The Specificity of 130-kDa Leech Sensory Afferent Proteins Is Encoded by Their Carbohydrate Epitopes

From early development through adulthood in the leech, sensory afferents, glial cells, and connective tissue express different epitopes located on a group of 130-kDa glycoproteins. The sensory epitope [reactive with monoclonal antibody (mAb) Lan3-2] is shared by the peripheral sensory afferents of different sensory modalities. In contrast, three other immunocytochemically distinct epitopes (reactive with mAbs Laz2-369, Laz7-79, and Laz6-212) differentiate these sensory afferents according to their sensory modalities. The glial epitope (mAb Laz6-297) is expressed on all macroglial processes, and the connective tissue epitope (mAb Laz9-84) is located on connective tissue surrounding the CNS, as well as in the peripheral tissues. The hydrophilic-hydrophobic nature of the 130-kDa sensory afferent and glial proteins was determined by phase separation with Triton X-114 and hypoosmotic extraction. They behave as peripheral membrane proteins. Deglycosylation of 130-kDa glycoproteins with N-Glycanase or preincubation of their respective mAbs with alpha-methylmannoside showed that the sensory epitope contains mannose, whereas the modality epitopes are of an undefined carbohydrate character. Immunoprecipitation and a peptide mapping experiment confirmed the existence of four distinct sensory afferent epitopes. Previous studies provided evidence that the mannose-containing Lan3-2 epitope mediates normal sensory afferent growth in the synaptic neuropile. We, therefore, postulate that the carbohydrate epitopes on sensory afferent glycoproteins participate in synapse formation.     

The radial glia antibody RC2 recognizes a protein encoded by Nestin

The RC2 antibody is widely used to label mouse radial glial cells in the developing central nervous system. While the antibody is known to recognize a 295-kDa intermediate filament proximal protein, the gene encoding the RC2 antigen remains to be identified. Here, we present evidences clearly demonstrating that Nestin encodes the RC2 antigen. First, the RC2 antigen and nestin have the same molecular weight and very similar tissue distribution. Second, genetic manipulations altering nestin expression also exert the same effect on the expression of the RC2 antigen. In particular, Nestin null mutation completely abolishes the RC2 immunoreactivity. Third, the expression of a truncated mouse nestin in Nestin−/− cells produces a small RC2 antigen whose size is the same to that of the truncated nestin. Furthermore, our data suggest that the RC2 antibody recognizes the C-terminal domain of nestin with unidentified posttranslational modification(s).     

Identification of several forms of the glial fibrillary acidic protein, or alpha-albumin, by a specific monoclonal antibody

Abstract: A monoclonal antibody (mAb), termed UIA/ NEU/I/G1 (G1), that reacted with the astroglial marker glial fibrillary acidic protein (GFAP), or α-albumin, is described. It was directed against a structural determinant of GFAP. The G1 mAb could be used for quantitative determination of GFAP in two-site radiometric assays and for histoimmunological demonstration of GFAP. The G1 mAb reacted with the GFAP from rat as well as from man. The presence of several different molecular weight forms of GFAP in aqueous and detergent extracts from human brain was shown with the G1 mAb. The possible meaning of these forms is discussed.     

Gliarin and macrolin, two novel intermediate filament proteins specifically expressed in sets and subsets of glial cells in leech central nervous system.

Using monoclonal antibodies, we have identified two novel intermediate filament (IF) proteins, Gliarin and Macrolin, which are specifically expressed in the central nervous system of an invertebrate. The two proteins both contain the coiled-coil rod domain typical of the superfamily of IF proteins flanked by unique N- and C-terminal domains. Gliarin was found in all glial cells including macro- and microglial cells, whereas Macrolin was expressed in only a single pair of giant connective glial cells. The identification of Macrolin and Gliarin together with the characterization of the strictly neuronal IF protein Filarin in leech central nervous system demonstrate that multiple neuron- and glial-specific IFs are not unique to the vertebrate nervous system but are also found in invertebrates. Interestingly, phylogenetic analysis based on maximum parsimony indicated that the presence of neuron- and glial cell-specific IFs in coelomate protostomes as well as in vertebrates is not of monophyletic origin, but rather represents convergent evolution and appears to have arisen independently.     

Gliarin and macrolin,two novel intermediate filament proteins specifically expressed in sets and subsets of glial cells in leech central nervous system

Using monoclonal antibodies, we have identified two novel intermediate filament (IF) proteins, Gliarin and Macrolin, which are specifically expressed in the central nervous system of an invertebrate. The two proteins both contain the coiled‐coil rod domain typical of the superfamily of IF proteins flanked by unique N‐ and C‐terminal domains. Gliarin was found in all glial cells including macro‐ and microglial cells, whereas Macrolin was expressed in only a single pair of giant connective glial cells. The identification of Macrolin and Gliarin together with the characterization of the strictly neuronal IF protein Filarin in leech central nervous system demonstrate that multiple neuron‐ and glial‐specific IFs are not unique to the vertebrate nervous system but are also found in invertebrates. Interestingly, phylogenetic analysis based on maximum parsimony indicated that the presence of neuron‐ and glial cell–specific IFs in coelomate protostomes as well as in vertebrates is not of monophyletic origin, but rather represents convergent evolution and appears to have arisen independently. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 244–253, 1999     

Identification of an epithelial protein related to the desmosome and intermediate filament network

Using a mAb, referred to as 08L, we have identified a protein, of M(r) approximately 140,000, associated with desmosomes of epithelial cells. The 08L antibody stained the intracellular side of lateral cell margins of monolayer epithelial cells but did not stain cell margins free of cell contact. Immunoelectron microscopy revealed that the 08L antigen was localized to the cytosolic surface of the desmosomal plaque near points of intermediate filament convergence with apparently little staining of the desmosomal plaque proper. Western blots revealed the 08L antigen to be a protein, of M(r) approximately 140,000, found in the Triton-X 100 insoluble pellet. High salt-containing buffers extracted the 08L antigen from the insoluble material. Examination of the assembly of 08L to the desmosome complex, in cells grown in low confluent culture or in calcium-switch assays, by double immunofluorescence with 08L and anti-desmoplakin antibody, revealed that 08L was recruited to morphologically identifiable desmosomes. 08L antigen may exist in a cytosolic pool prior to assembly to the cell surface. The solubility of 08L in low calcium and normal calcium conditions, however, was similar. 08L association to the desmosome was correlated with increased organization of the intermediate filament network. We suggest that the 08L antigen may be involved in the organization and stabilization of the desmosome-IF complexes of epithelia.     

Two different protein kinases act on a different time schedule as glial filament kinases during mitosis.

Glial fibrillary acidic protein (GFAP) is a component of glial filaments specific to astroglia. We now report the spatial and temporal distributions of four phosphorylated sites in the GFAP molecule during mitosis of astroglial cells, determined by antibodies which can distinguish phosphorylated epitopes from non-phosphorylated-epitopes. Immunofluorescence microscopy showed that the Ser8 residues in the entire cytoplasmic glial filament system are initially phosphorylated when the cells enter mitosis. In cytokinesis, the phosphoSer8 residues become dephosphorylated, whereas Thr7, Ser13 and Ser34 in glial filaments at the cleavage furrow become the preferred sites of phosphorylation. The cdc2 kinase purified from mitotic cells can phosphorylate GFAP at Ser8 but not at Thr7, Ser13 or Ser34, in vitro. These results suggest that cdc2 kinase acts as a glial filament kinase only at the G2-M phase transition while other glial filament kinases are probably activated at the cleavage furrow before final separation of the daughter cells.     

Novel monoclonal antibody mAb G3A5 recognizes 138-kDa glycoprotein localized on the Golgi membrane.

Partially purified Golgi membranes of HeLa cells were used as antigen to produce a novel monoclonal antibody (mAb G3A5). The mAb G3A5 specifically labeled Golgi apparatus of human and monkey cultured cells as ascertained by indirect immunofluorescence but did not stain those of bovine or mouse cells. Treatment with nocodazole and brefeldin A (BFA) induced fragmentation and redistribution of the staining. Western immunoblot analysis showed that mAb G3A5 was directed against a single polypeptide with an apparent molecular mass of 138-kDa (p138 antigen). The p138 antigen is an integral membrane protein of the Golgi apparatus, as assessed by several assays: protease protection, salt wash and flotation in sucrose density gradient centrifugation. The p138 antigen was purified using immunoaffinity chromatography. The apparent molecular mass of the p138 antigen decreased by 2 to 4 kDa after treatment with the peptide: N-glycosidase F, while digestion with ENDO F or Neuraminidase did not have this effect. Thus, p138 antigen is a glycoprotein containing asparagine-linked carbohydrates.     

Localization of carbonic anhydrase in identified glial cells of the leech central nervous system: application of histochemical and immunocytochemical methods

We localized the enzyme carbonic anhydrase (CA) in frozen sections of the leech (Hirudo medicinalis) central nervous system by two histochemical techniques and the indirect immunofluorescence technique. Hansson's cobalt precipitation method and the use of 1-dimethylamino-naphthalene-5-sulfonamide (DNSA) to build a fluorescent enzyme-substrate complex showed that glial cells are the sites of CA activity in the leech. Neuropil and connective glial cells surrounding the axons had strong CA activity, whereas packet glial cells, which surround neuron cell bodies, and neurons themselves remained unstained. Glial cells reacted markedly with FITC-coupled antibodies against CA isoenzyme II, but experiments with antibodies against CA isoenzyme I showed no reaction.     

Expression of a novel Müller glia specific antigen during development and after optic nerve lesion

To generate monoclonal antibodies, immunogen fractions were purified from embryonic chick retinae by temperature-induced detergent-phase separation employing Triton X-114. Under reducing conditions, the monoclonal antibody (mAb) 2M6 identifies a protein doublet at 40 and 46 x 10(3) Mr, which appears to form disulfide-coupled multimers. The 2M6 antigen is regulated developmentally during retinal histogenesis and its expression correlates with Müller glial cell differentiation. Isolated glial endfeet and retinal glial cells in vitro were found to be 2M6-positive, identified with the aid of the general glia marker mAb R5. mAb 2M6 does not bind to any other glial cell type in the CNS as judged from immunohistochemical data. Cell-type specificity was further substantiated by employing retinal explant and single cell cultures on laminin in conjunction with two novel neuron-specific monoclonal antibodies. MAb 2M6 does not bind either to neurites or to neuronal cell bodies. Incubation of retinal cells in vitro with bromodeoxyuridine (BrdU) and subsequent immunodouble labelling with mAb 2M6 and anti-BrdU reveal that mitotic Müller cells can also express the 2M6 antigen. To investigate whether Müller cell differentiation depends on interactions with earlier differentiating ganglion cells, transections of early embryonic optic nerves in vivo were performed. This operation eliminates ganglion cells. Müller cell development and 2M6 antigen expression were not affected, suggesting a ganglion-cell-independent differentiation process. If, however, the optic nerve of juvenile chicken was crushed to induce a transient degeneration/regeneration process in the retina, a significant increase of 2M6 immunoreactivity became evident. These data are in line with the hypothesis that Müller glial cells, in contrast to other distinct glial cell types, might facilitate neural regeneration.     

Detection of glial fibrillary acidic protein (GFAP) and vimentin (Vim) by immunoelectron microscopy of the glial cells in the central nervous system of the snail Megalobulimus abbreviatus

When examined under an electron microscope, the central nervous system of Megalobulimus abbreviatus showed two types of glial cells: firstly, protoplasmic glial cells which displayed a nucleus with peripheral heterochromatin, scanty or no intermediate filaments, a developed Golgi complex, rough and smooth endoplasmic reticula, mitochondria and polymorphic lysosomes that indicate phagocytic activity of debris from the extracellular space; and, secondly, fibrous glial cells which showed numerous glial fibrillary acidic protein (GFAP) and vimentin immunoreactive intermediate filament bundles, a discrete Golgi complex, mitochondria, endoplasmic reticulum, lipid droplets and lysosomes. The contacts between the glial cells consisted of desmosomes and puncta adherentia, while those between the glial cells and the basal lamina consisted of hemidesmosomes. Both glial cell types were located in the cortex and medullary regions, however, the protoplasmic glial cells prevailed in the cortical region, while the fibrous glial cells prevailed in the medullar region. As the nervous tissue is avascular, the passage of nutrients and waste products may be facilitated by the glial labyrinthic system which is located in the cortical region. Glial processes adjacent to large and giant neurones formed a trophospongium, which seemed to be involved in a metabolic exchange between these cells. Thus, this evidence suggests that glial cells of M. abbreviatus are involved in structural support, isolation of different ganglionic areas, the formation of a microcirculatory system and an intimate metabolic relationship with neurones.     

Susceptibility of astrocytes to class I MHC antigen-specific cytotoxicity

Cell-mediated immune mechanisms contribute to tissue injury within the central nervous system (CNS) in a number of experimental diseases, including experimental allergic encephalomyelitis and some viral infections, and may mediate lesion formation in multiple sclerosis. We investigated the conditions under which murine astrocytes can become susceptible targets of cytotoxic T cells. We demonstrate that mouse astrocytes in vitro can be susceptible targets of class I major histocompatibility complex (MHC)-specific cytotoxicity mediated by L3 cytotoxic T lymphocytes (CTL). Expression of appropriate class I MHC antigen on the astrocytes is a requirement, because only cells bearing the H-2d phenotype are susceptible to lysis by L3 cells. BALB/c-H-2dm2 astrocytes lacking the specific determinant recognized by L3 cells are not susceptible to lysis. Astrocyte lysis can, however, occur under culture conditions in which MHC antigen expression is immunocytochemically low or undetectable. Cytolysis can be inhibited by pretreatment of the effector L3 cells with either anti-Lyt-2 monoclonal antibody (mAb) or anti-clonotypic mAb and by preincubation of the glial target cells with an appropriate anti-H-2 antibody (anti-H-2Ld). mAb to lymphocyte function-associated antigen does not inhibit cytotoxicity of the L3 clone against glial cells. Knowledge regarding the role of CTL within the CNS, including the surface molecules involved in glial cell lysis, could further the development of immunotherapies designed to effect immune reactivity within the CNS.     

Molecular cloning of the cell surface antigen identified by the osteoprogenitor-specific monoclonal antibody,HOP-26

Bone is a highly organized structure comprising a calcified connective tissue matrix formed by mature osteoblasts, which develop from the proliferation and differentiation of osteoprogenitor cells. The osteogenic cell lineage is thought to arise from a population of uncommitted multipotential stromal precursor cells (SPC) which reside close to all bone surfaces, in the bone marrow spaces and the surrounding connective tissue. These SPC also give rise to related cell lineages which form cartilage, smooth muscle, fat, and fibrous tissue. Due to the lack of well defined cell surface markers, little is known of the precise developmentally regulated changes in phenotype which occur during the differentiation and maturation of human osteoprogenitor cells into functional osteoblasts and ultimately, terminally differentiated osteocytes. In order to identify antibody reagents with greater specificity for osteoprogenitors we generated a series of antibodies following immunization with freshly isolated human bone marrow stromal fibroblasts. One such antibody, HOP-26, reacts with a cell surface antigen expressed by SPC and developing bone cells. We now demonstrate that this mAb identifies a member of the tetraspan family of cell surface glycoproteins, namely CD63. Western blot analysis of human bone marrow stromal cells (HBMSC) has revealed that like a well defined CD63 mAb 12F12, HOP-26 interacts with a heavily glycosylated cell surface protein with an apparent molecular weight of 50-60 kD.     

The Dynamics of Filamentous Structures in the Apical Band, Oral Crescent, Fission Line and the Postoral Meridional Filament in Tetrahymena thermophila Revealed by Monoclonal Antibody 12G9

The ciliate Tetrahymena thermophila possesses a multitude of cytoskeletal structures whose differentiation is related to the basal bodies the main mediators of the cortical pattern. This investigation deals with immunolocalization using light and electron microscopy of filaments labeled by the monoclonal antibody 12G9, which in other ciliates identifies filaments involved in transmission of cellular polarities and marks cell meridians with the highest morphogenetic potential. In Tetrahymena interphase cells, mAb 12G9 localizes to the sites of basal bodies and to the striated ciliary rootlets, to the apical band of filaments and to the fine fibrillar oral crescent. We followed the sequence of development of these structures during divisional morphogenesis. The labeling of the maternal oral crescent disappears in pre-metaphase cells and reappears during anaphase, concomitantly with differentiation of the new structure in the posterior daughter cell. In the posterior daughter cell, the new apical band originates as small clusters of filaments located at the base of the anterior basal bodies of the apical basal body couplets during early anaphase. The differentiation of the band is completed in the final stages of cytokinesis and in the young post-dividing cell. The maternal band is reorganized earlier, simultaneously with the oral structure.The mAb 12G9 identifies two transient structures present only in dividing cells. One is a medial structure demarcating the two daughter cells during metaphase and anaphase, and defining the new anterior border of the posterior daughter cell. The other is a post-oral meridional filament marking the stomatogenic meridian in postmetaphase cells. Comparative analysis of immunolocalization of transient filaments labeled with mAb12G9 in Tetrahymena and other ciliates indicates that this antibody identifies a protein bound to filamentous structures, which might play a role in relying polarities of cortical domains and could be a part of a mechanism which governs the positioning of cortical organelles in ciliates.     

Autoantibodies recognizing proteins copurified with PCNA in patients with connective tissue diseases

Objective. Proliferating cell nuclear antigen (PCNA), one of the target antigen recognized by lupus sera, has been reported to be present as a subnuclear multi-peptide complex. But autoantibodies reacting with components of PCNA complex are poorly understood. To study the specificity of those autoantibodies, immunoreactivities of autoimmune sera against purified PCNA antigen were studied. Methods. PCNA antigens were purified from rabbit thymus extract by affinity column using murine monoclonal antibodies (mAbs) to PCNA, TOB7, TO17 and TO30. Immunoreactivities of autoimmune sera against purified PCNA were analyzed by WB. Results. PCNA antigen purified by serum AK predominantly showed a 34 kD band specific for PCNA in SDS-PAGE. When antigens were purified by anti-PCNA mAb TOB7 and TO30 which are known to be targeting different epitopes on PCNA antigen, SDS-PAGE analysis showed various mol. wt of proteins in addition to the 34 kD PCNA while both AK and mAbs reacted only with 34 kD PCNA in WB. In WB using PCNA purified by TOB7, various immunoreactivities were observed at 150, 66, 58, 48, 45, 37, 32 and 16 kDa in sera from patients with connective tissue diseases. Conclusions. These results suggested that many of the proteins copurified with PCNA were also targets of autoimmune responses and these autoantibody experssion may be induced through antigen-driven mechanisms.Abbreviations mAb monoclonal antibody - PCNA proliferating cell nuclear antigen - PCNA/AK PCNA affinity purified by antibodies from patient serum AK - PCNA/TO30 PCNA purfied by mAb TO30 - PCNA/TOB7 PCNA purified by mAb TOB7 - SLE systemic lupus erythematosus     

Different forms of 130 kD connective tissue protein are specific for boundaries in the nervous system and basement membrane of muscle cells in leech

The nervous system and muscle tissue of the leech express two different organ-specific forms of connective tissue protein. The nervous system-specific form appears in regional boundaries separating cell bodies, axonal tracts and areas of the neuropile during late embryogenesis. In contrast, the muscle-specific form appears earlier during development in the basement membrane of muscle cells. In extraction experiments both forms behave like extracellular matrix proteins and because of their molecular weight, are considered members of a group of cell type-specific 130 kD proteins (leech gp130s). How ever, the two forms differ in their posttranslational modification. As determined by Con A and lentil lectin affinity chromatography, only the nervous system-specific, but not the muscle-specific form, has fucosylated and high mannose N-linked carbohydrates. These differences in the developmental onset and glycosylation suggest that nervous system-specific and muscle-specific connective tissue proteins are regulated differently and participate in different molecular interactions. © 1993 John Wiley & Sons, Inc.     

Characterization of a monoclonal antibody reactive with a glycolipid antigen expressed by tumorigenic and certain immortalized,non-tumorigenic rat esophageal epithelial cell lines

A monoclonal antibody (mAb 5G) was produced against a tumorigenic rat esophageal epithelial cell line, designated B2T. Using an enzyme-linked immunosorbent assay, immunofluorescence assay (IFA), thin-layer chromatography (TLC) and immunoperoxidase staining, it was found that mAb 5G reacted specifically with a glycolipid antigen expressed by three tumorigenic rat esophageal epithelial cell lines, and two out of the three nontumorigenic, immortalized rat esophageal epithelial cell lines tested; but did not react with primary cultures of normal rat esophageal epithelial cells or fibroblasts. mAb 5G did not bind to rat respiratory tract carcinoma cell lines, to immortalized rat tracheal epithelial cell lines, or to primary cultures of normal rat tracheal epithelial cells. In addition, mAb 5G did not react with any of the human or mouse cell lines tested. In IFA experiments, mAb 5G stained imprints prepared from in vivo propagated B2T tumor tissues, but did not react with normal rat esophageal, tracheal, lung, liver, and kidney tissues. The antigen was identified by TLC as a neutral glycolipid, consisting of two bands, withR _F = 0.45 and 0.41, which migrated in proximity to the ceramide trihexoside standard on TLC plates. Densitometric scanning of the antigen bands indicated that the tumorigenic rat esophageal cell lines possessed 50%–90% more mAb-5G-reactive antigen than the nontumorigenic esophageal cell lines. The results show that mAb 5G reacts specifically with a glycolipid antigen expressed by tumorigenic and certain non-tumorigenic, immortalized rat esophageal epithelial cell lines that might be at the late stages of transformation and early malignancy.     

Calsensin: a novel calcium-binding protein expressed in a subset of peripheral leech neurons fasciculating in a single axon tract

The mAb lan3-6 recognizes a cytosolic antigen which is selectively expressed in the growth cones and axons of a small subset of peripheral sensory neurons fasciculating in a single tract common to all hirudinid leeches. We have used this antibody to clone a novel EF-hand calcium- binding protein, calsensin, by screening an expression vector library. A full-length clone of 1.1 kb identified by the antibody was isolated and sequenced. In situ hybridizations with calsensin probes and antibody staining using new polyclonal antisera generated against calsensin sequence demonstrate that calsensin indeed corresponds to the lan3-6 antigen. Calsensin consists of 83 residues with a calculated molecular mass of 9.1 kD that contains two helix-loop-helix domains. The calcium-binding domains are likely to be functional in vivo since a fusion protein derived from the calsensin clone binds 45Ca2+ in vitro. Immunoaffinity purification experiments with the lan3-6 antibody shows that a large 200,000 M(r) protein selectively copurifies with calsensin in two different leech species. These results suggest that calsensin may be functioning as a trigger protein which interacts with the larger protein. These data are consistent with the hypothesis that calsensin may mediate calcium-dependent signal transduction events in the growth cones and axons of this small group of sensory neurons which fasciculate in a single axon tract.     

Monoclonal antibody #3-9-16 recognizes one of the two isoforms of rabies virus matrix protein that exposes its N-terminus on the virion surface

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3-9-16, that recognized a linear epitope located at the N-terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Malpha) whose 3-9-16 epitope is hidden, and an N-terminal-exposed epitope-positive form (Mbeta). The Mbeta protein accounted for about 25-30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mbeta antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mbeta antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb-dependent complement-mediated cytolysis. Similarly, the Mbeta antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mbeta) of which exposes the 3-9-16 epitope located in the N-terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Malpha), probably through its intimate association with the Golgi area and/or the cell membrane.     

Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

HAb18G/CD147, a glycoprotein of the immunoglobulin super‐family (IgSF), is a T cell activation‐associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4⁺ and CD8⁺ T cells was up‐regulated. In vitro cross‐linking of T cells with an anti‐HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co‐stimulation inhibited T cell proliferation by down‐regulating the expression of CD25 and interleukin‐2 (IL‐2), decreased production of IL‐4 but not interferon‐γ. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti‐HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody–antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N‐terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co‐stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS.