A Non-Stimulatory Monoclonal Antibody Against the Inhibitory Immunoreceptor LAIR-1

Cell lines and antibodies

Mouse Sp2/0 myeloma cells, mastocytoma cell line P815, Jurkat cells, Burkitt lymphoma Raji cells, and green monkey kidney COS7 cells were all purchased from American Type Culture Collection (ATCC, Rockville, MD). The human erythroleukemia K562 cells were also purchased from ATCC (Rockville, MD) and stably transfected with human LAIR-1, as described previously.⁽¹³⁾ Mouse anti-LAIR-1 MAb (9.1C3) was purchased from Abcam (Cambridge, MA). Mouse anti-GST MAb (E43.1), anti-C-myc MAb (9E10), and anti-SEB MAb (B4) were generated previously in our laboratory.

Molecular cloning of LAIR-2 cDNA

Total RNA was isolated from peripheral blood mononuclear cells (PBMCs) of a patient with hemorrhagic fever and renal syndrome (HFRS) using a TRizol LS reagent kit (Life Technologies, Carlsbad, CA). The cDNA was obtained by an Advantage RT-for-PCR kit (Clontech, Palo Alto, CA). cDNA encoding LAIR-2 was amplified using the primers (P1: 5′-CAGGAGGGGGCCCTTCCC-3′; P2: 5′-TGGTGCATCAAATCCGGA-3′) and pfu DNA polymerase (Takara, Dalian, China) by PCR. The reaction mixture was subjected to pre-denaturation at 94°C for 5 min followed by 30 amplification cycles each consisting of denaturation at 94°C for 1 min, annealing at 58°C for 30 s and extension at 72°C for 1 min, and a final elongation cycle at 72°C for 10 min that was performed with Taq DNA polymerase (Takara, Dalian, China). Products of PCR were electrophoresed on 10 g/L agarose gels, purified using an Agarose Gel DNA Fragment Recovery kit (Takara), ligated into pMD18-T vector (Takara), and then transformed into Escherichia coli JM109 competent cells for sequencing.

Expression and purification of recombinant LAIR-2-GST and LAIR-2-myc-6×His fusion protein

The reading frame encoding LAIR-2 was amplified using the primers (LAIR-2-forward: 5′-CGGGATCCCAGGAGGGGGCCCTTCCC-3′; LAIR-2-reverse: 5'-CGGAATTCCTGGTGCATCAAATCCGGA-3′) with pfu DNA polymerase and subcloned into a pGEX-4T-3 vector (Amersham Pharmacia Biotech, Piscataway, NJ) by BamHI and EcoRI sites. The corresponding protein was expressed in E. coli BL21 cells as glutathione S-transferase (GST) fusion protein induced by isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma Chemical Co., Saint Louis, MO) and purified with Glutathione Sepharose 4B (Amersham Pharmacia). Similarly, recombinant LAIR-2-myc-6×His fusion protein was expressed in COS7 by eukaryotic expression vector pSecTag2 B (Invitrogen, Carlsbad, CA) and purified with Ni Sepharose (Amersham Pharmacia).

SDS-PAGE and Western blot analysis

To identify the expression of recombinant proteins, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were performed. For SDS-PAGE, the host cells were collected and lysed with ultrasound. Then the lysates with 2× sample loading buffer were resolved on SDS-PAGE under denaturing condition and stained with Coomassie Brilliant Blue R-250 (Sigma). For Western blot analysis, the fractionated proteins on the SDS-PAGE were transferred onto the nitrocellulose membrane (Amersham Pharmacia) in a semi-dry transblot apparatus. The nitrocellulose membrane was blocked with 10% defatted milk in PBS at 37°C for 2 h, and then incubated with the primary antibody for 45 min. Later, the blots were washed twice in PBST (PBS with 0.1% Tween-20) and once in PBS, further incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG for 45 min, followed by detection using ECL reagents (Boehringer Mannheim, Mannheim, Germany), according to the manufacturer's instructions, and exposed to photographic film (Kodak, Rochester, NY).

Hybridoma generation and screening

Monoclonal antibodies against LAIR-2 were generated from LAIR-2-GST-immunized BALB/c mice as described previously.⁽²⁷⁾ Briefly, female BALB/c mice (4 weeks old) were subcutaneously immunized at a couple of sites in the back with 10 μg purified recombinant LAIR-2-GST fusion protein emulsified in complete Freund's adjuvant (Sigma), followed by two sequential boosts every 3 weeks with the same dosage of LAIR-2-GST fusion protein, the former in incomplete Freund's adjuvant and the latter in normal saline. Ten days later, mice were bled from caudal vein and the serum titers were detected by indirect enzyme-linked immunosorbent assay (ELISA) with purified LAIR-2-myc-6×His. The mouse with the highest antibody titer to LAIR-2-myc-6×His was boosted with 50 μg of LAIR-2-GST by intravenous injection. Three days later, the mouse was sacrificed for spleen harvest and hybridoma generation. The splenocytes were fused with Sp2/0 mouse myeloma cells at a ratio of 5:1 in polyethylene glycol (PEG) 4000 (Merck, Rahway, NJ), according to the general procedure.⁽²⁸⁾ The fused cells were propagated in 96-well microculture plates (Nunc, Roskilde, Denmark) using hypoxanthine-aminopterin-thymidine (HAT) medium (Sigma). Twelve to 15 days later, culture supernatants were employed to screen the positive hybridomas using the indirect ELISA with LAIR-2-myc-6×His. Selected hybridomas were subcloned three times by limiting dilution prior to ascites production. The MAbs were purified using fast protein liquid chromatography (FPLC) according to previously published protocol,⁽²⁹⁾ and immunoglobulin (Ig) subclass was identified by using an isotyping kit (Pierce, Rockford, IL). Specificity of MAbs for LAIR-2 was assessed by Western blotting with purified LAIR-2-GST, LAIR-2-myc-6×His, and the chimeric protein composed of the extracellular domain of LAIR-1 fused with human immunoglobulin Fc fragment (LAIR-1-Fc; a gift from Dr. Ouyang,⁽²⁶⁾ Department of Immunology, Fourth Military Medical University, China).

Isolation of LAIR-1-expression cells by magnetic beads

LAIR-1-positive cells were isolated using immunomagnetic beads with 9.1C3 or 3G4, according to the routine method. Briefly, cells were harvested and washed twice with buffer (30% FCS in PBS) and then resuspended with the buffer (30% FCS in PBS) at a concentration of 1×10⁷cells/mL. After incubation with 9.1C3 MAb for 10 min at 37°C, goat anti-mouse IgG-conjugated magnetic beads (0.5 mg per 10×10⁶ cells; Immunotech, Marseille, France) were added and incubated for 10 min at 37°C. Cells bound to beads were captured with magnetic force. Unbound cells were collected for flow cytometry staining assay.

Flow cytometry staining assay

Cells to be evaluated were incubated with 9.1C3 or 3G4 at 4°C for 30 min followed by washing three times with FACS washing buffer (1% FCS and 0.1% NaN3 in PBS). Then cells were incubated with PE-conjugated goat anti-mouse IgG at 20 μg/mL. After washing three times, cells were analyzed using a flow cytometer (Becton Dickinson, San Jose, CA).

Antibody-induced redirected cytotoxicity assay for the effect of MAbs on cytotoxicity

To investigate the effect of 3G4 on cytotoxicity, antibody-induced redirected cytotoxicity assay (RCA) was performed according to the methods described previously.^(26,30) Human peripheral blood mononuclear cells pre-incubated with irradiated Raji cells were used as effector cells and FcγR-positive mouse-derived mastocytoma P815 were used as target cells. P815 cells were loaded with ⁵¹Cr via incubating with Na₂⁵¹CrO₄ (Amersham Pharmacia, Bucks, England) solution for 1 h at 37°C. Cells were then washed three times with PBS, resuspended in culture medium, and planted in triplicate in 96-well plates at 1×10⁴ cells/well. The effector cells (10⁶ cells in 100 μL PBS) were incubated with CD16 MAb, CD56 MAb, 9.1C3 MAb, or 3G4 MAb at 37°C for 30 min followed by one washing with RPMI 1640 medium containing 10% FCS. Then the effector cells were added to the ⁵¹Cr-loaded target cells in wells at indicated effector-target (E:T) ratios. After 4 h incubation in a CO₂ incubator at 37°C, the quantity of ⁵¹Cr released into the culture supernatants was determined by liquid scintillation counter (LS6000, Beckman, Fullerton, CA) immediately and the specific lysis was estimated using the following formula: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \rm {Specific \ lysis} \ (\%) = ({\rm experimental \ release-spontaneousrelease} ) / ({\rm total \ counts-spontaneousrelease} ) \times 100 \ \% , \end{align*} \end{document}

where the experimental release was defined as mean counts per minute (cpm) of gamma radiation released by target cells in the presence of effector cells and/or MAb and the spontaneous release was defined as the mean cpm released by target cells alone.

Effect of MAb 3G4 on binding of collagens to LAIR-1

To analyze the interference of 3G4 on the binding of collagens to LAIR-1, LAIR-1-transfected K562 cells were generated. The binding analysis was performed according to the previously published protocol with a minor modification.⁽¹³⁾ Briefly, 96-well flat-bottom plates (Nunc) were coated overnight at 4°C with purified collagens I and III (Sigma) or bovine serum albumin (BSA) in PBS, respectively, supplemented with 2 mM acetic acid. After washing twice, wells were blocked with BSA. K562 stably transfected with LAIR-1 were fluorescently labeled with 5 μM calceine AM (Invitrogen) in PBS for 30 min at 37°C. After washing twice with RPMI 1640 containing 1% FCS, cells were added to each well and plates were incubated at 37°C for 2 h. For the analysis of the interferential effect of 3G4, cells were preincubated with 3G4 for 30 min at room temperature. After four washings, their capacity to adhere to the collagens in the 96-well plates was determined by using a Fluoroskan Ascent fluorescent spectrophotometer (Thermo Labsystems, Franklin, MA).

Statistical analysis

All data were derived from at least three independent experiments. The statistical significance was analyzed using the Mann-Whitney test. Data were represented as mean±SEM. A value of p<0.05 was regarded as significant.

Expression and purification of LAIR-2-GST fusion protein

For expression of LAIR-2-GST fusion protein, a DNA fragment of LAIR-2 with GST tag was cloned into the expression vector pGEX-4T-3. After induction by IPTG, a protein with an apparent molecular mass of 43 kDa was overexpressed in E. coli BL21, which was consistent with the relative molecular weight (Mr) of the LAIR-2-GST fusion protein. The optimal conditions for LAIR-2-GST expression was culturing at 30°C and inducment by the addition of IPTG to a final concentration of 50 nM 2h before harvest. Western blotting with anti-GST MAb confirmed that this overexpressed band was LAIR-2-GST (data not shown). The LAIR-2-myc-6×His fusion protein was also successfully expressed in COS7 cells (data not shown).

Generation and characterization of MAbs to LAIR-2

After fusion of spleen cells from the LAIR-2-GST-immunized BALB/c mouse and myeloma cells, four hybridomas with high affinity to LAIR-2-myc-6×His were obtained from these clones using LAIR-2-myc-6×His fusion protein by indirect ELISA and were designated as 1A7, 3G4, 3G12, and 4A9. The titers of the ascitic fluid of the four hybridomas were all 1:10⁶ determined by indirect ELISA. Immunoglobulin class and subclass assay revealed that 3G4 was IgG1. Specificity of these MAbs for LAIR-2 was evaluated by their binding to LAIR-2-myc-6×His and LAIR-1-Fc. Western blot analysis showed that 1A7, 3G12, and 4A9 were all LAIR-2 specific. However, MAb 3G4 could react with both LAIR-2 and LAIR-1 fusion proteins (Fig. 1A).

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FIG. 1.

Characterization of MAbs to LAIR-2. (A) Specificity of 1A7, 3G12, 4A9, and 3G4 MAbs for LAIR-2 was analyzed by Western blotting. Anti-SEB MAb (B4) was used as negative control. The 1A7, 3G12, and 4A9 were all LAIR-2 specific; however, 3G4 could cross-react with both LAIR-2 and LAIR-1. PBMCs from healthy donor (B) and Jurkat cells (C) were incubated with 3G4, 9.1C3 (positive control), or B4 (negative control) and analyzed by flow cytometry. (D) LAIR-1-positive Jurkat cells were eliminated using 9.1C3-conjugated magnetic beads. The residual Jurkat cells were both 9.1C3 and 3G4 negative.

Further study by flow cytometry showed that 3G4 could stain both PBMCs from healthy donors and Jurkat cells with similar peaks to that of 9.1C3 (Fig. 1B,C). When the LAIR-1 positive Jurkat cells were eliminated using immunomagnetic beads with 9.1C3, the residual Jurkat cells demonstrated both 9.1C3 and 3G4 negative (Fig. 1D), suggesting that 3G4 and 9.1C3 recognize the same molecule on Jurkat cells.

MAb 3G4 demonstrated no inhibition of lymphocyte killing ability

It was previously shown that cross-linking of LAIR-1 on human NK cells or T cells delivers a potent inhibitory signal that is capable of inhibiting target cell lysis.^(4,31) In the current study, the potential role played by 3G4 in cytotoxicity was identified with RCA, which was a test to identify the function of a receptor on the cell surface with its specific MAb. CD16 and CD56 receptors were known as molecular markers of NK cells. CD16 was well known as a lysis receptor mediating direct NK cell cytotoxicity.⁽³²⁾ As shown in Figure 2, CD16 MAb administration significantly enhanced the cytotoxicity of PBMCs, while CD56 MAb exhibited no significant effect on the cytotoxicity. The 9.1C3 led to down-regulation of the activation effector cells as expected, even in the presence of CD16 MAb. However, 3G4, as another MAb to LAIR-1, showed an unanticipated result by exhibiting no effect on cytotoxicity although it could also bind to LAIR-1 as 9.1C3 MAb. This indicated that 3G4 binding did not activate LAIR-1 signaling.

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FIG. 2.

Effect of 3G4 on killing ability of effector cells was determined by RCA. The effector cells were incubated with various MAbs to be evaluated prior to co-incubating with ⁵¹Cr-loaded target cells. Specific lysis was assayed by detecting ⁵¹Cr release from P815 target cells. Data were represented as mean±SEM (n=3). The 3G4 demonstrated no inhibition to the cytotoxicity.

MAb 3G4 mildly prevented binding of LAIR-1 to collagen

As documented by Meyaard and colleagues,⁽¹³⁾ the K562 cells stably transfected with human LAIR-1 could bind firmly to plate-bound collagens I and III. To further investigate whether 3G4 treatment disturbs the interaction between LAIR-1 and its ligand, human LAIR-1-transfected K562 cells were employed in this study. As shown in Figure 3, preincubation of LAIR-1-transfected K562 cells with 3G4 did not fully block the interaction of LAIR-1 with collagens, but only mildly reduced the percentage of cells binding to collagen I or III in a dose-dependent manner. This indicated that the 3G4 treatment partly prevented the binding of LAIR-1 to collagen.

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FIG. 3.

The 3G4 mildly prevented the binding of LAIR-1 to collagen. 96-well plates were coated with collagens I (A) and III (B). K562 cells transfected with hLAIR-1 were fluorescently labeled and preincubated with indicated concentration of 3G4. Then cells were added to the plates coated with collagen for further incubation. Percentage of adhering cells was analyzed by a fluorescence plate reader. Data were represented as mean±SEM for three separate experiments.