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*
Department of Anatomy, University of Oslo, Oslo, Norway;
Veterans Administration Medical Center, University of California, San Francisco, CA 94121; and
Klinik für Abdominal und Transplantationschirurgie, Medizinische Hochscule, Hannover, Germany
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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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Less is known about the NK recognition system for class I in the rat than those in mice and man, and no rat MHC receptors have been identified to date. The existence of such receptors, however, can be inferred from functional studies of allogeneic lymphocyte cytotoxicity (ALC) (12, 13, 14, 15). Immunogenetic data show that rat NK cells can be both inhibited and activated by MHC molecules on target cells (15, 16, 17, 18). Here, we have exploited the observation that NK cells from the DA strain, while fully capable of killing xenogeneic lymphoblasts and tumor targets, are unable to lyse allogeneic leukocytes (19, 20). Recent evidence has suggested that this selective NK allorecognition defect correlates with the relative lack of Ly-49 transcripts in DA NK cells (6). In an attempt to raise mAb against MHC alloreceptors on rat NK cells, we immunized DA rats with alloreactivity-competent NK cells from MHC-matched PVG.1AV1 rats. Two mAb, STOK1 and STOK2, were obtained. They reacted with an NKC-encoded glycoprotein that has functional characteristics of an inhibitory MHC-binding receptor. These data, therefore, extend the observations of MHC receptor-mediated inhibitory regulation of NK cytotoxicity to the rat model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeding pairs from the AO, DA, PVG, PVG.1U, and PVG.1AV1 strains were obtained from Harlan Olac (Bicester, U.K.), whereas PVG.1L, PVG.1LV1, and PVG.1N were kindly provided by Dr. Geoffrey W. Butcher (Cambridge, U.K.). These strains were reared under conventional conditions in Oslo and screened for common rat pathogens. Individual F344, LEW, and BN rats were purchased directly from Møllegaard (Ejby Skensved, Denmark) and LOU/C from Harlan Olac (Bichester, U.K.).
Immunization and cell fusion
One DA (RT1av1)
rat was immunized twice s.c. with IL-2-activated NK cells
(106 and 5 x 106) from MHC-matched
PVG.1AV1 (RT1av1) rats, with an
interval of approximately 2 mo. The rat was boosted i.v. with 10
x 106 cells 3 days before fusion. Mononuclear spleen
cells, obtained by Lymphoprep centrifugation (Nycomed, Oslo, Norway),
were fused with NSO cells using Polyethylene Glycol 4000 (Merck,
Darmstadt, Germany; 1 g in 1.000 µl of H2O and 100
µl of DMSO). Supernatants obtained after culture in selection medium
(Iscove’s modified Dulbecco’s medium supplemented with 1 x
10-4 M hypoxanthine, 4 x 10-7 M
aminopterine, and 1.6 x 10-5 M thymidine) were
screened for binding to subsets of IL-2-activated PVG.1AV1 NK cells by
indirect immunofluorescence and flow cytometry, and mAb STOK1 and STOK2
were selected. They were both of the IgG2a, Ig 
isotype, as
determined by ELISA (93–9550; Zymed Laboratories, South San
Francisco, CA).
Generation of effector cells
IL-2-activated NK cells were generated from mononuclear spleen cells, depleted of T cells with the anti-CD3 mAb G4.18 and rabbit serum as a source of C, by positive selection of NKR-P1+ cells with M450 magnetic Dynabeads (Dynal, Oslo, Norway), as previously described (17). STOK2- cells were obtained from these cells by negative selection with sheep anti-rat IgG-coated magnetic Dynabeads, preincubated with STOK2 supernatant. Cell depletion was performed immediately before cytotoxicity assays, and the resulting populations were routinely not contaminated with STOK1- or STOK2-reactive cells as determined by flow cytometry (data not shown).
Cultures of STOK2+ NK cells were obtained from T cell-depleted mononuclear spleen cells (see above) by positive selection using streptavidin-coated M280 magnetic Dynabeads (Dynal), preincubated with STOK2-biotin. Selected STOK2+ cells were cultured in RPMI 1640 supplemented with 10% FCS, 1 mM glutamine, 1 mM Na pyruvate, 5 x 10-5 M 2-ME, antibiotics, and rat rIL-2 (obtained from the dialyzed cell culture supernatant of an IL-2 gene-transfected Chinese hamster ovary cell line) (21). After 1 to 2 wk in culture, the resultant cells were routinely approximately 90% STOK1+ and STOK2+.
Target cells and cytotoxicity assay
The generation of Con A-activated lymphoblasts as target cells and the 4-h 51Cr release assay were performed as previously described (16). In the Ab-blocking experiments, 20 µg of F(ab')2 of STOK2 or of serum IgG from nonimmunized rats (012-000-006, Jackson ImmunoResearch Laboratories, West Grove, PA) was added to the effector cells 20 min before adding the targets. The YAC-1, P815, and P388 cell lines were cultured in RPMI 1640 with 10% FCS and antibiotics. Spontaneous release was usually between 5 to 15% of the total cpm in the cells. The results are presented as median values from triplicate determinations for each E:T ratio.
Flow cytometry
Cells (50 µl, 0.2–2 x 107 cells/ml) were incubated with 50 µl of properly diluted Ab for 30 min on ice. After three washes, the cells were incubated with F(ab')2 of dichlorotriazinylaminofluorescein-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch). Nonviable cells, judged by their propidium iodide uptake, were excluded from analysis. Three-color staining of mononuclear spleen cells, depleted of Ig+ cells with sheep anti-rat IgG-coated magnetic Dynabeads (Dynal), was performed by a combination of FITC-conjugated 3.2.3 (anti-rat NKR-P1A (22), provided by Dr. J. C. Hiserodt (Pittsburgh, PA) and conjugated in our laboratory), phycoerythrin-conjugated G4.18 (anti-rat CD3 (23); PharMingen, San Diego, CA), and biotin-conjugated STOK1 or STOK2 (biotinylated according to standard procedures), followed by phycoerythrin-indodicarbocyanine (phycoerythrin-Cy5)-conjugated streptavidin (Dako, Glostrup, Denmark). The cells were analyzed on a Becton Dickinson FACScan (Mountain View, CA).
Immunoprecipitation and Western blotting
For surface labeling, cells were washed, resuspended in cold PBS, and incubated for 20 min on ice with 0.8 U of glucose oxidase (G 7141, Sigma Chemical Co., St. Louis, MO), 2.5 U of lactoperoxidase (L 2005, Sigma), 14 mM D-glucose, and 0.5 mCi of carrier-free 125I (Amersham, Aylesbury, U.K.). The cells were washed three times, suspended in cold lysis buffer (PBS, 1% bovine hemoglobin, 1 mM iodoacetamide, 1 mM PMSF, and aprotinin) containing 1% Nonidet P-40 or Triton X-100, and incubated for 60 min on ice. Lysates, obtained by 30-min microcentrifugation, were precleared with quenched Sepharose 4B beads, then immunoprecipitated with Sepharose 4B precoupled with STOK1, STOK2, or the isotype-matched control 2C7 for at least 3 h at 4°C. The coupling of cyanogen bromide-activated Sepharose 4B was performed at 5 mg of Ab/ml gel, according to the manufacturer’s instructions (17-0430-01, Pharmacia Biotech, Uppsala, Sweden). Precipitates were washed three times with buffer containing 0.1% Nonidet P-40 or Triton X-100, then once with 0.05 M Tris-buffer, pH 6.8, and finally resolved by 7.5–10% SDS-PAGE under reducing or nonreducing conditions. Gels were stained, fixed, and dried, and labeled protein was revealed by autoradiography. For Western blot analysis, unlabeled precipitates were transferred semidry to polyvinylidene difluoride membranes. The blots were blocked with PBS and 0.1% Tween-20 with 5% dry milk and incubated with 10 µg of purified STOK1, STOK2, or 2C7 control for 1 h at 4°C, followed by horseradish peroxidase-conjugated goat anti-rat IgG secondary Ab (112-035-008, Jackson ImmunoResearch), washed extensively with PBS and 0.1% Tween-20, and finally developed with enhanced chemiluminescence detection reagents (Amersham).
Deglycosylation
The 125I-labeled STOK2 precipitate was solubilized in 40 mM sodium phosphate, pH 6.0, with 1% Triton X-100 and 0.1% SDS, digested for 24 h at 37°C with 200 mU endoglycosidase F/N-glycosidase F (complemented with 20 mM EDTA with or without 1% 2-ME), 1 mU of O-glycosidase, 20 mU of Clostridium perfringens neuraminidase, or a combination of O-glycosidase and neuraminidase (all enzymes were from Boehringer Mannheim, Indianapolis, IN), and resolved by 8 to 9% SDS-PAGE. The gels were stained, dried, and fixed before autoradiography.
Generation of F(ab')2 of STOK2
Protein G-purified mAb STOK2 (2 mg/ml) was dialyzed against 0.1 M sodium acetate buffer at pH 4.0, centrifuged shortly, and digested with 0.1 mg/ml pepsin (enzyme to Ab ratio, 1:20) for 21 h at 37°C. The reaction was stopped with 2 M Tris base (diluted 1/20), dialyzed against acetate buffer, pH 5.0, and purified by protein G (which also binds Fab/F(ab')2 STOK2 under these conditions). Unfragmented STOK2 was removed from the reaction mixture by loading on a Sepharose 4B bead column that had been precoupled with the mouse mAb RG7/1.30 specific for the Fc portion of rat IgG2a Ab (24) (obtained from the American Type Culture Collection, Rockville, MD). The coupling of purified RG7/1.30 to cyanogen bromide-activated Sepharose 4B was performed at 5 mg of Ab/ml gel, according to the manufacturer’s instructions (17-0430-01, Pharmacia Biotech). The purity of the STOK2 fragments was confirmed by 10% SDS-PAGE followed by protein staining with Coomassie blue, as well as by Western blot analysis after transfer to polyvinylidene difluoride membranes and enhanced chemiluminescence detection; the immobilized STOK2 F(ab')2 were not reactive with an Fc-specific horseradish peroxidase-conjugated goat anti-rat IgG secondary Ab (112-035-008, Jackson ImmunoResearch) or with the mouse anti-rat IgG2a-Fc mAb RG7/1.30 (developed with a secondary goat anti-mouse IgG polyclonal Ab containing minimal cross-reactivity toward rat IgG, 115-035-100, Jackson ImmunoResearch; data not shown). However, a strong reaction was obtained with the Fab-specific mouse anti-rat IgG2a mAb RG9/6 (24).
RFLP analysis
Genomic DNA was isolated from liver tissue by overnight
incubation at 50°C in digestion buffer (10 mM Tris buffer pH 8.0, 100
mM sodium chloride, 25 mM EDTA, 0.5% SDS) containing 0.1 mg/ml
proteinase K (Boehringer Mannheim), followed by phenol extraction.
Eco-RI digested DNA was resolved by 0.8% TBE/agarose gel
electrophoresis and blotted onto nitrocellulose filters. Hybridizations
to radiolabeled NKR-P1 probe were performed overnight at 42°C in 50%
formamide, 6 x SSC, 5x Denhardt’s solution, 0.5% SDS, and 100
µg salmon testes DNA. The filters were washed twice for 5 min at room
temperature in 2 x SSC, 0.1% SDS, then once in 0.5 x SSC,
0.1% SDS for 30 min. at 42°C before autoradiography. The
p for nonlinkage was calculated with the
2 test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Spleen cells from one NK alloreactivity-deficient DA rat that had
been immunized with alloreactive NK cells from MHC-matched PVG.1AV1
rats, were fused with NSO myeloma cells. Hybridomas were screened for
reactivity with NK subsets by indirect immunofluorescence and flow
cytometry and two mAb, STOK1 and STOK2, were selected (both were of the
IgG2a, Ig isotype; data not shown). As discussed below, mAb STOK1
and STOK2 likely react with the same glycoprotein(s), which was
expressed by a substantial proportion (30–50%) of IL-2 activated NK
cells, but less than 1% of cervical lymph node T and B cells from
PVG.1AV1 (Fig. 1
). It could be noted that
relative numbers of STOK1/2+ cells consistently increased roughly
three- to fivefold as a result of IL-2 culture, as can be seen from a
comparison with fresh PVG.1AV1 NK cells (see below).
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Spleen cells from PVG.1AV1 rats were depleted of
Ig+ cells and stained with mAb STOK1 or STOK2, in
combination with anti-NKR-P1 and anti-CD3. As shown in Figure 2, STOK2 labeling was confined to the
NKR-P1+ but not NKR-P1- cells, and the same
was true also for STOK1 (data not shown). Thus, while 12% and 7,3%,
respectively, of the CD3-NKR-P1+ and
CD3+NKR-P1+ cells co-stained with STOK2,
CD3+NKR-P1-cells were negative. Similarly, in
the panel of inbred strains tested, only PVG, AO, and LOU/C stained
positive for STOK2 (and STOK1, not shown); DA, BN, F344, and LEW were
all negative (Table I). Among these
strains, it is noteworthy that only the STOK1/2+ strains
have NK cells with a broad allorecognition repertoire (19).
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STOK2+ NK cells from PVG were surface-iodinated
and lysed with Nonidet P-40. The cell-lysate was subject to
immunoprecipitation with mAb STOK1, STOK2, and the 2C7 isotype-matched
control, followed by SDS-PAGE and autoradiography. Broad bands
migrating at about 55 and 110 kDa were obtained under reducing and
nonreducing conditions, respectively, with both STOK1 and STOK2, while
there were no bands in the control (Fig. 3A and data not shown). Endo F
treatment of this molecule revealed bands at 35 (reduced) and 60 kDa
(nonreduced) indicating that this Ag contains abundant
N-linked carbohydrates. A smaller, but marked decrease in
m.w. was observed after neuraminidase digestion, indicating the
presence of terminal sialic acids, while O-glycosidase was
without effect (Fig. 3B). Importantly, immunoblot
analysis showed that both STOK1 and STOK2, but not control 2C7 reacted
with immobilized STOK2 glycoprotein (Fig. 3C). These
results, together with the concordant expression data described below,
indicate that mAb STOK1 and STOK2 are directed against the same
homodimeric glycoprotein, the STOK1/2 Ag.
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It has recently been shown that the inability of DA, as opposed to
PVG, NK cells to lyse allogeneic lymphocytes (allogeneic lymphocyte
cytotoxicity or ALC) is controlled by genes in the rat NKC on
chromosome 4 (6). Hence, it seemed likely that the DA anti-PVG mAb
STOK1 and STOK2 were reactive with a molecule coded for by this
complex. This was tested in a linkage analysis with NKR-P1 in a
backcross between the STOK1/2- LEW and
STOK1/2+ PVG.1L rat strains. (LEW x
PVG.1L)F1 animals, which were STOK1/2+ (data
not shown), were crossed with STOK1/2- LEW rats. Their
(LEW x PVG.1L)F1 x LEW offspring were typed for
STOK1 and STOK2 reactivity, as well as for the presence of a
PVG-specific RFLP for NKR-P1 after digestion of liver DNA with Eco-RI
(Fig. 4). Of the 11 backcrossed rats
tested, only the seven STOK1/2+ animals possessed the PVG
RFLP; the four STOK1/2- rats did not (data not shown).
Thus, independent assortment of these two characteristics did not occur
in meiosis, implying that the STOK1/2-encoding gene is linked to NKR-P1
(p < 0.01, by 2 test). Further
evidence for this was obtained in functional studies. Only NK cells
from the STOK1/2+, but not the STOK1/2-, rats
in the test cross between LEW and PVG.1L (both
RT1l) were able to kill
RT1av1 alloblasts (data not shown).
Since NK cells from PVG.1L, but not those from LEW, kill
RT1av1 blasts (17, 19), these data
indicate that the STOK1/2 locus may be related to the previously
characterized NK alloreactivity locus, nka, which is located
in the rat NKC, close to the Ly-49 cluster of genes (6).
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Cultures of STOK2+ and STOK2-
NK cells were generated from PVG
(RT1c) rats by positive
and negative selection with STOK2-coated magnetic Dynabeads, and these
subpopulations were tested for reactivity against a panel of allogeneic
lymphoblasts and tumor cells. It should be noted that the
STOK1+ subset was equally enriched or depleted as a result
of these separation procedures. Removal of STOK2+ cells
from the polyclonal population of
RT1c (PVG) NK cells consistently
resulted in the loss of almost all the cytolytic activity against
MHC-mismatched RT1l (PVG.1L) Con A
blasts as well as a substantial reduction of cytotoxicity against
RT1u (PVG.1U) and
RT1av1 (PVG.1AV1) allo-targets
(compare Fig. 5, B andA). STOK2 depletion also diminished killing of the tumor
targets YAC-1, P815, and P388, but less so than that of the three
allotargets (data not shown). Conversely, positively selected
STOK2+ NK cells displayed higher alloreactivity against all
three RT1 haplotypes compared with unselected control cells
(Fig. 5, C and A, respectively). Autologous
lymphoblasts, on the other hand, were spared from lysis by both
STOK2+ and STOK2- effectors. Thus, the
increased NK responsiveness to MHC allodeterminants, especially to
RT1l (PVG.1L), could not be explained
by a general increase in the cytolytic capability of
STOK1/2+ PVG NK cells and indicated that the STOK1/2 Ag
functions as an MHC receptor on these NK cells.
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This idea was further substantiated by studies of the influence of
self MHC genes on NK expression of the STOK1/2 Ag. As can be deduced
from Table II, relative proportions of
STOK2+ cells differed considerably, with the
RT1 haplotype in a set of MHC-congenic PVG strains. Thus,
while only about 2% of the CD3-NKR-P1+ NK
cells in RT1u were positive for
STOK2, approximately 10% of CD3-NKR-P1+ cells
from RT1av1 and
RT1lv1 rats stained with this mAb
(similar results were obtained with STOK1; data not shown).
Intermediate values were observed in the other haplotypes tested. Some
variations were also noted with the NKR-P1+ T cell subset,
although these were less pronounced and also somewhat different from
those for NK cells (Table II).
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Ab blocking experiments were performed to determine whether the
cytolytic activity of STOK1/2+ PVG NK cells
(RT1c) was provoked by
allo-MHC class I molecules or was inhibited by a self PVG class I Ag.
Addition of F(ab')2 of STOK2 to the cytotoxic culture
induced killing of syngeneic RT1c
lymphoblasts by the STOK1/2+ NK subset, almost to the level
seen with allogeneic RT1u targets
(Fig. 6A). The
cytolytic activity of STOK2- NK cells, on the other hand,
was not affected by the addition of mAb STOK2 (Fig. 6B), nor were there any effects on allogeneic
killing. These data therefore strongly indicate that the STOK1/2
molecule functions as an inhibitory receptor for an
RT1c-encoded class I molecule, and
that this receptor-ligand interaction is blocked by mAb STOK2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several features of the STOK1/2 Ag aligns it with the well-documented
Ly-49 receptor family for MHC class I in the mouse. The biochemical
characterization of STOK1/2 as a 110-kDa homodimer is as expected for a
lectin-like NK receptor. Furthermore, deglycosylated STOK1/2 showed up
as a band of about 35 kDa (reduced), and this corresponds well with the
estimate for the Ly-49 cDNAs cloned to date in the rat (6). The genetic
linkage with NKR-P1 established that STOK1/2 maps to the rat NKC, and a
relationship with the recently described nka locus seems
likely (6). The nka locus is within the Ly-49 portion of the
rat NKC and is associated with high NK alloreactivity, as is STOK1/2
(Fig. 5
). A similar correlation between Ly-49 expression and NK
alloreactivity has recently been provided by others in the mouse,
where, for example, the anti-B6 (C57BL/6) cytolytic activity of
Ly-49A+ NK cells is markedly higher than that of
Ly-49A- cells from D8 mice (B6 transgenic for
H-2Dd) (27). Also, the MHC
influence on STOK1/2 expression fits with previous studies of the mouse
Ly-49A molecule (28). Moreover, STOK1/2 apparently functions as an
inhibitory MHC receptor. As discussed above, the induction of syngeneic
NK lysis by F(ab')2 STOK2 (Fig. 6) is most likely due to
blockade of a self recognition event in the PVG strain. Analogous
results have been obtained for the mouse Ly-49 members Ly-49A, -C, and
-G2 (7, 8, 9). All these inhibitory Ly-49 molecules have in common a
binding motif for the tyrosine phosphatase, SHP-1, which inhibits
cellular activation in immune cells, including NK cells (10, 11).
Another Ly-49 member, Ly-49D, which lacks this motif, activates NK
cytotoxicity in response to an uncharacterized physiologic ligand
(29).
Our previous immunogenetic studies of rat alloreactive NK cells have indicated that these cells have a dual receptor system for MHC molecules; while classical class I (RT1.A) Ag are inhibitory (18), some other class I molecules, notably those encoded by the nonclassical class I region (RT1.C), may stimulate NK cells (16, 17, 18). In the present study we have identified a putative inhibitory class I receptor. What, then, is the stimulatory rat NK alloreceptor? One candidate could be the NK-activating receptor, NKR-P1 (22, 30). Although NKR-P1 may be involved in carbohydrate recognition (31), its natural ligand has not been identified, and it may not be class I. The only defined Ly-49 receptor activating NK cytotoxicity is Ly-49D in the mouse (29). However, identification of the NK alloreactivity locus nka amidst Ly-49 genes in the NKC (6) suggests that such a molecule eventually will be found in the rat.
It remains a paradox that the PVG NK subset possessing the inhibitory
self MHC receptor STOK1/2- is enriched for alloreactivity against more
than one allodeterminant (Fig. 5). Unless several other inhibitory MHC
receptors binding these allodeterminants are preferentially expressed
in the STOK1/2- NK subpopulation, this implies the
coexpression of activating MHC receptors with STOK1/2. Some precedence
for this has been provided in humans, in whom the KIR inhibits cellular
activation mediated by the p50 HLA receptor (32). If this is the case,
it probably results from the coselection of inhibitory and activating
MHC receptors during NK maturation due to inhibition apparently being
dominant over activation (18). The identification of other rat NK
receptors and the determination of the consequences of binding to
various MHC molecules in terms of activation vs inhibition are needed
to clarify this issue.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. T. Vaage, Department of Anatomy, P.O. Box 1105 Blindern, N-0317 Oslo, Norway.
3 Abbreviations used in this paper: NKC, natural killer gene complex; KIR, killer inhibitory receptor; ALC, allogeneic lymphocyte cytotoxicity; RFLP, restriction fragment length polymorphism.
Received for publication May 5, 1997. Accepted for publication September 17, 1997.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) PVG.1AV1
(RT1av1; 
), PVG.1U
(RT1u; 
), and PVG.1L
(RT1l; 
). Results are
presented as median values from four experiments.
and