 (Fc␥RIII) through which NK cells mediate
ADCC. As shown in Fig. 5, RMA target cells (H-2b) are resistant
to lysis by all three effector types. RMA cells coated with antiThy-1 Ab became susceptible to lysis. However, the lysis of coated
RMA cells by transgenic and mev-derived effectors was reproducibly somewhat less efficient (3-fold). Activation of dnSHP-1 and
mev-derived NK cells can thus occur via their Fc␥R.
dnSHP-1 transgenic mice reject ␤2m-deficient bone marrow
grafts
Since ␤2m-deficient T cell blasts are killed inefficiently by NK
cells from dnSHP-1 transgenic mice, we have thus tested whether
transgenic mice have retained the ability to reject class I-deficient
bone marrow grafts. Lethally irradiated dnSHP-1 transgenic mice
were challenged with a standard dose (5 ⫻ 106 cells) of ␤2mdeficient bone marrow cells. Marrow engraftment was monitored
by the incorporation of 125I-UdR in spleens of recipient mice,
which indicates donor cell proliferation (33). Whereas grafts were
accepted by ␤2m-deficient recipient mice, B6 dnSHP-1 and B6
mice rejected the ␤2m-deficient bone marrow grafts with similar
efficiency (Fig. 6). Thus, while natural cytotoxicity to ␤2m-deficient T cell blasts in vitro is greatly reduced in B6 dnSHP-1 transgenic mice, NK cells in these mice efficiently react to ␤2m-deficient bone marrow stem cells in vivo.
Discussion
To address the role of SHP-1 for NK cell function and development we have generated transgenic mice that express a dominantnegative form of SHP-1 in NK cells and T cells. Surprisingly, NK
cells expressed the dnSHP-1 transgene at significantly lower levels
than T cells (⬃4-fold). This may be due to an inferior activity of
the ␤-globin/CD2 LCR expression cassette in NK cells. Consistent
with this possibility, CD2 cell surface levels on T cells are ⬃2-fold
higher than on NK cells (data not shown). In addition, the FLAG
epitope of the transgenic SHP-1 may be less accessible for intracellular staining in NK cells compared to T cells.
The overexpression of the dnSHP-1 mutant used here has been
shown to prevent the transduction of MHC class I-dependent inhibitory signals in human NK cell clones (8, 9). Similarly, our
functional analysis suggests that Ly49A-mediated inhibition in NK
cells of Ly49A ⫻ dnSHP-1 double-transgenic mice is impaired,
but not completely abolished. Residual inhibition via Ly49A was
also reported in NK cells from homozygous mev and even from me
mice, which have 10 –20% of wild-type or no SHP-1 phosphatase
activity, respectively (10). These findings suggested that other effector molecules, such as SHP-2, are involved in mediating inhibition via Ly49 receptors (11). Indeed, SHP-2 has been shown to
be recruited to inhibitory Ly49 receptors (6). Because dnSHP-1
most likely acts by competing with endogenous proteins for ITIM
binding, it may also compete with SHP-2 binding. In addition, the
reduced phosphatase activity in transgenic NK cells suggests that
dnSHP-1 is also able to compete for SHP-1 substrates. However,
the residual Ly49A-mediated inhibition in dnSHP-1 mice suggests
that higher transgene levels may be required to completely block
the inhibitory pathway.
MHC class I-specific inhibitory receptors are important to prevent auto-aggression by mature NK cells (12–14). Thus, a reduced
capacity to transduce inhibitory signals due to dnSHP-1 expression
may affect NK cell development and/or function. However, reduced SHP-1 activity did not interfere with the generation of normal numbers of NK cells (Table I). Moreover, functional assays
demonstrate that these NK cells are self-tolerant since they do not
kill syngeneic, normal cells (Fig. 5). This raises the possibility that
they have somehow adapted to the reduced capacity to mediate
inhibition.
Indeed, we found that in the absence of MHC class I molecules
and thus MHC class I-mediated inhibition, NK cell activation via
non-MHC receptors was significantly impaired in transgenic mice.
A corresponding but even more pronounced phenotype was observed in NK cells derived from mev mice. Therefore, in two distinct models of impaired transduction of inhibitory signals, nonMHC-specific NK cell triggering pathways function inefficiently.
A comparison of the two mouse strains based on our and available
data (11) suggests that the more inhibition is impaired, the more
NK cell activation is reduced. The observed effects on NK cell
activation are reminiscent of NK cells that develop in mice with
targeted inactivation of the ␤2m or TAP genes, i.e., in the absence
of MHC class I molecules. These NK cells show even more drastically impaired NK cell activation, especially in response to untransformed target cells (25, 29, 34). Therefore, both the absence
of class I ligands as well as a reduced signal transduction capacity
by the respective inhibitory receptors results in NK cell activation
defects.
To our surprise, however, even though the lysis of ␤2m-deficient
target cells in vitro was significantly impaired, ␤2m-deficient bone
marrow grafts were efficiently rejected by B6 dnSHP-1 transgenic
mice. This represents one of only a few instances in which the
results of bone marrow graft rejection is not reflected by its in vitro
correlate (35, 36). It has been suggested that the rejection of bone
marrow grafts does not depend on the cytotoxic function of NK
cells (37). This process may thus reflect the capability of NK cells
to produce cytokines, which prevent stem cell proliferation. Compared to cytotoxicity, cytokine production may be less affected in
B6 dnSHP-1 transgenic NK cells. Alternatively, the two experimental systems may reflect NK cell activation via distinct triggering pathways. Only some of the pathways, which are used to activate NK cells in response to T cell blasts are significantly affected
in B6 dnSHP-1 transgenic mice.
Phenotypically, NK cells from mice deficient in class I expression tend to acquire more inhibitory Ly49 receptors per NK cell
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FIGURE 6. B6 dnSHP-1 transgenic mice reject a ␤2m-deficient bone
marrow graft. Rejection of bone marrow grafts by irradiated mice was
assessed using the splenic 125I-UdR incorporation assay. Low levels of
incorporation in B6 recipients reflects rejection of the graft. High levels in
␤2m-deficient recipients reflect graft acceptance. Four recipient mice were
used per experimental group; each symbol reflects an individual recipient
animal.
1319
1320
ROLE OF SHP-1 IN NK CELL DEVELOPMENT AND FUNCTION
4.
5.
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7.
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9.
10.
11.
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16.
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18.
19.
20.
21.
22.
23.
Acknowledgments
We thank P. Zaech for expert assistance with flow cytometry, and W.
Seaman for the C1498 and C1498.Dd cell lines. We also thank J.-C. Cerottini for helpful discussion and critical reading of the manuscript.
24.
25.
26.
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and express these at higher levels (29, 38, 39). NK cells from
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