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*
Laboratory of Experimental Immunology, Division of Basic Sciences, and
Intramural Research Support Program, Science Applications International Corporation-Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702;
University of Oklahoma, Health Sciences Center, Oklahoma City, OK 73104; and
§
Laboratory of Biological Chemistry, National Institute of Aging, National Institutes of Health, Baltimore, MD 21224.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and requires Syk/Zap70 family kinases and arginine 54 of Ly-49D,
suggesting that Ly-49D signals via association with TCR.
Coexpression studies in 293-T cells confirmed the ability of Ly-49D to
associate with TCR. In addition, we have used this model to study
the functional interactions between an inhibitory Ly-49 (Ly-49G2) and
an activating Ly-49 (Ly-49D). Ly-49G2 blocks activation mediated by
Ly-49D in an immunoreceptor tyrosine-based inhibitory motif
(ITIM)-dependent manner. In contrast, Ly-49G2 was incapable of
inhibiting activation by the TCR even though human killer cell
inhibitory receptor (KIR) (KIR3DL2(GL183)) effectively inhibits TCR.
Both the ability of Ly-49G2 to block Ly-49D activation and the failure
of Ly-49G2 to inhibit TCR signaling were confirmed in primary murine NK
cells and NK/T cells, respectively. These data demonstrate the dominant
effects of the inhibitory receptors over those that activate and
suggest an inability of the Ly-49 type II inhibitory receptors to
efficiently inhibit type I transmembrane receptor signaling in T cells
and NK cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Liver and splenic NK cells were isolated from C57BL/6 (B6) mice and grown for 7–10 days in 1000 U/ml Cetus recombinant IL-2 as previously described (1). Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985).
Antibodies
The following mAb were used: 4D11 (Ly-49G2; Ref.
3); 12A8 (Ly-49A/D; Ref. 1); and 4E5 (Ly-49D;
Ref. 14). Antisera to Fc
RI
and TCR were kindly
provided by Dr. J. P. Kinet and Dr. A. Weissman, respectively.
GL183 Ab (directed against the KIR3DL2) was a gift from Dr. Eric Long,
National Institute of Allergy and Infectious Diseases (NIAID). 4G10 Ab
to phosphotyrosine was purchased from Upstate Biotechnology (Lake
Placid, NY). Rabbit and goat cross-linking reagents were purchased from
ICN/Cappel Laboratories (Oxford, PA). T cell reagents anti-CD3
(OKT3) and anti-CD16 (3G8) were prepared from their hybridomas
(American Type Culture Collection, Manassas, VA) using standard
techniques. Goat anti-mouse or rabbit anti-rat were used as
cross-linking reagents (Cappel Laboratories). Syk Abs were purchased
from Santa Cruz Biotechnologies (Santa Cruz, CA). Additional Syk
Abs were the gift of Dr. Joseph Bolen, DNAX (San Francisco, CA). Zap-70
Abs have been previously described (15).
Flow cytometry analysis cytotoxicity assays
Cells were stained as previously described (12) and analyzed on a FACSort flow cytometer (Becton Dickinson, San Jose, CA). Cell sorting was performed using a Cytomation MoFlo (Cytomation, Ft. Collins, CO). Cells were directly stained using PE- and FITC-labeled primary Abs or indirectly stained using a primary Ab followed by an isotype-specific FITC- or PE-conjugated secondary or a biotinylated primary Ab followed by Streptavidin PerCp (Becton Dickinson) or Tricolor (Caltag, Burlingame, CA).
Targets
Tumor targets were maintained in culture as previously described (12). P815 is a mouse mastocytoma.
Cytotoxicity assays
Tumor targets were labeled with 51Cr and used in 6-h cytotoxicity assays as previously described (12).
Stimulation, immunoprecipitation, electrophoresis, and blotting
Cell stimulation was performed with cells at 1–5 x 106 per ml. Abs were added at a concentration of 1 µg/per 106 cells. Either rabbit anti-rat IgG (Zymed, San Francisco, CA) or goat anti-mouse IgG (cross-reactive to mouse and rat; Cappel Laboratories) was used to cross-link Abs at a concentration of 1 µg/per 106 cells. Preparation of lysates, immunoprecipitation, and immunoblotting were performed as previously described (1). Pervanadate stimulation of cells utilized 1 mM pervanadate for 5–15 min at 37°C.
Site-directed mutagenesis and transfection
A substitution mutant (Fig. 1
) was
generated within the Ly-49D transmembrane domain in which the arginine
at position 54 was mutated to a leucine
(Ly-49DR54L) (16). An Ly-49G2 ITIM
mutant was generated by changing VTY at position 6–8 to DTF
(Ly-49G2VTY/DTF). Membrane proximal tyrosines at
positions 36 and 39 were mutated to phenylalanine, altering the
parental YRKY to FRKY (Ly-49G2Y36F), YRKF
(Ly-49G2Y39F), or FRKF
(Ly-49G2Y36,Y39F) (17). Mutations
were performed with the Transformer Site-Directed Mutagenesis Kit
(Clontech, Palo Alto, CA) according to the manufacturer’s
instructions. The mutant constructs were confirmed by sequencing.
Jurkat cells were electroporated with mutated Ly-49s (see Fig. 1) as
previously described (11).
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Analysis of the changes in intracellular Ca2+ concentration ([Ca2+]i) were conducted using a FACSort Flow Cytometer (Becton Dickinson) and the calcium sensitive fluorochrome Fluo-3 (Molecular Probes, Eugene, OR). Briefly, cells (2 x 106/ml) were incubated at 25°C in DPBS without Ca2+ or Mg2+ containing 15 µg/ml Fluo-3. After 30 min, cells were washed in DPBS and held at room temperature in the dark until analysis. The intracellular Ca2+ concentration was monitored with the loaded cells (40 µl) diluted to 500 µl with DPBS containing Ca2+ and Mg2+, glucose, and sodium pyruvate. The cells were kept at 37°C during analysis. Baseline data were collected for 20–30 s; then the cells were stimulated with primary (10 µg/ml) mAb followed 20–30 s later by rabbit anti-rat Ab (10 µg/ml) or goat anti-mouse Ab (10 µg/ml). Data were analyzed using the MultiTime Kinetic Experiment Analysis Software (Phoenix Flow Systems, San Diego, CA) and was expressed as the percentage of responding cells relative to unstimulated baseline measurements.
Preparation and expression of vaccinia
Vaccinia expressing wild-type Ly-49D was prepared by insertion of the cDNA for Ly-49D into a pSport1 vector (16). The Ly-49D construct was expressed in a pVote construct driven by LacI-inducible early gene promoter that was inserted into a Not-1 site as previously described (18). WR strain vaccinia was used as a negative control.
Vaccinia infection, cell transfection, and cell lines
Cells were treated for 15 min in serum-free conditions with recombinant vaccinia virus at a multiplicity of infection (MOI) of 5–10. RPMI 1640 medium containing 10% serum was added, and the cells were cultured at 37°C for an additional 2–4 h. Jurkat cells infected with vaccinia expressing Ly-49D (JurkatvLy-49D) or KIR3DL2 (GL183) (Jurkat vGL183) were used. In addition, Jurkat T cells deficient in Syk (clone E6.1) (JurkatE61) or deficient in both Syk and Zap-70 (clone P116) (JurkatP116) were used as targets for vaccinia expression of Ly-49D (19). 293-T cells were transfected using the FuGene (Roche Diagnostics, San Jose, CA) transfection reagent as previously described (11).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies (11, 20) demonstrated that
Ly-49D is an activating NK receptor. However, the simultaneous
expression of multiple Ly-49 family receptors in fresh or IL-2 expanded
NK cells makes functional analysis of individual Ly-49 family members
complex. Therefore, we sought to develop a model system in which other
murine NK receptors would be absent. Jurkat T cells have been
extensively utilized to study various aspects of TCR signal
transduction. To determine whether Jurkat T cells could express a
functional mouse Ly-49 NK receptor, we expressed Ly-49D in Jurkat
(JurkatvLy-49D) using a vaccinia expression
system (expression exceeded 85%; not shown). We have reported that
Ly-49D mediates calcium mobilization in transfected P815; therefore, we
first analyzed JurkatvLy-49D for their ability to
mount a calcium flux in response to anti-Ly-49D Ab. As shown in
Fig. 2A, when
JurkatvLy-49D cells were treated with
anti-Ly-49D (4E5) or F(ab')2 fragments of
anti-Ly-49D Ab (12A8) and cross-linked with a rabbit anti-rat
IgG, increases in [Ca2+]i
were observed in 50–80% of the cells. The addition of 4E5 alone,
without cross-linking, resulted in a modest level of calcium
mobilization. Infection with these vaccinia constructs or WR strain
vaccinia does not effect CD3-induced calcium mobilization (data not
shown). Our recent studies have demonstrated that arginine 54 is
essential for Ly-49D signal transduction (11). To ensure
that this Jurkat model was functionally similar and specific, we
transiently transfected Jurkat with wild-type Ly-49D or the
Ly-49DR54L. Both transfections resulted in
45–50% of the Jurkat expressing Ly-49D (not shown). When these
receptors were evaluated for their ability to mediate calcium
mobilization, only wild-type Ly-49D (Fig. 2B) could mediate
calcium mobilization, whereas the mutant
Ly-49DR54L was indistinguishable from
control.
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Ly-49D signaling is dependent on its ability to interact with a
signal transduction chain via association with the transmembrane
Arg54 (Fig. 2
B) (11).
Jurkat T cells, however, do not express DAP-12, the molecule known to
be coupled to Ly-49D in NK cells (9, 10, 11). Therefore, we
sought to identify what signaling molecules were associated with Ly-49D
in Jurkat. Preliminary experiments showed that Ly-49D cross-linking
induced tyrosine phosphorylation of multiple cellular substrates within
1–3 min in JurkatvLy-49D. These studies showed
phosphorylation of both 70-kDa and 40-kDa proteins under nonreducing
conditions, masses consistent with Syk or Zap-70 and TCR,
respectively. Therefore, Jurkatwt and
JurkatvLy-49D were stimulated with either
anti-CD3 or anti-Ly-49D(4E5). The phosphorylation of TCR,
Zap-70, and Syk was directly assessed by sequential precipitation of
cell lysates, followed by anti-phosphotyrosine immunoblotting. As
expected, stimulation with CD3 resulted in strong TCR and Zap-70
phosphorylation (Fig. 3A).
Interestingly, when JurkatvLy-49D were stimulated
with 4E5 mAb, we detected tyrosine phosphorylation of Syk, Zap-70, and
TCR. These data suggest that Ly-49D ligation leads to
phosphorylation of TCR in JurkatvLy-49D and
results in activation of both Zap-70 and Syk. Jurkat infected with WR
strain vaccinia gave activation patterns identical to parental Jurkat
(not shown). To test whether Ly-49D could interact directly with
TCR, 293-T cells were cotransfected with combinations of
Ly-49Dwt, Ly-49DR54L and
DAP-12, or TCR (Fig. 3B). Cells were pervanadate
stimulated, immunoprecipitated with anti-Ly-49D, and blotted with
anti-phosphotyrosine. Coimmunoprecipitation of phosphorylated
DAP-12 and TCR (Fig. 3B, left panel) was
detected. The specificity of these interactions was demonstrated by the
failure of Ly-49DR54L to immunoprecipitate
associated phosphoproteins (Fig. 3B, right
panel). These data show the ability of Ly-49D to physically
associate with TCR, suggesting that this protein may be used in the
absence of DAP-12.
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A demonstrated that Ly-49D cross-linking
resulted in phosphorylation of TCR, to more directly show that
Ly-49D was associated with TCR, Jurkat cells were infected with
either WR strain or Ly-49D-expressing vaccinia. These cells then were
examined directly for TCR/Ly-49D association, and the results are
shown in Fig. 3D. When parental Jurkat was
immunoprecipitated with anti-TCR Ab, then blotted with
anti-TCR Ab, the expected band at 
18 kDa was observed under
reducing conditions. When either parental (uninfected) WR or
VacLy-49D Jurkat cells were immunoprecipitated
with anti-Ly-49D (4E5) and blotted with anti-TCR Ab, only
the Ly-49D-expressing Jurkat demonstrated Ly-49D-associated TCR.
NK cell tumor recognition (21) and Ly-49D ligation
delivers signals through Syk but not Zap-70 (22),
therefore we asked whether Ly-49D signals in Jurkat also required Syk.
Jurkat lacking Syk (E6.1) or both Syk and Zap-70 (P116) were evaluated
for Ly-49D-induced calcium mobilization (Fig. 3C). Ly-49D
cross-linking suggested that Syk, and to some extent Zap-70, were
involved. In several experiments, JurkatE6.1
showed a 30–70% reduction of inducible calcium mobilization, whereas
the P116 Jurkat demonstrated no significant calcium signal. In parallel
experiments with anti-CD3, JurkatE6.1 showed
intact calcium mobilization comparable to parental Jurkat, while Jurkat
P116 calcium mobilization was severely blunted (data not shown) as
previously reported (15, 23).
Utilization of Jurkat to examine the inhibitory motifs of Ly-49 receptors
Since JurkatvLy-49D appears to be a good
model to examine the activating murine NK receptors, we next analyzed
the biochemical interactions between activating and inhibitory
(Ly-49G2) NK receptors. Jurkat were first transiently transfected with
Ly-49G2. After 18 h, cells were infected for 3 h with
vaccinia expressing Ly-49Dwt. In all experiments,
>75% of the cells expressed both Ly-49G2 and Ly-49D. As shown in Fig. 4A, when both activating and
inhibitory mouse NK receptors were expressed, cross-linking with 4E5
(anti-Ly-49D) resulted in strong calcium mobilization. However,
when both Ly-49D and Ly-49G2 were simultaneously cross linked, over
80% of the inducible signal was lost. Since previous studies have
demonstrated the necessity of the Ly-49 ITIM, we evaluated mutated
Ly-49G2 NK receptors for their ability to function as inhibitory
receptors in Jurkat. In Jurkat expressing both Ly-49D and
Ly-49G2[VTY/DTF], this NK receptor failed to
inhibit Ly-49D calcium mobilization (Fig. 4A). In other
experiments, Ly-49G2Y36F,
Ly-49G2FRKY, and
Ly-49G2FRKF were coexpressed with vLy-49D and
tested for their ability to block calcium mobilization induced by
Ly-49D. These mutations failed to alter the inhibitory capacity of
Ly-49G2 (not shown). Therefore, the Ly-49G2 ITIM appears to be solely
responsible for the inhibition of Ly-49D NK receptor-mediated
signals.
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B),
using a wide range of doses (10–0.05 µg/ml; not shown) of
anti-CD3, no significant reduction in CD3-mediated calcium
mobilization was seen.
Since we have observed that Ly-49D signaling could be inhibited by
Ly-49G2, but CD3 could not, we examined whether Ly-49D signaling could
be blocked by a type I inhibitory KIR. We used the RNK-D cell line
(26) and expressed KIR3DL2 (GL183) using vaccinia. As can
be seen in Fig. 4C, simultaneous cross-linking of both
Ly-49D and KIR3DL2 did not diminish the Ly-49D signal. Expression of
GL183 was observed in 83% of the cells (not shown). In experiments not
shown, vaccinia expression of Ly-49A in RNK-D did diminish or ablate
calcium mobilization triggered through Ly-49D.
Biochemical evaluation of Ly-49G2 inhibition
Since Ly-49G2 inhibited Ly-49D signals but failed to block
CD3-mediated signals, we evaluated the ability of these receptors to
induce tyrosine phosphorylation. Fig. 4D shows whole cell
lysates of Jurkat expressing Ly-49D and Ly-49G2 stimulated with
anti-CD3 (Fig. 4D, left panel) or
anti-Ly-49D (Fig. 4D, right panel). Anti-CD3
stimulation induced the expected pattern of tyrosine phosphorylation,
including substrates consistent with TCR and Zap-70. Simultaneous
cross-linking of Ly-49G2 and CD3 did not alter this pattern.
Alternatively, cocross-linking of Ly-49D and Ly-49G2 totally abrogated
the Ly-49-induced phosphorylation. It should be noted, however, that
the magnitude of activation by Ly-49D appears to be less than that seen
with CD3. Therefore, lanes 1–3 of the right
panel of Fig. 4D represent the equivalent of 7 million
cells, where the CD3 positive control is 1 million cells.
Expression of inhibitory KIR in Jurkat cells
Since Ly-49G2 could not block TCR-mediated signals, we next
validated this system by confirmation that human KIR could block the
TCR signals. Jurkat expressing (>95% positive; not shown) the
inhibitory KIR3DL2 (GL183) after vaccinia infection were examined. As
shown in Fig. 5A, with
cocross-linking of KIR3DL2 (GL183) and anti-CD3, calcium
mobilization was significantly blocked. Control anti-CD16
cocross-linking had little effect. To confirm that this was the result
of reduction of CD3-mediated tyrosine phosphorylation, Jurkat cells
expressing KIR3DL2 (GL183) were evaluated using
anti-phosphotyrosine blotting. As shown in Fig. 5B,
CD3-induced tyrosine phosphorylation was totally blocked by GL183
cotreatment.
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Since Ly-49s are expressed in mouse NK cells, we wished to confirm
the functional and biochemical effects observed in Jurkat with primary
NK cells. Ly-49D+,
Ly-49G2+, and Ly-49A- NK
cells from C57BL/6 mice were sorted (>95% pure; not shown) and
cultured in IL-2 for 7–9 days. These cells then were evaluated for
their lytic ability (Fig. 6A).
Ly-49D delivers a positive signal for lysis, and Ly-49G2 delivers an
inhibitory signal. If Ly-49D is blocked with a
F(ab')2 Ab, lysis is inhibited; however, when
Ly-49G2 is blocked, lysis increases, indicating the loss of an
inhibitory signal. When both signals are delivered, inhibition is
dominant. When identical NK cells were then evaluated for calcium
mobilization (Fig. 6B), Ly-49D cross-linking resulted in
modest calcium mobilization, and cocross-linking of Ly-49G2 inhibited
this event. It should be noted, however, that, although >99% of the
mouse NK cells coexpress Ly-49G2 and Ly-49D, the calcium mobilization
seen by receptor cross-linking with 4E5 is never as strong as that seen
with cell lines. When primary CD3+, Ly-49G2+ T cells
(Fig. 6C) were evaluated for calcium mobilization, the
cross-linking of Ly-49G2 did not significantly block the CD3-mediated
signal, as compared with a control (4E5 not expressed on the primary
CD3+ T cells). Finally, these two primary
Ly-49G2+ cell types (T and NK) were evaluated
biochemically (Fig. 6D). When T cells were activated by CD3
cross-linking, numerous substrates became tyrosine phosphorylated.
Cross-linking of Ly-49G2 alone did not induce phosphorylation and did
not block anti-CD3-induced activation. When NK cells were
stimulated with anti-Ly-49D (Fig. 6D, right
panel) tyrosine phosphorylation of numerous substrates was
detected, and the simultaneous cocross-linking of Ly-49D and Ly-49G2
blocked most or all the Ly-49D-induced phosphorylation of proteins.
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, both Syk (Fig. 7A) and
Zap-70 (Fig. 7B) could reconstitute CD3 signaling, although
suboptimally. Interestingly, both signal pathways were blocked by
cross-linking GL183 and CD3, whereas no effect was observed by
cross-linking Ly-49G2 and CD3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although several of the general biochemical parameters of
Ly-49-mediated inhibition of mouse NK activity have been defined, the
expression of multiple species of Ly-49 molecules on any given NK cell
has hampered detailed study of these inhibitory or activating
receptors. In fact, mouse NK cells often express a combination of
inhibitory and activating receptors, some of which apparently bind the
same MHC ligands (27). Further confusion comes from the
fact that mAbs directed against one Ly-49 often cross-react with other
members of the family. Therefore, to further dissect the
structure/function relationships of activating and/or inhibitory Ly-49
proteins, we have developed a model that involves the expression of
Ly-49s in the human T cell line Jurkat. This model system allows for
the rapid expression of wild-type and/or mutants of Ly-49 proteins in
the total absence of other family members. In addition, the extensive
study of the signal transduction pathways in Jurkat, together with the
continuous development of somatic mutants of this line, provide an
unprecedented resource for the study of the signaling properties of
Ly-49s. We and others have previously reported that Ly-49D physically
interacts with a low m.w. polypeptide, DAP-12 (10, 11, 22). Cross-linking of Ly-49D in NK cells results in tyrosine
phosphorylation, and calcium mobilization (11, 22) (Figs. 2 and 3). Here, we demonstrate that cross-linking Ly-49D in Jurkat also
leads to protein tyrosine phosphorylation of multiple cellular
substrates and mobilization of intracellular calcium. Cross-linking of
inhibitory Ly-49 proteins does not. Moreover, mutation of
Arg54 to Leu ablated Ly-49D-mediated calcium
mobilization, confirming that, in Jurkat T cells, activating Ly-49s
require interaction with a signal transduction chain. The fact that
Jurkat T cells do not express DAP-12 suggests that Ly-49D is capable of
associating with some other signaling chain in these cells.
We hypothesized that Ly-49D function in Jurkat might be due to
interaction between Ly-49D and the TCR signal transduction
apparatus, specifically the TCR chain. Indeed, cotransfection
of Ly-49D and TCR into 293-T cells demonstrated that
tyrosine-phosphorylated TCR could be detected in Ly-49D
immunoprecipitates. In addition, stimulation of Ly-49D in Jurkat
resulted in tyrosine phosphorylation of TCR, as well as Syk and
Zap-70, and TCR can be coimmunoprecipitated from Jurkat with Ly-49D.
Together these findings suggest that, in Jurkat, Ly-49D is signaling
via TCR.
Similar to the Ly-49 family, human KIR that fail to become
phosphorylated contain charged residues in their transmembrane
receptors, and activation of NK cells has been reported
(4). The KIR associate with DAP-12 in NK cells
(9). However, activating KIR do not transduce biochemical
or functional signals in Jurkat T cells unless these cells are
cotransfected with DAP-12 (5). The contrast between the
ability of Ly-49 to function in Jurkat when activating KIR do not,
implies that, unlike Ly-49D, these KIR fail to associate with signal
transduction chains other than DAP-12. Thus, why might human KIR fail
to couple to the TCR apparatus? One possibility would be an inability
to physically interact with TCR. The interaction between activating
KIR and DAP-12 is appreciated only when using the low stringency
detergent digitonin, indicating that these KIR may not form high
affinity interactions. Perhaps the biochemical characteristics
responsible for the relatively weak interactions between DAP-12 and
activating KIR precludes any interaction between TCR and these
KIR.
Having determined that Ly-49D transmits positive signals in Jurkat even in the absence of DAP-12, we used this system to analyze the potential interaction between inhibitory and activating Ly-49 proteins. Jurkat cells easily coexpressed various forms of an inhibitory receptor (Ly-49G2) and an activating receptor (Ly-49D). Our analysis demonstrated that the negative Ly-49 receptor is dominant. In addition to an ITIM, Ly-49G2 has two other cytoplasmic tyrosine residues, Tyr36 and Tyr39. One of these, Tyr36, fits with the consensus for tyrosine phosphorylation. Our data show that, although the ITIM is required, mutation of Tyr36 and Tyr39 has no effect on Ly-49G2-mediated inhibition. To confirm these findings in NK cells, we cocross-linked Ly-49D and Ly-49G2. In each of several experiments cocross-linking of the inhibitory receptor blocked Ly-49D-mediated activation. Inhibition of a positive signaling Ly-49 by an inhibitory one is intriguing. Ly-49D and Ly-49G2 both have the same apparent ligand, H-2Dd, implying that Ly-49D would activate only NK cells that fail to express any other H2-Dd-binding receptors (26, 30, 31). The biological role of such tight regulation for Ly-49D activation remains unknown.
The ability of Ly-49G2 to inhibit Ly-49D, but not the Jurkat TCR, is in
contrast to the reported ability of human KIR to inhibit T cell
activation (24, 25). There are several possible
explanations for the lack of Ly-49G2-mediated inhibition of TCR. The
most obvious would be a general inability of Ly-49G2 to couple to the
inhibitory mechanism of human T cells. The ability of Ly-49G2 to block
Ly-49D-mediated activation, however, rules this out. So why does the
TCR fail to lead to phosphorylation of Ly-49G2 when these receptors are
cocross-linked? One possibility is that the kinase(s) activated by the
TCR is not efficient at inducing the phosphorylation of an Ly-49 ITIM.
Although the data to date suggest that KIR ITIMs are phosphorylated by
src family proteins, the kinases responsible for phosphorylation of
Ly-49s are unknown (3, 32). In fact, data presented here,
together with our previous studies, demonstrate that Syk is
preferentially activated by Ly-49D even when Zap-70 is present
(22). However, our experiments reconstituting
Jurkatp116 (Fig. 7) suggest that both CD3 signals and
GL183 inhibition can utilize either Syk or Zap-70. Yet another
possibility for the lack of Ly-49G2 inhibition of TCR is based on the
structural composition of the complex. Ly-49 proteins are type II
lectin-like proteins whereas the TCR chains are type I receptor
proteins. Do the type II inhibitory Ly-49s more efficiently block
signaling by type II-activating receptors? There are at least two
examples of type II ITIM-containing receptors blocking type I
receptor-mediated events. CD94/NKG2A complexes inhibit FcR signals in
human NK cells, and there is a report of Ly-49s inhibiting FcR-mediated
Ab-dependent cellular cytotoxicity (33). These
possibilities are currently under study. Finally, a possible
explanation could be signal strength. We (12) and others
(5) have shown that targets can induce inhibitory signals.
In the present study, we have used strong receptor cross-linking Abs
that engage the entire cell surface. This difference could reveal
inhibitory capacity present in CD3+ cells when
TCR signals are weak or suboptimal. This is consistent with our
previous data that, in primary T cells, target-induced cytokine
production could be blocked by Ly-49G2, whereas our anti-CD3
cross-linking was not (12).
In summary, we have demonstrated the utility of Jurkat T cells for the
study of Ly-49 proteins and have shown that inhibitory signals are
dominant. Ly-49D couples to TCR in these cells and leads to
phosphorylation of both Syk and Zap-70. In addition, we have
demonstrated an apparent discrepancy in the ability of Ly-49s to
inhibit activation signals. Signals derived from activating Ly-49s are
efficiently blocked whereas those delivered by the TCR are not.
Therefore, one must wonder what the targets are for Ly-49 inhibiting NK
receptors on murine T cells, since the TCR receptor appears not to be
easily regulated by these type II receptors. Further studies using this
model should provide further insight into the biochemical and molecular
interactions between receptors of the Ly-49 and/or KIR family and those
receptors involved in the activation of NK cells.
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Acknowledgments |
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RI and Dr. Allan
Weissman for rabbit antisera to the -chain of the TCR. |
Footnotes |
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2 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
3 Address correspondence and reprint requests to Dr. John R. Ortaldo, National Cancer Institute-Frederick Cancer Research and Development Center, Building 560, Room 31-93, Frederick, MD 21702-1201. E-mail address:
4 Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibitory motif; KIR, killer cell inhibitory receptor.
Received for publication May 5, 1999. Accepted for publication September 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells, and antigen-specific CTL. J. Immunol. 160:5239.This article has been cited by other articles:
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 J. R. Ortaldo, R. Winkler-Pickett, J. Wigginton, M. Horner, E. W. Bere, A. T. Mason, N. Bhat, J. Cherry, M. Sanford, D. L. Hodge, et al. Regulation of ITAM-positive receptors: role of IL-12 and IL-18 Blood, February 15, 2006; 107(4): 1468 - 1475. [Abstract] [Full Text] [PDF]
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S. E. Kirwan and D. N. Burshtyn Killer Cell Ig-Like Receptor-Dependent Signaling by Ig-Like Transcript 2 (ILT2/CD85j/LILRB1/LIR-1) J. Immunol., October 15, 2005; 175(8): 5006 - 5015. [Abstract] [Full Text] [PDF]
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G. Hart, L. Flaishon, S. Becker-Herman, and I. Shachar Tight Regulation of IFN-{gamma} Transcription and Secretion in Immature and Mature B cells by the Inhibitory MHC Class I Receptor, Ly49G2 J. Immunol., October 15, 2005; 175(8): 5034 - 5042. [Abstract] [Full Text] [PDF]
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 X. Xie, H. He, M. Colonna, T. Seya, T. Takai, and B. A. Croy Pathways Participating in Activation of Mouse Uterine Natural Killer Cells During Pregnancy Biol Reprod, September 1, 2005; 73(3): 510 - 518. [Abstract] [Full Text] [PDF]
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M. C. Rodriguez-Galan, J. H. Bream, A. Farr, and H. A. Young Synergistic Effect of IL-2, IL-12, and IL-18 on Thymocyte Apoptosis and Th1/Th2 Cytokine Expression J. Immunol., March 1, 2005; 174(5): 2796 - 2804. [Abstract] [Full Text] [PDF]
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H. I. McFarland, S. A. Hansal, D. I. Morris, D. W. McVicar, P. E. Love, and A. S. Rosenberg Signaling through MHC in transgenic mice generates a population of memory phenotype cytolytic cells that lack TCR Blood, June 1, 2003; 101(11): 4520 - 4528. [Abstract] [Full Text] [PDF]
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 R. B. Voyle, F. Beermann, R. K. Lees, J. Schumann, J. Zimmer, W. Held, and H. R. MacDonald Ligand-dependent Inhibition of CD1d-restricted NKT Cell Development in Mice Transgenic for the Activating Receptor Ly49D J. Exp. Med., April 7, 2003; 197(7): 919 - 925. [Abstract] [Full Text] [PDF]
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J. R. Ortaldo and H. A. Young Expression of IFN-{gamma} Upon Triggering of Activating Ly49D NK Receptors In Vitro and In Vivo: Costimulation with IL-12 or IL-18 Overrides Inhibitory Receptors J. Immunol., February 15, 2003; 170(4): 1763 - 1769. [Abstract] [Full Text] [PDF]
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J. Roger, A. Chalifour, S. Lemieux, and P. Duplay Cutting Edge: Ly49A Inhibits TCR/CD3-Induced Apoptosis and IL-2 Secretion J. Immunol., July 1, 2001; 167(1): 6 - 10. [Abstract] [Full Text] [PDF]
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J. R. Ortaldo, E. W. Bere, D. Hodge, and H. A. Young Activating Ly-49 NK Receptors: Central Role in Cytokine and Chemokine Production J. Immunol., April 15, 2001; 166(8): 4994 - 4999. [Abstract] [Full Text] [PDF]
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J. R. Ortaldo, R. Winkler-Pickett, and G. Wiegand Activating Ly-49D NK receptors: expression and function in relation to ontogeny and Ly-49 inhibitor receptors J. Leukoc. Biol., November 1, 2000; 68(5): 748 - 756. [Abstract] [Full Text]
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) and rat
IgG2a (
) were added at 30 s (event arrow 1o), and
rabbit anti-rat IgG cross-linked the Abs at 60 s (event arrow
2o). All Abs except 4E5 alone (x) were cross-linked. The
percentage of responding cells is shown as determined in the FACSort.
B, Mutation of the arginine residue of the Ly-49D
transmembrane domain ablates calcium mobilization in Jurkat. Jurkat
transfected and expressing the wild-type Ly-49D (
Ab. Lysates were made in Brij buffer and run on an
8% gel under reducing conditions. Proteins were transferred and
immunoblotted with TCR
)
is compared. Similar results were obtained with either parental Jurkat
triggered with Ly-49D Ab or with Ly-49D-expressing Jurkat treated with
rat IgG2a. Primary Abs were simultaneously added at 30 s (event
arrow 1o), and rabbit anti-rat IgG cross-linked the Abs
at 60 s (event arrow 2o). The percentage of responding
cells is shown as determined in the FACSort. B,
CD3-mediated calcium mobilization in Jurkat T cells is not blocked by
Ly-49G2. Similar to Fig. 3
