Identification of a putative antigen receptor on fish nonspecific cytotoxic cells with monoclonal antibodies

• Published in: The Journal of immunology (1950) Vol. 141; no. 1; pp. 324 - 332
• Main Authors: Evans, DL, Jaso-Friedmann, L, Smith, EE, Jr, St. John, A, Koren, HS, Harris, DT
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Am Assoc Immnol 01.07.1988; American Association of Immunologists
• Subjects: Animals; antibodies; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - physiology; antigens; Binding Sites, Antibody; Biological and medical sciences; Catfishes - immunology; Cytotoxicity Tests, Immunologic - methods; Cytotoxicity, Immunologic; Epitopes - immunology; Female; Freshwater; Fundamental and applied biological sciences. Psychology; Fundamental immunology; General aspects. Ontogeny. Phylogeny; Hybridomas - metabolism; Ictaluridae - immunology; Ictalurus punctatus; identification; Immunity, Cellular; Immunobiology; immunology; Immunosuppressive Agents - physiology; Kidney - cytology; lymphoid cells; Male; Mice; Mice, Inbred BALB C; monoclonal antibodies; receptors; Receptors, Antigen - immunology; Receptors, Antigen - isolation & purification; Spleen - cytology; Cell culture; Vertebrata; Membrane receptor; Pisces; Cytotoxicity; Identification; Cellular immunity; Ictalurus punctatus; In vitro; Effectory cell; Immunocytochemistry; Monoclonal antibody; Antigen; Characterization; Natural killer cell; Non specific immunity
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.141.1.324

In the present study mAb were derived against flow cytometry (FCM) purified fish (Ictalurus punctatus) nonspecific cytotoxic cells (NCC). mAb 5C6.10.4 and 6D3.2.10 produced 60 to 65% inhibition of lysis of NC-37 target cells (a human B-lymphoblastoid cell line) by unfractionated NCC. mAb 2B2.4.9 and 6D3.4.4 were noninhibitors of cytotoxicity. All mAb were the same isotype (IgM) and were cloned by limiting dilution (2x). Inhibitory activity was specific for the effector cells because the mAb had no effect on NCC cytotoxicity when only the target cells were treated. Inhibition could be produced by preincubation of the mAb with NCC or by no preincubation, and inhibition was not reversible. Killing by FCM-sorted NCC of NC-37 target cells was inhibited almost 100% by mAb 5C6.10.4. Inhibitor mAb also significantly reduced NCC killing of MOLT-4, K562, P815, U937, Daudi, YAC-1, and HL-60 cells. Experiments also were conducted to determine at which stage of the lytic cycle the mAb acted. Both inhibitor mAb significantly inhibited conjugate formation between effector and NC-37 target cells. The technique of FCM was combined with competitive binding experiments to determine that the Ag recognized by both inhibitor and noninhibitor mAb was found on the membranes of the same cells. These results were confirmed by demonstrating (by using FCM) that FITC-labeled inhibitor and biotinylated noninhibitor mAb bound to the same cells. FCM also was next used to determine mAb binding to various effector cell populations. Inhibitor and noninhibitor mAb bound to approximately 25% (5C6.10.4) and 39% (6D3.4.4) of fish anterior kidney cells; to 42% (5C6.10.4) and 54% (6D3.4.4) of fish spleen cells; and to 2.5% (5C6.10.4 and 6D3.4.4) of fish peripheral blood. mAb were used to purify the target cell binding structure found on NCC. Con A-Sepharose purified mAb were used as the fixed ligand for Affi-Gel-10 affinity chromatography experiments. FCM-purified NCC were solubilized and the receptor was purified by using this technique. Analysis of the NCC-purified receptor by 12% SDS-PAGE indicated that the mAb purified structure may be composed of a dimeric molecule consisting of 41 kDa and 38 kDa proteins. The same dimer was purified by using either inhibitory (6D3.2.10) or noninhibitory (6D3.4.4) mAb. Similar results were obtained with immunoprecipitation experiments by using mAb 5C6.10.4. These studies demonstrate that the Ag-binding receptor structure on fish NCC may be comprised of a dimeric complex.