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*
Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235; and
Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many patterns of NK-mediated rejection can be explained by the missing self hypothesis, which predicts the rejection of allogeneic or parental BMC grafts that do not express the full complement of class I molecules present on host cells 20 . Thus, F1 hybrids reject parental BMC, and class I normal hosts reject class I-deficient BMC 21, 22, 23 . H2-Dd transgenic D8 mice reject nontransgenic (but otherwise syngeneic) B6 BMC grafts in an NK-mediated fashion, demonstrating that lack of a single class I molecule expressed by the host but not by the target cells can result in susceptibility to rejection 24, 25, 26 . In vitro, Ly49C/I-expressing F1 (H2d/b) NK cells lyse BALB/c (H2d) lymphoblasts, but lyse B6 (H2b) lymphoblasts only if the interaction between Ly49C/I and H2Kb is interrupted by F(ab')2 of Abs against either of these two molecules 27 . This suggests that the inability of a target cell class I to interact with inhibitory Ly49 receptors expressed on a particular NK subset can account for rejection on the basis of the missing self hypothesis.
The missing self hypothesis cannot, however, account for all patterns of NK-mediated rejection. Of particular interest is the fact that B6 host NK cells reject D8 BMC grafts 25, 26, 28 . Since D8 BMC are not missing any class I molecules that B6 considers self, this result strongly suggests that B6 NK cells can positively recognize the allogeneic H2-Dd molecule. Based on the patterns of marrow graft rejection, it appears that Dd shares with Dr and Ld a cross-reactive antigenic motif that can be positively recognized by B6 NK cells 28 . Of additional interest, the rejection of D8 BMC can be reversed by administration of anti-Ly49D/A 12A8 mAb and its F(ab')2 to B6 hosts, suggesting that the Ly49D+ subset is responsible for the positive allorecognition of Dd 29 . In this report we have studied the alloreactivity of the Ly49D+ and Ly49D- subsets from B6 hosts in an in vitro cytotoxicity assay.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice were bred and maintained in the Microbiology Colony at
the University of Texas Southwestern Medical Center (Dallas, TX).
Derivation of the Dd transgenic D8 strain from C57BL/6 mice
and of class I-deficient TAP-/- mutant mice has been
described previously 30, 31 . The haplotypes of the various strains
used are given in Table I
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The unconjugated and biotinylated 4E5 (anti-Ly49D) and
FITC-conjugated 4D11 (anti-Ly49G2) mAbs as well as the 4D11 salt
cut and 12A8 mAbs were prepared as previously described 16, 17, 32 .
Biotinylated 4LO3311 (anti-Ly49C) was a gift from Dr. Suzanne
Lemieux (University of Quebec, Laval, Canada). FITC-conjugated
anti-NK1.1 and biotin-conjugated anti-Ly49A and 5E6
(anti-Ly49C/I) were purchased from PharMingen (San Diego, CA). The
anti-H2-Dd mAb, which is specific for the
12 domain 33 was derived from hybridoma
34-5-8S purchased from American Type Culture Collection (Manassas, VA;
catalog no. HB 102). The 5E6 mAb was derived as previously described
27 . The supernatants from hybridoma cells grown in serum-free medium
(HyClone, Logan, UT) were purified by affinity chromatography using
Affigel protein A-agarose (Bio-Rad, Hercules, CA) for mouse 34-5-8S and
5E6, or protein G-Sepharose 4 Fast Flow (Pharmacia LKB Biotechnology,
Piscataway, NJ) for rat 12A8 according to the instructions of the
manufacturer. F(ab')2 reagents were then generated.
Briefly, the purified mAbs were dialyzed twice against 0.5x PBS
buffer. Pilot digestions using pepsin (Sigma, St. Louis, MO) dissolved
in 200 mM sodium citrate buffer (pH 3.5) at a 1/40 pepsin to mAb ratio
were performed at 37°C for each Ab preparation to determine the
optimal length of digestion. F(ab')2 were then dialyzed
against PBS. The efficiency of digestion was checked by 4–20%
gradient SDS-PAGE.
Enrichment of splenic NK cells
Single cell suspensions of splenocytes were prepared aseptically
in complete RPMI 1640 (10% FBS, 100 U/ml streptomycin, 100 µg/ml
penicillin, 1 mM sodium pyruvate, 2 mM L-glutamine, and 0.1
mM nonessential amino acids) by gently crushing spleens between the
frosted edges of two glass slides. The cells were then washed,
resuspended at 50 x 106 cells/ml in PBS containing
2% FBS (PBS/FBS), and incubated with 5 µg/ml anti-FcR
III
(2.4G2) mAb to block the FcR. After washing, the cells were resuspended
at 60–100 x 106 cells/ml in PBS/FBS, then incubated
with StemSep murine NK enrichment mixture containing mAbs CD5, CD22,
Gr-1, and TER-119 (Stem Cell Technologies, Vancouver, Canada). After
washing and resuspending in PBS/FBS at the same concentration, the
cells were incubated with StemSep anti-biotin tetramer, then
incubated with magnetic colloid. All incubations were performed for 15
min at 4°C. The cells were then filtered onto a PBS-washed StemSep
0.6-in. column placed inside a VarioMACS magnetic field (Miltenyi
Biotec, Auburn, CA). The cells collected in the flow-through typically
stained 50–70% NK1.1 positive. The cells were then washed and
resuspended at 3 x 106 cells/ml in complete DMEM
supplemented with 2.25 x 10-5 M 2-ME and 500 U/ml
recombinant human IL-2 (Chiron, Wapole, MA) and were cultured overnight
in a 24-well plate at 37°C in a 10% CO2/air mixture.
Cell sorting and generation of effector cells
The NK-enriched cells described above were harvested, washed, then resuspended at 30 x 106 cells/ml in PBS/FBS. The FcR was blocked as described. Without washing, the cells were incubated with a 1/1000 dilution of biotinylated anti-Ly49D-specific 4E5 mAb. After washing, the cells were resuspended and incubated with 1 µg/ml PE-conjugated streptavidin (PharMingen, San Diego, CA) alone or with a 1/1000 dilution of FITC-conjugated 4D11. For generation of Ly49D+Ly49A/C/G2/I- and Ly49D+Ly49A/C/G2 and/or I+ subsets, cells were incubated with FcR block as described. Without washing, the cells were incubated with 5 µg/ml 5E6, 5 µg/ml anti-Ly49A, 8 µg/ml 4LO3311, and a 1/100 dilution of 4D11, all biotinylated. The cells were then washed and incubated with 1 µg/ml Red 670-conjugated streptavidin (Life Technologies, Gaithersburg, MD) and a 1/500 dilution of PE-conjugated 4E5. All incubations were performed for 15 min at 4°C. After washing, cells with forward and side scatter characteristics of lymphocytes were sorted on the FACStar Plus device (Becton Dickinson, Mountain View, CA). The recovered cells were cultured at 2.5 x 105 cells/ml and 5 x 104 cells/well in 96-well U-bottom plates in complete DMEM supplemented with 2.25 x 10-5 M 2-ME and 500 U/ml human rIL-2 for 5 days at 37°C in a 10% CO2/air mixture.
Generation of target cells
Splenocytes were prepared as described and resuspended at 4–6 x 106 cells/ml in complete RPMI 1640 with 6 µg/ml Con A (Sigma). The cells were cultured 2 ml/well in 24-well plates for 2 days at 37°C in a 5% CO2/air mixture. The cells were then harvested, washed once, resuspended in 2 ml complete RPMI 1640, layered on top of 4 ml of Ficoll-Hypaque solution (Pharmacia LKB Biotechnology), and centrifuged at room temperature for 20 min at 1300 x g. The buffy coat containing viable lymphoblasts was removed and washed once in complete RPMI 1640 before radiolabeling.
Cytotoxicity assay
Target Con A lymphoblasts (1.5–2 x 106) were
incubated for 1.5 h at 37°C in a total volume of 0.6 ml with
150–250 µCi of sodium chromate (51Cr; Amersham,
Arlington Heights, IL). Radiolabeled cells were washed once,
resuspended in 5 ml of complete RPMI 1640, and incubated an additional
1 h at 37°C. The cells were washed twice, and diluted to 500
targets/100 µl of medium. Effectors at constant or variable E:T cell
ratios in a final volume of 100 µl were added first to the wells of
96-well V-bottom plates. An identical volume of targets, except as
noted below, was added to the appropriate wells. The coincubation was
performed in triplicate groups at 37°C in a 5% CO2/air
mixture. Target cells were preincubated with mAb 34-5-8S
(anti-12 domain-specific
H2-Dd) F(ab')2 at 4°C for 15 min, while
effector cells were preincubated with 4E5 salt cut, 4D11 salt cut, 5E6
F(ab')2, and/or 12A8 F(ab')2 for 30–60 min at
37°C. For the cold target competition studies, unlabeled Con A
lymphoblasts were added to the plated effector cells in a volume of 50
µl before the addition of labeled (hot) D8 targets at various cold
target to hot target ratios. The 500 hot D8 targets were added in a
total volume of 50 µl of complete RPMI 1640. After 4 h of
incubation, 100 µl of supernatant was removed, and 51Cr
radioactivity was measured in a liquid scintillation counter. Specific
lysis, represented as the mean ± SEM, was calculated as follows:
percent specific lysis = 51Cr cpm [(ER -
SR)/(MR - SR)] x 100, where ER is the experimental
51Cr release in the presence of effector cells, SR is the
spontaneous 51Cr release in the presence of medium, and MR
is the maximum 51Cr release in the presence of 1.0% Triton
X-100. The percent inhibition of lysis is calculated as follows:
[(percent specific lysis of D8 hot target alone) - (percent specific
lysis of D8 target in the presence of cold targets)]/(percent specific
lysis of D8 hot target alone). Each cytotoxicity assay was performed at
least three times.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 12A8+ NK cell subset from B6 recipients reject
BALB/c (H2d) and D8 (H2b, Dd) BMC
grafts, and blocking experiments suggest that the 12A8 receptor
delivers positive signals from H2d, especially
Dd, in vivo 29 . mAb 12A8 reacts with at least two
receptors, Ly49D and Ly49A 16 . Since Ly49A is a known inhibitory
receptor for Dd 12 , the in vivo data imply that the Ly49D
receptor sends positive signals to host NK cells, resulting in the
rejection of H2-Dd+ BMC grafts. Another Ly49D-reactive mAb,
4E5, does not cross-react with Ly49A 17 . Two-color FACScan analysis
of magnetically enriched day 1 cultured splenocytes using
anti-NK1.1 and 4E5 revealed that approximately 50% of B10 NK cells
express the 4E5 Ag (Fig. 1A).
Based on the previous in vivo data, we hypothesized that the
4E5+ subset is responsible for the rejection of
H2-Dd+ BMC and lysis of H2d targets in vitro.
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If Ly49D delivers activating signals to NK cells as a result of
interactions with specific H2d class I ligands expressed on
the surface of target cells, we postulated that Ly49D+ NK
cells would lyse H2d target cells more efficiently than
Ly49D- cells. To test this hypothesis, freshly purified B6
(H2b) NK cells were sorted into 4E5+ and
4E5- subsets, cultured in IL-2, then tested for their
ability to lyse 51Cr-labeled B6, B10.D2, and B10.BR Con A
lymphoblasts. These three targets share a similar background but differ
at the MHC region. The results shown in Fig. 1
, B and
C, demonstrate that 4E5+ NK cells lyse B10.D2
(H2d) targets significantly more efficiently than
4E5- NK cells do. The increased cytolytic activity against
these targets does not reflect a general increase in lytic potential of
4E5+ NK cells, since neither the syngeneic B6 target nor
the allogeneic B10.BR (H2k) targets are lysed by this
subset compared with the 4E5- subset. Furthermore,
4E5- NK cells lyse target cells deficient in the
transporter associated with Ag processing (TAP-/-) as
efficiently as 4E5+ NK cells do, suggesting that the
4E5- subset has intact lytic machinery. This result also
demonstrates that while NK cells may recognize MHC molecules, the
presence of class I is not required for NK-mediated lysis. It is
presumed that the efficient lysis of H2-identical TAP-/-
targets is due to undefined triggering structures on Ly49D+
or Ly49D- NK cells in the absence of any negative signals
from the target cells. Thus, these data show that the
Ly49D+ subset specifically lyses H2d targets in
vitro.
Dr+ and Dsp2+ targets compete for lysis of the H2-Dd+ (D8) target by the 4E5+ subset
Previous in vivo data suggest that Dd, Dr,
and Ld share a cross-reactive antigenic motif that is
positively recognized by B6 NK cells 28 . Since the Ly49D+
cells are involved in the rejection of D8 BMC grafts by B6 hosts 29 ,
we predicted that Ly49D+ NK cells would lyse D8
lymphoblasts and that Dr and Ld might also be
stimulatory ligands for 4E5+ NK cells. To test this
possibility, we assayed the ability of a panel of unlabeled cold Con A
lymphoblasts to inhibit the lysis of 51Cr-labeled D8
targets by B6 4E5+ NK cells. Cold targets susceptible to
lysis by Ly49D+ cells would be expected to inhibit
cytolysis of the D8 target more efficiently than cold targets resistant
to lysis. D8 target cells were lysed efficiently (30%) at an E:T cell
ratio of 15:1 (data not shown). Unlabeled TAP-/- blasts,
previously shown to be susceptible to lysis by Ly49D+ NK
cells, fully compete for lysis of the hot D8 target (data not shown).
Unlabeled H2-Dd+ targets, such as B10.D2 and D8 Con A
lymphoblasts, also effectively compete for lysis of the D8 target,
while syngeneic B6 targets do not (Fig. 2). IntraH2 recombinants B6.R4
(Kb, Dr) and B10.R40 (Kb,
Dsp2) both effectively inhibit lysis of D8 targets by
4E5+ B6 NK cells, suggesting that Dr and
Dsp2 may also serve as ligands for positive signaling to
this subset. H2k (C3H) and Ld transgenic
H2k (C3H.Ld) targets did not compete for lysis
of D8, suggesting that H2k- and H2Ld-expressing
targets are not positively recognized by the 4E5+ subset.
The failure of C3H.Ld lymphoblasts to compete with the
lysis of the D8 target was unexpected 28 . This observation is
discussed below.
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In vivo administration of F(ab')2 of the
anti-Ly49D mAb 12A8 reversed rejection of H2-Dd+ BMC
grafts by B6 hosts 29 . This suggests that the Ly49D molecule is
involved in the recognition of H2-Dd. The previous data
presented here demonstrate that Ly49D+ cells specifically
recognize H2-Dd+, Dr+, and Dsp2+
targets in vitro (Fig. 2). To test the prediction that the Ly49D
receptor delivers stimulatory signals to NK cells upon interaction with
H2-Dd, Dr, or Dsp2 in vitro, we
preincubated 4E5+ effector NK cells with anti-Ly49D/A
12A8 F(ab')2 to block the interaction between Ly49D and its
target cell ligands (Fig. 3).
Preincubation with 12A8 F(ab')2 significantly reduced the
cytotoxic activity of 4E5+ NK cells against B10.D2, D8,
B6.R4, and B10.R40 targets. Such preincubation resulted in a similar
level of lysis of these targets as seen with 4E5-
effectors. Because the 12A8 mAb recognizes not only Ly49D but also
Ly49A, it could be argued that corecognition of the Ly49A+
subset within the Ly49D+ population may have influenced the
results. To remove this variable, a similar experiment was performed
with the Ly49D-specific 4E5 mAb (Table II). Preincubation of 4E5+
cells with the Ly49D-specific 4E5 mAb also reduced the lysis of D8
targets to the same extent that 12A8 F(ab')2 preincubation
did, while such preincubation did not affect the lysis of
TAP-/- lymphoblasts (Table II). This result also
demonstrates that 4E5 mAb or 12A8 F(ab')2 preincubation was
not cytotoxic to the effectors, because such treatment had no effect on
the lysis of syngeneic B6 or NK-sensitive TAP-/- targets.
Taken together, these data suggest that preincubation of effector cells
with an anti-Ly49D mAb interrupts the interaction between Ly49D and
specific target cell ligands, resulting in a blockage in the delivery
of positive signals. The significant reduction in the lysis of D8,
B6.R4, and B10.R40 target cell by 12A8-preincubated 4E5+
cells strongly suggests that Ly49D stimulates lysis upon interaction
with Dd, Dr, and Dsp2. The
differential sensitivity of the H2-Dd+ targets (B10.D2 and
D8) to lysis by these subsets will be addressed in later experiments
and in Discussion.
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Because it has been reported that negative signals often dominate
positive signals delivered to NK cells, it is likely that the subset
responsible for the lysis or rejection of Dd+ cells in vivo
receives only positive signals from the target cell alloantigen. Thus,
to detect lysis of H2d targets, there should exist a subset
of B6 Ly49D+ cells that does not coexpress Ly49G2 or Ly49A,
both of which are known to inhibit NK cytolysis upon interaction with
Dd. Two-color FACScan analysis of NK-enriched B6
splenocytes using 4E5 and anti-Ly49G2 (4D11) identifies two
partially overlapping subsets of NK cells (Fig. 4A). We predicted that the
4E5+4D11- subset would preferentially lyse
targets that expressed the Dd alloantigen.
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Approximately 50% of B6 Ly49D+ NK cells coexpress
Ly49G2, a known inhibitory receptor for class I Ags, in particular
Dd. Since negative signaling often dominates positive
signaling to NK cells, we hypothesized that targets capable of
delivering negative signals through Ly49G2 would be lysed by the
Ly49D+G2- subset. By contrast, targets
incapable of delivering such negative signals would be lysed equally
well by both Ly49G2- and Ly49G2+ subsets of
Ly49D+ cells. To test these predictions, we sorted B6 NK
cells into 4E5+4D11+ and
4E5+4D11- subsets and tested their ability to
lyse Con A lymphoblast targets (Fig. 4B). Ly49D+
cells that coexpress Ly49G2 were unable to lyse H2-Dd+
B10.D2 or D8 blasts, while Ly49D+G2- cells
efficiently lysed these targets. In keeping with the latter, only 25%
of Ly49D+G2- cells coexpressed Ly49A (data not
shown). Similarly, Dr+ B6.R4 targets were lysed
preferentially by the 4D11- subset of Ly49D+
effector cells. Thus, the presence of Ly49G2 abrogates the ability of
Ly49D+ NK cells to lyse Dd+ and Dr+
targets. B10.R40 (Kb, Dsp2) targets were lysed
by both subsets, demonstrating that Ly49G2 is not a negative signaling
receptor specific for H2-Dsp2. Although in Fig. 4B a difference is seen in the susceptibility of the B10.R40
target, most experiments do not show this difference. Neither of the
H2k-expressing targets (C3H and C3H.Ld) was
lysed by either subset. Taken together, these data show that
coexpression of the negative signaling Ly49G2 receptor on
Ly49D+ cells impairs the ability to lyse Dd+
and Dr+, but not Dsp2+, targets. It follows
that Ly49G2 is an inhibitory receptor for Dd and
Dr, but not Dsp2. It may be noted that while
Ly49D+G2- cells lysed the two
H2-Dd+ targets (B10.D2 and D8) quite efficiently, the level
of lysis of the D8 target was somewhat lower. This difference most
likely relates to the expression of H2b on D8, but not
B10.D2, cells. If the Ly49D+G2- cells express
negative signaling receptors for H2b, they will be
inhibited by D8, but not B10.D2, targets. That this is indeed the case
will be presented below.
Positive signaling to the 4E5+4D11- subset
Previous experiments demonstrated that blockage of the Ly49D
receptor with mAb 12A8 prevented lysis of targets normally susceptible
to killing by 4E5+ NK cells. Since the 4E5 subset that is
responsible for the lysis of these targets does not coexpress Ly49G2
(4E5+4D11-), we predicted that preincubating
this subset with 12A8 F(ab')2 would inhibit lysis of
targets that express positive signaling ligands for Ly49D. The ability
of 4E5+4D11- B6 NK cells to lyse D8, B6.R4,
and B10.R40 was abolished when the effectors were preincubated with
12A8 F(ab')2 (Fig. 5A). Such treatment also
greatly reduced the lysis of B10.D2 cells. B6, C3H, and
C3H.Ld targets were not lysed in the absence of blocking
reagent, and effector preincubation with 12A8 F(ab')2 had
no effect on the lysis of the NK-sensitive TAP-/- target.
These data suggest that Dd, Dr, and
Dsp2 specifically deliver positive signals to
4E5+4D11- B6 NK cells, resulting in target
cell lysis.
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Unlike their Ly49G2- counterparts,
Ly49D+G2+ NK cells do not lyse Dd+
or Dr+ targets efficiently (Fig. 4B). If target
cells express inhibitory ligands for Ly49G2 in addition to stimulatory
ligands for Ly49D, the net signaling outcome might result in inhibition
of target cell lysis. Alternatively, coexpression of Ly49G2 may render
Ly49D nonfunctional. To distinguish between these two possibilities,
4E5+4D11+ NK cells were tested for their
abilities to lyse a panel of lymphoblast targets in the presence or the
absence of 12A8 F(ab')2 and/or 4D11 mAbs (Fig. 5B). The resistance of H2-Dd+ targets (B10.D2
and D8) to lysis by this subset was reversed by preincubating the
effector cells with anti-Ly49G2 4D11 Ab. Cytolysis of B6 targets
was not enhanced by such effector treatment, demonstrating that 4D11
preincubation does not nonspecifically activate the lytic machinery of
B6 NK cells. Furthermore, when 4D11-blocked effectors were preincubated
with 12A8 F(ab')2, the boosted lysis of Dd+
targets was reduced, demonstrating that Ly49D function is intact in
this subset. However, the lysis of the B10.D2 target boosted in the
presence of 4D11 is not completely abrogated even when the effectors
are preincubated with 12A8 F(ab')2. This suggests that
there might exist other unidentified non-Ly49D-activating receptors on
Ly49D+G2+ NK cells. In addition, B6.R4
(Kb, Dr) targets were lysed by
Ly49D+G2+ effectors only in the presence of
4D11 Ab, suggesting that interactions between Dr and Ly49G2
result in inhibition of NK-mediated lysis. 12A8 F(ab')2
preincubation abrogated the boosted lysis of B6.R4, again demonstrating
that functional interactions between Dr and Ly49D activate
lysis. This subset lysed B10.R40 (Kb, Dsp2)
targets equally well in the presence or the absence of 4D11; the lysis
was reduced if the effectors were preincubated with 12A8
F(ab')2, suggesting that neither Dsp2 nor
Kb delivers inhibitory signals to NK cells upon interaction
with Ly49G2, but that Dsp2 does stimulate lysis through
interaction with Ly49D. Conversely, C3H and C3H.Ld targets
were only lysed in the presence of mAb 4D11, and 12A8
F(ab')2 preincubation of the effectors did not inhibit
their lysis, suggesting that Ly49G2 delivers inhibitory signals, but
Ly49D does not deliver stimulatory signals, upon interaction with
H2k alloantigens. Overall, these data support the
conclusion that Ly49D can function in a subset that coexpresses Ly49G2,
and that inhibitory signals delivered from one Ly49 can dominate
stimulatory signals from another.
Anti-H2-Dd F(ab')2 block positive signals to Ly49D+ NK cells
The previous genetic data strongly suggest that Ly49D interacts
with the Dd alloantigen, resulting in stimulation of
NK-mediated lysis. However, it remains possible that the Dd
Ag serves as a source of peptide that is presented in the context of a
different class I, and this complex served as the stimulatory ligand
for Ly49D. This would be similar to the ligand for human CD94, which
can be HLA-E complexed with various class I leader peptides 34, 35, 36 .
To test these possibilities, we assessed the cytolytic ability of
4E5+4D11- B10 NK cells against Dd+
D8 target cells preincubated with 12
domain-specific anti-H2-Dd F(ab')2 (Fig. 6). D8 lymphoblasts were lysed
efficiently, and this lysis was diminished when the effectors were
preincubated with 12A8 F(ab')2 and/or the target cells were
preincubated with anti-H2-Dd F(ab')2.
Furthermore, the reduction in lysis of D8 cells was similar in all
three Ab-treated groups. In control experiments, D8, but not B6, Con A
lymphoblasts stained with the anti-Dd reagent (data not
shown), and as expected, preincubation of the B6 target with
anti-H2-Dd F(ab')2 had no effect on its
lysis by this subset. Taken together, these data strongly suggest that
Ly49D interacts with Dd to deliver stimulatory signals to
B6 NK cells, resulting in target cell lysis.
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Since we have shown that Ly49-mediated inhibitory interactions can
dominate Ly49D-mediated stimulatory interactions, it is likely that the
precise B6 subset responsible for the rejection of D8 stem cells in
vivo and the lysis of D8 targets in vitro does not coexpress inhibitory
receptors specific for H2-Dd (Ly49A or Ly49G2) or
H2b (Ly49C or Ly49I) class I molecules. Two-color FACScan
analysis of NK-enriched B6 splenocytes using 4E5 and a mixture of 5E6
(anti-Ly49C/I), 4LO3311 (anti-Ly49C), 4D11 (anti-Ly49G2),
and A1 (anti-Ly49A) reagents identified a 4% subset that expresses
Ly49D, but not Ly49A, C, G2, or I (data not shown). We predicted that
this small subset would be able to efficiently lyse D8 targets in
vitro. To test this prediction, we assayed the ability of
Ly49D+A/C/G2/I- sorted B6 NK cells to lyse Con
A lymphoblast targets. As shown in Fig. 7, Ly49D+ NK cells that do
not coexpress any of the known inhibitory Ly49 receptors for
H2-Dd or H2b class I Ags lyse B6 targets
poorly, but are able to lyse B10.D2, D8, and TAP-/-
targets efficiently. Preincubation of these effectors with 12A8
F(ab')2 reduced lysis of the B10.D2 and D8 targets but not
the TAP-/- targets, demonstrating that Ly49D delivers
positive signals upon interaction with the H2-Dd
alloantigen. It is instructive to compare this experiment with others
(Figs. 3, 4B, and 5) in which Ly49D+ or
Ly49D+G2- effectors were employed. Under the
latter conditions, the ability of Ly49D+ cells to lyse
H2-Dd+ targets was noted, but the lytic activity against
B10.D2 targets was always higher than that against D8 T cell blasts. In
this experiment (Fig. 7), when Ly49D+ cells were selected
in a manner so that they did not express any known negative signaling
receptors specific for H2-Dd (shared by B10.D2 and D8) or
H2b (expressed only on D8), the previously noted difference
in the lysis of B10.D2 and D8 disappeared. These data demonstrate that
the B6 NK subset responsible for lysis of the H2-Dd
transgenic D8 target expresses Ly49D and that the full lytic potential
of the Ly49D+ population is revealed only by selecting
cells that do not express known inhibitory receptors for class I
molecules expressed on target cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B). The boost
in lysis was not due to a stimulatory effect of the Ab, since the lysis
of other targets, such as B6, B10.R40, and TAP-/-, was
unaffected. These data demonstrate that Ly49G2 is probably an
inhibitory receptor not only for Dd and Ld as
previously reported, but also for Dr and H2k.
Ly49G2 does not appear to be a negative signaling receptor for
H2-Dsp2 or H2b class I Ags.
The data presented here argue that Ly49D has at least three class I
ligands, Dd, Dr, and Dsp2, and that
interaction with these ligands stimulates NK-mediated lysis. Firstly,
Ly49D+ cells specifically and preferentially lyse targets
that express these class I molecules (Fig. 3). Secondly, lysis of these
targets is significantly reduced in the presence of anti-Ly49D
reagents (Fig. 3). Thirdly, lymphoblasts bearing these class I Ags
compete for lysis of the Dd+ D8 target cells by
Ly49D+ effectors (Fig. 2). Although unlikely, our data
cannot rule out the possibility that Ly49D functions as a coreceptor
with another molecule that determines the class I specificity of
positive signaling. In fact, previous binding studies could not
demonstrate direct binding of Ly49D to Dd 38 . However,
recent studies suggest that Ly49D-expressing rat leukemia cells
specifically lyse H2-Dd-expressing target
cells.4 It seems, therefore,
that the previous binding data 38 may have failed to detect the
Ly49D-Dd interaction.
Although the in vitro cytotoxicity assay system has supported in vivo
models of class I-induced negative signaling to NK cells, transplant
models for positive allorecognition could not be reproduced in vitro.
B6 BMC grafts or lymphoblasts are rejected or lysed, respectively, by
NK cells of the D8 strain, which is a B6 mouse transgenic for the
Dd alloantigen, presumably because B6 cells do not express
all the self class I molecules capable of inhibiting all D8 NK cell
subsets 24, 25, 26, 39 . For a similar reason, H2d/b
F1 hybrid NK cells reject parental BMC grafts in
vivo and lyse parental lymphoblasts in vitro 21, 22, 23, 27 . Strikingly,
however, B6 host NK cells mediate rejection of D8 BMC, a finding that
is not consistent with the missing self hypothesis but instead argues
that NK cell receptors can be triggered by non-self MHC molecules 25, 26, 28 . Administration of anti-Ly49D/A mAbs or F(ab')2
reverses the rejection, suggesting that Ly49D mediates positive
recognition of the Dd alloantigen 29 . Surprisingly,
despite the fact that approximately 50% of B6 NK cells express Ly49D
(Fig. 1a), previous studies failed to show lysis of D8
targets by unseparated B6 lymphokine-activated killer cells (LAKs) 28, 40 . Although in vitro positive allorecognition is readily detectable
in the rat and human 41, 42, 43, 44, 45 , no such allorecognition has previously
been demonstrated in the murine system. We now provide clear evidence
for allorecognition by murine Ly49D molecules in an in vitro system.
This became possible by sorting subsets of NK cells.
Although we sorted for Ly49D using the Ly49D-specific 4E5 mAb,
approximately 20% of these 4E5+ cells coexpress Ly49A
(data not shown). Since the mAb 12A8 cross-reacts with Ly49D and Ly49A,
we must consider the possibility that preincubation of effectors with
12A8 F(ab')2 interrupts the signaling potential of both
receptors 16 . Since it is known that Ly49A interaction with
Dd results in negative signaling, 12A8 blockage might boost
the potential of Ly49A+ cells to lyse D8 targets. However,
in all cases, 12A8-preincubated Ly49D+ effectors could not
lyse D8 targets. It is possible that even if
Ly49A+D+ cells can no longer receive negative
signals from Dd, blockage of Ly49D prevents positive
signaling interactions with the D8 target. This is supported by the
fact that preincubating Ly49D+ cells with either the
Ly49D-specific 4E5 mAbs or the Ly49D/A-specific 12A8
F(ab')2 reduced lysis of the D8 target to the same extent
(Table II).
Previous transplant data from our laboratory demonstrated that
Dd shares a common antigenic motif with Ld and
possibly Dr that is positively recognized by B6 NK cells
28 . Preliminary experiments have shown that the rejection of
C3H.Ld (H2k, Ld) and B6.R4
(Kb, Dr) BMC grafts can be reversed by
administering 12A8 mAb to B6 hosts, suggesting that the
Ly49D+ subset is responsible for the positive recognition
of Dr and Ld in addition to Dd
(data not shown). Because B6 Ly49D+ NK cells lyse the B6.R4
target unless the effectors are preincubated with 12A8
F(ab')2, we propose that Ly49D is a positive signaling
receptor for Dr (Fig. 3). The inability of B6 NK cells to
lyse H2k-expressing targets is not surprising because
H2k BMC grafts are very resistant to NK-mediated rejection
46 . The finding that C3H and C3H.Ld are susceptible to
lysis by Ly49D+G2+ cells when the Ly49G2
receptor is blocked suggests that cells within the
Ly49D+G2+ subset coexpress a positive signaling
receptor specific for a ligand expressed on C3H and C3H.Ld
lymphoblasts, presumably H2k alloantigens. The inability of
12A8 F(ab')2 to inhibit lysis of either of these two
targets suggests that a distinct positive signaling receptor specific
for C3H Ags exists within the Ly49D+G2+ subset.
Because Ly49D+G2- cells do not lyse C3H or
C3H.Ld targets, this subset either does not express the
putative stimulatory receptor for H2k, or negative signals
from other inhibitory receptors prevent positive signaling. Our
results, therefore, do not support or rule out positive signaling from
Ld. We are currently backcrossing the Ld
transgene onto an H2b background to prevent the
complicating effects of H2k negative signaling to NK cell
subsets.
Another in vivo model suggests that B6 host NK cells positively
recognize H2-Dsp2-bearing BMC grafts. Backcrossing
H2sp2 onto the B10 background produced several intraH2
recombinant mice, including the B10.R40 (Kb,
Dsp2) strain 47 . B6 NK cells are responsible for the
vigorous rejection of B10.R40 BMC grafts, suggesting that the
Dsp2 epitope is positively recognized in vivo 48, 49 .
Furthermore, 12A8 administration reverses the rejection of B10.R40 BMC
grafts by B6 hosts, suggesting that the Ly49D+ subset is
responsible for the positive allorecognition of Dsp2 (data
not shown). In vitro, blocking the Ly49D receptor abolishes the
preferential lysis of B10.R40 lymphoblasts by the B6 Ly49D+
subset, regardless of Ly49G2 coexpression (Figs. 3 and 6). These data
support the conclusion that the rejection of B10.R40 BMC grafts by B6
hosts results from positive signaling interactions between Ly49D and
Dsp2.
While it is clear that Ly49D+ NK cells positively recognize
and efficiently lyse B10.D2, B6.R4, and B10.R40 targets in vitro, this
subset lyses the D8 target only slightly better than the syngeneic B6
target. This is not surprising, since, as opposed to the other three
susceptible targets, the D8 lymphoblasts express all the MHC class I
molecules that the B6 NK cell considers self. In fact, approximately
60% of this subset coexpresses Ly49C and/or Ly49I, which are negative
signaling receptors specific for H2b class I molecules
(data not shown). Additionally, approximately 20% of these
4E5+ cells coexpress Ly49A, and >50% coexpress Ly49G2,
the known negative signaling receptors specific for Dd
(Fig. 4A and data not shown). Coexpression of any one of
these inhibitory Ly49 receptors can prevent lysis of the D8 target
(Fig. 4B). Only 4% of 4E5+ cells do not
coexpress known inhibitory ligands specific for D8 class I Ags. This
subset efficiently lyses B10.D2, D8, and TAP-/- targets
to a comparable extent, demonstrating that this particular B6 NK cell
subset is responsible for the lysis or rejection of D8 cells (Fig. 7).
By virtue of the fact that we sorted for expression of Ly49D and lack of expression of all other Ly49 receptors for which reagents are currently available, we can only hypothesize what other receptors are expressed by this subset. It is possible that other H2b-specific inhibitory receptors are expressed to ensure self tolerance. If this is the case, then the susceptibility of the D8 target to lysis by this subset implies that Ly49D-mediated positive signaling dominates in this instance. Alternatively, this subset may not express any H2b-specific inhibitory or stimulatory receptors. Self tolerance in this case would not require self class I-specific inhibition, and the alloantigenic class I transgenic target is susceptible because it expresses a stimulatory ligand (Dd) but no inhibitory ligand for receptors on the NK cell. Until more reagents become available, either explanation remains possible.
Thus, as previously observed 28, 40 , D8 target cells are resistant to
lysis by bulk B6 NK cells because the frequency of the single
Ly49D+ cells is very low. Selecting for Ly49D+
cells increases the frequency of these effectors to the point where
lysis of the D8 target is detectable but not impressive (Fig. 3).
Sorting away a major subset bearing inhibitory receptors specific for
the Dd transgene (Ly49D+G2-) again
increases the frequency of cells capable of lysing the D8 target, and
D8 targets are lysed to a moderate extent (Fig. 4B).
Finally, NK cells selected for expression of only the stimulatory
H2-Dd-specific Ly49D are able to lyse D8 targets as well as
B10.D2 and TAP-/- targets (Fig. 7). Thus, minor subsets
of NK cells can be responsible for the lysis of target cells that
differ from self only by the additional expression of a single class I
molecule.
The existence of functional class I-specific positive signaling Ly49 receptors within an NK cell repertoire adds a second layer of complexity to understanding the alloreactive specificity of a given NK cell. Thus, the resistance to NK-mediated lysis requires that the target cell either must express ligands for negative signaling receptors or must not express ligands for positive signaling receptors of the NK effector. Coexpression of ligands for both types of receptors also probably results in resistance to lysis. What function do class I-recognizing stimulatory receptors serve in physiological NK-mediated events? It is unlikely that the purpose of class I-specific stimulatory receptors is to facilitate rejection of allografts or lysis of allogeneic lymphoblasts. In fact, these types of receptors could theoretically jeopardize self tolerance. As originally suggested by Kärre 20 , the requirement for target cells to express ligands for class I inhibitory receptors provides a mechanism by which tumor cells or virally infected cells that evade T cell immunity by down-regulating or altering self class I may be rejected by NK cells. The selective loss of one negative signaling class I motif would allow rejection mediated by positive signaling receptors specific for a different self class I motif. Another, not mutually exclusive, possibility is inhibition of self NK cells requires stimulatory signals to phosphorylate the ITIM on inhibitory Ly49 receptors. Thus, normal self class I-expressing targets might cross-link both types of Ly49 receptors, resulting in SHP phosphatase recruitment and down-regulation of the activating kinase cascade. Abnormal targets unable to cross-link the inhibitory receptor would thus be susceptible to lysis. Presumably, generation of a self-tolerant NK cell repertoire requires that no NK cell should receive positive signals from self class I that does not coexpress another self-inhibited receptor. Whereas all NK subsets should be tolerant to self, some may not be tolerant to allogeneic cells. Thus, specific NK subsets are responsible for the lysis of allogeneic targets in vitro and for the rejection of BMC allografts in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Bennett, Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75235-9072.
3 Abbreviations used in this paper: BMC, bone marrow cell; ITIM, immunoreceptor tyrosine-based inhibitory motif; PE, phycoerythrin; LAKs, lymphokine-activated killer cells.
4 M. C. Nakamura, J. C. Ryan, and W. E. Seaman. Submitted for publication.
Received for publication August 25, 1998. Accepted for publication November 6, 1998.
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J. Liu, M. A. Morris, P. Nguyen, T. C. George, E. Koulich, W. C. Lai, J. D. Schatzle, V. Kumar, and M. Bennett Ly49I NK Cell Receptor Transgene Inhibition of Rejection of H2b Mouse Bone Marrow Transplants J. Immunol., February 15, 2000; 164(4): 1793 - 1799. [Abstract] [Full Text] [PDF]
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L. H. Mason, J. Willette-Brown, A. T. Mason, D. McVicar, and J. R. Ortaldo Interaction of Ly-49D+ NK Cells with H-2Dd Target Cells Leads to Dap-12 Phosphorylation and IFN-{gamma} Secretion J. Immunol., January 15, 2000; 164(2): 603 - 611. [Abstract] [Full Text] [PDF]
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J. R. Ortaldo, R. Winkler-Pickett, J. Willette-Brown, R. L. Wange, S. K. Anderson, G. J. Palumbo, L. H. Mason, and D. W. McVicar Structure/Function Relationship of Activating Ly-49D and Inhibitory Ly-49G2 NK Receptors ,2 J. Immunol., November 15, 1999; 163(10): 5269 - 5277. [Abstract] [Full Text] [PDF]
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M. C. Nakamura, C. Naper, E. C. Niemi, S. C. Spusta, B. Rolstad, G. W. Butcher, W. E. Seaman, and J. C. Ryan Natural Killing of Xenogeneic Cells Mediated by the Mouse Ly-49D Receptor J. Immunol., November 1, 1999; 163(9): 4694 - 4700. [Abstract] [Full Text] [PDF]
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D. H. Chung, J. Dorfman, D. Plaksin, K. Natarajan, I. M. Belyakov, R. Hunziker, J. A. Berzofsky, W. M. Yokoyama, M. G. Mage, and D. H. Margulies NK and CTL Recognition of a Single Chain H-2Dd Molecule: Distinct Sites of H-2Dd Interact with NK and TCR J. Immunol., October 1, 1999; 163(7): 3699 - 3708. [Abstract] [Full Text] [PDF]
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T. C. George, J. R. Ortaldo, S. Lemieux, V. Kumar, and M. Bennett Tolerance and Alloreactivity of the Ly49D Subset of Murine NK Cells J. Immunol., August 15, 1999; 163(4): 1859 - 1867. [Abstract] [Full Text] [PDF]
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 A. H. Idris, H. R. C. Smith, L. H. Mason, J. R. Ortaldo, A. A. Scalzo, and W. M. Yokoyama The natural killer gene complex genetic locus Chok encodes Ly-49D, a target recognition receptor that activates natural killing PNAS, May 25, 1999; 96(11): 6330 - 6335. [Abstract] [Full Text] [PDF]
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