Ly-49 receptor expression and functional analysis
in multiple mouse strains
John R. Ortaldo,* Anna T. Mason, Robin Winkler-Pickett,* Arati Raziuddin,†
William J. Murphy,† and Llewellyn H. Mason*
*Laboratory of Experimental Immunology, Division of Basic Sciences, NCI-FCRDC,
and †Intramural Research Support Program, SAIC-Frederick, Maryland
Abstract: We present data on the strain distribution and functional characteristics of the Ly-49
receptors A, C/I, D, and G2 on DX51 natural killer
(NK) cells. We have examined tyrosine phosphorylation of the Ly-49 molecules, regulation of NK
cytotoxic functions, and in vivo marrow rejection
capability. The flow cytometry results demonstrate
a diverse and complex pattern of expression of the
Ly-49 receptors in the 11 strains examined. The
vast majority of NK cells express Ly-49s, although
some NK1.11 CD31 cells also express these receptors. The results of our functional analysis indicate
that H-2Dd was able to inhibit the function of
Ly-49G21 NK cells, not only in B6 mice, but also
by NK cells derived from several haplotypes. The
examination of Ly-49 receptor tyrosine phosphorylation, which is a biochemical measure of inhibitory
function, was consistently observed in the 11 mouse
strains examined. In contrast, analysis of Ly-49D
function suggests its expression appears to be more
restricted and that H-2Dd is an activating ligand for
this receptor. In addition, the in vivo examination
of both inhibitory (Ly-49G2) and activating (Ly49D) receptors demonstrated regulatory roles of
these class I binding receptors in marrow transplantation. J. Leukoc. Biol. 66: 512–520; 1999.
H-2Dk [4, 5], and has been demonstrated to transmit a negative
signal to the NK cell [5], resulting in the inhibition of lysis.
The Ly-49G2 subset of NK cells also is inhibited by target
cells expressing H-2Dd and/or H-2Ld [6]. Studies with Ly49G21 NK cells have relied primarily on the reversal of target
cell inhibition by mAb 4D11 and mAb specific for H-2Dd and
H-2Ld. More recently this class I specificity has been demonstrated in vivo where Ly-49G21 NK cells reject H-2Db but do
not reject H-2Dd bone marrow grafts in lethally irradiated mice
[14]. The Ly-49C1 subset (defined by reactivity with SW5E6
antibodies) of NK cells has been shown to bind the class I
molecules H-2b, H-2d, H-2k, and H-2s [7, 8]. However, recent
data [15] indicating that SW5E6 reacts with both Ly-49C and
Ly-49I raises concerns regarding which Ly-49 family member
is responsible for these reported class I effects.
Recently, we have demonstrated that a functional subset of
murine NK cells expresses Ly-49D, an activating NK receptor
using rat monoclonal antibody 12A8 [3]. This rat monoclonal
antibody, although showing strong reactivity with Ly-49D, also
cross-reacted with Ly-49A. In this study we will describe two
new rat monoclonal antibodies, 12A1 (rat IgM) and 4E5 (rat
IgG2a), that react with Ly-49D and not with other reported
Ly-49 genes. We have utilized the pan-NK cell marker, DX-5
[16], to determine the percentage of NK cells expressing
Ly-49A, C/I, D, and G2 from multiple strains of mice. Cytotoxic
function as well as biochemical data on the phosphorylation of
these receptors from various mouse strains are also presented.
Key Words: flow cytometry analysis · natural killer · Ly-49 ·
tyrosine phosphorylation
MATERIALS AND METHODS
INTRODUCTION
Mice
Members of the Ly-49 gene family encode type II integral
transmembrane proteins and are primarily expressed on the
surface of murine natural killer (NK) cells. The Ly-49 gene
family encodes receptors that recognize class I MHC as self,
resulting in the inhibition of the lytic pathway by NK cells
[1, 2]. Recent studies also demonstrate that some Ly-49
receptors may activate [3], rather than inhibit [4–11], NK cells.
Recognition of class I molecules by Ly-491 NK cells has been
proposed as a regulatory mechanism to prevent lysis of host
cells [12, 13]. Characterization of Ly-49 expression and
function has most often been performed in C57BL/6 or BALB/c
mice with use of the prototype Ly-49A receptor. Ly-49A has
been shown to recognize the class I molecules H-2Dd and
512
Journal of Leukocyte Biology
Volume 66, September 1999
BALB/c (H-2d), C57BL/6 (H-2b), C3H/HeJ (H-2k), BALB/c 3 C57BL/6)
(CB6F1), DBA/2 1 AKR (AKH2), AKR, CBA/J, athymics, and SJL/J were
either obtained from the Animal Production Area, NCI-FCRDC, Frederick,
MD, or purchased from Jackson Laboratories, Bar Harbor, ME. All mice were
kept under specific-pathogen-free conditions until use at 8–16 weeks of age.
Animal care was provided in accordance with the procedures outlined in the
Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23,
1985).
Correspondence: Dr. John R. Ortaldo, NCI-FCRDC, Bldg. 560, Rm. 31-93,
Frederick, MD 21702-1201.
Received January 22, 1999; revised April 5, 1999; accepted April 5, 1999.
http://www.jleukbio.org
Flow cytometry analysis (FCA)
NK cells were stained as previously described and analyzed on a FACSort
(Becton-Dickinson, Mountain View, CA). Cell sorting was performed on either
an Epics 750 (Coulter Electronics, Hialeah, FL) or a FACStar (BectonDickinson). NK cells were stained with various directly fluorescein isothiocyanate (FITC)-labeled primary antibodies. Cells were sorted with CD3-biotin
followed by streptavidin-phycoerythrin (PE; Becton-Dickinson), NK1.1-PE,
and with FITC-labeled antibodies YE148 (Ly-49A), 5E6 (Ly-49C/I), 12A8
(Ly-49A/D), 4E5 (Ly-49D), and 4D11 (Ly-49G2).
NK and T cell isolation
Murine splenic or liver NK cells were isolated from nylon wool nonadherent
lymphocytes. The cells were stained with combinations of CD3, DX5, and
Ly-49s and were sorted for subpopulations expressing the desired phenotype.
Cells then were expanded for 7–10 days in RPMI-1640 medium supplemented
with 1,000 C.U. (Cetus units)/mL interleukin-2 (IL-2; Hoffman LaRoche,
Nutley, NJ), 10% fetal calf serum (FCS), nonessential amino acids, sodium
pyruvate, HEPES, b-mercaptoethanol, L-glutamine, and penicillin plus streptomycin.
Targets
Tumor targets were maintained in culture as previously described [3]. P815 is a
DBA/2 mouse mastocytoma. L5MF22 is a B cell line that naturally expresses
the H-2Db class I molecule. This line has been transfected with a neomycin
resistance gene alone (L5neo) or with the class I (H-2Dd) (L5cDd104)
expressing gene (kindly provided by Dr. I. Nakamura, Buffalo, NY).
Isolation of liver lymphocytes
Liver cells were isolated after mice were injected bidiurnally for 3 days with 33
µg recombinant IL-2 (generously provided by either Chiron, Emeryville, CA or
Hoffmann-LaRoche, Inc., Nutley, NJ). Liver-derived leukocytes then were used
immediately for functional studies as described previously [17].
Cytotoxicity assays
Tumor targets and concanavalin A (ConA) blasts were labeled with 51Cr or 111In
and used in 4-h cytotoxicity assays as previously described [6]. Assays
involving mAb included the specific antibodies at a concentration of ,2
µg/well for the duration of the cytotoxicity assay. Data are either presented as
lytic units per 107 cells or percent specific lysis.
Assays for BMC engraftment
Evaluation of BMC engraftment was performed as previously described in
detail [18]. Briefly, recipient mice were injected with NK-depleting Ly-49
antibody (80–200 µg) 2 days before lethal irradiation. BMC were injected
intravenously after irradiation of the host and evaluated for hemopoietic
engraftment by assessing hematopoietic progenitor content 7 days after transfer.
A soft agar colony assay was performed using predetermined amounts of
hematopoietic growth factors, IL-3, and granulocyte-macrophage colonystimulating factor (obtained from the repository, NCI-FCRDC, Frederick, MD)
[12]. All experiments had four mice per group and were performed at least two
times. A Wilcoxon rank sum test was performed to determine whether the
values were significantly (P # 0.05) different.
Anti-phosphotyrosine detection
Cells were stimulated with 0.1 mM pervanadate as described [19] for 15 min at
37°C, disrupted in lysis buffer (1.0% TX-100, 300 mM NaCl, 50 mM Tris, 2
mM EDTA, 0.4 mM sodium orthovanadate, plus protease inhibitors), then
centrifuged at 15,000 rpm for 30 min at 4°C. Lysates were immunoprecipated
for 3–4 h with specific mAb cross-linked to Protein G-Sepharose. Beads were
washed in wash buffer containing 0.2% TX-100 and proteins eluted in
non-reducing Laemmli buffer and separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels. Proteins were transferred to Immobilon-P (Millipore, Bedford, MA) and the blots were blocked with 5% bovine
serum albumin (BSA). Biotinylated 4G10 (Upstate Biotechnologies) was used to
detect phosphoproteins, followed by streptavidin-horseradish peroxidase [19].
Blots were exposed to enhanced chemiluminescence (Amersham, Arlington
Heights, IL).
RESULTS
Strain distribution of DX-51/Ly-491 NK cells
Ly-49s are class I receptors that are primarily restricted to NK
cells. The examination and study of these receptors has been
hampered by the inability to examine NK cells from most
strains of mice, because they do not express the pan NK marker,
NK1.1. Recently, a new monoclonal antibody, DX-5, has been
developed that reacts with murine NK cells from all strains of
mice. mAb DX-5 also appears to react with a subset of cells
similar to the one identified as CD31/NK1.11. In Figure 1, we
first examined the expression of DX-5 versus CD3 on six strains
of mice in order to determine the percentage of cells expressing
DX-5 alone compared with those co-expressing CD3. Figure 1A
demonstrates this reactivity on freshly isolated spleen cells,
whereas Figure 1B depicts 7- to 9-day ALAK cells. As can be
seen in all strains of mice, distinct populations of T, NK, and
DX-51 T cells can be observed, with the exception of athymic
mice. Our data clearly demonstrate that DX-5 primarily enumerates
CD32 NK cells, but also is found on a minor population of CD31
cells. In the C57BL/6 strain, DX-5 delineates a subset of cells highly
overlapping (.90%) with the NK1.11 CD31 subset that has been
previously identified [18, 20].
Using mAb DX-5 to enumerate NK cells, we next examined
the expression of Ly-49 molecules on DX-51, CD32 cells. The
Ly-49 histograms of these gated, DX-51 CD32 NK cells are
shown in Figure 2, A and B. Ly-49A is expressed at a high
mean channel fluorescence but on a low percentage of NK cells
on all strains except 129/J and SJL/J. In 129/J and SJL/J, we
observed a very weak shift over background upon staining for
Ly-49A. Ly-49C/I, detected with SW5E6, is expressed on all
strains except 129/J, AKR, and SJL/J. Low levels of Ly-49C/I
expression are observed on AKH2, CBA/J, and C3H/HeJ. Ly-49D is
only expressed on C57Bl/6, CB6F1, 129/J, and SJL/J. Ly-49D1
cells were observed in some, but not all, preparations of IL-2cultured NK cells from athymic/nude mice. Ly-49G2 is expressed
on all strains tested, but the percentage of positive cells varies
considerably between the strains, with CBA/J being very low and
C57BL/6 being the highest. The percentage of Ly-491 NK cells
expressed on CD32, DX51 fresh splenic lymphocytes is summarized in Table 1 (representative of two or more experiments).
C57BL/6 (H-2b) express high levels of Ly-49C/I, D, and G2, whereas
other strains (AKR and DBA/2) express low levels of Ly-49G2 and
virtually no Ly-49D, while retaining modest to high levels of
Ly-49C/I and Ly-49A. The lack of expression of some Ly-49s also
could be due to: (1) very low cell surface expression (like Ly-49A in
129/J mice or Ly49C/I in CBA/J); or (2) allelic differences that are
not recognized by the monoclonal antibody.
Ly-49 expression intensity in mouse strains
and C57Bl/10 recombinant haplotypes
Direct examination of expression of Ly-49s as shown in Figure
2 and Table 2 revealed some interesting differences. Previous
Ortaldo et al.
Ly-49 regulates murine NK cell functions
513
Fig. 1. Expression of DX-5 vs. CD3
receptors on T cells. Freshly isolated (A,
day 0) or IL-2-expanded (B; day 8) spleens
were examined for expression of CD3 and
DX-5 on selected strains. Values shown
are percent of lymphocytes (gated by forward and side scatter) that expressed the
phenotype in the various quadrants. The
percentage positive in the quadrants of
gated lymphocytes are shown. Dot plots
are used for fresh cells to better visualize
the minor populations of #2%. Experiment shown is representative of three or
more experiments.
studies with Ly-49A and Ly-49C/I have indicated that the
intensity of receptor expression is influenced by MHC expression. This receptor calibration has been previously demonstrated [21, 22] and is reiterated in Figure 3, where Ly-49A
expression intensity is high in C57Bl/6 (H-2b) mice and low in
BALB/c (H-2d) mice, consistent with the H-2Dd recognition by
Ly-49A. To the contrary, Ly-49C has been shown to demonstrate the converse. Examination of Ly-49D and Ly-49G2 in the
11 mouse strains indicated some noteworthy differences.
Similar to Ly-49A, C57Bl/6, CB6F1, and BALB/c mice exhibited an Ly-49G2 intensity consistent with H-2Dd recognition.
However, the examination of other H-2b strains, e.g., 129/J and
most H-2k or H-2d strains or their F1 hybrids indicate a low
intensity of Ly-49G2. Examination of Ly-49D in the four
expressing strains did not demonstrate a strong modification of
expression, although Ly-49D, based on 4E5 or 12A8, is not
expressed in the H-2d or H-2k strains examined. Ly-49D was
514
Journal of Leukocyte Biology
Volume 66, September 1999
modestly diminished in CB6F1s. To more closely examine the
possible role of MHC complex on the expression of Ly-49G2
and Ly-49D, we utilized the C57Bl/10 recombinant haplotypes
that express H-2b,H-2d, or H-2k within their K-D loci. The
results of these analyses are shown in Figure 4. The expression
of Ly-49G2 and Ly-49D is only modestly changed in mice
expressing KkDk (B10.BR), Kd (B10.D2), or Dd (B10.5R). None
of the C57Bl/10 recombinant haplotypes demonstrated the
strong Ly-49G2 expression changes seen in H-2d or H-2k mice
in Figure 3. Collectively these data would suggest that the
regulation that is the result of MHC expression may reside
outside the K-D boundary.
Functional inhibition of Ly-49G1 NK cells from
different strains of mice
The Ly-49G2 receptor has been shown to inhibit NK cell
functions through its interaction with H2-Dd and Ld using
http://www.jleukbio.org
Fig. 2. Distribution of Ly-49 receptors on DX-51 CD32 NK cells in spleen. Freshly isolated spleen lymphocytes were evaluated by three-color analysis using cells
stained with appropriate Ly-49 (FITC) and CD3 (anti-CD3-biotin-avidinPerCP) and DX-5 (with anti-rat IgM-PE). Histograms of the DX-51 CD32 NK cells are
shown. Ly-49 receptors were enumerated with the following antibodies: Ly-49A (YE1/48-F); Ly-49C/I (5E6-F); Ly-49A/D (12A8-F); Ly-49D (4E5-F); Ly-49G2
(4D11-F); control (rat IgG2a-F). Experiment shown is representative of three or more experiments.
antibody-blocking studies [6]. A typical experiment using
C57Bl/6 mouse NK cells sorted for Ly-49G21 and Ly-49G22
cells is shown in Figure 5A. Ly-49G22 NK cells demonstrate
lysis of P815, an H2-Dd tumor line. Contrary to this lysis,
Ly-49G21 NK cells failed to lyse these tumor cells. When
either intact or F(ab’)2 antibodies to Ly-49G2 (4D11) were
included, the Ly-49G2/H-2Dd interaction is blocked and lysis
of P815 was observed. Inclusion of these same antibodies with
the Ly-49G22 NK cells had little or no effect. To confirm that
the class I molecule H-2Dd is the ligand for Ly-49G2, we
obtained a B cell line, L5MF22 (H-2Db), and stable H-2Dd
transfectants of this parental line in clones D104 and D4 or
H2-Ld (Fig. 5B). As is shown in Figure 5B, Ly-49G21 cells from
B6 mice failed to demonstrate significant lysis against both
H2-Dd transfected lines. Lysis of the H-2Ld targets also was
significantly reduced compared to the parental target cell line.
Ortaldo et al.
Ly-49 regulates murine NK cell functions
515
Ly-49 Expression on DX51 Cells from Different
Mouse Strains
TABLE 1.
DX51 % Ly49
Strain
Haplotype
C57B1/6
DBA/2
AKH2
AKR
CBA/J
129
BALB/c
CB6F1
C3H/HeJ
Athymics
SJL/J
H-2b
H-2d
H-2d/k
H-2k
H-2k
H-2b
H-2d
H-2b/d
H-2k
H-2s
CD32
DX51
CD32
Ly49A1
CD32
Ly49C/I1
CD32
Ly49D1
CD32
Ly49G1
7.6
3.5
6.1
7.2
6.9
4.6
8.0
10.3
7.4
16.9
1.5
16
12
12
16
3
19w
4
9
5
5
0
47
47
46
0
13w
0
55
53
10
28
19w
50
0
0
0
0
30
0
28
0
13
27
54
26
28
31
10
50
21
39
22
23
30
Ly-49 expression on CD32, DX51 subsets from normal splenic lymphocytes.
Freshly isolated spleen cells were analyzed with the indicated antibodies based
on a lymphocyte gate (forward and side scatter). w indicates that some
antibodies/strain combinations gave weak shifts in expression that were not
easily evaluated (see Figure 2).
Next we evaluated the lytic potential of Ly-49G21 NK cells
from three different strains of mice (Fig. 5C) against P815. The
Ly-49G21 cells from BALB/c, C3H/HeJ, and DBA/2 mice do
not lyse P815 targets, whereas lysis was observed by the
unsorted and Ly-49G2-negative populations. Figure 5D demonstrates that antibodies to Ly-49G2 are capable of restoring the
lytic potential of the Ly-49G21 cells against P815 target cells.
To confirm the specificity of the Ly-49G2/H-2Dd interaction
against the P815 cell line, we examined the ability of Ly-49G21
NK cells from four strains of mice (C57BL/6, BALB/c, DBA/2,
and C3H/HeJ) to mediate lysis of the parental L5MF22 targets
and their transfected L5M-D104 counterparts after pretreatment with anti-Ly-49G2 antibodies (Fig. 5, E and F). The
addition of anti-Ly-49G2 antibodies to all four strains resulted
in reversal of inhibition when H2-Dd-expressing targets were
employed, but not with the H2-Db parental B cell line.
After demonstrating that Ly-49G21 NK cells from multiple
strains of mice subserved an inhibitory function on engaging
the class I ligand, H-2Dd, we examined the known Ly-49
TABLE 2.
Strain
Tyrosine Phosphorylation of Ly-49 Receptors
from Different Mouse Strains
Haplotype
C57B1/6
DBA/2
AKH2
AKR
CBA/J
129/J
BALB/c
CB6F1
C3H/HeJ
Athymics
SJL/J
H-2b
H-2d
H-2d/k
H-2k
H-2k
H-2b
H-2d
H-2b/d
H-2k
H-2s
Ly-49A
Ly-49C/I
Ly-49D
Ly-49G
1
1
1
1
1
1
1
1
1
1
1?
1
1
1
N.E.
1
N.E.
1
1
1
1
N.E.
2
N.E.
N.E.
N.E.
N.E.
2 or w1
N.E.
2
N.E.
1/2
2 or w1
1
1
1
1
1
1
1
1
1
1
1
Antibodies employed: Ly-49A (YE148); Ly-49C/I (5E6); Ly-49D (4E5);
Ly-49G2 (4D11). N.E., Not expressed, denotes marker is not expressed on
indicated strains based on flow cytometric analysis; 1/2, expression was
variable in different strains.
516
Journal of Leukocyte Biology
Volume 66, September 1999
receptors biochemically for their ability to be phosphorylated
by kinases. Figure 6 demonstrates a representative experiment
with selected strains. Figure 6A examines C57BL/6 (H-2Db)
and BALB/c (H-2Dd) as well as CB6F1 effectors (H-2dxb]. NK
cells were stimulated with pervanadate, immunoprecipitated
with anti-Ly-49s, and examined by Western blotting with
anti-phosphotyrosine. Immunoprecipitation with mAb YE1/48
and 4D11 demonstrated that Ly-49A and G2 are phosphorylated in all strains. However, mAb SW5E6 demonstrated weak
phosphorylation of Ly-49C/I in six mouse strains tested and in
CB6F1 mice. Antibodies specific to Ly-49D (4E5) did not
detect receptor phosphorylation, although mAb 12A8 (anti-Ly49A/D reactive) did react with Ly-49A1 NK cells in C57BL/6
mice. When Ly-49A2 NK cells were used (not shown), no
receptor phosphorylation was seen after immunoprecipitation
with mAb 12A8. In Figure 6B, C3H/HeJ (H-2k), DBA/2 (H-2d),
and AKR (H-2k) NK cells were analyzed. The phosphorylation
of the inhibitory Ly-49s (Ly-49A, C/I, and G2) was seen in all
relevant strains. Small differences in the size of the inhibitory
Ly-49s were observed and were probably due to glycosylation
differences in NK receptors from the different mouse strains. In
all experiments, B6 mice were evaluated as a positive control
for receptor phosphorylation of Ly-49A, C/I, and G2.
The Ly-49 receptor phosphorylation data from NK cells of
different mouse strains are summarized in Table 2. As can be
seen, Ly-49 phosphorylation is consistent within strains expressing the individual Ly-49 molecules. These results indicate that
Ly-49G2 appears to be inhibitory, based on its phosphorylation,
in all 11 strains examined; however, our functional studies have
not been extended to all strains (see Fig. 5). Therefore, Ly-49
inhibitory and activating receptors demonstrate consistent
biochemical signaling patterns based on the fidelity of the
phosphorylation observed among the various strains. In contrast
to Ly-49A and G2, Ly-49D is not strongly phosphorylated on
any expressing strains, but weakly phosphorylated proteins
were detected in SJL/J and 129/J mice. Examination of SJL/J
and 129/J mice has demonstrated that 4E5 reacts primarily
with the activating Ly-49D (based on its precipitation of the
phosphorylated DAP12 signaling molecule), but does show a
weak 97-kDa phosphorylated protein in these two strains only.
Studies are currently examining the nature of this reactivity.
Overall, the Ly-49D receptor phosphorylation is consistent with
its activating nature in all strains examined.
DISCUSSION
Previous studies have usually examined Ly-49s on selected
strains of mice, e.g., BALB/c or C57BL/6. In this study, the
expression and functional comparison of Ly-49G2 and Ly-49D
were performed on NK cells from a variety of mouse strains. The
flow cytometric analysis of Ly-49s in 11 strains of mice yielded
a complex and interesting pattern of expression. Ly-49G2 was
expressed at various levels on NK cells from all strains of mice
examined. This report demonstrates that Ly-49G2 functions as
an inhibitory receptor on NK cells from all the mouse strains
examined, even in strains that normally express the proposed
ligand (H-2Dd) for Ly-49G2, such as BALB/c mice. Studies
http://www.jleukbio.org
TABLE 3.
Treatment
Reactivity
Bone Marrow Engraftment After In Vivo Depletion of Ly-49D1 NK Cells
Recipient
Donor
Isotype
of mAb
Total CFU-c/
spleen (106)
Experiment 1
NRtS
12A1
SW5E6
PK136
None
Ly-49D
Ly-49C
NK1.1
C57BL/6
C57BL/6
C57BL/6
C57BL/6
BALB/c
BALB/c
BALB/c
BALB/c
rIgG
rIgM
rIgG2a
mIgG
0.0 6 0.0
333.3 6 91.6a
19.5 6 5.6
312.6 6 157.8
Experiment 2
NRtS
12A8
12A1
4E5
PK136
None
Ly-49A/D
Ly-49D
Ly-49D
NK1.1
C57BL/6
C57BL/6
C57BL/6
C57BL/6
C56BL/6
BALB/c
BALB/c
BALB/c
BALB/c
BALB/c
rIgG
rIgG2a
rIgM
rIgG2a
mIgG
0.0 6 0.0
292.8 6 89.2a
209.3 6 50.2
292.7 6 77.3a
273.3 6 93.1a
Experiment 3
NRtS
12A1
4D11
NRbS
asGM1
None
Ly-49D
Ly-49G2
None
GM1
BALB/c
BALB/c
BALB/c
BALB/c
BALB/c
C57BL/6
C57BL/6
C57BL/6
C57BL/6
C57BL/6
rIgG
rIgM
rIgG2a
rbIgG
rbIgG
56.2 6 34.1
33.3 6 18.6
351.6 6 107.8a
8.2 6 2.0
209.0 6 62.5a
Mice received a single injection of 0.5 mg of SW5E6 i.p., 0.2 mg of 4D11 i.p., 0.2 mg of 12A8 or 12A1 or 4E5 i.p.; 0.5 mg of PK136 i.p.; 20 µL of ASGM1 i.v.; 0.2
mL of NRts i.p. or 0.2 mL of Nrbs i.v., 2 days before lethal irradiation. Recipient mice C56BL/6 (experiments 1 and 2) and BALB/c (experiment 3) were irradiated at
1,000 and 850 cGy, respectively, followed by inoculation of 5 3 105 BMC, i.v. The proliferation of donor cells in the spleens of recipient mice was assessed 8 days
later by colony assay in soft agar.
a Values significantly (P , 0.3) greater than control mice receiving normal rat or rabbit serum in each experiment.
Fig. 3. Intensity of Ly-49 class I binding receptors on DX-51 CD32 NK cells in spleen. Freshly isolated spleen lymphocytes were evaluated by three-color analysis
using cells stained with appropriate Ly-49 (FITC) and CD3 (anti-CD3-biotin-avidinPerCP) and DX-5 (with anti-rat IgM-PE). Ly-49 receptors were enumerated with
the following antibodies: Ly-49A (YE1/48-F); Ly-49C/I (5E6-F); Ly-49D (4E5-F); Ly-49G2 (4D11-F); control (rat IgG2a-F). The intensity of B6 NK cells was
normalized in all experiments. The mean channel flourescence is of CD32, DX51 gated lymphocytes. Experiment shown is representative of three or more
experiments. Control IgG mean channel flourescence was 175–200 in all experiments.
Ortaldo et al.
Ly-49 regulates murine NK cell functions
517
Fig. 4. Intensity of Ly-49 class I binding receptors on DX-51 CD32 NK cells in C57BL/10 spleen. See Figure 3 legend for details.
examining receptor intensity in 11 strains and in C57Bl/10
recombinant haplotypes resulted in a complex pattern of
expression levels. Overall, the level of Ly-49G2 was diminished
in H-2d and H-2k strains of mice. However, the examination of
B10 recombinants indicated that H-2K/D changes were not
sufficient to explain these variances, thus supporting influences
distal to this MHC region. Examination of Ly-49D did not yield
any distinguishable pattern of H-2 influence on receptor
expression, however, only a few strains express this receptor.
Our findings that no H-2k or H-2d strains express Ly-49D
support this hypothesis. Ly-49D is an activating receptor and
its expression may be regulated differently from the inhibitory
Ly-49 molecules. Our findings with C57Bl/10 recombinant
haplotypes and with Ly-49D suggest and support a complex
model for NK repertoire selection [19, 21–23]. It should be
noted that some of the reactivities of these anti-Ly-49s could be
cross-reactivities with yet to be discovered Ly-49s. Without
detailed functional analysis, this possibility could not be ruled
out.
We also performed a more extensive evaluation of Ly-49
inhibitory receptor phosphorylation patterns in NK cells from a
number of different mouse strains. Recent studies have demonstrated that rapid receptor phosphorylation of Ly-49A and G2
occur [19, 24] after cells are stimulated by receptor interaction
or pharmacological means. Phosphorylation of the immune
receptor tyrosine-based inhibitory motif (ITIM) is a hallmark of
numerous inhibitory NK receptors in both the human and the
518
Journal of Leukocyte Biology
Volume 66, September 1999
mouse. Examination of the Ly-49 inhibitory receptors A, C, and
G2 on NK cells from all strains tested revealed that these
inhibitory Ly-49 receptors are capable of being phosphorylated,
consistent with their proposed biological functions.
In addition to the inhibitory Ly-49 receptors that have been
characterized and studied, recent reports have elucidated some
unique activating NK receptors both in the human KIR [25–27]
and mouse Ly-49 gene families (Ly-49D and H). Our recent
studies have demonstrated that Ly-49D is an activating receptor on mouse NK cells that can mediate reverse ADCC [3].
Ly-49D lacks an ITIM motif and is not phosphorylated, but it
does associate with a signaling moiety that is phosphorylated on
activation of Ly-49D [28, 29] and has recently been cloned and
named DAP12 [26]. Our present studies examining NK cells
from numerous mouse strains have revealed that only a few
strains express Ly-49D based on two mAbs, 4E5 and 12A8.
Previous studies have indicated that 12A8 cross-reacts with
Ly-49A [6], whereas 4E5 appears to be specific for Ly-49D.
Recent studies with Ly-49D have elucidated a ligand for this
activating Ly-49 receptor. These studies in both RNK cells and
primary NK cells [30, 31] present several lines of evidence that
Ly-49D recognizes H-2Dd as a positive ligand. Antibody
blocking studies have shown that blocking Ly-49D results in
inhibition of killing, rather than reversal of inhibition commonly seen with the inhibitory Ly-49s. Likewise, these studies
demonstrated that blocking of Dd, but not Db or Dk also inhibits
killing by Ly-49D1 but not Ly-49D2 NK subsets. Finally, in
http://www.jleukbio.org
Fig. 5. In vitro inhibitory function of Ly-49G2. (A) C57Bl/6, IL-2-expanded Ly-49G2-positive or -negative NK cells were evaluated against P815. The values are the
lytic units at 30% lysis per 10 million cells and standard error of lytic unit. Antibodies were added in microtiter wells at 10 µg/mL. (B) The lytic potential of C57Bl/6,
IL-2 expanded Ly-49A2, Ly-49G2-positive or -negative NK cells directed against L5MF22 clones; expressing H-2Dd and H-2Ld and the parental L5MF22 cells
(H-2Db). The values are the lytic units at 30% lysis per 10 million cells. (C) IL-2 expanded unsorted or Ly-49G2-positive or -negative NK cells from three different
mouse strains. (D–F) Ability of anti-Ly-49G2 (Fab’)2 antibodies to reverse inhibition in four mouse strains. IL-2 expanded Ly-49G2-positive or -negative (Ly-49A2)
NK cells were evaluated against P815 [D], L5MF22 parent [E], and L5M-D104 [F] with addition of control or anti-Ly-49G2 antibodies in microtiter wells at 10
µg/mL. Values are expressed at percent specific lysis at a 10:1 effector-to-target ratio.
this study, a marrow rejection model where H-2b mice reject
H-2d bone marrow, all three antibodies to Ly-49D, 4E5, 12A8
(that cross-reacts with Ly-49A), and 12A1 mAbs resulted in an
engraftment of H-2d bone marrow after in vivo elimination of
Ly-49D-expressing NK cells (Table 3). These studies indicate
that Ly-49D-positive cells recognize H-2d. Collectively, these
studies are consistent with and strongly support recognition of
H-2d, presumably Dd, by Ly-49D receptors. Other recent in vivo
studies [11] by Raziuddin using the chimeric D8 mouse
(expressing Dd) supports this contention in the bone marrow
transplant system. However, these studies do not rule out
positive recognition of other class I or non-class I moieties.
The recent discovery of a novel signaling molecule (DAP12)
that is associated with human KIR [23] and mouse Ly-49s
[27–29] provides a basis for understanding the diversity and
complexity of NK cell recognition and regulation. Ly-49D
represents only one of the proposed activating NK receptors
identified to date, whereas the function of Ly-49H has yet to be
examined. The association of DAP12 with Ly-49D and its
demonstrated activating properties in cytotoxicity, calcium flux,
and downstream biochemical signaling events presents a
complex yet promising future for studies of the mouse NK
receptors. Our study provides further evidence that H-2Dd is
one of the ligands for the activating NK receptor Ly-49D.
ACKNOWLEDGMENTS
Fig. 6. Tyrosine phosphorylation of Ly-49 receptors. NK cells expanded with
IL-2 for 7–10 days were washed, rested for 1 h, and stimulated with
pervanadate. Cells were lysed and immunoprecipitated with anti-Ly-49 antibodies. The immune complexes were run on a non-reducing 10% gel, transferred,
and blotted with 4G10 (anti-phosphotyrosine antibody) to evaluate the tyrosine
phosphorylation status of the receptors. Representative blots are shown for
selected strains. Experiment shown is representative of two or more experiments.
This project was funded in whole or in part with funds from the
National Cancer Institute, National Institutes of Health, under
contract N01-CO-56000. We would like to thank Eric Darby
and Louise Finch from the Clinical Services Program, SAIC, for
their expert technical assistance in cell sorting, and Susan
Charbonneau and Joyce Vincent for typing and editing the
manuscript.
Ortaldo et al.
Ly-49 regulates murine NK cell functions
519
REFERENCES
1. Smith, H. R. C., Karlhofer, F. M, Yokoyama, W. M. (1994) Ly-49 multigene
family expressed by IL-2-activated NK cells. J. Immunol. 153, 1068–1079.
2. Wong, S., Freeman, D., Kelleher, C., Mager, D., Takei, F. J. (1991) Ly-49
multigene family. New members of a superfamily of type II membrane
proteins with lectin-like domains. J. Immunol. 147, 1417–1423.
3. Mason, L. H., Anderson, S. K., Yokoyama, W. M., Smith, H. R.,
Winkler-Pickett, R., Ortaldo, J. R. (1996) The Ly-49D receptor activates
murine natural killer cells. J. Exp. Med. 184, 2119–2128.
4. Karlhofer, F. M., Ribaudo, R. K., Yokoyama, W. M. (1992) MHC class I
alloantigen specificity of Ly-491 IL-2-activated natural killer cells. Nature
358, 66–70.
5. Daniels, B. F., Nakamura, M. C., Rosen, S. D., Yokoyama, W. M., Seaman,
W. E. (1994) Ly-49A, a receptor for H-2Dd, has a functional carbohydrate
recognition domain. Immunity 1, 785–792.
6. Mason, L. H., Ortaldo, J. R., Young, H. A., Kumar, V., Bennett, M.,
Anderson, S. A. (1995) Cloning and functional characteristics of murine
large granular lymphocyte-1: A member of the Ly-49 gene family
(Ly-49G2). J. Exp. Med. 182, 293–303.
7. Brennan, J., Mager, D., Jefferies, W., Takei, F. (1994) Expression of
different members of the Ly-49 gene family defines distinct natural killer
cell subsets and cell adhesion properties. J. Exp. Med. 180, 2287–2295.
8. Yu, Y. L. L., George, T., Dorfman, J. R., Roland, J., Kumar, V., Bennett, M.
(1996) The role of Ly49A and 5E6 (Ly49C) molecules in hybrid resistance
mediated by murine natural killer cells against normal T cell blasts.
Immunity 4, 67–76.
9. Murphy, W. J., Raziuddin, A., Mason, L., Kumar, V., Bennett, M., Longo,
D. L. (1995) NK cell subsets in the regulation of murine hematopoiesis I.
5E61 NK cells promote hematopoietic growth in H-2d strain mice. J.
Immunol. 155, 2911–2917.
10. Sentman, C. L., Hackett, J., Jr., Kumar, V., Bennett, M. (1989) Identification of a subset of murine natural killer cells that mediates rejection of
Hh-1d but not Hh-1b bone marrow grafts. J. Exp. Med. 170, 191–202.
11. Raziuddin, A., Longo, D. L., Mason, L., Ortaldo, J. R., Bennett, M.,
Murphy, W. J. (1998) Differential effects of the rejection of bone marrow
allografts by the depletion of activating versus inhibiting Ly-49 natural
killer cell subsets. J. Immunol. 160, 87–94.
12. Stoneman, E. R., Bennett, M., An, J., Chestnut, K. A., Wakeland, E. K.,
Sheerer, J. B., Siciliano, M. J., Kumar, V., Mathew, P. A. (1995) Cloning and
characterization of 5E6 (Ly-49C), a receptor molecule expressed on a
subset of murine natural killer cells. J. Exp. Med. 182, 305–313.
13. Ljunggren, H. G., Karre, K. (1990) In search of the ‘‘missing self’’: MHC
molecules and NK cell recognition. Immunol. Today 11, 237–244.
14. Raziuddin, A., Longo, D. L., Mason, L., Ortaldo, J. R., Murphy, W. J. (1996)
Ly-49G21 NK cells are responsible for mediating the rejection of H-2b
bone marrow allografts in mice. Int. Immunol. 8, 1833–1839.
15. Brennan, J., Lemieux, S., Freeman, J. D., Mager, D. L., Takei, F. (1996)
Heterogeneity among Ly-49C natural killer (NK) cells: Characterization of
highly related receptors with differing functions and expression patterns. J.
Exp. Med. 184, 2085–2090.
16. Moore, T. A., von Freeden-Jeffry, U., Murray, R., Zlotnik, A. (1996)
Inhibition of gamma delta T cell development and early thymocyte
maturation in IL-7-/- mice. J. Immunol. 157, 2366–2373.
520
Journal of Leukocyte Biology
Volume 66, September 1999
17. Fogler, W. E., Volker, L., McCormick, K. L., Watanabe, M., Ortaldo, J. R.,
Wiltrout, R. H. (1996) NK cell infiltration into lung, liver, and subcutaneous B16 melanoma is mediated by VCAM-1/VLA-4 interaction. J.
Immunol. 156, 4707–4714.
18. Yankelevich, B., Knobloch, C., Nowicki, M., Dennert, G. (1989) A novel
cell type responsible for marrow graft rejection in mice. T cells with NK
phenotype cause acute rejection of marrow grafts. J. Immunol. 142,
3423–3430.
19. Mason, L. H., Gosselin, P., Anderson, S. K., Fogler, W. E., Ortaldo, J. R.,
McVicar, D. W. (1997) Differential tyrosine phosphorylation of inhibitory
versus activating Ly-49 receptor proteins and their recruitment of SHP-1
phosphatase. J. Immunol. 159, 4187–4196.
20. Yoshimoto, T., Paul, W. E. (1994) CD41, NK1.11 T cells promptly produce
interleukin 4 in response to in vivo challenge with anti-CD3. J. Exp. Med.
179, 1285–1295.
21. Held, W., Raulet, D. H. (1997) Ly49A transgenic mice provide evidence for
a major histocompatibility complex-dependent education process in natural killer cell development. J. Exp. Med. 185, 2079–2088.
22. Held, W., Dorfman, J. R., Wu, M. F., Raulet, D. H. (1996) Major
histocompatibility complex class I-dependent skewing of the natural killer
cell Ly49 receptor repertoire. Eur. J. Immunol. 26, 2286–2292.
23. Hoglund, P., Sundback, J., Olsson-Alheim, M. Y., Johansson, M., Salcedo,
M., Ohlen, C., Ljunggren, H. G., Sentman, C. L., Karre, K. (1997) Host
MHC class I gene control of NK-cell specificity in the mouse. Immunol.
Rev. 155, 11–28.
24. Nakamura, M. C., Niemi, E. C., Fisher, M. J., Shultz, L. D., Seaman, W. E.,
Ryan, J. C. (1997) Mouse Ly-49A interrupts early signaling events in
natural killer cell cytotoxicity and functionally associates with the SHP-1
tyrosine phosphatase. J. Exp. Med. 185, 673–684.
25. Campbell, K. S., Cella, M., Carretero, M., Lopez-Botet, M., Colonna, M.
(1998) Signaling through human killer cell activating receptors triggers
tyrosine phosphorylation of an associated protein complex. Eur. J.
Immunol. 28, 599–609.
26. Long, E. O., Burshtyn, D. N., Clark, W. P., Peruzzi, M., Rajagopalan, S.,
Rojo, S., Wagtmann, N., Winter, C. C. (1997) Killer cell inhibitory receptors:
diversity, specificity, and function. Immunol. Rev. 155, 135–144.
27. Lanier, L. L., Corliss, B. C., Wu, J., Leong, C., Phillips, J. H. (1998)
Immunoreceptor DAP12 bearing a tyrosine-based activation motif is
involved in activating NK cells. Nature 391, 703–707.
28. Mason, L. H., Willette-Brown, J., Anderson, S. K., Gosselin, P., Shores,
E. W., Love, P. E., Ortaldo, J. R., McVicar, D. W. (1998) Characterization of
an associated 16-kDa tyrosine phosphoprotein required for Ly-49D signal
transduction. J. Immunol. 160, 4148–4152.
29. Smith, K. M., Bakker, A. B., Phillips, J. H., Lanier, L. L. (1998) Ly-49D
and Ly-49H associate with mouse DAP12 and form activating receptors. J.
Immunol. 161, 7–10.
30. George, T. C., Mason, L. H., Ortaldo, J. R., Kumar, V., Bennett, M. (1999)
Positive recognition of MHC class I molecules by the Ly49D receptor of
murine NK cells. J. Immunol. 162, 2035–2043.
31. Nakamura, M. C., Linnemeyer, P. A., Niemi, E. C., Mason, L. H., Ortaldo,
J. R., Ryan, J. C., Seaman, W. E. (1999) Mouse Ly-49D recognizes
H-2Dd and activates natural killer cell cytotoxicity. J. Exp. Med. 189,
493–500.
http://www.jleukbio.org