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Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto, Ontario, Canada
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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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and NKR-P1 is essential for activation of NK
cells via the NKR-P1 molecule (10). The genes encoding Ly49A and NK1.1 are on distal mouse chromosome 6, where they are separated by only 0.4 cM. Since both Ly49 and NK1.1 glycoproteins are NK-specific, of type II transmembrane protein orientation, share homology with C-type lectins, and are functionally active in NK-mediated lysis, this genetic region has been termed the NK gene complex (11). It is further suggested that there are multiple members of the NK gene complex. Many members of the Ly49 family, in addition to Ly49A, have been identified (Ly49A-I) and are highly polymorphic (12). Although Ly49A is an inhibitory receptor, this does not apply to all Ly49 genes: Ly49D does not contain an immunoreceptor tyrosine-based inhibitory motif (ITIM), and has been demonstrated to be an activation receptor (13, 14). For mouse NKR-P1, three transcripts (NKR-P1-A, -B, and -C) have been identified in C57BL NK cells (15). NKR-P1A shares the highest identity with the rat NKR-P1 molecule, an activation receptor that has some target specificity (16) and that interacts with the N-terminal domain of p56lck via a cytoplasmic Cys-X-Cys-Pro sequence (17). NKR-P1C (but not NKR-P1A) is identified to be the gene encoding the NK1.1 molecule recognized by PK136 mAb (18). Due to the lack of serological reagents against other members of the mouse NKR-P1 gene family, the NKR-P1C gene is the only mouse NKR-P1 gene known to be expressed and to have function. In particular, neither surface expression nor function(s) of the NKR-P1B gene product has been shown, although it has been noted that the cytoplasmic tail contains an ITIM, suggesting that it may be inhibitory (19).
In an attempt to identify NK activation receptors, we immunized BALB/c mice with (BALB/c x C57BL/6)F1 (CbyB6F1) NK lymphokine-activated killer (LAK) cells and made B cell hybridomas. These were then screened for mAb that activated NK cells to kill P815 targets (relatively resistant to NK lysis) in the AIRL assay. Four hybridomas (1C10, 1F10, 2D10, and 4G4) were selected for further characterization. They were able to bind to and activate NK cells from B6 and CByB6F1 mice in FACScan analysis and in the AIRL assay, respectively (our manuscript in preparation). We have also tested whether these mAbs plus PK136 mAb (20), which recognizes NK1.1 on B6, could "cross-react" with NK cells from other mouse strains: SJL/J, DBA/2, 129/J, C3H, and BALB.K. None showed any reactivity except 1C10 and PK136, both of which reacted with some cells in SJL/J, with the PK136-staining subset being entirely included in the larger 1C10-staining subset. Here, we report on the functions of the putative NK receptors (as identified by PK136 and our novel 1C10 mAbs) on SJL/J NK cells and provide evidence that the 1C10 mAb recognizes a yet to be identified NK activation receptor on SJL/J NK cells, whereas the PK136 mAb recognizes the SJL allele of the NKR-P1B gene product. We provided direct evidence to support the notion that the NKR-P1B gene product is an NK inhibitory receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J (B6), DBA/2, SJL/J, and (BALB/c x B6)F1 (CByB6F1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were kept in a specific pathogen-free animal colony in the Ontario Cancer Institute (Toronto, Canada). In most experiments, 6- to 10-wk-old female mice were used (although either sex gave similar results).
mAbs and flow cytometry
Hybridomas PK136/HB191 (NK1.1), TIB 207 (anti-mouse CD4),
TIB105 (anti-mouse CD8), and W6/32/HB95 (anti-HLA-A, B, C) were
obtained from American Type Culture Collection (ATCC; Manassas, VA).
W6/32 mAb (IgG2a, 
) was used as an isotype control for
PK136 mAb, where indicated. mAbs were purified from culture
supernatants by protein G (Sigma, St. Louis, MO) chromatography.
Purified normal mouse Ig Abs were purchased from Sigma. FITC-conjugated
Pan-NK (DX5), PE-conjugated anti-NK1.1 (PK136), biotinylated PK136,
and H-2Dd (clone 34-2-12) mAbs were purchased from
PharMingen (San Diego, CA). Fluorescence-conjugated Abs anti-CD3
FITC (clone 29B) and anti-CD3 PE (clone 29B) were purchased from
Sigma. Labeling Abs with FITC was performed as described (21). FACScan
analysis was done as described previously (22). In particular, Fc
receptors were blocked with a mixture of normal mouse serum, normal
mouse Ig, and 2.4G2 mAb as described (22). Intracellular staining was
performed according to the instruction manual for the Cytofix/Cytoperm
staining kit (PharMingen).
Preparation of splenocytes
Spleens were pressed through a wire mesh screen with a
disposable syringe plunger into complete medium (CM), which was 
-MEM
(Life Technologies, Burlington, Ontario, Canada) supplemented with 10%
FCS (Life Technologies), 50 µM 2-ME, and 10 mM HEPES. Released cells
were layered over 5 ml of 6% BSA in PBS and centrifuged to remove cell
debris. To obtain nylon wool-nonadherent (NWNA) splenocytes,
splenocytes were resuspended in 5 ml CM, underlaid with 5 ml of
lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada), and
centrifuged at 500 x g for 20 min to remove RBC and
dead cells. After one wash in CM, the cells were resuspended in 2 ml of
CM and then loaded onto a nylon wool column (0.6 g nylon wool in a
12-ml syringe, autoclaved, preequilibrated with 30 ml of warm 1%
BSA/PBS). After 75 min of incubation, the nonadherent cells were eluted
from the column with 1% BSA/PBS. The NWNA cells were washed and
resuspended in CM.
T cell depletion by Dynal magnetic bead separation
NWNA splenocytes were prepared as described. The cells were resuspended in CM at 10 x 106/ml. To deplete CD4+ and CD8+ T cells, anti-CD4 and anti-CD8 ascites were added to the cell suspension at 1:200 (v/v) dilution. The mixture was rocked gently for 1 h at 4°C. Excess Abs were removed by washing the cell pellet twice in 1% BSA/PBS. The cell pellet was resuspended in CM at 10 x 106/ml, and sheep anti-rat Dynabeads (Dynal, Oslo, Norway) were added to the cell suspension at a ratio of 1:1 (bead:cell). The mixture was rocked gently for 1 h at 4°C. At the end of incubation, the immunocomplex was removed by magnetic separation. The unbound fraction was collected, washed, and resuspended in CM. Efficiency of T cell depletion was confirmed by staining the unbound fraction with anti-CD3 mAb and analyzing by FACScan. Depletion of T cells was routinely 75–90%.
Preparation of LAK cells
Briefly, 2–10 x 106 splenocytes, from normal inbred F1 mice or athymic F1 nude mice, were suspended in 10 ml CM, supplemented with 500 U/ml murine rIL-2 obtained from a cell line transfected with the mouse IL-2 gene, kindly provided by Dr. H. Karasuyama (University of Tokyo, Tokyo, Japan) (23). The cells were incubated at 37°C, 10% CO2/air atmosphere for 72 h. LAK cells were then collected, resuspended in 5 ml of CM, underlaid with 5 ml of lympholyte-M (Cedarlane Laboratories), and centrifuged at 500 x g for 20 min to remove dead cells. To obtain highly enriched NK-LAK, NWNA splenocytes from F1nude mice or T cell-depleted (TCD) NWNA, splenocytes were cultured with 500 U/ml murine rIL-2 for 48–72 h as described above.
Production of B cell hybridomas
BALB/c mice were immunized i.p. with 2–3 x 106 CByB6F1 LAK once a month for 3 mo. Splenocytes were obtained for cell fusion from the immunized mouse 4 days after the last immunization. Log phase Sp2/0 murine myeloma cells (1 x 107) (CRL 1581, ATCC) were transferred to a 50-ml centrifuge tube that contained 1 x 108 splenocytes. The tube was filled with CM and centrifuged at 200 x g for 5 min. The cell pellet was washed once in 50 ml of CM and warmed in a 37°C water bath for 2 min. The splenocytes and Sp2/0 cells were fused by polyethylene glycol (PEG) solution (50% w/v PBS; Sigma) as follows. Over the first 1 min, 1 ml of PEG solution was added to the pellet at 37°C with gentle mixing. Over the next 2 min, the cells were centrifuged at 100 x g. Over the next 3 min, 4.5 ml of CM was added to the mixture, followed by another 5 ml of CM over the next 2 min. The tube was then filled with CM, centrifuged at 100 x g for 5 min, and the cell pellet was gently resuspended in 35 ml of CM. The fused cells (in 100-µl suspension per well) were cultured in the flat-bottom wells (precultured with macrophages) of a 96-well plate; only the 60 inner wells were used. For selection, started at 24 h, 100 µl of culture supernatant was removed from each well and replaced by 100 µl of 2x hypoxanthine/aminopterin/thymidine (HAT; Sigma). The cultures were replenished with fresh 1x HAT medium every other day until, at 2 wk, hybrid cells covered 10–50% of the surface area of positive wells. Positive hybridomas were then switched to HT (hypoxanthine-thymidine, Sigma) and were maintained in HT until the completion of two cloning procedures.
Screening, cloning of hybridoma cell lines by limiting dilution, and isotyping
Hybridomas were screened by the AIRL assay as described below. Briefly, F1 LAK effectors were mixed with 51Cr-labeled P815 at an E:T ratio of 5:1. Culture supernatant (100 µl) from each hybridoma-containing well was transferred directly to the mixture of LAK cells and target cells. Specific lysis was determined in a 4-h 51Cr release assay. Positive clones were identified by their ability to increase the specific lysis of the P815 targets. Positive hybridomas were cloned at the single cell level by limiting dilution. Briefly, hybridomas from each positive well were grown to the desired cell density by culturing them in a 96-well plate, and then in a 24-well plate (if necessary, a 6-well plate). Actively growing hybridoma clones were washed, counted, and seeded into the 96-well plate (which was previously cultured with mouse macrophage feeder cells 1 day before the limiting dilution experiment) at a density of 1 cell/well. When the cells grew up to 10–50% of the surface area of the wells, they were screened and cloned by limiting dilution until all the wells that contained hybridoma were positive in the screening assay. Isotypes of various hybridomas were determined with a mouse mAb isotyping kit, IsoStrip (Boehringer Mannheim, Indianapolis, IN).
Ab-induced redirected lysis assay
LAK cultures were prepared as described previously (8). P815 (murine mastocytoma, TIB64) target cells (1 x 107) were labeled with 0.4 mCi of sodium [51Cr]-chromate (Dupont Chemicals, Mississauga, Ontario, Canada) for 1 h. After three washes in CM, the target cells were added to 96-well V-bottom microtiter plates at 2 x 103 cells/well in 100-µl aliquots (103 cells per 100-µl aliquot when resting NK were used). LAK effectors were mixed with 51Cr-labeled P815 at various E:T ratios with or without 1 µg per well of purified PK136 mAb or mAbs of interest. Specific lysis was determined as described previously. The error bars in the cytotoxicity assays represent SEM of the specific lysis. Each killing assay has been independently replicated at least three times.
Flow cytometric analysis of calcium flux by fluo-3-acetoxymethyl ester (fluo-3-AM)
Resting NK cells (107/ml) were washed once with 1% FCS in HBSS, once with HBSS, and were then incubated in HBSS alone (without FCS) containing fluo-3-AM and Pluronic F-127 stock solution (Molecular Probe, Eugene, OR) at a final concentration of 2 µM and 3 µl/ml, respectively, for 20 min at room temperature. The cells were then diluted to 2 x 106 cells/ml in 1% FCS/HBSS, and were incubated in a 37.5 ± 0.1°C water bath for another 40 min (24). Fluo-3-loaded cells were washed two or three times in 1% FCS/HBSS. After the wash, nonspecific binding to NK cells was blocked by adding 4 µl of FcR blocking mixture (normal mouse serum, anti-FcR, and normal mouse Ig at 20:1:5 v/w/w) we routinely use in flow cytometry to the cells (0.2 x 106 cells in 0.1 ml of buffer) for 5–10 min on ice. Afterwards, 5 µg of the mAb of interest was added to the cells for 20 min at 4°C. The cells were washed with 1% FCS/HBSS and were resuspended in 0.5 ml 1% FCS/HBSS. The sample was incubated in a 37.5°C water bath for 5 min before it was run on a FACScan apparatus. Green fluorescence emission of fluo-3 was detected at 530 nm (DF 530/30 dichronic bandpass filter), and was displayed on a linear scale (1024 channels for best accuracy) with the time parameter activated on a scale of 512s (500 ms/channel). After the baseline of the Ca2+ signal was measured, 5–10 µg of rabbit anti-mouse Ab (Jackson ImmunoResearch, West Grove, PA) was added to the reaction tube to cross-link the primary mAb. The Ca2+ flux induced by receptor cross-linking was then measured. If necessary, 3 µl of 1 µg/ml of the calcium ionophore A23187 (Sigma) was added to the 0.4-ml sample as a positive control.
Immunoprecipitation
LAK cells were washed in ice-cold PBS twice to remove serum proteins. The cell pellet (10 x 107 cells) was then resuspended in 6 ml of sulfo-NHS-biotin solution (Pierce, Rockford, IL) in PBS, at a final concentration of 0.5 mg/ml, for 30 min at 4°C. At the end of the reaction, 1 M NH4Cl (at 10 µl/ml) was added to the reaction. Excess sulfo-NHS-biotin was removed by centrifugation at 500 x g for 10 min. Cell surface-biotinylated cells were incubated in ice-cold serum-free CM for 10 min, washed twice with ice-cold PBS, and quick frozen in liquid nitrogen. Frozen pellets were stored at -70°C until use. Surface biotinylated F1 or SJL LAK cells (1–2 x 107 cells/ml) were lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 10 mM sodium pyrophosphate (Sigma), 10 mM sodium fluoride (Sigma), 10 mM EDTA (Sigma), 1 mM sodium orthovanadate (Sigma), 1.25 mM PMSF (Sigma), 10 ng/ml aprotinin (Sigma), and 10 µg/ml leupeptin (Boehringer Mannheim) on ice for 30 min. BSA (0.1% final) was added to the cell lysate to block nonspecific binding. PK136 mAb (5 µg) was added to the cell lysate to immunoprecipitate the NK1.1 Ag. W6/32 mAb was used as the isotype control. The mixture was incubated at 4°C on a mixer for 3 h before the addition of 40 µl of protein A/G Plus-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA). After 2 h of incubation at 4°C, the immunocomplexes were collected by centrifugation at 2500 rpm for 5 min, and then washed three times with 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Triton X-100, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 0.5 ng/ml aprotinin, and 0.5 µg/ml leupeptin. Captured Ags were eluted by boiling the agarose beads in 80 µl of nonreducing sample buffer for 5 min. The reaction mixture was then vortexed vigorously and microcentrifuged at full speed for 10 s. Supernatant was collected and saved as a nonreduced sample. To prepare samples under reducing condition, 5 µl of 0.9 M DTT was added to 40 µl of the nonreduced sample, and the mixture was boiled for another 5 min. Both reduced and nonreduced samples were analyzed under SDS/PAGE, followed by Western blotting. The blot was incubated in 10% nonfat milk powder in 1% Tween 20 in PBS (PBST) for 1 h, and was washed once in the wash buffer (1% nonfat milk powder in PBST). To detect biotinylated proteins on the blot, HRP-conjugated streptavidin (1/2000 dilution in the wash buffer; Pierce) was added to the blot. After 1 h of incubation at room temperature, the blot was washed three times in the wash buffer. The autoradiograph was developed in Renaissance Enhanced Luminol Western Blot chemiluminescence reagents as described in the user manual (NEN Life Science, Boston, MA). Apparent m.w. was determined by comparison to Benchmark prestained protein ladder (Life Technologies).
Coimmunoprecipitation of SHP-1 with the NK1.1 Ag
A total of 1–2 x 107 SJL/J TCD, NWNA LAK cells were allowed to react with 10 µg biotinylated PK136 mAb (PharMingen) at 4°C for 2 h. The cells were washed once in 1% BSA/PBS to remove unbound mAbs and were then resuspended in 2 ml of PBS containing 0.1 mM sodium orthovanadate. Then, 100 µl Dynabeads M-280 Streptavidin (Dynal) was added to the cell suspension to allow binding and cross-linking of the surface-bound biotinylated PK136 mAb. After 3–4 h incubation at 4°C, the Dynabeads/cell complexes were isolated by magnetic separation, washed once in 1% BSA/PBS, incubated in a 37°C water bath for 1–10 min, and lysed as described previously. Dynabeads/immunocomplexes were separated from the cell lysate by magnet, and the cell lysate was centrifuged at 13,000 rpm for 15 min at 4°C to remove cell debris. The cleared lysate supernatant was allowed to react with the Dynabeads/immunocomplexes for an additional 1–2 h at 4°C. Afterwards, the Dynabeads/immunocomplexes were washed three times in the washing buffer as described previously. Captured Ags were eluted by boiling the Dynabeads/immunocomplexes in 35 µl of reducing sample buffer for 5 min. The reaction mixture was then vortexed vigorously and microcentrifuged at full speed for 10 s. Supernatant was collected and analyzed in Western blotting. For the control reaction, the same number of SJL/J TCD, NWNA LAK cells were incubated with 100 µl of Dynabeads M-280 streptavidin in the absence of biotinylated PK136 mAb for 3–4 h of incubation at 4°C. The Dynabeads and the unbound cells were separated by magnet. The cells were washed once in 1% BSA/PBS, incubated in a 37°C water bath for 1–10 min, and lysed. The cell lysate was centrifuged at 13,000 rpm for 15 min at 4°C to remove cell debris. Isotype control biotinylated mAb (2.5–5 µg, H-2Dd, clone 34-2-12; PharMingen) was added to the lysate supernatant, and was allowed to react with the Dynabeads for 1–2 h at 4°C. Afterwards, the Dynabeads/immunocomplexes were washed and boiled as described previously. For Western blotting, the blot was incubated in 10% nonfat milk powder in 1% PBST for 3 h and was washed once in the wash buffer (1% nonfat milk powder in PBST). The blot was incubated with polyclonal anti-SHP-1 Abs (at 1/5000 dilution) at room temperature for 1 h. Unbound Abs were removed by three washes of the membrane with the wash buffer at room temperature for 5 min. To detect bound anti-SHP-1 Ab on the blot, HRP-conjugated anti-rabbit Ig Abs (1/5000 dilution in the wash buffer) were added to the blot. After 1 h of incubation at room temperature, the blot was washed three times in the wash buffer. The autoradiograph was developed in Renaissance Enhanced Luminol Western Blot chemiluminescence reagents as described previously.
RT-PCR analysis
Total RNA was isolated from TCD, NWNA LAK cells (B6, CByB6F1, or SJL/J) using the Trizol RNA isolation protocol (Life Technologies). RT-PCR was performed using the GeneAmp RNA PCR kit (Perkin-Elmer, Branchburg, NJ) on an automated GeneAmp 2400 thermocycler (Perkin-Elmer). Reverse transcription of RNA (<1 µg) using random hexamers was performed at 42°C for 1 h, 99°C for 5 min. RT product (1 µl) was used for PCR (touchdown) amplification with a hot start at 94°C for 3 min, followed by 30 cycles of 15-s denaturation at 94°C, 30-s annealing reaction at 60.5°C with 0.5°C decrement in each subsequent cycle, and 30-s at 72°C for 8 cycles, and a final extension at 72°C for 10 min. Gene-specific primers (based on the published C57BL NKR-P1 GenBank sequences) used for PCR were as follows: Gene 2/NKR-P1A (5'), ACA ATG GAC ACA GCA AGG GTC TAC; Gene 2/NKR-P1A (3'), CTT TGT CTC CTG AGA TAG CA; Gene 34/NKR-P1B (5'), CAA CAA CAC TGG TCT ATG CA; CTG GTC TAT GCA GAT TTA AA; Gene 34/NKR-P1B (3'), GGA CAG GGG AGA GAT GGA GAT; Gene 40/NKR-P1C (5'), TTA AGT GTT GCA TCT CCT GTG; Gene 40/NKR-P1C (3'), TCT GAA GCA CAG CTC TCA GG. In addition, the following sets of primers were used to obtain PCR products that encode the full open reading frame: NKR-P1A (5'), ATG CAT CTC CTA TGC ACA ATG; NKR-P1A (3'),GAG TTC AGT GTC CAT AAC CCA; NKR-P1B (5'), TCT ACA ATG GAT TCA ACA ACA; NKR-P1B (3'), GGA CAG GGG AGA GAT GGA GAT; NKR-P1C (5'), TTA AGT GTT GCA TCT CCT GTG; NKR-P1C (3'), GAT GGG ATT CGC AGT CAG GA. These PCR products were ligated onto mammalian expression vector, using the Eukaryotic TOPO TA cloning kit (Invitrogen, Carlsbad, CA) or green fluorescense protein (GFP) fusion TOPO cloning kit (Invitrogen). Nucleic acid sequencing was performed by the DNA sequencing facility, the Centre for Applied Genomics (Hospital for Sick Children, Ontario, Canada).
DNA transfection
NKR-P1 plasmid DNA (30 µg, in pcDNA3.1/CT-GFP-TOPO vector;
Invitrogen), together with 6 µg of the pcDNA3.1/CT-GFP plasmid
(Invitrogen), was transfected into Jurkat cells (3–4 x
106 cells in 0.25 ml of 20% FCS/RPMI 1640) by
electroporation using a BTX (San Diego, CA) ECM600 at 300V, 186
,
1600 µF in 4-mm gap cuvettes (Bio-Rad, Richmond, CA). Transfected
cells were then cultured in 20% FCS/RPMI 1640 for 6–7 h and analyzed
for protein expression by FACScan analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have generated four new mAbs that recognize NK activation
receptors in B6 mice (our manuscript in preparation). As shown in Fig. 1
A, three of these new mAbs
(1C10, 1F10, and 2D10), together with PK136 mAb, recognize a small
population of NWNA lymphocytes in B6 mice. However, only 1C10 and PK136
mAbs identified cells in SJL/J. Thus, such surface staining could not
be found if NWNA splenocytes of DBA/2 were used (Fig. 1A).
In an AIRL assay using LAK cells prepared from SJL/J NWNA splenocytes,
only the 1C10 mAb was able to activate SJL/J LAK cells to kill P815
targets (Fig. 1B). To control for the specificity of the
1C10 mAb, other mAbs (1F10, 2D10, or 4G4) were tested and shown to have
no effect on lysis (Fig. 1B). In addition, the 1C10 mAb had
no effect on the activation of DBA/2 NWNA LAK (that were negative for
1C10 mAb surface staining; Fig. 1A) in the same AIRL assay
(Fig. 1B). Taken together, data from the FACScan analysis
and the AIRL assay are consistent with the notion that the 1C10 mAb
specifically recognizes a lymphocyte population in SJL/J mice, and that
1C10 can induce AIRL, suggesting that the cells recognized by the mAb
are NK cells.
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A, the small
1C10+ population was enriched when the NK cell population
was enriched. The majority of the 1C10+ cells were also
NK1.1+ (identified by PK136 mAb). Down-regulation of
surface expression of the 1C10 Ag was consistently observed in more
than 10 independent measurements upon rIL-2 activation of SJL/J NK
cells (Fig. 2A). The 1C10 mAb was able to activate SJL/J NK
LAK cells in the AIRL assay (Fig. 2B,
right). These LAK cells were >74% DX5+ NK
cells and <11% CD3+ T cells (Fig. 2B, left). Furthermore, in an AIRL assay using
freshly prepared cells from SJL/J spleen cells (enriched for NK cells
by depletion of nylon wool-adherent cells and TCD), 1C10 mAb was able
to induce redirected lysis (data not shown), most likely due to its
action on unactivated NK cells.
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,
top, PK136 mAb and the two of our new hybridomas tested
(1C10 and 1F10) were able to induce a transient Ca2+ flux
in CByB6F1 NK cells 1–2 min after the rabbit
anti-mouse Ab was added. As expected, no Ca2+ flux
could be detected if a mAb against an inhibitory receptor (5E6 mAb
against Ly49C/I molecule; 26) was used in the assay. We used the
same assay to test the ability of our mAbs to induce a Ca2+
flux in resting SJL/J NK cells. Cross-linking the 1C10 mAb (but not the
1F10 or PK136 mAb) resulted in a weaker but reproducible
Ca2+ flux in the SJL/J resting NK cells (Fig. 3, bottom). In agreement with the AIRL data (Fig. 2
), these
data suggest that 1C10, but not 1F10 or PK136, can mediate an
activation signal in SJL/J NK cells.
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SJL/J NK cells have a relatively high level of surface expression
of the "NK1.1" Ag (recognized by PK136 mAb) (Figs. 1 and 2),
comparable to that of CByB6F1 NK cells (data not shown).
However, PK136 mAb was not able to activate SJL/J NK cells in the AIRL
assay or to trigger a calcium flux in these cells (
Figs. 1–3). In
fact, it appeared that addition of PK136 mAb to the AIRL assay
inhibited NK cytotoxicity (Figs. 1 and 3). These data were in contrast
to the findings obtained when the PK136 mAb was used in other
well-documented systems (B6 or CByB6F1 NK cells) (7, 8, 22, 27). To formally compare the effect of PK136 mAb on B6, SJL/J, and
CByB6F1 LAKs in the AIRL assay under identical experimental
conditions, we used day 3 LAK cultures prepared from whole splenocytes
of these mice. As expected, the PK136 mAb as well as our new mAbs (1C10
and 1F10) were able to activate B6 and CByB6F1 LAK to kill
P815 targets, and the 1C10 mAb (but not 1F10 mAb) activated SJL/J LAK
(Fig. 4). However, the same PK136 mAb
failed to activate SJL/J LAK (Fig. 4).
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, 1C10 mAb activated SJL/J LAK to kill
P815 targets, while PK136 mAb inhibited the lysis. The presence of
PK136 mAb suppressed the activation effect of the 1C10 mAb. It should
be noted that the same amount of isotype control mAb was used in
replacement of 1C10 and PK136 mAb, respectively, in the PK136 and 1C10
experimental groups to eliminate the possibility of mAb competition for
the binding of FcR on the P815 targets. As a further control,
NK1.1+CD3- LAK cells were sorted from B6 mice
and assayed under identical experimental conditions. Unlike SJL/J NK
cells, PK136 mAb activated B6 LAK cells to kill P815 targets in the
presence or absence of 1C10 mAb (Fig. 5).
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81 kDa (42 kDa when reduced). It
was slightly different from the NKR-P1C gene product of
CByB6F1 NK cells (a homodimer of 81 kDa, 39 kDa when
reduced) (Fig. 6A).
Interestingly, upon cross-linking of the "NK1.1" Ag on SJL/J LAK
cells with PK136 mAb, SHP-1 was found to coimmunoprecipitate with the
"NK1.1" protein, but not with the isotype control mAb (Fig. 6B). As a further control, biotinylated PK136 mAb was added
to the lysate of NK LAK cells that had not been cross-linked with PK136
mAb before lysis. Immunocomplexes of this control reaction were
captured by streptavidin-Dynabeads for a coimmunoprecipitation
experiment, as described in Fig. 6B. Under these conditions,
we did not detect any SHP-1 proteins (data not shown),
suggesting that cross-linking/phosphorylation of the "NK1.1" Ag is
essential for SHP-1 recruitment.
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Primers specific for C57BL NKR-P1A, -B, and -C genes were designed
and used to detect the presence of their mRNA transcripts, using total
RNAs from B6, CByB6F1, and SJL/J NK cells in RT-PCR
reactions. As shown in Fig. 7A, PCR products of sizes of
700 bp and 1 kb, as expected for the NKR-P1A and NKR-P1B gene
products, respectively, were detected in all three mouse strains (B6,
CByB6F1, and SJL/J). A PCR product of size 700 bp as
expected for the NKR-P1C gene product, was detected in both B6 and
CByB6F1 total RNAs, but was not detected in the SJL/J LAK
total RNAs under identical conditions (Fig. 7A). The same
observations were obtained using a different set of primers specific
for NKR-P1A, two other 5'-primers specific for NKR-P1B, and another
3'-primer specific for NKR-P1C (data not shown). Nucleotide sequencing
of the SJL/J RT-PCR product obtained by the use of our C57BL
NKR-P1B-specific primers confirmed the identity of this SJL/J gene as
NKR-P1B. It is fully identical to the C57BL sequence published by
Giorda and Trucco (15) at the nucleotide level (data not shown) as well
as the deduced amino acid level, thus containing a recently identified
LxYxxL ITIM motif (Fig. 7B, and 28). Surprisingly, our
B6 NKR-P1B sequence was found to be different from the published C57BL
sequence (Fig. 7B, see Discussion).
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). In addition, PK136 mAb showed
positive staining of the SJL/J NKR-P1B transfectants but not B6
NKR-P1B, SJL/J NKR-P1B antisense, or SJL/J NKR-P1A transfectants (Fig. 8). The lack of PK136 mAb staining in the B6 NKR-P1A and -B
transfectants was not due to a lack of protein expression in these
transfectants because 1C10 mAb was found to stain all of the B6
NKR-P1A, -B, and -C transfectants in a separate experiment (our
manuscript in preparation).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In contrast, the "NK1.1 Ag" on
SJL/J NK cells (recognized by PK136 mAb), unlike the well-documented
activation receptor identified by the same mAb in F1 or B6
NK cells, was found to be a surface receptor that shares features in
common with NK inhibitory receptors. First, PK136 mAb inhibited SJL/J
NK cells (resting or rIL-2-activated) from killing P815 targets in the
AIRL assay (Fig. 1, 2, 5). Secondly, resting SJL NK cells could not be
induced to trigger a Ca2+ flux upon Ab cross-linking of the
"NK1.1" Ag (Fig. 3). Third, co-cross-linking of the "NK1.1" Ag
and the 1C10 Ag, in the AIRL assay, inhibited the NK cell activation
seen when 1C10 alone was cross-linked (Fig. 5). Fourth, receptor
cross-linking of the "NK1.1" Ag recruited SHP-1 (Fig. 6), a
protein-tyrosine phosphatase associated with inhibition of activation
pathways in hematopoietic cells (29, 30, 31). Flow cytometric analysis of
transient transfectants identified the NKR-P1B gene product of SJL/J
mice as the receptor that was recognized by PK136 mAb (Fig. 8). The NKR-P1 gene was first identified on rat NK cells by the mAb 3.2.3 (32, 33). The presence of an NKR-P1 gene family has been more extensively defined in the mouse. When a cDNA library from C57BL LAK cells was screened with the rat NKR-P1 cDNA probe, three sets of positive clones (referred to as genes 2, 34, and 40) were identified (15). Nucleotide sequence analysis of these genes suggested that they are closely related but distinct genes, coexpressed in mouse NK cells (15). Rat NKR-P1 shares 72–74% amino acid identity to both genes 2 (NKR-P1A) and 40 (NKR-P1C); however, the 3'-untranslated region of rat NKR-P1 is more closely related to NKR-P1A than to NKR-P1C (15). Gene 34 (NKR-P1B) is the gene that shares the lowest amino acid identity with rat NKR-P1 (61%), gene 2 (74%), or gene 40 (80%). Further analysis of the deduced amino acid sequences from the cDNA indicates that gene 40 resembles gene 2 in the 5' flanking region and through exon 1, 2, and 3, whereas the predicted carbohydrate recognition domain and 3' untranslated portions of gene 40 instead resemble gene 34 (34).
In mice, the NK1.1 Ag is thus far the best marker for defining NK cells
in those mouse strains that express it (20). In such strains (e.g.,
B6), all NK1.1+CD3- NK cells are capable of
lysing protoypic NK targets (35). The mouse NK1.1 molecule is a
disulfide-linked homodimer of 80 kDa (39 kDa when reduced) and
has been demonstrated to be an activation receptor in NK recognition
(7, 18). Expression of the B6 NKR-P1C gene in Sf9 insect cells restores
the expression of the NK1.1 Ag (recognized by PK136 mAb) in both flow
cytometry and immunoprecipitation (18), thus establishing that the B6
NKR-P1C cDNA encodes the NK1.1 Ag. SJL/J mice have been shown to have
low NK activity and frequency of NK cell progenitors (36). However, an
increasing fraction of SJL/J spleen cells stained for the "NK1.1"
Ag (with PK136 mAb) could be observed as the NK population was enriched
(as T cells and B cells were depleted) (
Figs. 1–3), correlating with
the published work on NK cell frequency. Despite the high level of
NK1.1 surface expression in flow cytometry analysis, no NK activation
was observed when we used the PK136 mAb in either the AIRL assay or the
calcium flux assay. Furthermore, concurrent cross-linking of the
surface receptors by PK136 mAb and 1C10 mAb showed that the "NK1.1"
molecule on SJL/J NK cells was inhibitory. All these data are in
contrast to previous findings obtained when PK136 mAb and B6 NK cells
were used in similiar experiments. Therefore, we hypothesized that the
"NK1.1" Ag on SJL/J NK cells is not identical with the Ag encoded
by the B6 NKR-P1C gene. Using C57BL NKR-P1A- and NKR-P1B-specific
primers in RT-PCR, we isolated cDNA of SJL/J NKR-P1A and NKR-P1B genes.
Nucleotide sequencing of the SJL/J RT-PCR product obtained by the use
of C57BL NKR-P1B-specific primers confirmed the identity of this SJL/J
gene as NKR-P1B. In addition, it is fully identical to the C57BL
sequence published by Giorda and Trucco (15) at both the nucleotide
level (data not shown) and the deduced amino acid level (Fig. 7B). Expression of the SJL/J NKR-P1B gene demonstrated that
this cDNA encodes the novel "NK1.1" Ag on SJL/J NK cells (Fig. 8).
The existence of the LxYxxL ITIM in SJL/J NKR-P1B gene thus supports
the notion that the "NK1.1" Ag on SJL/J NK cells is an inhibitory
receptor that recruits SHP-1 phosphatase upon activation.
It was surprising that PK136 mAb recognized the SJL/J NKR-P1B gene
product but not the B6 NKR-P1B gene product (Fig. 8) when our deduced
SJL/J NKR-P1B amino acid sequence and the published C57BL NKR-P1B
sequence (15) are fully identical (Fig. 7B). Therefore, we
sequenced our B6 NKR-P1B cDNA and compared it to the published NKR-P1B
sequence (Fig. 7B). It was found that our deduced B6 amino
acid sequence and the published sequence were identical in the
cytoplasmic and transmembrane regions. However, there were 15 amino
acid differences in the extracellular domains. This may provide a
molecular basis for the lack of reactivity of PK136 mAb on the B6
NKR-P1B gene product in our transient transfectants (Fig. 8). Yokoyama
and Seaman’s statement (11) that PK136 mAb does not react with B6
NKR-P1A and -B gene products is in indirect support of the observations
we report here. In fact, the observation that PK136 mAb recognizes only
the NKR-P1C (but not the NKR-P1B) gene product in B6 in our study, is
fully consistent with the notion that the PK136 mAb activates B6 NK
cells through the NK1.1 Ag. At this point, the lack of a serological
reagent specific for the B6 NKR-P1B gene product makes it difficult to
determine whether the NKR-P1B gene product is actually expressed on B6
NK cells.
We cannot reconcile the differences between our B6 NKR-P1B sequence and
the NKR-P1B sequence published by Giorda and Trucco (15), except that
the source of the C57BL mice might be different. We do not think that
the differences we observed are experimental artifacts because: first,
the differences we observed in the B6 NKR-P1B gene were reproducible in
two independent sequencing reactions; secondly, our SJL/J NKR-P1B gene
is identical to the published sequence; and thirdly, we have also
sequenced our B6 NKR-P1A and -C genes and compared them to the
published sequences (15). With the exception of a single nucleotide
(C->T) difference that translates into a Ser to Leu difference at
position 45 (Fig. 7B), our B6 NKR-P1A sequence is identical
to that published for NKR-P1A. Our B6 NKR-P1C gene sequence is also
identical to the published NKR-P1C sequence except for a 9-base (ATT
GTT CAG, Asp-Cys-Ser) insertion at amino acid position 93 (Fig. 7B). It is interesting to note that Ryan et al. (18) have
reported a 9-base deletion at the same position in their mNKR-P1.9
nucleotide sequence.
SJL/J NK cells have been demonstrated to have low NK activity toward
YAC-1 targets (when cultured at no or low concentration of IL-2) (37)
and low responsiveness to IFN and IFN inducers (38). Our finding that
the "NK1.1" Ag on SJL/J NK cells is inhibitory (rather than the
well-documented NK activation observed in B6 NK cells) might provide
insight into the cellular basis of the NK defect in SJL/J mice. In
addition, identification of the 1C10 Ag on SJL/J NK cells appears to
reveal a previously unidentified SJL/J NK activation receptor. Western
blotting of SJL/J lysate showed that 1C10 mAb recognizes a protein of
70 kDa (nonreduced) (data not shown). It remains to be determined
whether this 70-kDa protein is a monomer or dimer as attempts to
further characterize this protein by Western blotting (reduced
condition) and immunoprecipitation with the 1C10 mAb have failed. We
have separately transfected B6 NKR-P1A, -B, -C, SJL/J NKR-P1A, and -B
cDNAs into Jurkat cells and tested for the reactivity of 1C10 mAb
toward these members of the NKR-P1 family. It was found that 1C10 mAb
recognized all the three NKR-P1A, -B, and -C gene products of B6 but
not NKR-P1A or -B of SJL/J (our manuscript in preparation), supporting
our conclusion that 1C10 mAb recognizes a novel activation receptor in
SJL/J mice. This yet to be identified receptor might represent a new
member of the NKR-P1 family or a receptor that is closely related to
the NKR-P1 family. Our inability to generate an NKR-P1C PCR product of
SJL/J, using C57BL NKR-P1C-specific primers, suggests that either there
is no NKR-P1C mRNA in SJL/J or that the NKR-P1C gene of SJL/J is
substantially different from the B6 NKR-P1C gene.
Taken together, we have provided formal evidence to support the notion that the NKR-P1B gene product is an inhibitory receptor that shares functional features in common with Ly49A. The ligand for some (perhaps all) inhibitory receptors previously characterized is associated with class I MHC molecules (39). To this end, NK inhibitory receptors can recognize a trimeric MHC class I complex composed of the heavy chain, ß2-microglobulin (ß2m), and a bound peptide (although the composition of the bound peptide does not appear to influence the recognition) (40); an "empty" dimer of class I heavy chain and ß2m (41); or a trimeric HLA molecule composed of HLA-E heavy chain, ß2m, and a hydrophobic leader segment derived from HLA-A, -B, -C, or -G molecules (42). It remains to be determined whether the NKR-P1B receptor also recognizes class I MHC molecules or whether it recognizes carbohydrate, as has been suggested for the rat NKR-P1 gene (43). Nevertheless, our findings show that the NKR-P1 gene family, like the murine Ly49 family and human KIR family, includes as members both activation and inhibitory receptors (e.g., Ly49A and Ly49D in the Ly49 family; KIR2DL and KIR2DS2 in the KIR family) (14, 25, 44).
It is well documented that a single NK cell contains multiple receptors (both activation and inhibitory) on its surface. The outcome of an NK cell-target interaction seems to depend on the types of receptors that are engaged by the specific ligands on the targets (2, 3). Interestingly, most of these NK receptors are encoded by genes linked within the NK gene complex, a region originally defined by the NKR-P1 and Ly49 gene families. It now appears that both of these families can each encode closely related activation and inhibitory receptors. What is the evolutionary advantage? Perhaps, it is a primitive way to generate diversity of NK specificities out of a limited number of genes. Another mutually nonexclusive possibility is that it is a way to define a threshold for NK activation or inhibition. It has been suggested that carbohydrates are the likely ligands for NKR-P1 (43), whereas many (all?) Ly49 receptors recognize H-2 molecules (12). If all members of the NKR-P1 and Ly49 families recognize carbohydrates and H-2, respectively, expression of various activation and inhibitory member of each family might represent a balance of the activation and inhibitory signals of the same ligands (carbohydrate or H-2), thereby defining a threshold for the recognition of carbohydrate moieties and H-2 levels, respectively. Occupation of varying number of activation and inhibitory receptors across different families might further define a threshold for activation set by the sum total of all positive and negative signals received (41).
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology and Medicine, University of California Los Angeles, AIDS Institute, Los Angeles, CA 90095.
3 Address correspondence and reprint requests to Dr. Richard G. Miller, Department of Medical Biophysics, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada M5G 2 M9. E-mail address:
4 Abbreviations used in this paper: AIRL, Ab-induced redirected lysis; NWNA, nylon wool nonadherent; TCD, T cell-depleted; ITIM, immunoreceptor tyrosine-based inhibitory motif; LAK, lymphokine-activated killer; CM, complete medium; GFP, green fluorescence protein.
Received for publication November 25, 1998. Accepted for publication March 2, 1999.
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is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NK1.1+ T cells. J. Exp. Med. 186:1957.This article has been cited by other articles:
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