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*
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and
Swiss Institute for Experimental Cancer Research (ISREC), Epalinges, Switzerland
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 3, 4, 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, 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, 13, 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 cells that are able to efficiently react to the absence of MHC class I from normal cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 ß-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'-CATGCAGGGCCCATCATTGTGCATTCCTGCGCTGGC-3'; and 3' FLAG, 5'-CTTGTCATCGTCGTCCTTGTAGTC-3'.
Ly49A transgenic mice (line no. 2) were described before (18). Ly49A x 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). ß2-microglobulin (ß2m)-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.
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 x 106 cells/ml for 48 h in DMEM supplemented with 10% FCS and 2.5 µg/ml Con A (Sigma, Buchs, Switzerland). Before use as targets in a standard 4-h 51Cr-release 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, 22, 23, 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 x 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 µl of assay buffer containing 25 mM para-nitrophenyl phosphate (p-NPP) at 30°C. The reaction was terminated by the addition of 200 µl 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 µCi of 51Cr for 1 h at 37°C. After three washes, 5 x 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 Vß8) at a concentration of 20 µg/106 cells. For Ab-dependent cell-mediated cytotoxicity (ADCC) assays (20), target cells, after 51Cr labeling, were incubated with 10 µg/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 x 106 bone marrow cells from ß2m-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 µCi of 125I-UdR, and 1 day later the spleens were removed. After rinsing with PBS, whole spleen radioactivity was measured in a gamma-counter.
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Results |
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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 lymphocyte-restricted
transgene expression, the dnSHP-1 construct was inserted into an
expression cassette which is driven by the human ß-globin promoter
and a CD2 locus control region (17) (Fig. 1
A). 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, 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).
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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 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.
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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.
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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 x
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 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 (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.
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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.
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NK cell self-tolerance may also be ensured by the modulation of
triggering pathways (25, 28, 29, 30, 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 contrast, IC-21 cells were reproducibly somewhat more
resistant to lysis by mev-derived NK cells
(Fig. 4).
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). These data suggest that NK cells with
low SHP-1 activity are self-tolerant. Interestingly, dnSHP-1 transgenic
and mev-derived NK cells were very inefficiently
lysing ß2m-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.
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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
anti-Thy-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 FcR. dnSHP-1 transgenic mice reject ß2m-deficient bone marrow grafts
Since ß2m-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 x
106 cells) of ß2m-deficient 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 ß2m-deficient recipient mice, B6 dnSHP-1 and
B6 mice rejected the ß2m-deficient bone marrow grafts
with similar efficiency (Fig. 6). Thus,
while natural cytotoxicity to ß2m-deficient T cell blasts
in vitro is greatly reduced in B6 dnSHP-1 transgenic mice, NK cells in
these mice efficiently react to ß2m-deficient bone marrow
stem cells in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4-fold). This may be due to an inferior activity of the
ß-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 x 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, 13, 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, non-MHC-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 ß2m 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 ß2m-deficient target cells in vitro was significantly impaired, ß2m-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 and express these at higher levels (29, 38, 39). NK cells from dnSHP-1 transgenic and mev mice also showed a tendency toward increased usage of certain inhibitory Ly49 receptors. In contrast to class I-deficient mice, however, expression levels of inhibitory Ly49 receptors were not increased in dnSHP-1 NK cells. If anything, Ly49C/I expression levels were decreased in mev NK cells. However, we cannot exclude that some of these effects are secondary to the chronic activation of macrophage/myeloid populations in mev mice. The interpretation of these results is also complicated by the fact that mAb 5E6 reacts with two distinct Ly49 receptors, which could be differentially affected by low SHP-1 activity. Nevertheless, the results indicate that the frequency of usage and the cell surface levels of Ly49 receptors can be differentially affected. This is consistent with the notion that Ly49 receptor acquisition is a developmentally regulated process that results in stable Ly49 receptor expression patterns (40, 41). In contrast, Ly49 cell surface levels can be rapidly modulated depending on MHC ligand availability (42).
In contrast to class I-deficient mice mev and
dnSHP-1, transgenic mice express class I MHC molecules normally (data
not shown). MHC-specific activating receptors will therefore encounter
ligands in the latter mouse strains. Analysis of the expression of the
Dd-specific activating receptor Ly49D revealed that
Ly49D-positive NK cells were under-represented in
mev mice. However, no changes were observed in
dnSHP-1 transgenic mice. The low capacity to transduce inhibitory
signal in mev mice may thus also influence the
generation of NK cell subsets expressing activating MHC receptors.
However, the function of Ly49D was not significantly affected due to
reduced SHP-1 activity. This is based on the normal lysis of xenogeneic
CHO cells via Ly49D (Fig. 3), which recognizes Dd on murine
target cells (43, 44). In addition,
mev and dnSHP-1 NK cells can be activated via
FcR, although not as efficiently as B6 NK cells. It is therefore
possible that CD16 signaling is reduced. However, an effect on ADCC may
also reflect the fact that inefficient NK cell activation via non-MHC
specific receptors and ADCC are superimposed and only the former is
affected by low SHP-1 activity.
In this paper we have shown that reduced SHP-1 activity results in multiple abnormalities in the NK cell compartment. Strikingly such NK cells show defective natural killing of class I-deficient target cells. SHP-1 may thus play a positive role in some NK cell activation pathways. However, based on similar effects of class I deficiency it seems more likely that a reduced capacity to mediate inhibitory signals during NK cell development is compensated by down-modulating activation pathways. These findings imply an important role for SHP-1 in the process of self-tolerance induction. Besides refining our understanding of this mechanism, dnSHP-1 transgenic mice may be useful to identify downstream targets of SHP-1 in the inhibitory MHC receptor signaling pathway.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Werner Held, Ludwig Institute for Cancer Research, Lausanne Branch, Ch. de Boveresses 155, 1066 Epalinges, Switzerland.
3 Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibition motif; ADCC, Ab-dependent cell-mediated cytotoxicity; B6, C57BL/6; ß2m, ß2-microglobulin; dn, dominant-negative; LCR, locus control region; me, motheaten; mev, viable motheaten; p-NPP, para-nitrophenyl phosphate; PTP, protein tyrosine phosphatase; SHP, SH2-containing protein tyrosine phosphatase.
Received for publication December 22, 1999. Accepted for publication May 17, 2000.
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ßTCR population in i-IEL with an extrathymic maturation. Int. Immunol. 4:699.This article has been cited by other articles:
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) or in the presence of Ly49A-specific mAb A1 (
) and
isotype-matched control antibody (F23.1) (
). 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 x dnSHP-1 double
transgenic) of NK1.1+CD3- NK cells,
respectively.
