Isolation and characterization of a tumor-specific T suppressor factor from a T cell hybridoma.
In our laboratory we have described a monoclonal antibody, B16G, which has been shown to bind to suppressive T cell factors (TsF) in DBA/2 mice. Therefore, B16G was used as a probe to identify T cell hybridomas secreting putative TsF. Hybridomas were obtained by the fusion of DBA/2 thymocytes stimulated in vivo by P815 tumor membrane extracts with the thymoma BW5147. One such hybridoma, A10, was selected and used for additional studies. From both the supernatants and ascites fluid of this hybrid a factor could be obtained that could specifically bind to both B16G and P815 antigen immunoadsorbent columns, and that scored positively with B16G in an ELISA after elution. Such reactivity could not be obtained from A10 supernatants or ascites absorbed over irrelevant columns, nor was it obtained from supernatants or ascites from other T cell hybrids that had scored B16G nonreactive in the original screening. In vivo studies indicated that affinity-purified A10 material injected into DBA/2J mice enhanced significantly the growth of P815 tumor cells, but not the growth of other DBA/2 syngeneic tumor lines such as L1210 or M-I. Additionally, this material did not inhibit the in vitro mixed leukocyte reaction (MLR) between DBA/2 splenocytes and allogeneic B10.BR target cells (unlike B16G purified material from whole DBA/2 spleens, which has been demonstrated to be suppressive in this type of MLR). Biochemical analysis of this tumor-specific TsF from A10 was undertaken; the native m.w. was found to be in the region of 80,000 and 90,000. Under reducing conditions, affinity-purified A10 TsF was found to resolve in SDS-PAGE as what appeared to be a heterodimer of 45,000 and 43,000. In most preparations, an associated molecule resolving at about 25,000 was observed. The implications of these observations are discussed.
Steele
J K
JK
Stammers
A T
AT
Levy
J G
JG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000951:Antigens, Neoplasm / Q000276:immunology*; D001666:Binding Sites, Antibody; D000939:Epitopes / Q000032:analysis; D005260:Female; D006825:Hybridomas / Q000276:immunology*; D007939:Leukemia L1210 / Q000276:immunology; D008222:Lymphokines / Q000302:isolation & purification* / Q000502:physiology; D012515:Mast-Cell Sarcoma / Q000276:immunology; D051379:Mice; D008811:Mice, Inbred DBA; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000276:immunology*; D012513:Sarcoma, Experimental / Q000276:immunology; D013491:Suppressor Factors, Immunologic; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000939:Epitopes; D008222:Lymphokines; D013491:Suppressor Factors, Immunologic