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*
Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada; and
Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1/2 domain is required
for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling
and transfection. Thus, Ly-49P is the second activating Ly-49 receptor
demonstrated to induce NK cytotoxicity by recognizing a class I MHC
molecule. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Ly-49 family also includes Ly-49D and -H. These receptors lack ITIMs, do not recruit SHP-1, and are unable to function as inhibitory receptors. Instead, they associate with the disulfide-bonded immunoreceptor tyrosine-based activation motif-containing signaling adapter molecule DAP12 and can stimulate transmembrane signaling initiated by tyrosine phosphorylation events (13, 14). Association of Ly-49D or -H with DAP12 is through a charge interaction between a conserved arginine in the transmembrane segment of Ly-49D and -H and a glutamic acid in the transmembrane segment of DAP12 (13). Engagement of Ly-49D or Ly-49H on NK cells with FcR-mediated target cell-bound Abs induces NK-mediated target cell lysis by the mechanism of reverse Ab-dependent cell-mediated cytotoxicity (rADCC) (15, 16). Ly-49D has been shown to recognize the H-2Dd class I MHC molecule, and this event results in NK activation (17, 18, 19, 20). Thus, an activating Ly-49 receptor recognizes a class I MHC molecule. The Ly-49D+ NK subset of some strains of mice has also been shown to mediate MHC-specific and Ly-49D-dependent allogeneic bone marrow rejection (21). These findings potentially complicate interpretation of the missing self hypothesis, since they would not be readily predicted within the context of this model. Recently, the Ly-49D molecule was also shown to recognize undefined xenoantigens, resulting in NK lysis of certain xenogeneic target cells (22, 23). Although able to stimulate NK cells, the role of activating Ly-49 receptors in regulating NK cell function remains unclear.
The Ly-49 family may be substantially larger than the commonly known members, Ly-49A through Ly-49I. Complex Southern blot hybridizations obtained with Ly-49-specific probes using genomic DNA from inbred mouse strains suggest that additional Ly-49 family members may remain to be identified and characterized (24). Recently, genes or gene segments of five potential additional Ly-49s, tentatively designated J, K, L, M, and N, have been identified in the C57BL/6 (B6) genome (25). In addition, two Ly-49 cDNAs, one encoding an inhibitory receptor designated Ly-49O and another encoding an apparent activating receptor designated Ly-49P, were recently cloned from the 129 mouse strain (26). The Ly-49P receptor is closely related to Ly-49A in its carbohydrate recognition domain (CRD), associates with DAP12, and triggers transmembrane signaling (26). These results suggest that Ly-49P may serve as an activating receptor. In this report we describe the independent cloning of Ly-49P from nonobese diabetic (NOD) and nonobese diabetes-resistant (NOR) NK cells and provide evidence that it can activate NK cell cytolytic activity by recognizing H-2Dd. Before this report, Ly-49D was the only activating member of the Ly-49 family shown to recognize a class I MHC molecule. Thus, Ly-49P joins Ly-49D in this capability. Understanding the diversity, ligand specificity, activation potential, and expression of activating Ly-49 members may provide insight toward understanding their function(s).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five- to 8-wk-old female BALB/c (H-2d), C57BL/6 (H-2b), NOD (H-2Kd,Db), NOR (H-2Kd,Db), B10.BR (H-2k), B10.D2 (H-2d), B10.S (H-2s), BALB.B10 (H-2b), and BALB.K (H-2k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Additional 5- to 8-wk-old NOR and NOD mice were obtained from the University of Alberta mouse breeding facility (Edmonton, Canada).
Hybridomas and Abs
Hybridomas producing the following Abs: A1(IgG2a),
anti-Ly-49A (27); MK-D6(IgG2a),
anti-IAd (28); 11-4.1(IgG2a),
anti-H-2Kk (29); 14-4-4S(IgG2a)
anti-IEk (30); BBM.1(IgG2b),
anti-human ß2-microglobulin
(31); B27 M1(IgG2a), anti-HLA-B7,27 (32);
Y3(IgG2b) anti-Kb (33);
34-2-12S(IgG2a), anti-H-2Dd (3 domain)
(34, 35); 34-5-8S(IgG2a),
anti-H-2Dd (1/2 domain) (34, 35); and 2.4G2(rat IgG2b) anti-mouse Fc-
receptor
(36); were obtained from American Type Culture Collection
(Manassas, VA), except A1, which was obtained from Dr. James Allison
(University of California, Berkeley, CA). Abs were prepared by ammonium
sulfate precipitation and PBS dialysis of tissue culture supernatants
obtained from hybridomas grown in protein-free hybridoma medium.
Purified G28 (IgG2a) anti-rat CD8 (37), F23.1(IgG2a)
anti-mouse Vß 8.1 and 8.2 TCRs (38, 39); DX5 (rat
IgM, PE-coupled), which recognizes NK cells (3); 145-2C11
(hamster IgG, PerCP-coupled), which recognizes the CD3
chain
(40); and isotype control Abs, G155-178 (mouse IgG2a,
fluorescein-coupled), anti-TNP, A19-3 (hamster IgG) anti-TNP,
R4-22 (rat IgM, PE-coupled) of unknown specificity, and A23-1 (rat
IgG2c) of unknown specificity were purchased from PharMingen (San
Diego, CA). The Ly-49A-reactive YE1/48 (rat IgG2c) Ab
(24), was provided by Dr. Fumio Takei (Terry Fox
Laboratories, Vancouver, Canada). Tissue culture supernatants were also
obtained from hybridomas grown in RPMI 1640, 8% heat-inactivated FCS,
20 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100
µg/ml streptomycin for use in cell surface staining. FITC-coupled rat
anti-mouse IgG, goat anti-rat IgG, and mouse anti-rat IgG
were purchased from Jackson ImmunoResearch Laboratories (West
Grove, PA).
Rat tumor cell lines
RNK-16, a spontaneous F344 rat strain NK cell leukemia cell line (41) was provided by Dr. Mary Nakamura (University of California, San Francisco, CA). The RNK-16 cells were maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine, penicillin, streptomycin, and 5 x 10-5 M 2-ME (RNK medium). YB2/0 is a rat myeloma (42) we obtained from American Type Culture Collection, and it was maintained in RPMI 1640 supplemented with L-glutamine, penicillin, streptomycin, and 1 mM sodium pyruvate (YB2/0 medium).
COS-7 cells
COS-7 SV40-transformed African green monkey kidney cells were provided by Dr. John Elliott (University of Alberta, Edmonton, Canada).
PCR cloning of Ly-49 transcripts
IL-2-activated NK cells were prepared from spleen cells harvested from 6- to 10-wk-old female mice as described by Smith and co-workers (2). Total cellular RNA was isolated from NOD and NOR IL-2-activated NK cells with TRIzol reagent (Life Technologies, Burlington, Canada), and reverse transcribed using SuperScript II (Life Technologies) with an oligo(dT) primer. Ly-49 transcripts were amplified with Advantage cDNA Polymerase (Clontech, Palo Alto, CA) using the sense cloning primer 5'-CCCAAGATGAGTGAGCAGGAGG-3' and the antisense cloning primer 5'-GAGAGTCAATGAGGGAATTTATCC-3'. The PCR products were purified using QIAquick spin columns (Qiagen, Santa Clarita, CA) and directly ligated into the TA cloning vector p123T (Mo Bi Tec, Gottingen, Germany). Individual cDNA clones were divided into two groups: ITIM-encoding clones, which could be PCR-amplified using the antisense cloning primer with the sense primer 5'-GGAGACTCAAGGGCCCAGAG-3', and ITIM-lacking clones, which could be PCR-amplified by using the antisense cloning primer with the sense primer 5'-CTRRAAAAGCT GGCCTCAGAGTY-3' (where R = A/G and Y = C/T).
To verify the nucleotide sequences of the ends of the open reading frames and to obtain sequence of the noncoding gene flanking regions for cloning, we performed 5'- and 3'-rapid amplification of cDNA ends (5'- and 3'-RACE) for NOR Ly-49A and Ly-49P. Poly(A)+ RNA was isolated from total RNA obtained from IL-2-activated NK cells using Oligotex spin columns (Qiagen) and used to synthesize a cDNA library with the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). The following gene-specific primers were used: for Ly-49P 5'-RACE, 5'-CTTGAGGAGACGCTGAAGCCTAG-3'; and for Ly-49P 3'-RACE, 5'-GCCAGC TTTTCTAGGCTTCAGC-3'. Ly-49A required nested primers: for 5'-RACE, 5'-TGGTGAGATTTCATAAATCTGCAGGAT-3' followed by 5'-TGGCTACAGAAGGTGTTCATTCCAC-3'; for 3'-RACE, 5'-TCCTGCAGATTTATGAAATCTCACCAT-3' followed by 5'-ATGAACTTCCAGTGGAATGAACACCT-3'. With the sequence data thus acquired, we designed the following cloning primers to obtain both NOD and NOR full length cDNA clones by RT-PCR: for Ly-49A, 5'-TTCCTCCACCAGAACCACTTCTTG-3' (sense) and 5'-AGAAGATCTGTCCAGTCTCAACA GGG-3' (antisense); for Ly-49P, 5'-TTTAAAAGAGAACATACTCTACATCCTC-3' (sense) and the same antisense primer as Ly-49A. Ly-49D was cloned by RT-PCR from NOD and NOR first strand cDNA using the same primers and conditions as were used to previously clone Ly-49D from C57BL/6 cDNA (43). Sequencing reactions were performed using a dideoxy terminator method and analyzed on an ABI 373A automated sequencer (Applied Biosystems, Foster City, CA).
Transfections
COS-7. COS-7 cells were grown in Opti-MEM I (Life Technologies, Grand Island, NY) containing 5% heat-inactivated FCS (Medicorp, Montreal, Canada). DNA containing the coding regions of Ly-49A and Ly-49P cDNA were inserted into the XbaI-EcoRI sites of the mammalian expression vector pCI-neo (Promega, Madison, WI). Sequence encoding the mature form of the mouse DAP12 protein was inserted into the pFLAG-CMV-1 expression vector (Sigma-Aldrich, Oakville, Canada) to create an epitope-tagged chimeric protein. Vectors were then transfected into COS cells using Lipofectamine (Life Technologies).
YB2/0 cells.
A cDNA encoding H-2Dd cloned from S49.1 T
lymphoma cells was inserted into the expression vector pCDNA3 and
electroporated into YB2/0 using 180 mV and 960 µF. A chimeric class I
molecule, referred to as
Kb/Dd, was produced by
ligating the 1/2 domain portion of H-2Kb to
the 3, transmembrane, and cytoplasmic portion of
H-2Dd at the common FspI site. This
chimeric cDNA was inserted into the expression vector pCI-neo and
electroporated into YB2/0 as described above. Transfected cells were
immediately cloned in 96-well microtiter plates in YB2/0 growth medium
supplemented with 1 mg/ml of G418 for drug selection (Life
Technologies).
RNK cells.
The coding region of Ly-49P was inserted into the
XhoI-XbaI sites of the bicistronic expression
vector BSREN, which was provided by A. Shaw, Washington University
(St. Louis, MO), and transfected into RNK-16 cells using the protocol
described by Nakamura et al. (10). Four million cells were
transfected with 20 µg of linearized plasmid by electroporation at
180 mV and 960 µF. Transfected cells were cloned in 96-well
microtiter plates in RNK medium supplemented with 1 mg/ml G418 for drug
selection.
Preparation of nylon wool-nonadherent spleen cells and generation of IL-2-activated LAK
Single-cell suspensions of spleen cells from 5- to 7-wk-old C57BL/6 and NOD mice were prepared with tissue grinders. After osmotic lysis of RBC, the cell suspensions were passaged over nylon wool columns. The nylon wool-nonadherent (NWNA) spleen cells were either analyzed immediately by FACS or cultured for 6 days at a concentration of 2 x 106 cells/ml in RPMI 1640/10% FCS supplemented with sodium pyruvate, nonessential amino acids, 5 x 10-5 M 2-ME, and 1000 U/ml human rIL-2 expressed in and isolated from Escherichia coli, before FACS analysis.
Flow cytometry
Approximately 48 h after transfection, COS-7 cells were incubated for 30 min with A1 or isotype control mAb MK-D6. The cells were washed with PBS, incubated with FITC-labeled secondary Ab, washed again with PBS, and then fixed with 2% formaldehyde for analysis on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). YB2/0 cells as well as the H-2Dd and H-2Kb/Dd transfectants of YB2/0 were incubated with either rat IgG (Sigma) or normal rat serum before staining with the mAbs 34-2-12S or 14-4-4S, followed by FITC-conjugated rat anti-mouse IgG. YB2/0 transfectants expressing the chimeric class I molecule H-2Kb/Dd were also analyzed by FACScan with the Y3 H-2Kb-specific mAb and the BBM.1 isotype control after blocking with normal rat serum, followed by FITC-conjugated secondary rat anti-mouse IgG and formaldehyde fixation. RNK-16 transfectants were incubated with normal rat serum for 30 min before addition of the mouse IgG2a mAbs A1 and B27 M1 followed by FITC-conjugated rat anti-mouse IgG, then fixed and analyzed by FACScan. Cells were gated for forward and side scatter.
Three-color analysis of NWNA cells
Freshly isolated NWNA spleen cells or 6-day IL-2-activated NWNA
spleen cells were preincubated with normal mouse serum to block
nonspecific Ab binding to Fc receptors before Ab incubation. Abs used
for analysis were PerCP-coupled 2C11 (anti-mouse CD3),
PE-coupled DX5 (anti-mouse pan-NK cell), and either
fluorescein-coupled A1 or YE1/48 with a fluorescein-coupled mouse
anti-rat second Ab. Isotype control Abs used were A19-3-PerCP,
R4-22-PE, G155-178-fluorescein, and A23–1. Cells were gated for
forward and side scatter.
Generation of Con A-activated blast target cells
Con A-activated T cell blasts were prepared from spleen cells of various mouse strains. Fifteen million spleen cells were cultured at 5.0 x 106 cells/ml in RPMI 1640 with 10% heat-inactivated FCS, 2-ME, and 3.0 µg/ml Con A for 48 h. Blast cells were recovered either after three washes in RPMI 1640 medium or following Ficoll-Hypaque separation.
Cytotoxicity assays
Target cells were labeled at 37°C with 100–150 µCi of Na51CrO4 (51Cr; Mandel/NEN Life Science Products, Guelph, Canada), for 1 h if tumor cells or for 1.5 h if Con A blast cells. Following extensive washes, 1 x 104 51Cr-labeled target cells were incubated for 4–5 h at 37°C in V-bottom microtiter plates with RNK-16 cells or RNK-16 cells transfected with Ly-49P at various E:T cell ratios in triplicate. After the incubation, plates were centrifuged for 5 min, and 100 µl of supernatant was removed and counted in a gamma counter. The percent specific lysis was determined as [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. To perform the rADCC experiments, untransfected RNK-16 cells and the 1B9 Ly-49P transfectant of RNK-16 were preincubated for 15 min with 5 µg/ml of the A1, G28, or F23.1 Abs or with medium alone before addition of FcR-expressing YB2/0 target cells and subsequent 4-h cytotoxic assay as described above. For Ab inhibition experiments, Abs were incubated with soluble PA (2 µg/10 µg of mAb; Sigma, St. Louis, MO) for 30 min before addition to effector or target cells. Effector or target cells were preincubated with the mAb and PA for 15 min before the cytotoxicity assay. The mAb were present throughout the cytotoxicity assay at a final concentration of 5 µg/106 effector cells or 5 µg/ml when the mAb was directed against a target cell Ag. All cytotoxicity assays were repeated a minimum of three times.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Ly-49 family includes both activating and inhibitory receptors (1). Collectively, Ly-49 molecules may contribute to a balance of positive and negative signals that control murine NK activities as well as those of certain subsets of T cells (3, 7, 8, 9, 18). Most studies of Ly-49 gene family members have used the B6 strain as a prototype. It is clear from recent studies, however, that the diversity and expression of Ly-49 members can differ among strains (44). Furthermore, complex hybridization patterns obtained in Southern blot analyses of genomic DNA from multiple inbred mouse strains using Ly-49A and Ly-49C cDNA probes suggest that additional Ly-49 members may exist (24). We chose to examine Ly-49 receptor expression and function in a mouse strain other than B6, one in which immune dysregulation can occur, resulting in spontaneous disease. The NOD mouse spontaneously develops autoimmune disease at an early age (45), while the NOR mouse does not, although it shares many, but not all, NOD genetic loci (46). We designed oligonucleotide primers to clone cDNAs encoding putative activating and inhibitory Ly-49 family members by RT-PCR and used RNA obtained from IL-2-activated NK cells of NOD and NOR mouse strain origin as sources of transcripts. In addition to cDNAs encoding well-characterized Ly-49 family members, we cloned a noninhibitory Ly-49 family member that possessed high amino acid identity in its CRD with the inhibitory Ly-49A molecule. Sequence identities with a newly described activating Ly-49 present in the 129 mouse strain, designated Ly-49P by Makrigiannis et al. (26), indicate that we have cloned the NOD strain allele of Ly-49P. The NOR strain expresses Ly-49P identical in sequence to Ly-49PNOD.
We also cloned a single cDNA sequence from both NOD and NOR NK cells
encoding a novel allele of Ly-49A, since it has a very high
identity of 99.1 and 99.6% on the nucleotide level, and 98.9 and
99.2% on the amino acid level, with B6 and BALB/c Ly-49A
alleles, respectively (data not shown; Fig. 1
A). The NOD (and NOR)
Ly-49A allele is closer in sequence to both B6 and BALB/c
alleles than they are to each other, with 99.0% nucleotide (not shown)
and 98.1% amino acid identities (Fig. 1A). The predicted
Ly-49PNOD amino acid sequence lacks an ITIM
sequence and thus is not an inhibitory receptor, yet a comparison of
the Ly-49PNOD sequence with
Ly-49AB6 indicates an amino acid identity of
83.3% for the entire molecule and 92.9% for the CRD (Fig. 1A). Similarly, a comparison of the
Ly49PNOD sequence with
Ly-49ANOD shows an overall amino acid identity of
82.5%, and 92.1% for the CRD region (Fig. 1A). In addition
to the full-length Ly-49P we have designated P1, there are two other
major PCR products amplified with Ly-49P cloning primers from NOD
IL-2-activated NK cells. These smaller products are apparent splice
variants of Ly-49P encoding in-frame transcripts containing the
complete CRD region. Using the gene organization of Ly-49A as a
reference (47), one Ly-49P variant lacks exon 4, while the
other omits exons 3 and 4 (Fig. 1A). We designate the three
Ly-49P splice variants by size as P1, P2, and P3. The first amino acid
residue encoded by exon 5 of the P2 splice variant is predicted to
differ from P1 and P3. This difference is a common result when two or
more different RNA upstream donor sites are spliced to the same
acceptor site. This occurs in transcripts of many genes, including CD94
and NKG2B (48, 49). In the case of P2, fusion of the first
base of the codon ATT (Ile) at the end of exon 3 to the second and
third bases of the codon GGC (Gly) at the beginning of exon 5 forms the
codon AGC (Ser) without interruption of the established reading frame.
For the P3 splice variant, the first base of the codon GTG (Val) at the
end of exon 2 is fused to the second and third bases of the codon GGC
(Gly) at the beginning of exon 5 to reform the glycine codon at the
start of the CRD, again without interruption of the established frame.
We have been unable to clone a Ly-49P-related cDNA from IL-2-activated
B6 NK cell RNA with primers used for cloning
Ly-49PNOD. It appears that Ly-49P does not exist
in the B6 genome, is not expressed, or is divergent in sequence from
Ly-49PNOD.
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B). Ly-49D cloned from NOR mice is identical in
sequence to Ly-49DNOD. High nucleotide (98.5%)
and amino acid sequence identity (96.6%) between
Ly-49DNOD and Ly-49DB6
confirm that these are very likely to be alleles (data not shown; Fig. 1B). The nucleotide and amino acid sequence identities
between Ly-49DNOD and
Ly-49PNOD are 84.8 and 85.6%, respectively (not
shown; Fig. 1B), indicating that these are unlikely to be
alleles. We are confident of the nucleotide and predicted amino acid
sequences on the very ends of Ly-49PNOD, since we
performed 5'- and 3'-RACE reactions to determine them, whereas
sequences at the ends of Ly-49P129, reported
previously (26), were primer derived and thus may contain
minor sequence differences; nevertheless, a comparison of the entire
Ly-49PNOD and Ly-49P129
nucleotide and predicted amino acid sequences (98.2 and 95.4%
identity, respectively), strongly supports the conclusion that these
are indeed alleles. There are only 14 nucleotide differences found
between these two cDNAs, resulting in 12 amino acid changes (data not
shown and Fig. 1B). Another notable aspect of Ly-49P relates
to potential post-translational processing; whereas Ly-49A and Ly-49D
each have three predicted N-linked glycosylation sites,
Ly-49P129 has two, and
Ly-49PNOD has only one (Fig. 1). The influence of
N-linked glycosylation on Ly-49 expression and function
remains to be determined. Finally, consistent with other noninhibitory
Ly-49 family members, Ly-49PNOD possesses a
charged residue (Fig. 1B), arginine, in its putative
transmembrane segment that is required for activating Ly-49 receptors
to associate with the immunoreceptor tyrosine-based activation
motif-containing signaling adaptor protein DAP12 (13). The
sequences of Ly-49ANOD/NOR,
Ly-49DNOD/NOR, and
Ly-49PNOD/NOR were not artifacts of RT-PCR, since
they were confirmed in clones derived from different RT-PCR reactions
with different sets of primers, and the genomic sequence of Ly-49P has
been shown to exist (26). The A1 and YE1/48 Abs recognize Ly-49PNOD
The A1 Ab was previously reported to recognize a polymorphic
epitope expressed only by the B6 allele of Ly-49A, since it was shown
not to react with other known alleles of Ly-49A or other known Ly-49
family members (24). Since the noninhibitory
Ly-49PNOD possesses high sequence identity with
Ly-49A, particularly in the CRD, where the A1 Ab is predicted to bind
Ly-49A (24), the reactivity of the A1 Ab and that of a
second Ly-49A-specific Ab, YE1/48, with Ly-49PNOD
were examined. We expressed Ly-49AB6,
Ly-49ANOD, or Ly-49PNOD in
COS cells for Ab staining. Ly-49AB6 reacted with
the A1 and YE1/48 Abs, as did Ly-49ANOD and
Ly-49PNOD (Fig. 2A). There was some expression
of Ly-49P detected with both Abs when Ly-49P is expressed in the
absence of DAP12; however, coexpression of DAP12 substantially augments
Ly-49P expression, as indicated by enhance staining with both Abs (Fig. 2A). No staining of COS cells with the A1 Ab was observed in
the absence of Ly-49A or Ly-49P expression or with the expression of
DAP12 alone (data not shown). Coexpression of DAP12 was necessary for
optimal expression of Ly-49PNOD (Fig. 2A), similar to Ly-49P129
(26). Thus, the A1 Ab does not exclusively react with the
B6 allele of Ly-49A, but also recognizes the NOD allele of Ly-49A and a
noninhibitory/putative activating receptor, Ly-49P.
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Cross-linking of Ly-49P129 results in
transmembrane signaling, including tyrosine phosphorylation and
Ca2+ flux (26), suggesting that it
can function as an activating receptor. We were interested in the
ability of Ly-49P to activate NK cytotoxicity. To this end, we
transfected Ly-49PNOD into the heterologous rat
NK leukemia RNK-16, which displays NK activity (10).
RNK-16 has been used successfully to demonstrate the NK-activating
function of transfected Ly-49D (17) and 2B4S, an
activating form of an Ig-domain containing receptor expressed by murine
NK cells (50). This experimental system avoids the
complication of other mouse activating and/or inhibitory receptor
coexpression and cross-reaction of the A1 Ab with Ly-49 receptors other
than Ly-49P. We show three Ly-49P transfectant clones that were
positive for A1 Ab staining (1B9, 1A3, and 1G4), while RNK-16 was
negative (Fig. 2B), indicating that the transfectants
express Ly-49PNOD. These transfectant clones
expressed different mean densities of Ly-49P (Fig. 2B).
Transfectant 1A3 expressed quite a high density of Ly-49P, suggesting
that possessing only one predicted N-linked carbohydrate
attachment site is not a limitation for proper folding and cell surface
expression of this molecule. RNK-16 cells and transfectants
constitutively express the rat CD8 molecule, as indicated by G28 Ab
staining (Fig. 2B, right panels, and data not
shown).
To determine whether Ly-49P can function as an NK activating receptor,
we examined the ability of Ly-49P transfectants to perform rADCC. The
rat myeloma YB2/0 expresses Fc receptors and can be rendered
susceptible to lysis when bound to an Ab directed against heterologous
NK-activating receptors expressed on RNK-16 cells by transfection
(17). YB2/0 is normally sensitive to RNK-16 lysis (Fig. 3A); however, upon
transfection of RNK-16 with mouse noninhibitory NK receptors such as
Ly-49D (17) and Ly-49PNOD (Fig. 3B), cytotoxicity is reduced substantially. This may be due
to the heterologous activating receptor competing successfully for a
limiting signaling component, such as the rat DAP12, with endogenous
rat activating receptor(s) responsible for YB2/0 recognition and lysis.
An Ab directed against Ly-49P, but not rat CD8, stimulated rADCC of the
FcR-bearing YB2/0 cells by the Ly-49P RNK-16 transfectant 1B9 (Fig. 3B). In contrast, the A1 Ab was unable to enhance the
cytotoxicity of untransfected RNK-16 cells even at lower E:T cell
ratios where cytotoxicity is limiting (Fig. 3A). An
additional isotype control Ab, F23.1 also did not affect lysis of YB2/0
by either effector cell (Fig. 3). These results indicate that Ly-49P
can transmit stimulating signals resulting in NK-mediated cytotoxicity
and can be considered an NK-activating receptor.
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A number of inhibitory Ly-49 family members have been shown to
recognize class I molecules as ligands, including Ly-49A, -C, and –G,
in their apparent role of inhibiting NK cells unless there is reduction
of self-MHC expression (7, 8, 9). Much less is understood
regarding the ligand specificity and role of activating Ly-49 members.
Ly-49D is the only activating Ly-49 subfamily member demonstrated to
recognize a class I MHC molecule (17). Since Ly-49P, like
Ly-49D, is an activating Ly-49 receptor for which there are serological
reagents, we attempted to determine whether Ly-49P also recognizes
class I MHC molecules as ligands. We first compared the ability of
RNK-16 cells and three Ly-49P transfectants to lyse NK-susceptible Con
A T cell blast target cells from five different inbred mouse strains,
BALB/c, B6, B10.BR, B10.D2, and B10.S (Fig. 4A). Lysis of all five strains
was negligible for RNK cells, but each of the Ly-49P transfected RNK-16
cells lysed BALB/c and B10.D2 Con A-activated blasts, and the
transfectant with the highest Ly-49P expression, 1A3, also lysed B10.BR
blasts, but to a much lesser extent (Fig. 4A). No lysis of
C57BL/6 or B10.S Con A blasts was observed. Taken together, these
results suggest that Ly-49P may recognize an H-2d
product and possibly an H-2k product.
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B). A
low level of lysis is also observed with the BALB.K target cells in the
case of the 1A3 transfectant (Fig. 4B). We conclude from
these experiments that an H-2d product, possibly
Kd or Dd, is a potential
ligand for the Ly-49P activating receptor. It is also possible that an
H-2k product may be a much lower affinity ligand.
Finally, we examined whether Ly-49PNOD is capable
of activating RNK-16 lysis of NOD and NOR Con A blasts. Although B10.D2
Con A blasts were lysed, no lysis of NOD or NOR Con A blasts was
observed using the Ly-49PNOD transfectants (Fig. 4C). This suggests that the NOD/NOR MHC molecules,
H-2Kd and Db, which are
identical with the Kd and
Db alleles of BALB/c and B6 mice, respectively,
are apparently not detectable ligands for
Ly-49PNOD/NOR. Considering all the results
together, they suggest that Ly-49P, like Ly-49D, may recognize the MHC
molecule, H-2Dd.
The foregoing results suggested that Ly-49P triggers RNK-16-mediated
lysis of Con A blasts by recognizing H-2Dd. To
more directly assess this possibility we attempted to block lysis
mediated by the Ly-49 RNK transfectants, 1B9 and 1A3, by including the
A1 Ab, which recognizes Ly-49P, or the 34-5-8S Ab, which recognizes the
1/2 domain of H-2Dd expressed by the Con A
blast targets, in the cytolytic assays. Soluble PA was used to bind the
Fc portion of the Abs to prevent ADCC. The A1 Ab, but not one directed
against rat CD8 expressed by the RNK-16 cells, was able to completely
block lysis of B10.D2 Con A blast targets by 1B9 (Fig. 5A) and 1A3 (Fig. 5B), indicating that Ly-49P is the receptor responsible for
activating lytic potential of RNK-16 cells. In a similar fashion, the
34-5-8S Ab, but not an isotype control Ab 11-4.1, completely blocked
lysis of the B10.D2 Con A blasts by a representative Ly-49P
transfectant, 1B9 (Fig. 5C). Similar effects were observed
with 34-5-8S, but not 11-4.1, using the 1A3 transfectant and B10.D2 Con
A blasts (data not shown). No effect of soluble protein A alone was
observed in the assays (Fig. 5). These results suggest that Ly-49P
recognizes H-2Dd, possibly at a location within
its 1/2 domains, as has been reported for Ly-49A and Ly-49D
(10, 17, 51, 52).
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12 domain by Ly-49P
stimulates cytotoxic function in RNK-16 cells
The previous Ab-blocking experiments suggested that the 1/2
domain of H-2Dd is recognized by Ly-49P. To
directly assess this possibility we transfected rat YB2/0 cells with
genes encoding either wild-type H-2Dd or a class
I chimera referred to as
Kb/Dd, in which the 3
domain of Dd remains, but the 1 and 2
domains of Kb are substituted for the same
domains of Dd for use as targets of Ly-49P
transfected RNK-16. Two Dd and two
Kb/Dd transfectant clones
were shown to express comparable densities of class I using the
34-2-12S Ab, which recognizes an epitope on the
Dd 3 domain shared by both molecules (Fig. 6, upper panels). Expression
of the Kb 1/2 domain on the
Kb/Dd chimeric YB2/0
transfectants is confirmed by Y3 Ab staining (Fig. 6, lower
panels). A substantial increase in lysis is observed with the
wild-type Dd YB2/0 transfectants over that
observed with YB2/0 using a representative Ly-49P transfectant of
RNK-16, 1B9 (Fig. 7). No increase in
lysis over that found with YB2/0 was detected using the YB2/0
transfected with the Kb/Dd
chimeric gene (Fig. 7). These results indicate that Ly-49P can activate
NK-mediated lysis as a consequence of interacting with the 1/2
domain of H-2Dd. This specificity is shared with
the inhibitory Ly-49A receptor and the activating receptor Ly-49D.
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We have demonstrated that NOD IL-2-activated NK cells express
Ly-49A and Ly-49P at the RNA level and that Ly-49P can serve as an
NK-activating receptor by recognizing a class I MHC molecule. To
determine whether these receptors may be expressed by NK cells of the
NOD mouse, we performed three-color FACS analysis on freshly isolated
NWNA and IL-2-activated NOD spleen cells. Because the NOD mouse strain
does not express the NK1.1 epitope, we used the DX5 Ab to identify NK
cells (3, 44).
DX5+CD3- cells (Fig. 8, top panels) were analyzed
for staining with YE1/48 and A1 Abs (Fig. 8, middle and
bottom panels). A substantial portion of freshly isolated
NOD NK cells express A1 and YE1/48 recognized epitopes (Fig. 8, left panels), and two populations of YE1/48 and A1
positively stained fresh NOD NK cells apparently exist; a small
population constituting 2.5–4% of the total NK cells that are
strongly stained and a larger population of more weakly stained cells,
most effectively seen with the YE1/48 Ab, that constitute 50–80% of
the total NOD NK cells. This pattern contrasts with C57BL/6 NK cells,
where 
20% express A1 and YE1/48 epitopes that uniformly stain with
high fluorescence intensities, similar to those observed for the
smaller A1+ and YE1/48+ NOD
NK subset(s) (44) (data not shown). The
DX5+CD3- IL-2-activated
NOD NK cells (Fig. 8, right panels) show similar numbers of
cells staining with the A1 and YE1/48 Abs compared with their freshly
isolated NK counterparts, with perhaps one notable difference (Fig. 8).
The IL-2-activated NOD NK expressing the YE1/48- and A1-recognized
epitopes appear to fall into less discrete populations than fresh NOD
NK cells, with increased numbers of cells demonstrating moderate to
high levels of A1 and YE1/48 staining. Together, these results indicate
that Ly-49A and/or Ly-49P are expressed at the surface of freshly
isolated and IL-2-activated NOD NK cells. Since
Ly-49ANOD and Ly-49PNOD are
both recognized by YE1/48 and A1 Abs, the staining pattern does not
allow us to readily discern which cells express Ly-49A and/or
Ly-49P.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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McQueen et al. have described the existence of genes encoding putative activating Ly-49s designated K, L, and N (25). Ly-49K and N have highest homology to Ly-49C in the CRD, whereas the Ly-49L CRD most resembles Ly-49D (25). Ly-49K and N transcripts contain premature stop codons and/or defective alternative splices and do not encode functional protein products (53). We show here that full-length Ly-49P transcripts expressed in NOD and NOR mice can encode a viable protein product. This was also found to be the case for Ly-49P expressed in the 129 strain (26). We identified two additional alternatively spliced forms of Ly-49P in NOD/NOR NK cells. One splice variant, P2, encodes a Ly-49P protein that would lack the membrane-proximal stalk portion, but is otherwise not different from full-length Ly-49P. Transcripts encoding other NK C-type lectin receptor subunits, such as NKG2A and NKG2C, can undergo identical splicing (48, 54, 55). The smallest splice variant, P3, removes a portion of the cytoplasmic domain, the transmembrane segment, and the stalk region. This transcript encodes a protein that would probably remain cytosolic. Equivalent splice variants to P3 have been identified for several NK C-type lectin superfamily members (53). The P1, P2, and P3 messages appear to be expressed in approximately equivalent amounts judging from the intensity of PCR-amplified products (data not shown). It remains to be determined whether proteins encoded by Ly-49P2 and Ly-49P3 transcripts are expressed as stable polypeptides and what function they may perform.
Strain 129 NK cells, although expressing transcripts for Ly-49P, do not express transcripts for Ly-49A (26). In contrast, C57BL/6 strain NK cells express Ly-49A, but no transcripts for Ly-49P are found (26) (data not shown). It is not clear whether the Ly-49A gene is absent in the 129 strain or is simply silent, and the same question is pertinent for Ly-49P in the B6 strain. It could be speculated that the genes for Ly-49A and Ly-49P are found in separate mouse strains, or expression of these two genes may be mutually exclusive in a strain-specific manner. However, we were able to clone Ly-49A and Ly-49P cDNAs from both NOD and NOR strain IL-2-activated NK cells, suggesting that Ly-49A and Ly-49P genes can reside in the same genome and can be expressed at the same time. We identified two subsets of freshly isolated NOD NK cells and possibly IL-2-activated NOD NK cells that stained with the YE1/48 and A1 Abs, a small subset that stains relatively strongly and a larger subset that stains weakly. Since Ly-49A positive cells from several mouse strains stain strongly with these Abs (44), it is possible that the strongly stained NOD NK subset we have observed expresses Ly-49A. The level of YE1/48 staining observed with the weakly stained NOD NK cells reported here resembles the weak level of YE1/48 staining previously reported with 129 strain NK cells that express Ly-49P but not Ly-49A (26). By analogy, it is possible that the weakly staining NOD NK cells express Ly-49P. It is conceivable, therefore, that Ly-49A and Ly-49P may be expressed on separate fresh NOD NK subsets, but further studies will be necessary to resolve this possibility.
It has been suggested that the Ly-49P gene arose during evolution from recombination of two Ly-49 genes (26). This conclusion was based on a comparison of nucleotide sequences of Ly-49P, -A, and -D (26). An interval within exon 4 of Ly-49P129 may have been a site of recombination between the inhibitory Ly-49A gene and an activating Ly-49 gene. In any event, Ly-49P DNA sequences exist in the mouse genome (26). Nucleotide sequences of Ly-49PNOD transcripts are also consistent with some form of recombination (data not shown). Determining the identity of the activating Ly-49 donor gene contributing the 5' portion of the Ly-49P gene, however, may require full characterization of the activating Ly-49 gene repertoire. Activating Ly-49 genes unable to produce viable transcripts may be an additional source of such sequences for recombination (53). Recombination between Ly-49 genes may provide a mechanism for creating additional functional diversity within the Ly-49 gene family. Since Ly-49A and Ly-49P genes can be expressed by the same mouse strain, this suggests that if recombination was the origin of Ly-49P, Ly-49A gene duplication may have occurred before the recombination event. Gene conversion is another possible mechanism by which Ly-49P arose without involving Ly-49A gene duplication.
Ly-49A and Ly-49D are receptors that recognize
H-2Dd (10, 17). Ly-49P shares amino
acid sequence identities with both these receptors, particularly with
the CRD of Ly-49A, where there are only nine amino acid differences in
this domain, which consists of 127 residues (Fig. 1). Despite being
highly related in sequence to Ly-49A and -D, the specificity of Ly-49P
was not assured. We have demonstrated that Ly-49P does indeed recognize
H-2Dd in NK functional studies. It is possible
that Ly-49P may also recognize an H-2k product;
however, should this be the case, our data suggest that it is probably
with substantially lower affinity. It is worth noting that Ly-49A has
been reported to recognize Dk, although less
effectively than Dd (56, 57),
whereas Ly-49D can recognize Dd, but does not
appear to be able to interact with H-2k products
(17, 18). It is possible that Ly-49P may share the class I
specificity of Ly-49A, but its ability to also recognize
H-2k products still remains to be confirmed. In
our studies, Ly-49PNOD did not recognize NOR or
NOR Con A blasts, suggesting that this NK-activating receptor, or this
allele of Ly-49P, does not recognize self MHC molecules in the NOD
mouse.
The crystal structure of Ly-49A bound to H-2Dd
has recently been resolved to 2.3Å (58). The Ly-49A CRD
folds into seven ß strand and two helix secondary structure
elements. Given the high degree of sequence identity between Ly-49A and
Ly-49P in the CRD, it is likely that the Ly-49P CRD would form a very
similar structure. In the crystal complex, Ly-49A binds
H-2Dd at two sites: site 1 straddles the
1/2 domain boundary, and site 2 involves the cleft formed by the
3 domain, ß2-microglobulin, and the bottom
of the platform supporting the peptide binding groove where a portion
of CD8 has also been shown to bind (58). It remains to be
determined whether site 2 on Dd participates in
Ly-49A binding when in trans, i.e., during an interaction
with Dd expressed on a target cell. We show that
the Dd 1/2 domain is required for Ly-49P
recognition, which is consistent with participation of site 1. In fact,
Ly-49P conserves all 12 of the Ly-49AB6 contact
residues for this site (58) (Fig. 1A). Ly-49P
also conserves 24 of the 25 residues of Ly-49A that may interact with
site 2 (58) (Fig. 1A).
The NOD mouse strain from which we cloned Ly-49PNOD has immunodeficiencies and develops type I diabetes (45, 59). Adoptive transfer experiments have demonstrated that T cells are required for the induction of diabetes in the NOD mouse (reviewed in Ref. 45). NK cells as well as T cells and B cells are found in the cellular infiltrates of pancreatic insulitis in the NOD mouse, but the role of NK cells, if any, in the disease process remains unclear (60). NK cells of NOD mice have been described as functionally deficient. This characteristic is attributed as a result of observations that NOD NK cells lyse the prototype NK target cell, Yac-1, less efficiently than NK cells of other mouse strains (61, 62). Determining the extent of the NOD NK deficiency will require a comprehensive analysis of NK lytic potential and cytokine production in response to additional target cell types. We have shown that NOD mouse NK cells express viable transcripts for the NK activator molecule Ly-49P, and subsets of NOD NK cells stain with Abs reactive with Ly-49P and Ly-49A, suggesting that Ly-49P may be expressed at the cell surface of some NOD NK cells. Ly-49P and other NK activator molecules may be relevant for NOD NK recognition of targets other than Yac-1. The identification of NK activator molecules and the development of serological reagents specific for these receptors may aid in the characterization of NOD NK functional efficiency as well as in determining the contribution, if any, of NK cells to the development of diabetes in the NOD mouse.
The NOD/SCID mouse has shown particular utility in studying human tumor
growth and development as well as human hemopoiesis, since it accepts a
broader spectrum of human xenografts than SCID or nude mice (59, 63). Human graft acceptance by NOD/SCID mice has been suggested
to be due to the NK functional deficiency of the NOD mouse in
combination with a lack of B cells and T cells related to the SCID
mutation (59, 63). Mice lacking the common -chain,
which is required for signaling through a number of cytokine receptors,
including IL-2R and IL-15R, do not develop functional NK cells
(64). Mice lacking the common -chain coupled with the
SCID mutation accept various human xenografts much more readily than
NOD/SCID mice (64). This difference may suggest that NOD
NK cells have an ability, although possibly diminished relative to that
of other strains, to reject xenografts. Since the NK-activating
receptor Ly-49D of C57BL/6 origin has demonstrated xenoreactivity
against cells of rat and hamster origin, albeit not against the
limited number of human target cell types tested as yet (22, 23), perhaps Ly-49D, Ly-49P, or additional NK-activating Ly-49
receptors expressed by the NOD mouse are responsible for some of the
residual barriers to human xenografts exhibited by the NOD/SCID
mouse.
NK cells are not the only cells that express Ly-49 receptors, as some T
cell subsets can also express these receptors (3). NK T
(NKT) cells, for example, express TCR using a conserved TCR -chain
and are regulatory T cells that rapidly secrete large quantities of
IL-4 or IFN- in response to glycolipid Ags presented by the
nonclassical class I-like CD1 molecules (65). As the name
implies, NKT cells express various receptors normally found on NK
cells, including NKRP.1 and Ly-49 molecules. Interestingly, NKT cell
numbers are reduced in the diabetes-prone NOD mouse relative to other
mouse strains, and manipulations that substantially increase the number
of NKT cells result in protection from diabetes (66, 67).
Evidence provided by Exley et al. (68) suggests that the
NK receptor NKR-P1A, expressed on human NKT cells, may function as a
costimulator in NKT cell activation. It remains to be determined
whether mouse NKT cells express activating Ly-49 family members and
what role these receptors may play in NKT cell function.
The significance of Ly-49P expression in NOD and NOR mice with respect to immune regulation is unclear. However, it is perhaps notable that some of the pancreas-infiltrating T cells of both strains of mice express Ly-49P transcripts and the A1 epitope by FACS analysis (data not shown). Whether cells of NOD or NOR mouse tissues other than splenic T cells express ligands for Ly-49P in normal or disease states remains to be determined.
|
Acknowledgments |
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EN expression vector, and Dr. Michael Durairaj (University of
Alberta, Edmonton, Canada) for the Kb/Dd
chimera cDNA. We thank Dr. John Elliott (University of
Alberta) for provision of NOD and NOR mice and COS-7 cells, and Dr.
Fumio Takei (Terry Fox Laboratories, Vancouver, Canada) for provision
of the YE1/48 Ab. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Kevin P. Kane, Department of Medical Microbiology and Immunology, 660 HMRC, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
3 Abbreviations used in this paper: SHP-1, SH2 domain-containing tyrosine phosphatase; 51Cr, Na51CrO4; CRD, carbohydrate recognition domain; ITIM, immunoreceptor tyrosine-based inhibitory motif; NKT cells, NK T cells; NOD, nonobese diabetic; NOR, nonobese diabetes resistant; NWNA, nylon wool nonadherent; PA, protein A; PerCP, peridinin chlorophyll protein; rADCC, reverse Ab-dependent cellular cytotoxicity; RACE, rapid amplification of cDNA ends.
Received for publication January 11, 2000. Accepted for publication May 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion. J. Immunol. 164:603./ß antigen receptor. J. Immunol. 138:815.[Abstract]
ß T cell receptors. J. Immunol. 142:2736.[Abstract]
2 domain of H-2Dd restricts the allelic specificity of the murine NK cell inhibitory receptor Ly-49A. J. Immunol. 160:5971. chain. Br. J. Haematol. 103:335.[Medline]
14-J281 transgenic nonobese diabetic mice against diabetes. J. Exp. Med. 188:1831.24Q T cell receptor chains. J. Exp. Med. 188:867.This article has been cited by other articles:
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M. S. Osman, D. N. Burshtyn, and K. P. Kane Activating Ly-49 Receptors Regulate LFA-1-Mediated Adhesion by NK Cells J. Immunol., February 1, 2007; 178(3): 1261 - 1267. [Abstract] [Full Text] [PDF]
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K. J. Lavender and K. P. Kane Cross-Species Dependence of Ly49 Recognition on the Supertype Defining B-Pocket of a Class I MHC Molecule J. Immunol., December 15, 2006; 177(12): 8578 - 8586. [Abstract] [Full Text] [PDF]
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 M. S. Osman, E. T. Silver, J. C. Varghese, C. S. Chang, D.-E. Gong, G. F. Audette, B. Hazes, and K. P. Kane Epitope mapping of Ly-49G and G-like receptors: CK-1 antibody defines a polymorphic site of functional interaction with class I ligand J. Leukoc. Biol., May 1, 2005; 77(5): 644 - 651. [Abstract] [Full Text] [PDF]
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K. J. Lavender, B. J. Ma, E. T. Silver, and K. P. Kane The Rat RT1-A1c MHC Molecule Is a Xenogeneic Ligand Recognized by the Mouse Activating Ly-49W and Inhibitory Ly-49G Receptors J. Immunol., March 15, 2004; 172(6): 3518 - 3526. [Abstract] [Full Text] [PDF]
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B. J. Ma, E. T. Silver, B. Hazes, and K. P. Kane Reciprocal Transfer of Class I MHC Allele Specificity between Activating Ly-49P and Ly-49W Receptors by Exchange of {beta}4-{beta}5 Loop Residues J. Immunol., November 15, 2003; 171(10): 5337 - 5344. [Abstract] [Full Text] [PDF]
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L. H. Mason, J. Willette-Brown, S. K. Anderson, W. G. Alvord, R. L. Klabansky, H. A. Young, and J. R. Ortaldo Receptor Glycosylation Regulates Ly-49 Binding to MHC Class I J. Immunol., October 15, 2003; 171(8): 4235 - 4242. [Abstract] [Full Text] [PDF]
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C. Carnaud, J.-M. Gombert, O. Donnars, H.-J. Garchon, and A. Herbelin Protection Against Diabetes and Improved NK/NKT Cell Performance in NOD.NK1.1 Mice Congenic at the NK Complex J. Immunol., February 15, 2001; 166(4): 2404 - 2411. [Abstract] [Full Text] [PDF]
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