NORMAL HUMAN LYMPHOCYTE MEDIATED CYTOTOXICITY AGAINST AN ESTABLISHED CELL LINE (KU-2) DERIVED FROM HUMAN RENAL CELL CARCINOMA

ROLE OF T & B CELLS AS EFFECTOR

Abstract

In order to solve the problems related to the resistance of human being against hypernephroma, in vitro cytotoxicity of the peripheral blood lymphocytes (PBL) from healthy persons was examined using an established cell line (KU-2), which was derived from human renal cell carcinoma, as the target cells.
The original of the KU-2 was a metastatic hypernephroma in the lung, which was capable of producing ervthropoietin, and the KU-2 has been established in our Department as a monolayer cell-line from the tumor successively transplanted in nude mice.
As the effector cells in the cytotoxicity assay, PBL were isolated by Ficoll-Conary method from the blood drawn from 3 healthy male and one healthy female adults. T cells and B cells separated from the PBL after removing macrophages by the carbonyl iron-powder treatment were also used as the effector cells. T cells were obtained as sheep erythrocyte-rosette (ER) forming cells, and B cells as non-ER forming cells. PBL, T cells, and B cells thus obtained were used as the effector cells either as such or after stimulation with phytohemagglutinin (PHA). In vitro cytotoxicity of the effector cells was assayed by % cytotoxicity in the microcytotoxicity assay and also by morphological observation of time lapse cinematographs. Identification of the effector cells responsible for the spontaneous lymphocyte-mediated cytotoxicity (SLMC) was made by scanning electron microscopic observation and by immunofluorescence technique for detection of surface Ig and Fc receptors on the cells.
Cytotoxicity against KU-2 of PBL, T and B cells as such and after stimulaton with PHA showed (1) the highest Cytotoxicity by PHA stimulated PBL and PHA stimulated T cells (PHA induced cytotoxicity), (2) high cytotoxicity by individually isolated non PAH-stimulated B cells (SLMC), absence of cytotoxicity by individually isolated non PHA-stimulated T cells, and (3) discrepancy between the cytotoxic effects demonstrated under time lapse cinematography and % cytotoxicity determined by microcytotoxicity assay with regard to cytotoxicity by individually isolated PHA stimulated B cells. Furthermore, (4) presence of long microvilli on the surface of B cells connected closely with KU-2 cells as target cells was demonstrated under scanning electronmicroscopic observation, and (5) presence of surface Ig and Fc receptor as surface markers on B cells playing major role in SLMC was observed by immunofluorescence technique.
Above mentioned findings indicated strong possibility that cytotoxicity generated by both PHA stimultaed T cells and non PHA-stimulated B cells were responsible for high cytotoxicity generated by PHA-stimulated PBL.