The Ly-49D Receptor Activates Murine Natural Killer Cells
By
L.H.
Mason,*
S.K.
Anderson,§
W.M.
Yokoyama,
H.R.C.
Smith,
R.
Winkler-Pickett,*
and
J.R.
Ortaldo*
From the * Laboratory of Experimental Immunology, Division of Basic Sciences, and
Intramural
Research Support Program, SAIC Frederick, National Cancer Institute-FCRDC, Frederick,
Maryland 21702-1201; § Division of Rheumatology, Washington University, St. Louis, Missouri
63110
Summary
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
References
Summary
Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to
inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we
present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has
generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members
of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the
Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immunoprecipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from
those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and
concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their
susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex
class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate
that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc
R+ target cells in
a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/IxYxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has
been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first
member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather
than inhibiting NK cell function.
Members of the Ly-49 gene family encode type II integral transmembrane proteins and are primarily expressed on the surface of murine NK cells. Several members of the Ly-49 family of proteins can bind to class I
MHC and transmit inhibitory signals to NK cells. When
expressed on target cells, selected class I proteins can prevent NK cells from delivering their lethal hit. Recognition of class I molecules by Ly-49+ NK cells has been proposed
as a regulatory mechanism to prevent lysis of normal host
cells. However, NK cell lysis can proceed upon downregulation of host class I after transformation or viral infection
(1). Recent studies have identified eight Ly-49 gene family
members in NK cells from B6 mice (2, 3). The prototypic member of the Ly-49 family, Ly-49A, has been shown to
recognize the class I molecules H-2Dd and H-2Dk (4, 5).
The interaction of Ly-49A with H-2Dd has been postulated to transmit a negative signal to the NK cell. This hypothesis has been formulated because Ly-49A+ NK cells
are apparently not capable of mediating antibody-dependent cellular cytotoxicity or lectin-induced cytotoxicity
against H-2Dd-expressing target cells. Upon addition of
mAb A1, which recognizes Ly-49A+ NK cells (6), enhanced lysis of target cells that is not FcR dependent is observed (4). Studies have also shown that Ly-49A can recognize carbohydrate expressed on the surface of target cells,
which may contribute to the interaction of Ly-49A and
class I proteins (7).
The Ly-49G2 subset of NK cells also has been shown to
be inhibited by target cells expressing H-2Dd and/or H-2Ld
(8). Studies with Ly-49G2+ NK cells have relied primarily
on the reversal of target cell inhibition by mAb 4D11
(anti-Ly-49G2) and mAb specific for H-2Dd and H-2Ld.
The Ly-49C+ subset of NK cells has been shown to bind
the class I molecules of the H-2b, H-2d, H-2k, and H-2s
haplotypes (9). Recent data from Yu et al. (10) demonstrates that Ly-49C+ NK cells from BALB/c and BALB.B
mice are inhibited by H-2d and H-2Kb class I antigens. The
authors in this study concluded, however, that not all Ly49C+ NK cells function the same way in all mouse strains,
and suggest that allelic differences may regulate class I recognition by these cells (10). Previous data by members of
this group have shown that 5E6+ NK cells can reject bone
marrow grafts expressing H-2d but not H-2b (11). These
results suggest that Ly-49C binding to its H-2d ligand may
not be inhibitory in the strains studied. In H-2d strain mice,
Ly- 49C+ NK cells may be responsible for promoting hematopoiesis through the upregulation of GM-CSF, as demonstrated by Murphy et al. (12), implying further that some
Ly-49 family members may upregulate NK cell function.
The Ly-49 gene family now consists of eight distinct
molecules in a single inbred stain. The original Ly-49 gene
has been renamed Ly-49A, and the others have been designated Ly-49B-H (2, 3, 9). mAb specific for the Ly-49A, C,
and G2 molecules have helped provide significant information on their functional attributes. Functional characterization of other Ly-49 family members has been hampered by
the lack of antibodies that specifically recognize each molecule. Ly-49D is of particular interest because it has a cytoplasmic domain that is significantly different from other Ly49 family members. In this report, we describe mAb 12A8, which reacts with the Ly-49D subset of murine B6 NK
cells. Although transfection studies with the known members of the Ly-49 cDNA family of genes indicates that it reacts with both Ly-49D and Ly-49A, the Ly-49D+ subset of
NK cells can be isolated. Here, we characterize the functional characteristics of Ly-49D+ NK cells and suggest that
the Ly-49D receptor on NK cells is an activating rather
than an inhibitory receptor.
Materials and Methods
Mice and Rats.
All mice and rats were obtained from the Animal Production Facility, FCRDC. Mice were between 8 and 20 wk old when killed.
Generation of mAb 12A8.
IL-2-cultured B6 NK cells were
used to immunize Fischer 344 rats. Approximately 5-10 × 106
cells were injected subcutaneously into rats and boosted every 2 wk
for a total of four boosts. After the final boost, the rat spleen was
harvested and spleen cells were fused with the SP2/0 hybridoma
by PEG. Supernatants were first screened for rat IgG by ELISA,
followed by a live cell ELISA assay against IL-2-propagated B6
NK cells, as previously described (13). mAb 12A8 was isolated and the hybridoma was cloned by limiting dilution. mAb 12A8 is of the rat IgG2a isotype.
Flow Cytometry Analysis.
NK cells were stained as previously
described and were analyzed on either a FACScan® or FACSort®
(Becton Dickinson & Co., Mountain View, CA). Cell sorting
was performed on either an Epics 750 (Coulter Electronics, Hialeah, FL) or a FACStar® (Becton Dickinson). NK cells were either stained with the primary antibody followed by an isotypespecific, FITC-conjugated secondary reagent, or directly with a
FITC-labeled primary antibody. Cells sorted for mAb 12A8 were
stained with biotinylated 12A8, followed by Streptavidin PE
(Becton Dickinson). Antibodies used in these experiments included 2.4G2 (Fc
RII/III), PK136 (NK-1.1), A1 (Ly-49A), SW5E6 (Ly-49C), and 4D11 (Ly-49G2). All antibodies were semipurified by salt fractionation (2×) of ascites fluid. RM21 antibody
against murine CD2 was affinity purified before use.
NK Cell Isolation.
Murine splenic NK cells were isolated as
described previously (13). Essentially, nylon wool-nonadherent
(NWNAD)1 cells were obtained and subjected to mAb plus complement (mAb + c
) depletion of T cells using anti-CD4 and
CD8 mAbs. Partial depletion of Ly-49A, C, and G2+ NK cells
was obtained by including mAb SW5E6 and 4D11 to the cocktail
of anti-CD4 and CD8 mAbs. Lysis of NK cells with mAb 5E6
and 4D11 accomplished two goals: the first was to reduce the number of NK cells expressing known Ly-49 receptors such as C and G2 (and coincidentally, those cells coexpressing Ly-49A), and
the second was to enrich the NK cell population for cells expressing 12A8 (Ly-49D) to make sorting these cells less time consuming. Treatment of NWNAD cells with this cocktail resulted in an
NK cell population that was enriched for 12A8+ (Ly-49D+) NK
cells, but generally <10% Ly-49A, C, and G2+. The remaining B
cells were further removed by immunoadsorbance to anti-mouse
IgG- coated petri plates. After culture for 6-12 d in 1,000 U/ml
IL-2, the resulting cell populations were >95% NK-1.1+.
Tumor Targets and Con A Blasts.
Tumor targets were maintained in culture as previously described (13). Splenic Con A
lymphoblasts were essentially prepared by the method of Chadwick and Miller (14). Blasts were prepared and frozen at
70°C
before labeling.
Cytotoxicity Assays.
Tumor targets and Con A lymphoblasts
were labeled with 51Cr and used in 4-h cytotoxicity assays, as described previously (13). 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 the percent of specific lysis.
Immunoprecipitation.
IL-2-cultured NK cells were 125I-surface
labeled with lactoperoxidase and lysed in 0.5% Triton X-100
containing protease inhibitors, as described previously (13). A single lysate was subjected to sequential immunoprecipitation using
an mAb cross-linked to protein G-Sepharose. The order of immunoprecipitations was control rat IgG2a, A1, 4D11, SW5E6,
and 12A8. Approximately equal amounts of radioactivity (except
for the control antibody in which the entire eluate was used) was
applied to each well of a 10% SDS-PAGE gel under nonreducing
and reducing conditions. Gels were fixed, dried, and autoradiography was performed.
Transfection of Cos-7.
cDNA plasmids encoding Ly-49A, D,
E, F, G1, and G2 were initially transfected into Cos-7 cells using
the DEAE-dextran method (15). Cells were harvested with
versene (GIBCO BRL, Gaithersburg, MD) 3 d after transfection
and were analyzed by flow cytometry analysis (FCA) for binding
of mAb 12A8. Reactivity was confirmed as shown in Fig. 2 by
using Cos-7 cells transfected with Lipofectamine (Life Technologies, Gaithersburg, MD). A 1:60 dilution of Lipofectamine and 5 µg of Ly-49 plasmid cDNA was used according to the manufacturer's instructions. Transfection was performed for 6 h at 37 °C,
followed by washing, trypsinization, and replating cells in a T-150
flask. After 72 h of culture at 37°C, the cells were removed using
HBSS without Ca++ and Mg++ and with 1 mM EDTA. FCA
was performed as described previously.
Fig. 2.
mAb 12A8 stains Cos-7 cells transfected with Ly-49A and Ly-49D. Cos-7 cells were transfected with either Ly-49A or Ly-49D using Lipofectamine and were harvested after a 72-h culture at 37°C for protein expression. Cells transfected with Ly-49A (A and B) and Ly-49D (C and D) were
stained with mAb A1 (A and C) or mAb 12A8 (B and D). mAb 12A8 staining was observed on both Ly-49A and Ly-49D transfectants, whereas mAb A1
stained only Cos-7 cells tranfected with Ly-49A. The unshaded histogram represents staining with FITC-GAM (mAb A1) or FITC-GART (mAb 12A8).
Shaded histograms represent staining observed with a primary antibody (A1 or 12A8) plus FITC-GAM or FITC-GART.
[View Larger Version of this Image (42K GIF file)]
Apoptosis.
Highly enriched populations of NK cells were obtained from C57BL/6 splenocytes as described previously. The
methods used to measure apoptosis have been developed by Ortaldo et al. (15a). 106 NK cells were cultured for 24 h in RPMI
1640 + 10% FBS + 103 U/ml IL-2 in 24-well plates. NK cells
were collected, and viable cells were obtained after passage over
Lympholyte-M (Cedarlane Labs., Ontario, Canada). Cells were
then cultured in 48-well plates at 106 cells/well, to which a variety of mAb were added, including A1, 12A8, 4D11, 5E6, and
PK136 at a concentration of 40 µg/well in a total volume of 100 µl. After a 15-min incubation at room temperature, 40 µg of a
secondary goat anti-mouse or anti-rat antibody was added to
each well. Plates were incubated at 37°C for 6 and 12 h. NK cells
were harvested and stained with propidium iodide for FCA. Flow
cytometry was performed on a FACSort® using Lysis II software
(Becton Dickinson), which analyzed the percentage of cells incorporating propidium iodide vs. forward scatter.
Results
mAb 12A8 Identifies a Unique Subset of NK Cells.
mAb
12A8 was generated by fusing spleen cells from Fischer 344 rats that had been immunized with C57BL/6-IL-2-cultured
NK cells, with the SP2/0 myeloma. Screening of lymphoid
cells by FCA revealed that mAb 12A8 did not react with
thymocytes, T cells, or B cells from B6 mice (data not
shown). mAb 12A8 did react, however, with a unique subset of B6 NK cells. Fig. 1 A shows the staining profile of
mAb 12A8 on highly enriched populations of freshly isolated NK cells. Two-color FCA of 12A8 vs. NK-1.1 demonstrated that virtually all 12A8+ cells were NK-1.1+, and
that 12A8+ cells represented ~63% of the NK-1.1+ cells.
The staining profiles and reactivity of mAb 12A8 on freshly isolated and IL-2-cultured NK cells indicated that this antibody may be similar to the previously characterized mAbs,
4D11 (13) and 5E6 (11). Two-color FCA was used to examine this possibility. Fig. 1 presents the staining profiles of
mAb 12A8 vs mAb A1 (Ly-49A; Fig. 1 B), mAb 4D11
(Ly-49G2; Fig. 1 C), and mAb 5E6 (Ly-49C; Fig. 1 D).
These staining profiles revealed that mAb 12A8 reacted with a unique subset of B6 NK cells, and that it did not
demonstrate reactivity similar to mAb 5E6 or 4D11. However, an interesting staining profile was observed on NK
cells when costained with mAb 12A8 vs. A1 (Fig. 1 B). It
was apparent that all cells that stained with mAb A1 were
also 12A8+. One explanation for the expression of mAb
12A8 on all A1+ cells could simply be that all A1+ cells express both Ly-49A and another molecule recognized by mAb 12A8. An alternative explanation would be that mAb
12A8 cross-reacts with both Ly-49A and another highly
homologous member of the Ly-49 gene family. Blocking
studies were performed with mAbs 12A8 and A1 to determine if binding of one mAb could block the reactivity of
the alternate mAb. Binding of mAb 12A8 to NK cells does
reduce the staining intensity (mean channel fluorescence)
of FITC A1; however, mAb A1 does not block binding of
FITC 12A8 (data not shown). Control blocking studies revealed that mAb 12A8 did not affect staining by mAb
4D11 or 5E6. The results of our FCA data suggested that
mAb 12A8 identified a unique molecule on the surface of
NK cells with possible cross-reactivity to Ly-49A.
Fig. 1.
mAb 12A8 identifies a distinct subset of C57BL/6
NK cells. Freshly isolated
C57BL/6 splenic NK cells were
enriched by passage over nylon
wool columns, depleted of T and
B cells, and prepared for FCA as
follows: (A) PK136 plus biotinylated 12A8 followed by FITC-
goat anti-mouse IgG2a and
Steptavidin-PE, (B) FITC-A1 plus biotinylated 12A8 followed
by Streptavidin PE, (C) FITC4D11 plus biotinylated 12A8 followed by Streptavidin PE, and
(D) FITC-5E6 plus biotinylated
12A8 followed by Streptavidin PE.
[View Larger Version of this Image (41K GIF file)]
mAb 12A8 Recognizes Ly-49D and Ly-49A on Transfected
Cos-7 Cells.
Cos-7 cells were transfected with cDNA for
Ly-49A or Ly-49D, and stained for reactivity with mAb A1
(Ly-49A) or mAb 12A8. Fig. 2 presents the profiles of these
staining reactivities. These results clearly reveal that cells
transfected with Ly-49A react with both mAb A1 and
12A8 (Fig. 2, A and B), whereas cells transfected with Ly49D only react with mAb 12A8 (Fig. 2 D). Cos-7 cells
transfected with the cDNAs for Ly-49E, F, G1, and G2 did
not react with mAb 12A8 (data not shown). Furthermore,
transfection of Cos-7 cells with Ly-49B, C, and H did not
reveal any cells staining positive with mAb 12A8 (data not
shown). These transfection studies explain our FCA results
(Fig. 1 B), in which all A1+ cells were also 12A8+, and they
conclusively demonstrated 12A8 binding to Ly-49D. Therefore, mAb 12A8 is the first reported antibody to bind to Ly49D and should be a useful reagent to help understand the
biological role of Ly-49D+ NK cells.
mAb 12A8 Immunoprecipitates a 50-kD Homodimer on NK
Cells.
Considering the unique phenotypic characterization of the 12A8+ subset of NK cells and its reactivity with
Ly-49D, we decided to examine the biochemical characteristics of the antigen recognized by this antibody. IL-2-cultured NK cells from B6 mice were surface labeled with 125I,
lysed, and subjected to immunoprecipitation with an mAb
linked to protein G-Sepharose. Preliminary experiments
demonstrated that mAb 12A8 immunoprecipitated a glycoprotein of ~100 kD (nonreduced) and ~50 kD (reduced). To confirm our transfection data and to determine
if mAb 12A8 could immunoprecipitate a unique Ly-49
glycoprotein, we performed sequential immunoprecipitation
(IP). A single lysate from 125I-labeled NK cells was subjected to IP, first with an isotype control mAb, followed by
mAbs A1, 5E6, 4D11, and 12A8, respectively. IP with mAb
A1 was performed with an excess of protein G-Sepharose- linked antibody, and was allowed to react at 4°C overnight
to remove cross-reactive Ly-49A+ glycoproteins. Figs. 3, A
and B, presents the results of these IP experiments. Despite
the highly glycosylated nature of the Ly-49 proteins that
result in diffuse bands during SDS-PAGE analysis, clear differences between the IPs were observed. IP with mAb A1 (16), 5E6 (11), and 4D11 (13) resulted in bands under both nonreducing and reducing conditions that were consistent
with previously published reports. mAb 12A8 immunoprecipitation yielded proteins with noticeably higher molecular weight bands than those seen with mAb 4D11 and
slightly higher than those seen with mAb A1. 5E6 IP,
however, resulted in proteins that were slightly larger than
those seen with mAb 12A8. These results support our FCA
data on both NK cells and transfected Cos-7 cells, that
mAb 12A8 recognizes a unique glycoprotein on B6 NK
cells.
Fig. 3.
Immunoprecipitation
using mAb 12A8 reveals a unique
disulfide-linked homodimer. B6
NK cells were cultured for 7 d in
high dose IL-2. Cells were radiolabeled with 125I and lysed in
0.5% Triton X-100. Sequential
IP was performed on a single lysate by the following mAb crosslinked to protein G-Sepharose:
Control rat IgG2A, A1, 5E6,
4D11, and 12A8, respectively. Approximately equal CPMs
were applied to a 10% SDSPAGE gel and electrophoresed
under both nonreduced (A) and
reduced (B) conditions.
[View Larger Version of this Image (44K GIF file)]
Isolation of Ly-49D+ NK Cells and Lysis of Tumor Targets.
Murine NK cells from C57BL/6 mice are heterogenous in their expression of Ly-49 gene family members
(Fig. 1), where individual NK cells are likely to express
multiple Ly-49 gene products. To examine the functional
properties of Ly-49D+ NK cells, it was necessary to isolate
cells expressing this marker from those NK cells expressing
other Ly-49 proteins such as Ly-49C, G2, and particularly
Ly-49A, with which it cross-reacts. Splenic NWNAD cells
were depleted of T and B cells along with partial depletion
of the Ly-49C+ and Ly-49G2+ subsets of NK cells using
the appropriate mAb + C
. Coexpression of Ly-49A on
Ly-49C+ and G2+ NK cells resulted in the removal of the
majority of Ly-49A+ NK cells during the lysis step. These
depletion experiments resulted in a population of NK cells
expressing <10% Ly-49A, C, and G2+ cells. Ly-49A+ NK
cells (that cross-react with mAb 12A8) were separated from Ly-49D+ NK cells by two-color cell sorting. NK cells
were stained using biotinylated mAb 12A8 vs. FITCmAbA1 and sorted. From this population, the Ly-49D+/A
and Ly-49D
/A
cells were collected and cultured in IL-2.
Upon completion of cell sorting, these subsets were >98%
12A8+ or 12A8
for their respective phenotypes.
The purified NK cell subsets were examined for their
cytolytic properties against various class I target cells. A
panel of tumor target cells was examined for susceptibility
to lysis by Ly-49D+/A
and Ly-49D
/A
NK cells. Table 1
presents the lytic data on the ability of the input population
(Ly-49A, C, and G2 reduced), Ly-49D+/A
, and Ly49D
/A
NK cells to lyse tumor target cells expressing different class I phenotypes. It is apparent from this data that
Ly-49D+/A
NK cells are capable of lysing tumor target
cells expressing H-2a, H-2b, H-2d, and H-2k. Lysis by Ly49D+ NK cells was equivalent to, if not greater than, Ly49D
/A
NK cells and the input population. These results
indicate that lytic activity of Ly-49D+ cells is not inhibited
by these class I-bearing tumor targets. Previous studies in
our laboratory have demonstrated that both P815 and
WEHI-164 (H-2d targets) are not lysed efficiently by Ly49A+ or G2+ NK cells when compared to the Ly-49A
and G2
subsets (data not shown).
Ly-49D+ NK Cells Lyse Con A Blasts of Varied H-2 Haplotypes.
In further attempts to establish any possible class I
restriction of the lytic capacity of Ly-49D+ NK cells, Con
A lymphoblasts from seven different H-2 haplotypes were
examined. Ly-49D+ cells were isolated as described above
and expanded in IL-2 for 6-10 d. Splenic Con A lymphoblasts were generated for 48 h and labeled with either 51Cr
or 111In, and were used as targets in a 4-h cytotoxicity assay.
The data presented in Fig. 4 demonstrates that targets expressing H-2a, H-2b, H-2d, H-2k, H-2s, H-2q, and H-2p are
all lysed by Ly-49D+ NK cells. In general, the patterns of
lysis observed indicated that Ly-49D+ NK cells are comparable to both the Ly-49D
and input cells against the majority of the blast targets. These results support the lytic
data using tumor targets that Ly-49D+ NK cells do not appear to be inhibited by any of the class I-expressing target
cells tested to date. One conclusion from this data is that
the glycoprotein encoded by the Ly-49 gene may not interact with class I MHC. If recognition does occur between Ly-49D-encoded proteins and class I, signals that inhibit NK lysis are not generated.
Fig. 4.
Ly-49D+/A
and Ly-49D
/A
NK cells
lyse Con A blasts of multiple H-2 haplotypes. Ly49D+/A
NK cells were sorted from Ly-49D
/A
NK cells after partial depletion of the Ly-49C+/A+ and
G2+ cells, as described in Materials and Methods. Cells
were cultured for 6 d in high dose IL- 2. Con A lymphoblasts were prepared from a variety of H-2 haplotypes representing H-2a, H-2d, H- 2k, H-2s, H-2a, and
H-2q. Blasts were labeled with 51Cr and used as targets
in a 4-h cytotoxicity assay. E/T ratios of 20:1, 7:1, 2:1,
and 0.7:1 were assayed, and the percent of specific lysis
at 20:1 is shown. Spontaneous release of targets varied
from 19 to 35%. Results also are presented for the input population that was partially depleted of Ly-49A,
C, and G2+ cells. The resulting Ly-49 phenotypes of
these populations are the same as in the legend to Table
1. These results represent one of two such experiments
performed.
[View Larger Version of this Image (38K GIF file)]
Ly-49D+ NK Cells Mediate Reverse Antibody-dependent
Cellular Cytotoxicity (RADCC) in the Presence of mAB 12A8.
Since Ly-49D+ NK cells failed to exhibit any pattern of
class I restriction, we decided to examine mAb 12A8 for its
ability to activate the lytic potential of Ly-49D+ NK cells.
A panel of different tumor target cells, including both
Fc
R+ and Fc
R
targets, were examined for susceptibility to lysis by Ly-49D+ and Ly-49D
NK cells in the presence of various antibodies. Fig. 5 presents the data obtained
from a representative redirected lysis (RADCC) assay. The
addition of mAb 12A8 to Ly-49D+, but not Ly-49D
, NK
cells, results in augmented lysis of Fc
R+ target cells. Both
P815 (Fig. 5 A) and WEHI-3 (Fig. 5 B) targets (Fc
R+)
are lysed much more efficiently in the presence of mAb
12A8 when compared to either media alone, a negative
control mAb (R
2A), or mAbs reactive with NK cells
(e.g., RM21 and 2.4G2). This RADCC effect has been
reported for mAb PK136, which detects the NK-1.1 molecule (17), and can be blocked by the addition of mAb
2.4G2, which is specific for Fc
RII/III (18). As a further
control in these experiments, it can be seen that mAb
PK136 is capable of augmenting the lysis of both the Ly49D+ and D
subsets (>95% NK-1.1+) against the Fc
R+
targets P815 and WEHI-3. The Ly-49D
subset, however,
is not augmented in the presence of mAb 12A8 to lyse
these same targets. These results also reveal that the augmented lysis observed in the presence of mAb 12A8 can be
abrogated upon addition of mAb 2.4G2 (identical to that
seen using PK136 + 2.4G2). Similar results have been obtained upon pretreatment and washing of Ly-49D+ NK
cells with mAb 12A8 before performing cytotoxicity assays (data not shown). Fc
R
target cells such as SST (Fig. 5 C)
and WEHI 164 (Fig. 5 D) do not demonstrate any significant augmentation of lysis by Ly-49D+ NK cells in the
presence of mAb 12A8. The data shown in Fig. 5 (C) shows
that mAb 12A8 weakly augments killing by Ly-49D+ NK
cells against the SST target. This finding has not been reproducible in other experiments using this target and was
not considered significant. Based on the above experiments,
we conclude that mAb 12A8 engagement of the Ly-49D
molecule augments the lytic function of NK cells. These
results establish for the first time that a member of the Ly-49
gene family may upregulate NK lytic function upon binding the appropriate ligand.
Fig. 5.
mAb 12A8 mediates RADCC of Fc
R+ target cells by Ly-49D+, but not Ly-49D
, NK cells. Splenic NK cells were depleted of cells expressing Ly-49A, C, and G2. Ly-49D+ and Ly-49D
cells were sorted (using biotinylated 12A8 followed by streptavidin PE) into populations that were
>98% 12A8+ (Ly-49D+) or 12A8
(Ly-49D
). These subsets were cultured in high dose IL-2 for 9 d, washed, and tested against the indicated targets in
a 4-h 51Cr-release assay at E/T ratios starting at 10:1. mAbs were added at a final concentration of 2 µg/well. Data are presented as lytic units per 107 cells
at 30% lysis. This experiment is one of at least three representative assays (NT, not tested). Both the Ly-49D+ and Ly-49D
populations were <5% Ly49A, C, and G2+ at the time of testing.
[View Larger Version of this Image (36K GIF file)]
mAb 12A8 Induces Apoptosis of Ly-49D+ NK Cells.
The ability of mAb 12A8 to mediate RADCC in Ly-49D+
NK cells presents functional data for involvement of the
Ly-49D molecule in activating the lytic mechanism of NK
cells. We also have obtained evidence that the Ly-49D
molecule may be involved in triggering apoptotic events in
NK cells that lead to cell death. The results shown in Fig. 6
demonstrate that mAb 12A8 induces apoptosis in ~40% of
bulk NK cells by 6 h. Although some variability was observed in different experiments, none of the other Ly49-specific antibodies induced any consistent or potent apoptosis. Since mAb 12A8 stains ~60% of bulk NK cells, of
which up to 20% may represent cross-reacting Ly-49A+
cells, it appears that most Ly-49D+ NK cells are susceptible
to mAb 12A8-induced apoptosis. Of interest, mAb PK136
that recognizes the NK-1.1 antigen (mouse NKR-P1) was not very effective at inducing apoptosis. Only 10-12% of
NK cells demonstrated PI uptake after the addition of
PK136. These results support the hypothesis that Ly-49D
represents an activation molecule on NK cells. Although
Ly-49A, C, and G2 have been demonstrated to be class I
inhibitory receptors on NK cells, cross-linking of their specific receptors does not induce apoptotic associated events
in NK cells. Therefore, intracellular signaling events mediated by Ly-49D appear to be different from those mediated by Ly-49A, C, and G2. Apoptotic-associated events also
have been observed using the CellFit program (Becton
Dickinson) to measure the DNA index of murine NK cells
after appropriate stimulation and cross-linking of the Ly-49
mAb. Using this methodology, we have observed increases
in abnormal DNA content in NK cells treated with mAb
12A8, but not with 5E6 or 4D11 (data not shown).
Fig. 6.
mAb 12A8 induces apoptosis in Ly-49D+ NK cells. This
graph presents the data obtained from a representative experiment in
which percent cell death (PI uptake) is calculated after 6 and 12 h of antibody treatment. Cell death is calculated over that observed with media
alone (33, 35 & 38% respectively). Maximum cell death was observed after 6 h of treatment with mAb 12A8 and cross-linking. This is a representative experiment of eight similar experiments performed.
[View Larger Version of this Image (17K GIF file)]
Discussion
Eight distinct members of the Ly-49 gene family have
been identified (Ly-49A-H), although specific mAbs that
recognize Ly-49A (A1/YE1-32 and 48), Ly-49C (5E6),
and Ly-49G2 (4D11) are the only ones available. The
availability of these mAbs has helped to further define the
functional role of NK cells that express these receptors. The importance of generating mAbs against the remaining,
undefined members of the Ly-49 gene family is paramount
to understanding the functional role that these molecules
play in NK cell biology. Investigation of the proposed class I
inhibitory properties mediated by members of the Ly-49
gene family may be facilitated by acquiring such reagents.
The prototype molecule, Ly-49A, when expressed on NK
cells, has been shown to bind the class I molecules H2-Dd
and Dk. This recognition of class I ligand by the Ly-49A
receptor has been shown to inhibit the lytic properties of
Ly-49A+ NK cells. A similar recognition process has been
proposed for Ly-49G2+ NK cells. Inhibition of lysis by Ly49G2+ NK cells, however, has only been observed against
selected target cells that appear to express high levels of
H2-Dd and/or Ld (8, 18). Experiments to determine if Ly49G2 specifically binds to H2-Dd and/or Ld are now in
progress. Brennan et al. (9) have observed binding of Ly49C+ cells to multiple H-2 haplotypes, including H-2d,
H-2k, H-2s, and H-2b. Furthermore, Ly-49C+ NK cells
have been demonstrated to be the subset of NK cells that is
responsible for rejecting H-2d, but not H-2b, bone marrow
allografts (11). These experiments imply that Ly-49C+ NK
cells also may be capable of upregulating NK cell function upon appropriate receptor/ligand interaction. A recent report by Stoneman et al. (19) demonstrates that glycosylation of the Ly-49C core protein varies in different mouse
strains. They have implied that the extent to which the Ly49C molecule is glycosylated may explain the functional
discrepancies observed by Ly-49C+ NK cells from different
mouse strains. Of further interest is the recent data from Yu
et al. that describes the role of Ly-49A+ and C+ NK cells in
mediating hybrid resistance (10). These authors conclude
that class I inhibition of Ly-49A+ and C+ NK cells support
the "missing self" and not the "hemopoietic histocompatibility antigen" hypothesis for hybrid resistance. The data
from this group also suggested that allelic differences in Ly49C+ NK cells may control which class I molecules inhibit
their lytic function.
Our laboratory has generated an mAb designated 12A8
that recognizes the Ly-49D molecule expressed on the
surface of B6 NK cells. Functional analysis of Ly-49D+ NK
cells demonstrate that they do not appear to be restricted in
their lytic capacity against either tumor targets or Con A blasts of different H-2 haplotypes. More significantly, we have demonstrated that the protein receptor encoded by Ly-49D
is capable of enhancing the lytic potential of NK cells. In
the presence of Fc
R+ target cells, mAb 12A8 triggers Ly49D+ NK cells to lyse these targets in a RADCC fashion
similar to that which occurs with the mAb PK136 against
the NK-1.1 (NKR-P1) molecule (17). Furthermore, crosslinking of mAb 12A8 on the surface of NK cells using a
secondary anti-rat antibody induces IL-2-activated NK cells
to undergo apoptotic events resulting in programmed cell
death. Studies with both T cells (20, 21) and human NK
cells (22) have revealed that IL-2-activated cells can undergo apoptosis when stimulated through activating surface receptors such as CD3 and CD16, respectively. The significance of this finding is that mAb specific for the Ly-49A,
C, and G2 proteins did not induce positive signaling events
leading to apoptosis. Therefore, our data provide strong
evidence that Ly-49D is not an inhibitory receptor on NK
cells as are Ly-49A, C, and G2. Ly-49D appears capable of
enhancing lytic function, and possibly signals NK cells
through alternate pathways.
A question arises as to whether or not the observed
RADCC results obtained with mAb 12A8 on Ly-49D+
NK cells could be an intrinsic function of the mAb itself,
rather than that of the Ly-49D receptor. It would be
tempting to determine if Ly-49A+ NK cells could be induced to mediate RADCC by mAb 12A8 because of its
cross-reactivity. The results of such an experiment could not be interpreted, however, because the percentage of Ly49A+ NK cells that coexpress Ly-49D is unknown. Until
an antibody is produced that specifically reacts with Ly49D, we must at least entertain the possibility that mAb
12A8 could have some intrinsic capacity to activate Ly-49
molecules.
Because of the unique activating properties of Ly-49D+
NK cells compared to the inhibitory properties observed in
Ly-49A, C, and G2+ NK cells, we compared the amino
acid homology of these proteins. Fig. 7 A is a schematic
comparison of Ly-49D vs. Ly-49 A, C, and G2, based on
the proposed amino acid sequence of these molecules (3).
There is obviously a high degree of homology between Ly49D and A that is highest in the extracellular domain (86%)
and least homologous in the cytoplasmic domain (51%).
The structural homology between Ly-49A and D in their
extracellular domains probably accounts for the cross-reactivity that is observed with mAb 12A8, since it can bind to
both Ly-49D and Ly-49A. Furthermore, we can also speculate that the lack of homology in the cytoplasmic region of these molecules accounts for the different signaling
properties of these proteins. The cytoplasmic domains of
Ly-49A, C, and G2 all share at least two potential serine/
threonine phosphorylation sites (P). These three molecules
also share similar sites for tyrosine phosphorylation (Y) in
their intracellular domains. A significant divergence in
phosphorylation motifs is seen in the cytoplasmic domain
of Ly-49D. Ly-49D contains only one potential serine/
threonine phosphorylation motif, and none of the tyrosine phosphorylation motifs found in either Ly-49A, C, or G2.
Fig. 7 B presents a 13-amino acid consensus sequence
comparing the Ly-49A, C, G2 and D molecules with a
similar cytoplasmic region of the human p58 inhibitory receptors. Long et al. (23) have recently described a V/IxYxxL consensus sequence in p58 proteins that may represent an immunoreceptor tyrosine-based inhibitory motif
(ITIM). This V/IxYxxL motif is present in the Ly-49 inhibitor receptors A, C, and G2. However, the Ly-49D receptor does not contain this ITIM motif, which further
supports our functional data that Ly-49D is an activation
receptor on murine NK cells.
Fig. 7.
(A) Amino acid homology of Ly-49D vs. Ly-49A, C, and G2.
This figure is a schematic representation of the proposed amino acid sequences
of Ly-49D, A, C, and G2, as described by Smith et al. (3) The highly conserved
extracellular cysteine residues are represented (C) along with the proposed glycosylation sites (CHO). Potential serine/threonine phosphorylation sites are labeled in the cytoplasmic region (P). Possible tyrosine phosphorylation sites (Y)
are also designated in the cytoplasmic domain. Open spaces in these schematics
represent areas of homology between these four proteins, whereas the perpendicular lines shown in Ly-49A, C, and G2 represent individual amino acid differences compared to Ly-49D. (B) Homologous regions from the intracellular domains of the four Ly-49 molecules compared to the human p58 inhibitory
receptor. Dashes represent amino acids identical to the p58 sequence. A consensus sequence (V/IxYxxL) for a possible immunoreceptor tyrosine inhibitory
motif found in p58 is also present in Ly-49A, C, and G2, but not in Ly-49D.
[View Larger Versions of these Images (31 + 24K GIF file)]
In summary, the present study describes the first member
of the Ly-49 family of NK receptors that appears to positively regulate NK function, rather than to inhibit it. The
detailed structure/function relationship of the ITIMs of
Ly-49 family members will greatly aid in the understanding
of the mechanism of action of these novel NK cell receptors.
Footnotes
Address correspondence to Dr. Llewellyn Mason, National Cancer Institute-FCRDC, Bldg. 560, Room
31-93, Frederick, MD 21702-1201.
Received for publication 13 May 1996
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.
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).
1Abbreviations used in this paper: ab+c
, antibody plus complement; FCA,
flow cytometry analysis; ITIM, immunoreceptor tyrosine-based inhibitory motif; IP, immunoprecipitation; NWNAD, nylon wool-nonadherent (cells); PI, propidium iodide; RADCC, reverse antibody-dependent cellular cytotoxicity.
We would like to thank Jeanette Higgins 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 this manuscript.
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