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Trem-like transcript 2 (TLT-2), one of the TREM family members, which is expressed on B cells, T cells, and macrophages, plays a critical role in immune response mechanism. In this study, two novel mouse anti-human TLT-2 monoclonal antibodies (MAbs) were prepared using hybridoma technology and their immunological characteristics were determined. The results showed that the two MAbs (clones 10F5 and 8C10) were both IgG1 (κ) and bound specifically to human TLT-2. Furthermore, 10F5 and 8C10 seemed to recognize a different site (epitope) of TLT-2 by competition assay. MAb 10F5 was proven in Western blot analysis to specifically bind to denatured TLT-2 protein while both MAbs were proven in dot blot analyses and immunofluorescence to specifically bind to natural TLT-2 protein. In addition, crosslinking of TLT-2 with MAb 8C10 markedly blocked TLT-2 positive signal and T cell proliferation. Taken together, these two monoclonal antibodies might be of great value as tools for further exploration of the expression and function of TLT-2.
The innate immune system is an ancient form of host defense that relies upon a wide range of pattern recognition receptors,(1–4) such as toll-like receptors (TLR),(5,6) G-protein coupled receptors, mannose receptors, and Fc receptors, which are expressed on neutrophils, monocytes-macrophages, NK cells, and other immune cells. These receptors can recognize and bind pathogens, then mediate effector cells playing an immune function. In addition to these receptors, effector immune cells express other pattern recognition receptors, which often include stimulate and inhibitory receptors.(7–13) Triggering receptors expressed by myeloid cells (TREM) are an example of one of these kinds of receptor.(14)
Trem-like transcript 2 (TLT-2) gene is one of the conserved TREM family members, which is expressed on B cells,(15) T cells , macrophages, and neutrophils.(16) The mouse TLT-2 is suggested to play an important role in the immune system, for it is not only highly expressed in inflammation, but also promotes the proliferation of T cells and the production of cytokines. However, the biological function of TLT-2 remains unclear and is still controversial. Masaaki and colleagues have shown that TLT-2 interacts with B7-H3 expressed on accessory cells and that this promotes enhanced T cell activation.(17,23,24) But Leitner and colleagues found that there is no evidence for B7-H3 and TLT-2 interaction.(18) At the same time, the biological function of the B7H3 is also in dispute.(17–24)
Although several studies have reported on the generation of some monoclonal antibodies (MAbs) against human TLT-2,(23) up until now only two of these were found to reveal agonistic or antagonistic functions and most of these antibodies can only be applied to flow cytometry, immunohistochemistry, or Western blot analysis.(24) In this study we successfully generated two mouse anti-human TLT-2 monoclonal antibodies (MAbs 10F5 and 8C10). When the biological character of these two MAbs was studied, we proved that the epitope recognized by 10F5 and 8C10 is different by competition assay. TLT-2 was elucidated and widely and distinctively expressed on many tumor cell lines. Flow cytometric analysis showed that TLT-2 is highly expressed on monocytes and B cells and weakly expressed on T cells. The significance of TLT-2 expression on tumor cells and the underlying mechanisms need further investigation. Therefore, specific MAbs against the TLT-2 molecule are valuable tools for modulation of TLT-2 signal pathway. This not only provides a new perspective for a thorough understanding of the delicate regulation of the immune system, but also plays a potential role in clinical application.
Six-week-old BALB/c mice were purchased from the Department of Experimental Animal, Shanghai Institute of Biological Products, Ministry of Health of China. Murine myeloma cell line SP2/0, murine fibrous cell line L929, and all the human lines (Raji, Daudi, THP-1, M431, M231, Jurkat) were originally obtained from American Type Culture Collection (Manassas, VA). Human peripheral blood mononuclear cells (PBMCs) were prepared from healthy donors by Ficoll-Hypaque (Hengxin Chemistry Company, Shanghai, China) density-gradient centrifugation. Then the T cells were enriched from PBMCs by EasySep and the purity was >90%. The above cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) or standard DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT), 100U/mL penicillin, 100μg/mL streptomycin, and 2mM L-glutamine. Cells were cultured in a 5% CO2, 37°C incubator. Cell counting kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Goat anti-human TLT-2 polyclonal antibody (clone AF3259) was purchased from R&D Systems (Minneapolis, MN). PE-conjugated goat anti-mouse IgG, biotinylated mouse IgG1, agonistic mouse anti-human CD3 MAb (clone UCHT1), and mouse IgG1 (clone 679.1Mc7) were all purchased from Immunotech (Marseille, France). Functional mouse anti-human CD28 MAb was prepared in our laboratory.(25)
The complete cDNA encoding human TLT-2 was cloned from human tumor cell line Daudi by the reverse transcription polymerase chain reaction (RT-PCR) method with special primers (forward: 5′–CCTCTCGAGATGGCCCCAGCCTTCCTGC-3′; reverse: 5′–TCGGATCCAGTAGACTTCCACATAGG-3′) and was inserted into vector pIRES2-EGFP. The recombinant pIRES2-EGFP-TLT-2 was then transformed into chemically competent TOP 10 cells and selected by kanamycin resistance. The positive clones were then extracted. L929 cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) reagent according to the manufacturer's protocol. Selected by G418 (Invitrogen) at 800μg/mL, the TLT-2 transfected L929 cells (L929/TLT-2) were identified by RT-PCR, Western blot analysis, and flow cytometry. Empty vector-transfected L929 cell line (L929/mock) was also obtained. In addition, CD28 and B7-H3 transfectants were constructed by the same procedure.
The MAbs were prepared using classical hybridoma technology. Six-week-old BALB/c mice were immunized a total of four times with injections of 1×107 mitomycin-treated L929/TLT-2 cells in 0.5mL phosphate-buffered saline (PBS) per mouse at 21-day intervals. The first subcutaneous injection was accompanied by complete Freund's adjuvant (Sigma-Aldrich, St Louis, MO). Four days after the final boost injection, the splenocytes of immunized mice were fused with murine myeloma SP2/0 cells, according to the method described by Groth and Zhang. After hybridomas were selected by hypoxanthine-aminopterin-thymidine (HAT, Sigma-Aldrich), flow cytometry (Altra, Beckman Coulter, Miami, FL) was performed to screen positive clones. Hybridomas secreting mouse anti-human TLT-2 MAbs were obtained by their strong reactivity with L929/TLT-2 cells, but not with L929/mock, L929/CD28, or L929/B7-H3 transfectants. The hybridomas were injected intraperitoneally into pristane-primed BALB/c mice for 8–10 days. The MAbs were purified from ascites of BALB/c mouse using protein G-sepharose affinity columns (Pharmacia, Uppsala, Sweden).
For flow cytometric analysis, the transfected cells (1×106) mentioned above were incubated with MAb 10F5 or 8C10 for 30min at 4°C and washed with PBS. For indirect staining, PE-conjugated goat anti-mouse antibody was added and incubated for another 30min. After being washed with PBS, the stained cells were analyzed by flow cytometry and the Beckman-Coulter's Expo32 Multicomp software. The Ig isotypes were identified with a rapid test paper (Roche, Mannheim, Germany), according to the method provided by the manufacturer. The competition assay showed that MAbs 10F5 and 8C10 identified different epitopes. The L929/TLT-2 cells (1×106) were incubated with 10F5 and 8C10 (0μg/10μL, 0.5μg/10μL, 1μg/10μL, 2μg/10μL) for 45min at 4°C. After being washed with PBS, the cells were incubated with 1μg biotinylated identical MAbs, followed by streptavidin-PE for 30min for flow cytometry to ascertain the saturating amounts of each MAb.
For flow cytometry, the cells mentioned above (1×106/mL) were incubated with the Mabs 10F5 and 8C10, respectively, for 30min at 4°C and washed. For indirect staining, PE-labeled goat anti-mouse antibody as secondary antibody was incubated for another 30min at 4°C and washed. The results were analyzed by flow cytometry facility and Beckman-Coulter's Expo 32 multicomp software.
To further analyze the binding ability of the antibodies to TLT-2 protein, the transfectants were lysed by RIPA Lysis Buffer (Cell Signaling Technology, Boston, MA) in an ice incubator for 2h. Next, dot-blot and Western blotting assay were used to analyze the binding ability of the two MAbs to recombinant TLT-2. Briefly, drops of the lysates were spotted onto circled dot (5μm in diameter) on nitrocellulose membranes (Bio-Rad, Hercules, CA). After 30min of drying at room temperature (RT), the membrane was blocked for 2h at RT with 5% non-fat dry milk in PBS (pH 7.2) on a rocking platform. The membrane was then washed three times with PBS containing 0.1% Tween-20 (PBST), and incubated with PBS, 5μg/mL 8C10, or 10F5 or mouse IgG1 for 2h at RT. After being washed, membrane was sealed in 5% milk containing 1:2000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and incubated at RT for 1h with agitation. The membrane was subjected to enhanced chemiluminescence kit (ECL, Roche) and then exposed to film.
For Western blotting, 10μg of lysates was mixed with loading buffer and boiled at 95–100°C for 5min followed by separating on 10% SDS-polyacrylamide gels under reducing conditions. Gel-loaded proteins were transferred onto a nitrocellulose membrane, which was treated as described above for dot-blot. Western blot analysis was performed to analyze the binding capacity of 10F5 and 8C10 MAbs to TLT-2 protein on L929/TLT-2. L929/mock cells were set as negative control.
For immunofluorescence analysis, the cells mentioned above (1×106/mL) were incubated with the MAbs 10F5 and 8C10, respectively, for 30min at 4°C and washed. For indirect staining, PE-labeled goat anti-mouse antibody as secondary antibody was incubated for another 30min at 4°C and washed. The results were analyzed by fluorescence microscope.
Seeded peripheral blood mononuclear cells (PBMCs) were plated into 96-well plates pre-coated with LPS (100ng/mL), anti-CD3 MAb (0.5μg/mL)+IgG (1μg, 2.5μg, 5μg, 10μg/well), and anti-CD3 MAb (0.5μg/mL)+8C10 (1μg, 2.5μg, 5μg, 10μg/well) and cultured at 37°C, 5% CO2. After 3 days in culture, the reagent of cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan) was added to the culture system and incubated for 6h. The absorbance of every well was measured at 450nm by a microplate reader (Bio-Rad) using enzyme-linked immunosorbent to assay the concentration of IL2, IL10, and INF-γ in culture supernatant. The procedure was performed as described previously.(26) Student's t test was applied for statistical analysis among the groups.
As shown in Figure 1, the L929/TLT-2 transfectants expressed on their membrane high amounts of target molecules in comparison with L929/mock cells. To generate MAbs specific for human TLT-2, the splenocytes of mice immunized by L929/TLT-2 cells were fused with myeloma SP2/0 cells. After repeated screening and cloning, two MAbs, named 10F5 and 8C10, were obtained. Figure 2 shows that 10F5 and 8C10 could recognize transfected cells L929/TLT-2, but not L929/mock, L929/CD28, and L929/B7-H3 cells, indicating that 10F5 and 8C10 are specific for TLT-2. The isotype of 10F5 and 8C10 were IgG1 (κ).
For further analysis, we conducted Western blotting and dot-blot to confirm the specificity of the two MAbs and explore whether structural folding is indispensable for their recognition of TLT-2. As shown in Figure 3, only 10F5 MAbs could recognize the denatured TLT-2 protein, and both MAbs were proven in dot-blot to specifically bind to natural TLT-2 protein as compared with PBS and mouse IgG1 staining (negative control). The MAbs were also identified by immunofluorescence. The results indicated that both 10F5 and 8C10 can identify the L929/TLT-2 gene transfection cell surface expression of TLT-2 molecules, but cannot identify L929/mock gene transfected cells (Fig. 4). Thus, the two novel MAbs against human TLT-2 reported in this work provide additional reagents for the functional characterization of TLT-2.
Next, competition assay was performed to further determine whether the two antibodies recognize the same, overlapping, or distinct epitopes of human TLT-2 antigen. At first the L929/TLT-2 cells (1×106) were incubated with 10F5 (0μg/10μL, 0.5μg/10μL, 1μg/10μL, 2.5μg/10μL) for 45min at 4°C. After being washed with PBS, the cells were followed by staining with the detecting amounts (1μg/test) of biotin-conjugated identical antibody to ascertain the saturating concentration at which all binding sites on L929/TLT-2 were occupied by this MAb. As shown in Figure 5A, graded amounts of unlabeled 10F5 blocked subsequent binding of biotinylated MAb to L929/TLT-2 cells in a dose-dependent way and 2μg was sufficient for antibody to saturate their interacting sites in TLT-2, respectively. Secondly, competition assay between 10F5 and 8C10 was performed using the same protocol. The results showed that 8C10 could not be blocked in the 10F5 saturated L929/TLT-2 cells (Fig. 5B). Taken together, these data indicated that these two MAbs (10F5 and 8C10) seemed to recognize different epitopes.
The expression of TLT-2 was examined on different human tumor cell lines using flow cytometry (Fig. 6). Results showed that TLT-2 could be highly expressed on tumor cell lines of Raji, Daudi, THP-1, monocytes, B cells, and activated T cells; weakly expressed on M431, M231, and T cells; but not expressed on Jurkat, Hct, SW480, Hela; also the two MAbs we generated could be used in FCM analysis. With the expression of TLT-2 in these tumor cell lines detected by these new MAbs 8C10 and 10F5, it may be assumed these two MAbs may have great value for further study of the tumor stem cell.
To assess whether the blocking MAbs 8C10 exhibited any functional effect on T cells, an in vitro direct proliferative assay was performed in the presence of MAbs 10F5 or 8C10. Compared with the control groups, the addition of MAbs 8C10 inhibited T cell proliferation and inhibited the function of T cell in a dose-dependent manner that could be measured by CCK-8 and ELISA within 72h of incubation (Fig. 7). Therefore, MAbs 8C10 is a functional antibody that can inhibit TLT-2 positive signals on T cells.
The fluctuant balance between co-stimulatory and co-inhibitory signals that a T cell receives participates in the initiation, effectivity, and termination of an immune response.(27,28) Excessive activation and immune reaction of T cells may result in autoimmune diseases and host immune injury. TLT-2 was elucidated widely and distinctly expressed on many tissues and tumor cell lines. The significance of TLT-2 expression on tumor cells and the underlying mechanisms need further investigation. Therefore, the specific MAbs against TLT-2 molecule, valuable tools for modulation of TLT-2 positive signal pathway, not only provides a new perspective for a thorough understanding of the delicate regulation of the immune system, but also plays a potential role in clinical application.
Although several articles have reported on the generation of monoclonal antibodies against human TLT-2, only two of these were found to reveal agonistic or antagonistic functions.(24) Thus developing more efficient and specific monoclonal antibodies may be important for further disclosure of TLT-2. The choice of an appropriate immunization strategy and screening assay is the most important part of hybridoma production. In order to avoid an immune response against heterogeneous antigen in this study, mouse original fibrous cell line L929 was selected to carry the target antigen. Human TLT-2 antigen presented by the L929 cells can produce a more specific immune response and decrease the probability of non-specific MAb generation. All the hybridomas were screened by flow cytometry, whose constraints on screening of assays are reliable, fast, and simple. According to these considerations, we successfully obtained two desired hybridomas secreting mouse anti-human TLT-2 MAbs by one fusion operation.
After purification, the two novel mouse MAbs against human TLT-2 were generated, identified, and subjected to strict verification by immunofluorescence dot-blot and Western blot analysis. Both 10F5 and 8C10 specifically bound to natural TLT-2 protein in dot-blot and immunofluorescence while in conditioned Western blotting the denatured TLT-2 protein could be immunoblotted with10F5. This indicated that 10F5 could bind to a linear epitope. Competition assay showed that the two MAbs recognize different epitopes of TLT-2.
Additional experimental evidence showed that TLT-2 could be highly expressed on tumor cell lines of Raji, Daudi, THP-1, monocytes, B cells, and activated T cells; weakly expressed on M431, M231, and T cells; and not expressed on Jurkat, Hct, SW480, and Hela. Moreover, the blocking MAbs 8C10 exhibited functional effect on T cells. Engagement of human TLT-2 by the 8C10 MAbs could inhibit T cell proliferation, suggesting that 8C10 may be useful for blocking theTLT-2 positive signal.
With their high specificity and few side effects, monoclonal antibodies have recently been unanimously accepted as ideal medicaments in targeting therapy in some refractory diseases.(29) With the expression of TLT-2 on the cells above, detected by the new MAbs 10F5 and 8C10, these two MAbs may have great value for the further study of diseases. Therefore, 10F5 and 8C10 have a potential value in further studying tumor escape, autoimmune diseases, and infectious diseases.
In conclusion, we have prepared two specific mouse MAbs against human TLT-2. They recognize different epitopes. One of the MAbs acts as an adjuvant to inhibit proliferation on purified human T cells. This makes 8C10 a valuable tool for further exploration of TLT-2 function. Furthermore, another anti-TLT-2 MAb will be generated, so a sandwich ELISA technology may be developed based on these MAbs to analyze TLT-2 in view of its important regulatory functions.
This work was supported by grants from the National Natural Science Foundation of China (grant no. 30901313).
The authors have no financial conflict of interest to disclose. The generated antibodies were distributed solely to non-profit research organizations for research purposes only.