title: Functional analysis of mononuclear cells infiltrating into tumors. IV. Purification and functional characterization of cytotoxic cell-generating factor.
abstract: Rat cytotoxic cell-generating factor (CGF) was purified from cell-free supernatants of a T cell hybridoma (6B2-B8) that constitutively produces CGF. CGF activity was assessed by its ability to generate cytotoxic cells against 51Cr-labeled T-9 cells from spleen cells of T-9-immunized rats. The purification scheme consisted of ammonium sulfate precipitation, AcA 54 gel permeation, Mono Q anion exchange chromatography, Superose 12HR 10/30 gel permeation, SDS-PAGE with subsequent electroelution, and ProRPC HR5/10 reverse phase column chromatography. Overall, CGF was purified approximately 13,000-fold, with a maximum 2.5% recovery of activity, and the sp. act. of the purified CGF was approximately 19,000 U/mg. The purified CGF is distinct from the other lymphokines such as IL-1, IL-2, IL-3, IL-4, T cell-replacing factor/IL-5, IL-6, and IFN-gamma. It is capable of promoting the generation of cytotoxic T cells from R1-10B5 (+) spleen cells of T-9-immunized rats and also stimulates a W3/25 (+) T cell hybridoma to express the IL-2R. The CGF has an apparent m.w. of 28,000 under non-reducing and 14,000 and 16,000 under reducing conditions. 125I-labeled CGF binds to normal thymocytes as well as splenic T cells. The highest level of binding of CGF was detected on splenic T cells derived from T-9-immunized rats that were previously shown to contain CTL precursors. The binding analysis with 125I-labeled CGF demonstrated that CGF binds to a specific cell surface molecule with an approximate m.w. of 60,000 to 70,000.
authors.affiliation: Department of Pathology, Sapporo Medical College, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Murakami
authors.forename: T
authors.initials: T
authors.lastname: Uede
authors.forename: N
authors.initials: N
authors.lastname: Shijubo
authors.forename: Y
authors.initials: Y
authors.lastname: Torimoto
authors.forename: T
authors.initials: T
authors.lastname: Ide
authors.forename: K
authors.initials: K
authors.lastname: Sugimura
authors.forename: I
authors.initials: I
authors.lastname: Azuma
authors.forename: Y
authors.initials: Y
authors.lastname: Takai
authors.forename: K
authors.initials: K
authors.lastname: Kikuchi
mesh_terms: D000818:Animals; D002465:Cell Movement; D002474:Cell-Free System; D003602:Cytotoxicity, Immunologic; D005260:Female; D006825:Hybridomas / Q000032:analysis; D055646:Killer Factors, Yeast; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D009368:Neoplasm Transplantation; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D051381:Rats; D011916:Rats, Inbred F344; D011917:Rats, Inbred Lew; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism / Q000502:physiology
chemical_list: D055646:Killer Factors, Yeast; D011506:Proteins