Functional analysis of mononuclear cells infiltrating into tumors. IV. Purification and functional characterization of cytotoxic cell-generating factor.
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.
Murakami
T
T
Department of Pathology, Sapporo Medical College, Japan.
Uede
T
T
Shijubo
N
N
Torimoto
Y
Y
Ide
T
T
Sugimura
K
K
Azuma
I
I
Takai
Y
Y
Kikuchi
K
K
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
D055646:Killer Factors, Yeast; D011506:Proteins