Identification and characterization of a tumor-derived immunosuppressive glycoprotein from murine melanoma K-1735

Summary A suppressive immunoregulatory factor (IRF) produced by murine melanoma K-1735 M3 has been identified. Extracts from tissue or cultured cells grown in serummedium were prepared by 3 M KCl extraction and partially purified by low-salt precipitation. IRF extracted from fresh tumor, cultured cells, and spent medium from the K-1735 cell line suppressed ³H-thymidine incorporation by splenocytes during mitogen stimulation. Cell viability was not impaired by IRF. IRF suppressed splenocyte proliferation, protein synthesis, murine IL-2-mediated blastogenesis, and mixed splenocyte responses. However, in vitro generation of allogenic cytotoxic cells was not suppressed. Significant inhibitory activity could not be extracted from normal tissues. IRF activity was reduced by treatment with proteolytic enzymes and neuraminidase and was bound by lentil lectin, indicating that the factor is a glycoprotein. IRF was heat-stable, yet labile to treatment with acid, base, or 2-mercaptoethanol. Inhibitory activity was partially characterized by preparative isoelectric focusing (pI 3.5–5.8), and the active moiety had a molecular size of 10–12 K according to HPLC. The HPLC-purified active fraction of IRF did not contain the immunosuppressive retroviral antigen p15(E). Splenocytes from animals treated with IRF in vivo demonstrated reduced responses to Con A and PHA in vitro. Suppressor cells were not identified. We have identified a low-molecular-weight glycoprotein from a murine melanoma, which suppresses a variety of immunologic responses in vitro and in vivo. IRF appears to be a potent mediator of tumor-induced immunosuppression in this model.Abbreviations ACK ammonium chloride-potassium erythrocyte lysing buffer - BSA bovine serum albumin - CM complete medium - Con A concanavalin A - HBSS Hank's balanced salt solution - HPLC high-performance liquid chromatography - HS hepes sucrose - IL-2 interleukin II (lectin-free) - IRF immunoregulatory factor - Leu L-leucine - LPS lipopolysaccharide - M3 metastasis from murine melanoma K-1735 - MASH multiple automated sample harvester - PBS phosphate-buffered saline - PIEF preparative isoelectric focusing - PHA phytohemaglutinin - PWM pokeweed mitogen - Tdr thymidine