The cytolytic protein of human lymphocytes related to the ninth component (C9) of human complement: isolation from anti-CD3-activated peripheral blood mononuclear cells.

摘要

A 70-kDa channel-forming protein has recently been isolated from human large granular lymphocytes maintained in interleukin-2-dependent culture. The protein was shown to be immunochemically related to the ninth component of complement (C9) and was therefore designated C9-related protein (C9RP). Using the procedure that was developed for the isolation of C9RP from large granular lymphocytes--i.e., affinity chromatography employing anti-human C9 linked to Sepharose, a cytolytic protein has now been isolated from OKT3-activated human peripheral blood mononuclear cells. Nineteen to 40 micrograms of active protein was obtained from 1 X 10(9) human peripheral blood mononuclear cells after the cells were cultured for 3 days with OKT3 (monoclonal antibody to cell surface antigen T3). During this period, a marked increment occurred in the amount of the cytotoxic protein contained per cell, indicating that OKT3 induced de novo synthesis of the protein. By NaDodSO4/PAGE the molecular mass was determined to be 70 kDa. By ELISA the isolated protein and C9RP of large granular lymphocytes reacted to the same extent with anti-C9RP. Using K-562 or M21 human melanoma cells as targets, the cytotoxic activity of the isolated protein, in the presence of 5 mM Ca2+, was comparable to that of C9RP. The same cytolytic protein was isolated from peripheral blood mononuclear cells that were depleted of CD16+ cells prior to OKT3 activation and that consisted primarily of CD4+ and CD8+ T lymphocytes. These results suggest that the cytolytic protein of OKT3-activated cytotoxic T lymphocytes is identical with C9RP of interleukin-2-stimulated large granular lymphocytes.