Epitope Determination of Anti Rat Thy-1 Monoclonal Antibody That Regulates Neurite Outgrowth

Abstract

Glycosylphosphatidylinositol-anchored protein Thy-1 is abundantly expressed on the cell surface of neurons and T lymphocytes in rodents. Although Thy-1 is known to bind integrins as a ligand and to mediate neurite outgrowth and immune responses, its precise function is not fully understood. Previously we produced several anti rat Thy-1 monoclonal antibodies and identified one, 2E11, which induces PC12 cell neurite outgrowth. Here we screened antibodies that inhibit 2E11-induced neurite outgrowth and stimulate or inhibit rat thymocyte aggregation. Since Thy-1 lacks an intracellular region, it requires other membrane-bound molecules for the signal transduction. Hence these antibodies are hypothesized to play key roles in the interaction between Thy-1 and signaling molecules. To elucidate the mechanisms of antibody-induced Thy-1 functions, antibody characterization and epitope determination were carried out. Thy-1 cleavage and mutation revealed that the antibodies recognize not only amino acid sequences, but also the three-dimensional structures consisting of immunoglobulin-like domains. Two antibodies were suggested to bind spatially close to the integrin binding site and crosslink Thy-1 molecules, while a third antibody is believed to inhibit Thy-1 crosslinking and subsequent Thy-1 signaling. The antibodies reported here may therefore function as crosslinkers, agonists, or antagonists that modify Thy-1 signaling.

Introduction

Thy-1 (CD90) is a 22–37 kDa glycosylphosphatidylinositol (GPI)-anchored protein consisting of immunoglobulin-like domains, N-linked glycosylations, and an integrin binding sequence.^(1,2) It is abundantly expressed in the rodent brain and thymus, human neurons, fibroblasts, and endothelial cells,^(3,4) and localizes on the cell surface cholesterol-rich lipid raft microdomain.^(5,6) Extensive research has shown that Thy-1 is involved in numerous cellular functions such as neurite outgrowth, immune responses, morphology changes, migration, adhesion, proliferation, and death.^(7,8) Its versatility may be due to associated proteins since it lacks transmembrane and intracellular regions; however the detailed mechanisms remain unclear.

Thy-1 expression and stimulation result in neuron development and neurite outgrowth in rats,^(9,10) murine thymocyte aggregation,⁽¹¹⁾ and T lymphocyte activation, including regulatory T cells^(12–14); while Thy-1 deficiency in mice results in reduced neuronal functions,⁽¹⁵⁾ T cell maturation, and proliferation.⁽¹⁶⁾ However, Thy-1 exerts discrepant functions depending on experimental conditions. For example, it is down-regulated during rat dorsal ganglia neuron development and sciatic nerve regeneration, then increases with maturation,^(17–19) while Thy-1 deficient mouse T cells show augmented TCR-regulated proliferation.⁽¹⁶⁾ Moreover, Thy-1 is regarded as a marker that distinguishes hematopoietic stem cells from bone marrow,^(20,21) which implies a negative role in stabilizing immature or inactive cells. Thy-1 also acts as a ligand for integrins; for example, Thy-1 expressing endothelial cells bind αMβ2-integrin (Mac-1) bearing leukocytes and mediate cell adhesion in humans,⁽²²⁾ while rat neuronal Thy-1 binds to αVβ3-integrin expressing astrocytes and activates ATP release, P2X7 activation, RhoA signaling, cytoskeleton assembly, and focal adhesion formation.^(23,24)

Using rat adrenal pheochromocytoma-derived cell line PC12⁽²⁵⁾ as an antigen, we previously cloned several monoclonal antibodies (MAbs) against PC12 cell surface molecules, including anti rat Thy-1 MAb 2E11 that induces PC12 neurite outgrowth.⁽²⁶⁾ To investigate this mechanism of 2E11-induced neurite outgrowth, we screened anti Thy-1 MAbs that inhibit 2E11 function and further characterized selected MAbs. Thy-1 crosslinking was shown to be crucial for activation of PC12 neurite outgrowth and rat thymocyte aggregation, and antibody binding sites were located spatially close to the integrin binding motif, suggesting that they block or mimic the ligand for Thy-1. We propose that anti Thy-1 MAbs against specific epitopes could be useful tools for modulating Thy-1 activities.

Materials and Methods

Materials and animals

Dulbecco's modified Eagle's medium (DMEM), RPMI1640 medium, fetal bovine serum (FBS), horse serum (HS), and o-phenylenediamine (OPD) were obtained from Sigma (St. Louis, MO). 2-Mercaptoethanol (2-ME) and cyanogen bromide (BrCN) were from Wako (Osaka, Japan). Peptide N-glycosidase F (PNGaseF) and bovine serum albumin (BSA) were purchased from Roche Diagnostics (Mannheim, Germany).

BALB/c mice and Wistar rats were purchased from Sankyo or CLEA Japan (Tokyo, Japan). Animal experiments were carried out in a humane manner after receiving approval from the Institutional Animal Experiment Committee of Tokyo University of Science and Jichi Medical University, in accordance with the Institutional Regulation for Animal Experiments and Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the Jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology.

Screening of MAbs that inhibit 2E11-induced PC12 neurite outgrowth

Hybridoma cells secreting antibodies against PC12 cell surface molecules were maintained in RPMI 1640 containing 10% FBS. For neurite outgrowth, PC12 cells were cultured 3 to 5 days on Poly-D-Lysin (PDL)-coated 96-well plates (Becton Dickinson, Bedford, MA) in DMEM containing 10% FBS, 5% HS, and anti rat Thy-1 MAb 2E11⁽²⁶⁾ with or without the culture supernatants of hybridomas tested. Inhibition of 2E11-induced PC12 neurite outgrowth were estimated by β_III-tubulin quantification using cell ELISA, a modified enzyme-linked immunosorbent assay (ELISA) as previously described.⁽²⁷⁾ In brief, MAb-treated PC12 cells in 96-well plate were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and blocked with PBS plus 0.1% (w/v) BSA, then incubated with biotinylated anti β_III-tubulin MAb SDL.3D10 (Sigma). Each well was gently washed three times with PBS and reacted with horseradish peroxidase (HRP)-conjugated streptavidin (Zymed, San Francisco, CA). After washing three times with PBS, HRP activity was detected with 10 mM OPD–0.01% H₂O₂–50 mM sodium acetate buffer (pH 5.0) and the optical density at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA). Neurite length was measured following immunofluorescent staining. Briefly, PC12 cells cultured for 5 days with 10 μg/mL 2E11 in the presence or absence of inhibitor MAb in 96-well plates were fixed with 4% PFA-PBS. After blocking with BSA-PBS, each well was reacted with Goat Anti-Mouse IgG-Alexa Fluor 488 (Invitrogen, Carlsbad, CA). Subsequently, neurite length was calculated from photomicrographs of each well using IN Cell Analyzer 1000 and IN Cell Developer Toolbox Software (ver. 1.9, GE Healthcare UK, Buckinghamshire, United Kingdom).

Because clones 10A5 and 13H7 were shown to inhibit 2E11-induced PC12 neurite outgrowth and to recognize rat Thy-1, we decided to focus on anti Thy-1 MAbs. Hybridomas were intraperitoneally injected into BALB/c mice to obtain ascitic fluids. MAbs were purified from ascites using affinity column chromatography through a Hi-Trap protein G column (GE Healthcare). Purified MAbs 2E11, 10A5, and 13H7 were conjugated to biotin or HRP as previously described.⁽²⁸⁾ MAbs 2E11 and 10A5 were treated with 1 mg/mL papain and 10 mM mercaptoethylamine, and applied to a Sephadex-50 (GE Healthcare) gel filtration column to obtain Fab’ fragments. The titration of Fab’ fragment binding to Thy-1 was carried out by immunoblotting.

Immunochemical procedures

Thy-1 protein was prepared from rat brain or cultured cells as described previously.⁽²⁹⁾ In brief, samples were homogenized using Teflon homogenizer and centrifuged at 15,000 g for 30 min at 4°C. Precipitated membrane fraction was lysed with 2% deoxycholate–10 mM Tris-HCl (pH 8.0). Immunoblotting was performed as described previously.^(28,30) Briefly, samples were heat denatured at 95°C for 5 min with SDS-PAGE sample buffer in the absence of reducing reagent, unless noted otherwise. After separation of the proteins by SDS-PAGE and transfer to a PVDF membrane (Millipore, Bedford MA), the membrane filter was blocked in 10 mg/mL of skim milk in PBS containing 0.05% (w/v) Tween-20 (PBS-T). Immunodetection was carried out with antibodies diluted in skim milk–PBS-T for 1 h, then rinsed three times with PBS-T. The filter was reacted with the HRP-labeled Goat Anti-Mouse IgG secondary antibody (Zymed) for 30 min, and protein bands were detected using the Enhanced Chemiluminescent (ECL) system (GE Healthcare).

Competitive ELISA was carried out as described previously, with a slight modification.⁽²⁸⁾ Briefly, PC12 cells previously fixed overnight with 4% PFA-PBS on the 96-well plate were blocked with 0.5% BSA–PBS for 1 h. Each well was reacted with HRP-labeled 2E11 with or without increasing concentrations of non-labeled antibodies for 1 h at room temperature. HRP activity was measured after washing three times with PBS-T. For competitive immunoblotting, the Thy-1 protein-immobilized PVDF membrane filter was cut into strips after the blocking with skim milk–PBS-T, and each strip was reacted with biotinylated MAbs plus excess non-labeled antibodies. After rinsing three times with PBS-T, the bindings of biotin-conjugated MAbs were detected using streptavidin-HRP conjugate followed by ECL reagents.

Thymocyte aggregation

Rat thymus was removed from a Wistar rat sacrificed under anesthesia, and thymocytes were suspended in RPMI1640 plus 10% FBS, then cultured for 24 h at 5×10⁵/well in 96-well plates with or without various concentrations of MAbs. The size and number of formed aggregates were calculated from the photographs of each well taken by IN Cell Analyzer 1000. Inhibition of thymocyte aggregation was carried out by culturing rat thymocytes with 0.5 μg/mL 2E11 or 13H7 in the presence or absence of 10A5 for 24 h. For MAb crosslinking, rat thymocytes were cultured overnight with 5 μg/mL anti-mouse secondary antibody in addition to each anti Thy-1 MAb.

Thy-1 processing

To reduce disulfide bonds, Thy-1 protein was treated with various concentrations of 2-ME in SDS-PAGE sample buffer for 5 min at 95°C. Deglycosylation was carried out as described previously.⁽³¹⁾ In brief, the samples were heat denatured for 5 min in 20 mM sodium phosphate buffer (pH 7.3)–0.1% SDS–10 mM EDTA. Thereafter, TritonX-100 and PNGaseF were added at the final concentrations of 0.5% and 12.5 U/mL, respectively, and incubated overnight at 37°C. BrCN cleavage at the C-terminal of the methionine residue was carried out by reacting Thy-1 protein with 1 mg/mL BrCN in 70% formic acid overnight at room temperature in the dark. Samples were then processed for SDS–PAGE and subsequent immunoblotting using each MAb.

Rat-mouse chimeric Thy-1

Thy-1 cDNA was synthesized from rat brain total RNA by RT-PCR and inserted into the GFP fusion protein expression vector pQBI25-fc2 (QBiogene, Carlsbad, CA). Chimera Thy-1 proteins that were partially converted into mouse Thy-1 amino acid sequences (mutations 1–5) were generated by PCR using primers with mouse Thy-1 genomic sequences. PCR primer sequences used are listed in Supplementary Table S1. Introduction of the expression vectors into HeLa cells was carried out using Lipofectamine 2000 (Invitrogen). Plasmid-transfected HeLa cells were collected 48 h after the transfection, and membrane fractions were processed for immunoblotting with each MAb or Rabbit Anti-GFP polyclonal antibody (Torrey Pines Biolabs, East Orange, NJ).

Results

Screening of anti rat Thy-1 antibodies inhibiting 2E11 function

Screening of MAbs inhibiting 2E11-induced PC12 neurite outgrowth showed that two anti Thy-1 MAb clones, 10A5 and 13H7, inhibited neurite outgrowth to control levels (Fig. 1A, B). The 2E11 Fab’ fragment did not allow neurite growth, suggesting that Thy-1 crosslinking is crucial for neurite extension. Moreover, 10A5 Fab’ also inhibited neurite outgrowth, showing that divalency is not necessary for the inhibition of neurite extension (Fig. 1A).

FIG. 1.

Inhibition of PC12 cell neurite outgrowth. (A) PC12 cells were cultured for 4 days with (a) medium only, (b) 20 μg/mL 2E11, (c) 2E11 plus 10A5, (d) 2E11 plus 13H7, (e) 2E11 Fab’ fragment, and (f) intact 2E11 plus 10A5 Fab’. Scale bars indicate 50 μm. (B) Neurite length measurement. PC12 cells were cultured for 5 days with (slashed bar) or without (white bar) 10 μg/mL 2E11 alone, 2E11 plus 50 □g/mL 10A5 (gray), and 2E11 plus 13H7 (black). Each experiment was carried out in triplicate. Photomicrographs of 16 fields were taken from each well, and the average neurite length was calculated using IN Cell Analyzer.

Two competition assays were carried out to clarify whether the three MAbs inhibited the binding of each other with Thy-1. Competitive ELISA and immunoblotting showed that 13H7 dose-dependently inhibited 2E11 binding, whereas 10A5 did not compete with either 2E11 or 13H7 (Fig. 2A, B). This suggested that 2E11 and 13H7 have close epitopes, and that 13H7 physically interferes with 2E11 binding and neutralizes its physiological function. On the other hand, 10A5 was suggested to have a distant epitope from the others and to inhibit the function of 2E11 in a different manner from 13H7.

FIG. 2.

Competition analysis of the MAbs. MAbs were reacted with Thy-1 in the presence of increasing concentrations of non-labeled competitor antibodies. (A) Competitive ELISA. HRP-labeled 2E11 was added to the ELISA plate coated with PC12 cells on its surface. Relative HRP activities were measured by Cell ELISA. (B) Competitive immunoblotting. Rat brain Thy-1 was separated and transferred onto a PVDF membrane filter. Biotinylated 2E11 or 10A5 with non-labeled 13H7 were reacted and detected by streptavidin-HRP. Data shown are representative of three independent experiments.

Effect of anti rat Thy-1 antibodies on thymocyte aggregation

In order to estimate the effect on T cell activity, rat thymocytes were cultured in the presence of MAbs. MAb 2E11, but not 10A5, dose-dependently aggregated thymocytes (Fig. 3A, B). However, 10A5 clustered thymocytes only in the presence of a secondary antibody (Fig. 3D), indicating that Thy-1 crosslinking is crucial for cell adhesion. Interestingly, 13H7 stimulated thymocyte aggregation, despite its ability to inhibit PC12 neurite outgrowth. On the other hand, 10A5 dose-dependently abrogated 2E11-induced thymocyte aggregation as well as neurite outgrowth (Fig. 3D), but it did not alter 13H7-induced thymocyte aggregation. Taken together, 10A5 and 13H7 appear to differentially modify Thy-1 functions in a cell-type dependent manner.

FIG. 3.

Thymocyte aggregation. (A) Rat thymocytes were cultured for 24 h at 5×10⁵/well in 96-well plates with indicated MAb concentrations. Scale bar indicates 100 μm. (B) Average numbers and areas of the aggregates were calculated using IN Cell Analyzer from 16 fields of view per well. (C) Inhibition of thymocyte aggregation. Thymocytes were cultured overnight with indicated concentrations of 10A5 and 0.5 μg/mL 2E11 or 13H7. (D) Crosslinking of MAbs. Thymocytes were cultured with or without MAbs in the presence or absence of 5 μg/mL anti mouse secondary antibody.

Epitope determination of anti Thy-1 monoclonal antibodies

Epitope mapping of anti Thy-1 MAbs was carried out using a number of enzymes, chemicals, and mutants. The schematic models of mouse and rat Thy-1 and cleaved rat Thy-1 are shown in Figure 4A. Immunoblotting revealed that each clone recognizes rat but not mouse Thy-1 (Fig. 4B, lanes 1, 2). The clones did not bind to 2-ME reduced Thy-1, suggesting that disulfide bonds are critical for antibody binding (Fig. 4B, lanes 2–4).

FIG. 4.

Immunodetection of cleaved rat Thy-1. (A) Schematic models of Thy-1 proteins. (a) Mouse Thy-1. Blank parts indicate non-conserved domains between rat Thy-1. Rat Thy-1 that is native (b), cleaved with 2-ME (c), PNGaseF (d), and BrCN (e). (B) Membrane fractions from mouse (lane 1) and rat (lane 2) brains were separated by SDS-PAGE and transferred onto a PVDF membrane filter that was treated with anti Thy-1 MAbs 2E11 (upper), 10A5 (middle), and 13H7 (lower). Arrow indicates the position of Thy-1. Molecular weight markers are shown on the right side. For the reduction of the disulfide bond, rat brain membrane lysate was left untreated (lane 2) or reduced with 2-ME at 95°C for 5 min with SDS-sample buffer (lanes 3 and 4 treated with 10 and 100 mM of 2-ME, respectively). Deglycosylation with PNGaseF. Open arrowhead indicates the position of deglycosylated Thy-1 (lane 5). BrCN-cleaved Thy-1 (lane 6).

Rat Thy-1 has three N-glycosylation sites, and deglycosylation with PNGaseF resulted in a single 13 kDa core protein band. 2E11 and 10A5 bindings to deglycosylated Thy-1 were partially attenuated, suggesting that N-linked glycosylations affect the affinity of 2E11 and 10A5 but are not necessary for their binding (Fig. 4B, lane 5). MAbs 2E11 and 10A5 faintly bound BrCN-cleaved Thy-1 whereas 13H7 binding was not altered. We speculate that 2E11 and 10A5 epitopes are close to the methionine residue at amino acid position 84 (Met84) (Fig. 4B, lane 6), and that the 13H7 epitope is distant from the others.

GFP fusion rat-mouse chimeric Thy-1 proteins were expressed (Fig. 5A) and detected by immunoblotting. The Thy-1 band detected by each MAb was expected to disappear only when following mutation of the epitope. Of the five mutations, 10A5 did not react with mutant number two (mt2) that was converted into the mouse Thy-1 sequence at amino acid positions 60 to 66 (aa60–66) (Fig. 5B, middle panel, lane 4). 2E11 and 13H7 bindings against mt4 with the mouse Thy-1 sequence at aa88–95 was clearly shown to disappear, suggesting that the mutated region contains 2E11 and 13H7 epitopes. The bindings against mt3 that codes phenylalanine instead of Met84 were attenuated, whereas the bindings against mt5 were unaltered (Fig. 5B, upper and lower panels, lanes 5–7). The anti-GFP polyclonal antibody detected each GFP-fused Thy-1 mutant. Although the calculated molecular weight of GFP fusion Thy-1 without the mutation is approximately 40.5 kDa, its actual size will be larger because of the presence of glycosylation.

FIG. 5.

Epitope mapping of GFP fusion rat-mouse chimeric Thy-1 proteins. (A) Amino acid sequences of rat Thy-1 and mutants. Mouse Thy-1 sequences converted from rat sequences are boxed with dashed lines and written in bold. The mutation numbers are shown above. The integrin binding RLD sequence is indicated in the black box with white lettering. (B) Mutant Thy-1 expressing HeLa cell membrane was processed for SDS-PAGE and immunoblotting. Immunodetection was carried out using anti-Thy-1 MAbs and anti-GFP polyclonal antibody as shown on the left. Each lane indicates membrane fraction of HeLa cells transfected with plasmids coding native rat Thy-1 (lane 1), GFP-Thy1 fusion protein without mutation (lane 2), GFP-Thy1 with mutations 1–5 (lanes 3–7, respectively). Arrow and arrowhead indicate the positions of GFP-Thy1 and native Thy-1, respectively.

Discussion

Anti Thy-1 MAbs 10A5 and 13H7 inhibited 2E11-induced neurite outgrowth in PC12 cells (Fig. 1), and the bindings of 10A5 and 13H7 were non-competitive and competitive to 2E11, respectively (Fig. 2). They detected rat Thy-1 but not murine Thy-1 (Fig. 4). Attenuated 2E11 and 10A5 bindings to Thy-1 after PNGaseF digestion of the N-glycosylation, 2-ME reduction of the disulfide bonds, and BrCN cleavage between Met84-Cys85 suggest that the epitopes for these agonistic and antagonistic MAbs are close to the disulfide bond between Cys85 and Cys19. It is noteworthy that 13H7 binding seemed relatively resistant to this treatment (Fig. 4). However, no Thy-1 derivatives were detected, presumably because of the damage to the three-dimensional (3-D) structure following complete digestion with trypsin, lysyl endopeptidase, endoproteinase Asp-N, V8 protease, or N-Bromosuccinimide (data not shown).

MAbs, particularly functional ones, are highly sensitive to conformational changes in antigen proteins. In the present case, it appears that the 3-D structure around the disulfide bonds might be crucial for antibody recognition. In order to overcome the difficulty presented by the changeable Thy-1 structure,⁽⁵⁾ we used the rat-mouse chimeric proteins with rat Thy-1 amino acid sequences partially converted into mouse Thy-1.2 sequences (Fig. 5A). Plasmids expressing these GFP-fusion Thy-1 mutants should limit major conformational changes, since rat and mouse Thy-1 share 82% identical amino acid sequences, and have similar functions; for example, Thy-1 bearing mouse thymoma cell line EL-4 binds to both mouse and rat integrin-expressing cells.⁽²⁾ Using this system, each MAb successfully reacted with most of the mutants, but not with a chimera protein carrying a mutation within the putative epitope. Based on the result of mutation number 4 (mt4), 2E11 and 13H7 epitopes were suggested to be located between two disulfide bonds (Fig. 5B). Competition assays revealed that 10A5 has an epitope distant from the others, which was narrowed down to aa60–66 after mt2 clearly abrogated 10A5 binding (Fig. 6).

FIG. 6.

Schematic model of Thy-1 protein and putative MAb epitopes. Rat Thy-1 amino acids that differ from those of mouse Thy-1 are in gray lettering. The putative MAb binding sites of 10A5, 2E11, and 13H7 are enclosed in dashed lines, and the integrin binding RLD motif is shown in the black box with white lettering.

10A5, a non-competitive inhibitor of 2E11-induced PC12 neurite outgrowth, also inhibited rat thymocyte aggregation by 2E11. However, 13H7, a competitive inhibitor of neurite outgrowth, stimulated thymocyte aggregation as well as 2E11, and it was not inhibited by 10A5 (Fig. 3). This discrepancy may be because of the slight difference in epitope locations that influences the attachment of the cell surface signaling molecules to Thy-1, and besides, Thy-1 associated proteins in PC12 cells and thymocytes might be different. In summary, 2E11 and 13H7 epitopes are so close that the two MAbs compete with each other and elicit the same function from thymocyte-Thy-1, but are still distant enough to react differently to PC12 cells or 10A5-treated thymocytes. 2E11 and 13H7 reaction to 10A5-treated thymocytes and rat Thy-1 cleaved or mutated at Met84 showed that 13H7 binding site seems to be further than 2E11 epitope from Met84 and 10A5 epitopes.

As schematically illustrated in Figure 6, the epitopes of 2E11 and 13H7 are spatially very close to the integrin binding RGD-like motif RLD, so they might block or mimic the ligand for Thy-1. Although the effect of integrins against Thy-1 expressing cells is not yet fully understood, mature rat astrocytes reportedly suppress neurite outgrowth via Thy-1,⁽³²⁾ while αVβ3-integrin expressing astrocytes directly interacts with neuronal Thy-1.⁽²³⁾ This suggests a negative role of integrins, such that a blockade of their binding may trigger neurite extension. It remains to be determined whether Thy-1 can interact in cis with integrin on an identical PC12 cell surface.

Importantly, the 2E11 Fab’ fragment did not induce PC12 neurite outgrowth (Fig. 1A), suggesting that Thy-1 crosslinking is important for signal transduction. Thy-1 crosslinking has been suggested to aggregate thymocytes by activating adhesion molecules and cytoskeleton assembly,⁽¹¹⁾ and such crosslinking with MAbs may accumulate not only Thy-1 but also binding proteins on the lipid raft, including Src family kinases Lck, Fyn, Lyn, and/or Csk-binding protein,^(33–37) thus amplifying intracellular signaling. It is possible that components of the Thy-1 signaling complex vary among cell types, tissues, and the MAb clones used, and that a different set of signaling molecules would attribute the diversity of Thy-1 activity. Detection of Thy-1 associated proteins by immunoprecipitation using these MAbs will be intriguing. Interestingly, 10A5 did not aggregate rat thymocytes unless it was crosslinked by second antibody, suggesting that it is not capable of crosslinking two Thy-1 molecules on its own, and perhaps preventing 2E11 from Thy-1 multimerization and the accumulation of signaling molecules. Therefore, the 10A5 epitope (aa60–66: NLFSDRF) could be a target for silencing Thy-1 signals by inhibiting T or Treg cell aggregation and adhesion onto antigen presenting cells, which may also be applicable for the inhibition of cancer metastasis,⁽³⁸⁾ when anti human Thy-1 MAb recognizing aa60–66 (NFTSKYH) is produced in the future. Furthermore, synthesized peptides corresponding to each MAb epitope may abolish antibody bindings if they maintain the functional conformations.

In conclusion, the anti Thy-1 MAbs with specific epitopes are able to modify Thy-1 activity and may provide further information regarding the binding partners of Thy-1 and intracellular signaling.

Footnotes

Acknowledgments

The authors thank Ms. Sawako Yamamoto, Mr. Tetsusato Nomoto, Mr. Sozo Ichikawa, Mr. Shin-ichi Kobayashi, Ms. Chiyoko Kato, Ms. Eriko Ohta, and Ms. Noriko Oshima (GE Healthcare) for their excellent technical assistance. We are also thankful to Dr. Nobuo Watanabe (Tokyo University of Science) and the laboratory members for valuable discussions.

Author Disclosure Statement

The authors have no financial interests to disclose.

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