(Journal of Leukocyte Biology. 2000;68:583-586.)
© 2000
by Society for Leukocyte Biology
Recognition of CHO cells by inhibitory and activating Ly-49 receptors
Llewellyn H. Mason
Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute-FCRDC, Frederick, Maryland
Correspondence: Dr. L. Mason, Laboratory of Experimental Immunology, DBS, NCI-FCRDC, Bldg. 560, Rm. 31-93, Frederick, MD 21702-1201. E-mail: masonl{at}mail.ncifcrf.gov
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ABSTRACT
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Upon ligand recognition, members of the murine Ly-49 receptor family
can transmit inhibitory or activating signals that regulate NK cell
function. Ly-49A, G, and D have been shown to recognize the murine
class I molecule H-2Dd as a potential ligand. Recent
studies also have demonstrated also that Ly-49D+ NK cells
can lyse CHO cells, although the ligand responsible for this
recognition was not identified. Because allorecognition by NK cells may
be important in bone-marrow transplantation and because of the
overlapping class I recognition by these receptors, recognition of CHO
cells by Ly-49G and A was investigated. Our data suggest that Ly-49G
and probably A transmit inhibitory signals in response to CHO cells.
Receptor inhibition was assessed by examining NK lytic function,
IFN-
secretion, and DAP12 phosphorylation in response to CHO cells
by sorted subsets of Ly-49D vs. G B6 NK cells. Our results suggest that
CHO cells may express a common ligand(s) that is capable of engaging
Ly-49D, G, and possibly A in C576BL/6 NK cells. In addition to our
findings that Ly-49 inhibitory receptors also recognize CHO cells,
activating receptors other than Ly-49D are present in B6 mice that can
lyse CHO cells.
Key Words: Ly-49 • NK • CHO • IFN- • DAP-12
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INTRODUCTION
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Natural killer (NK) cell killing appears to be regulated by a
combination of inhibitory and activating cell-surface receptors.
Although structurally distinct, functionally similar families of
receptors exist on human (KIR/KAR) [1
, 2
]
and mouse (Ly-49) [3
, 4
] NK cells. Although
the Ly-49 family contains multiple inhibitory receptors (Ly-49A, B, C,
E, F, G, and I-L), only two activators, Ly-49D [5
] and
H, are known to be expressed on NK cells. Ly-49A and G are inhibitory
receptors that recognize H-2Dd, and the activating Ly-49D
receptor also recognizes this ligand [6
7
8
]. Recognition
of "self" can be explained readily by the inhibitory Ly-49s in the
context of the "missing self hypothesis," where these inhibitory
receptors function normally to down-regulate NK cell activity against
self major histocompatibility complex (MHC). However, recognition of
self by activating Ly-49 receptors is more difficult to reconcile.
Ly-49D has been characterized as an activating receptor on murine NK
cells because Ly49D+ NK cells: 1) mediate redirected lysis
of FcR+ target cells in the presence of specific antibody
to Ly-49D [5
], 2) mediate rejection of H-2d
bone marrow cells [6
], 3) show enhanced lysis of
H-2d concanavalin A (Con A) lymphoblasts
[7
], and 4) demonstrate enhanced lysis of
H-2d-transfected target cells [8
].
Furthermore, Ly-49D associates with the signaling moiety DAP12 that is
phosphorylated on tyrosine residues upon receptor cross-linking
[9
10
11
]. Recently, we have shown enhanced secretion of
interferon (IFN)- and tyrosine phosphorylation of DAP12 upon
Ly49D/H-2Dd interaction [12
]. Together, this
evidence strongly suggests that Ly-49D recognizes H-2Dd,
resulting in NK cell activation.
Recent data by Idris et al. [13
] suggest that
Ly-49D+ NK cells recognize Chinese hamster ovary (CHO)
cells and are responsible for their elimination in vivo. In
this study, most but not all lysis of CHO cells could be blocked by
monoclonal antibody (mAb) to Ly-49D. We were interested in whether the
class I inhibitory receptors Ly-49G and/or A might recognize CHO cells.
Our data suggest that Ly-49G and possibly A can recognize CHO cells,
because their inhibitory functions can be blocked by the addition of
mAb to these receptors. Our results demonstrate that although Ly49D
recognizes CHO cells in B6 mice as activating, Ly-49G also recognizes
CHO cells as inhibitory. Furthermore, activating receptors other than
Ly-49D are capable of recognizing CHO cells, leading to their
destruction.
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MATERIALS AND METHODS
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NK isolation, cell sorting, and antibodies utilized
Splenic NK cells were isolated from C57BL/6 mice, as previously
described [14
]. Fluorescein isothiocyanate (FITC)-4D11
(Ly-49G), FITC-YE1-48 (Ly-49A), and phycoerythrin (PE)-4E5
(Ly-49D) were used to separate
Ly-49D+G-A- cells from Ly-
49D+G+ and Ly-49G+D-
cells on a MoFlo Cell Sorter (Cytomation, Ft. Collins, CO). NK subsets
were cultured for 9–10 days in 1000 U/ml interleukin (IL)-2 (Chiron,
Emeryville, CA). The Ly-49D+G-A-
subset consisted of >95% Ly-49D+ and <5%
Ly-49G/A+ cells; the Ly-49D+G+
cells contained >95% Ly-49D+G+ and 27%
Ly-49A+ cells; and the Ly-49G+D-
subset contained >90% Ly-49G+ and <3%
Ly-49D+ cells and 40% Ly-49A+ cells. Control
antibodies consisted of a rat immunoglobulin G (IgG)2A isotype negative
control and PK-136 (NKR-P1C), reactive with all B6 NK cells.
IFN- assays
NK cells were pretreated with mAb at concentrations of 5
µg/5 x 105 cells in 0.5 ml RPMI 1640 + 5%
fetal bovine serum (FBS) for 15 min at room temperature in 24-well
plates. CHO cells (2.5x105) were added to each well, and
the plates were incubated for 6 h at 37°C, after which
supernatants were collected and analyzed for IFN- by enzyme-linked
immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN).
Cytotoxicity assays
NK cell subsets were plated into 96-well plates to yield various
E:T ratios, starting at 20:1 (in triplicate), pretreated with mAb at 2
µg/well for 15 min at room temperature, and assayed against
51Cr-labeled CHO cells in a 4-h cytotoxicity assay.
Ly-49D-mediated DAP12 phosphorylation
After each population was chilled on ice for 5 min, NK cells
(3x106) were combined with CHO cells
(1.5x106) in 1.5 ml microcentrifuge tubes in a total of
100 µl RPMI 1640 + 0.5% bovine serum albumin (BSA). The
cells were centrifuged for 5 sec at 5000 rpm at 4°C. Cell
combinations were lysed immediately in ice-cold 1% TX-100 (0') or
incubated at 37°C for the indicated times and then lysed. Cell
lysates were cleared by centrifugation and immunoprecipitated with mAb
4E5 plus protein G sepharose. Proteins were separated by
electrophoresis, transferred to immobilon, and blotted with
antiphosphotyrosine mAb (Bio 4G10) [9
]. A rabbit
antiserum prepared against the DAP12 peptide (TB5/20) was used to
confirm the presence of DAP12 [11
].
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RESULTS
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Ly-49G inhibits Ly-49D-mediated DAP12 phosphorylation by CHO cells
Recently, our laboratory has described the phosphorylation of
Ly-49D-associated DAP12 when
Ly-49D+G-A- NK cells are combined
with YB20 target cells transfected with H-2Dd
(YB/Dd) [12
]. Phosphorylation of DAP12 could
not be detected upon immunoprecipitation with mAb 4E5 and required the
use of rabbit antisera prepared against Ly-49D/DAP12 immune complexes.
In this study, DAP12 phosphorylation was observed after a 1-min
incubation of Ly-49D+G-A- NK
cells and YB/Dd targets, after which it was no longer
detectable. Furthermore, Ly-49D+G+ cells did
not respond to YB/Dd targets with Ly-49D-associated DAP12
phosphorylation, consistent with the inhibitory role of Ly-49G for
H-2Dd. Here, we examined the ability of CHO cells to
mediate phosphorylation of Ly-49D-associated DAP12 using similar NK
cell subsets. Figure 1
demonstrates that CHO cells are potent mediators of
Ly-49D-associated DAP12 phosphorylation as compared with our previous
studies using the YB/H-2Dd-transfected cell line. The
following observations suggest that Ly-49D interacts strongly with CHO
cells as compared with our previous studies using
H-2Dd-transfected YB20 target cells: (1) Phosphorylation of
DAP12 is prolonged significantly up to 20 min following CHO cell
stimulation. (2) Much lower numbers of NK cells can be used to
demonstrate DAP12 phosphorylation. (3) DAP12 phosphorylation can be
detected using mAb 4E5. (4) DAP12 phosphorylation can be demonstrated
in the Ly-49D+G+ subset of NK cells, although
with a somewhat abbreviated response. However, because phosphorylation
of DAP12 in the Ly-49D+G+ cells was attenuated
when compared with the Ly49D+G-A-
cells and no longer observed after 20 min of incubation, we examined
Ly-49G for its ability to inhibit NK cell lysis and IFN- secretion
in the presence of CHO cells.
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Figure 1. DAP12 phosphorylation in response to CHO cells.
Ly-49D+G-A- and
Ly-49D+G+ B6 NK cells were combined with CHO
cells for the indicated times at 37°C and examined for
phosphorylation of Ly- 49D-associated DAP12 upon immunoprecipitation
with mAb 4E5. These data are representative of at least three such
experiments performed.
|
|
Blocking Ly-49G augments lysis of CHO cells
Figure 2
confirms the data of Idris et al. [13
]
that NK subsets containing Ly-49D+ cells apparently contain
most of the lytic activity against CHO cells. The
Ly-49D+G-A- subset and the
Ly-49D+G+ subset lysed CHO cells, and the
Ly-49G+D- subset did not lyse these targets.
However, because the lysis of CHO cells by the
Ly-49D+G+ subset was much less efficient than
the Ly-49D+G-A- subset, Ly-49G
could recognize a ligand on CHO cells as inhibitory. The addition of
antibodies to Ly-49G resulted in a three-fold increase in lysis of CHO
cells by the Ly49D+G+ subset and a 20-fold
increase in lysis by the Ly- 49G+D- subset.
This augmentation of lysis by antibody suggest strongly that Ly-49G
recognizes a ligand on CHO cells as inhibitory and that blocking this
inhibitory receptor/ligand interaction allows lysis to occur. As
demonstrated previously, antibodies to Ly-49D blocked lysis of CHO
cells by both Ly-49D+ subsets, whereas PK-136, which binds
to NKR-PIC, had no effect on lysis by any subset. Therefore, Ly-49D and
G appear to recognize CHO cells, consistent with their common
recognition of H-2Dd.
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Figure 2. Lysis of CHO cells by Ly-49D vs. G subsets. B6 NK cell subsets were
pretreated with the indicated mAb and tested for their ability to lyse
CHO cells in a 4 h 51Cr-release assay. Lytic unit
calculations at 30% lysis per 107 cells are presented.
These data are representative of at least three experiments
performed.
|
|
Although the percentage of Ly-49A+ NK cells in the
Ly-49D+G+ and
Ly-49G+D- subsets was low (27% and 40%,
respectively), addition of antibodies to Ly-49A resulted in some
increase in lysis of CHO cells. The augmented lysis observed with
antibodies to Ly-49G was increased further when combined with
antibodies to Ly-49A (unpublished results). Because the percentage of
Ly-49A+D-G- cells in bulk NK cell
populations is very low, sorting these cells was not performed to
confirm that recognition of CHO cells by Ly-49A occurs. Of particular
concern in these assays was the possibility that a small population of
Ly-49D+ cells remained in our
Ly-49G+D- subset (usually 2–3%), which,
potentially, could account for lysis of CHO cells in this population.
Our best efforts to remove Ly-49D+ cells from the
Ly-49G+ population resulted in a Ly-49G+ subset
containing 1% Ly-49D+ cells. As seen in Figure 3
, this population of NK cells appeared to have little, if any,
innate ability to lyse CHO cells. However, upon addition of antibodies
to Ly-49G, CHO cell lysis was enhanced greatly. Furthermore, addition
of antibodies to Ly-49G and D resulted in substantial lysis of CHO
cells, although almost 50% of this restored ability to lyse CHO cells
appeared to be mediated by the Ly-49D+ cells present. Our
results suggest strongly that Ly-49G+D- NK
cells not only recognize CHO cells as inhibitory but express receptors
other than Ly-49D that can recognize these targets and deliver
activation signals to NK cells.
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Figure 3. Lysis of CHO cells by purified Ly-49G+D- NK
cells. Ly-49G+D- NK cells containing only 1%
Ly-49D+ cells were assayed against CHO cells as in Figure 1
.
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Blocking Ly-49G augments IFN- secretion
To confirm the lytic data, CHO cells were combined with NK cell
subsets and examined for secretion of IFN- in the presence or
absence of mAb to Ly-49D and/or Ly-49G. Figure 4
demonstrates that
Ly-49D+G-A- cells secrete large
amounts of IFN- when cocultured with CHO cells, and the addition of
antibodies to Ly-49D inhibited IFN- secretion. However, the
production of IFN- by the Ly-49D+G+ and
Ly-49G+D- subsets was much lower in the
presence of CHO cells. These data correlated well with our cytotoxicity
data, suggesting that Ly49G may recognize a ligand on CHO cells.
Addition of antibodies to Ly-49G resulted in a four-fold increase in
IFN- secretion by the Ly-49D+G+ subset and a
10-fold increase in IFN- secretion by the
Ly-49G+D- cells. These results complemented
those of our cytotoxicity assays and further support the fact that
Ly-49G recognizes a ligand on CHO cells as inhibitory.
|
DISCUSSION
|
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Our findings demonstrate that Ly-49G (and possibly Ly-49A)
recognize CHO cells and deliver inhibitory signals to NK cells. These
results have been confirmed using three different assays: 1) augmented
lysis of CHO cells in the presence of antibodies to Ly-49G; 2)
augmented IFN- secretion in the presence of antibodies to Ly-49G;
and 3) attenuated levels of Ly-49D-associated DAP12 phosphorylation in
Ly-49D+G+ NK cells when combined with CHO
cells.
It has been shown that Ly-49D can recognize H-2Dd,
Dr, and Dsp2 [8
,
9
]. The findings of Idris et al.
[13
] and this study demonstrate that CHO cells are
potently lysed by Ly-49D+ NK cells. The ligand recognized
by Ly-49D on CHO cells is speculated to be hamster class I protein.
Therefore, the multiple target specificity of Ly-49D leaves open the
question as to the physiological ligand for this receptor.
The observation that Ly-49D+ NK cells from B6 mice are
responsible for recognition and lysis of CHO cells, and therefore
represent the Chok locus, was significant in that it
identified a unique NK cell-activating receptor that could mediate the
rejection of a specific tumor-target cell [13
,
15
]. Although blocking of Ly-49D-mediated lysis of CHO
cells with mAb 4E4 greatly reduced the lytic potential of
Ly-49D+ NK cells in this study, it did not abrogate killing
completely. This residual lysis could reflect simply a technical
limitation in the ability to totally block lysis but probably
represents additional activating receptors that recognize CHO cells
(e.g., Ly-49H). The hypothesis that B6 NK cells contain activating
receptors that recognize CHO cells other than Ly-49D is supported by
our data with the Ly-49G+D- subset of NK
cells. Ly-49G+D- NK cells can lyse CHO cells
effectively in the presence of antibodies to Ly-49G and secrete
significant amounts of IFN-. Furthermore, experiments with BALB/c NK
(Ly-49D-) cells did not demonstrate lysis of CHO cells
upon addition of antibodies to Ly-49G when bulk populations of NK cells
were used (unpublished results). Comparable NK cell populations from B6
mice did mediate lysis of CHO cells in the presence of this antibody.
These results support the findings of Idris et al.
[13
] that Ly-49D+ NK cells from B6 mice do
lyse CHO cells and also demonstrate that other Ly-49 receptors,
specifically Ly-49G and A, recognize these cells. In addition, our data
suggest that additional receptors, which have the potential to lyse CHO
cells, exist on B6 NK cells. Whether Ly-49A, G, and D recognize the
same ligand on CHO cells or separate ligands remains to be resolved.
|
ACKNOWLEDGEMENTS
|
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This project has been funded in whole or in part with federal funds
from the National Cancer Institute, National Institutes of Health,
under Contract No. NO1-CO-56000. 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, 1995). 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. The author
thanks Drs. Dan McVicar, Steve Anderson, John Ortaldo, and Andrew
Makrigiannis for their critical review of this manuscript.
|
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ABSTRACT
INTRODUCTION
