RESUMEN
To produce large quantities of recombinant CD58 (rCD58) glycoproteins for biochemical and functional studies, a cDNA clone containing the phosphatidylinositol-linked form of human CD58 was expressed in insect cells using the baculovirus system. Gel filtration showed rCD58 to form soluble oligomeric aggregates which were functionally, antigenically, and biochemically similar to their natural counterpart. Sequence analysis of the amino- and carboxy-terminal ends of released rCD58 protein revealed that the 28 amino acid signal peptide was accurately removed. In contrast, the hydrophobic C-terminal peptide was not removed. rCD58 binds to its natural ligand CD2 with a dissociation constant Kd = 5 x 10(-8) M, which is equivalent to the affinity of physiological T cell adhesion mediated by the membrane bound CD2-CD58 receptor-ligand pair. Rosette formation of human T lymphocytes with sheep and human erythrocytes was completely abrogated. In addition, the mixed lymphocyte reaction was significantly inhibited by rCD58. Moreover, cytotoxicity of human NK clones (CD2+CD3-) was inhibited by rCD58 similar to inhibition by CD58 mAbs. In contrast, rCD58 synergized with mitogenic CD2R mAbs in T cell triggering. These data demonstrate that rCD58 might serve as a biological immunomodulator which influences T cell adhesion and activation.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Receptores Inmunológicos/fisiología , Linfocitos T/citología , Secuencia de Aminoácidos , Antígenos CD/química , Baculoviridae/genética , Secuencia de Bases , Antígenos CD2 , Antígenos CD58 , Adhesión Celular , Células Cultivadas , Clonación Molecular , Citotoxicidad Inmunológica , Humanos , Tolerancia Inmunológica , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/química , Formación de Roseta , Solubilidad , Linfocitos T/inmunologíaRESUMEN
The interaction between the E-rosette receptor (CD2) expressed on the cell surface of T lymphocytes and its natural ligand CD58 (LFA-3), a broadly distributed cell surface molecule, represents a potential target for immune modulation. To explore this possibility, we have expressed a soluble recombinant form of human CD58 which exerts potent biological effects in that it acts as a competitive inhibitor for cell surface CD58 in its binding to CD2 positive lymphocytes. Thus, recombinant CD58 blocks NK-mediated cytotoxicity, the mixed lymphocyte reaction, and the T lymphocyte adhesion to CD58 positive cells. In contrast, recombinant CD58 synergized with mitogenic CD2R monoclonal antibodies (anti-T11(2/3) or 9.1) in T cell triggering.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Células Asesinas Naturales/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos CD2 , Antígenos CD58 , Pruebas Inmunológicas de Citotoxicidad , Humanos , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/metabolismo , Unión Proteica , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Solubilidad , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
CD58 (LFA-3), a heavily glycosylated protein of 40-70 kDa, is expressed on a broad range of hematopoietic and non-hematopoietic cells. It serves as a physiological ligand of the CD2 receptor, present on T cells and natural killer cells, and plays, thus, an important role in lymphocyte adhesion and T cell activation through CD2. Whereas several epitopes and their respective function are known for CD2, a similarly detailed characterization of CD58 is still lacking. We raised a panel of novel murine monoclonal antibodies (mAb) against recombinant human CD58 and describe here the identification of six structurally and/or functionally distinct epitopes on the CD58 molecule. All epitopes were found to be present in equal numbers on a wide range of CD58+ cells, none of them being differentially up-regulated following cell activation or malignant transformation. Two of these epitopes represent functionally relevant sites, involved in binding of CD58 to CD2 and T cell activation via CD2. One further epitope appears to be selectively involved in CD58-mediated activation, whereas the other three displayed no functional effects. The new mAb allow for the first time the detection of CD58 in enzyme-linked immunosorbent assays and immunofluorescence while bound to its receptor CD2 in human serum or on freshly isolated blood cells. Finally, one mAb was found to specifically cross-react with T11TS, the equivalent of CD58 in sheep.
Asunto(s)
Antígenos CD/inmunología , Epítopos/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos CD2 , Antígenos CD58 , Citotoxicidad Inmunológica , Epítopos/fisiología , Humanos , Activación de Linfocitos , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Receptores Inmunológicos/fisiología , Linfocitos T/inmunologíaRESUMEN
The human adhesion receptor CD58 (LFA-3) is expressed on most human cell types. Here we report on a soluble form of CD58 (sCD58) in human serum, human urine, and culture supernatants of several cell lines. sCD58 partially purified from human serum, from supernatant of the Hodgkin cell line L428, and purified sCD58 from human urine were found to have a molecular mass of 40-70 kDa under denaturating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting). However, gel filtration of sCD58 purified from human urine gave a molecular mass of 118-166 kDa, suggesting a noncovalent homotrimer conformation or its association with other molecules. Using an enzyme-linked immunosorbent assay specific for CD58 we found that sera from patients suffering from different forms of hepatitis contained elevated sCD58 levels (n = 108). Accordingly, there was a fivefold increase of supernatant sCD58 when the hepatocellular carcinoma cell line Hep G2 was incubated with 25 ng/ml recombinant tumor necrosis factor-alpha in vitro. In contrast, sCD58 serum levels of 337 additional patients suffering from various other immunological disorders were not found to be raised. At high concentrations sCD58 binds to CD2-positive cells and inhibits rosette formation of human T cells to human erythrocytes. Thus, local release of large quantities of naturally occurring sCD58 may interfere with intercellular adhesion in vivo.