No abstract

The immunoglobulin class of the complement-dependent cytotoxic antibodies in serum from D23 hepatoma-bearing rats (D23 TBS) for D23 hepatoma cells was analysed. When studied by affinity chromatography with concanavalin A, protamine, or staphylococcal protein A conjugated to Sepharose, the cytotoxic activity bound to the former two but not protein A. The binding fractions were further characterized by column chromatography on Sepharose CL-4B. The cytotoxic activity was recovered exclusively in the high molecular weight fractions corresponding to human IgM. Monitoring with IgG- or IgM-specific rabbit antibodies indicated that these high molecular weight cytotoxic fractions contained both IgG and IgM. However, fractionation of D23 TBS at low pH suggested that cytotoxicity was due to IgM antibodies rather than to immune-complexed IgG antibodies. This was supported by the findings that rabbit antirat IgM antibodies inhibited the cytotoxicity of TBS completely when added at high dilutions.

Summary.-3M-KC1 extracts of the hepatoma D23 contain antigens that inhibit the complement-dependent cytotoxicity for D23 hepatoma cells of serum from D23 tumour-bearing rats (D23 TBS). Inhibition was not due to a general anticomplementary activity of the extracts. Although a minor part (25%) of the protein of D23-KC1 extract was insoluble in PBS, this part contained most of the inhibitory activity. Fractionation of the PBS-soluble material of the extract on Concanavalin A-Sepharose showed that the inhibitory activity did not bind to the lectin. Analysis of D23-KC1 extracts on a Sepharose CL-4B column showed that the antigens involved in the cytotoxicity were heterogeneously distributed in the high -mol. wt region ( > 200,000). Precipitation with 10% trichloroacetic acid (TCA) of D23 KC1 extracts revealed that most of the antigenicity was insoluble in TCA. Heating of D23 KC1 extracts at 100°C did not affect the antigenicity. Enzyme treatment of D23 extra nuclear membranes (D23 ENP) revealed that the inhibitory activity was not sensitive to proteolytic digestion, while treatment with phospholipase A2, C or D abrogated partly the inhibitory activity. The lipid nature of the antigenicity was indicated by its solubility in organic solvents as chloroform or n-butanol.

The complement-dependent cytotoxicity of antibodies in tumour-bearer serum (TBS) from rats carrying the chemically induced D23 hepatoma was investigated. Target cells were D23 cells from solid tumours (D23sol), from ascites tumours (D23asc), or from in vitro growing cell cultures (D23cc). The D23asc and D23cc cells were not lysed when used as target cells in the assay, although they evoke cytotoxic antibodies when growing in vivo. The D23 ascites cells became susceptible to complement-dependent lysis after trypsin treatment. This was, however, not due to unmasking of target antigens, since untreated D23 ascites cells absorbed cytotoxic antibodies as efficiently as trypsinized cells. No increase in susceptibility to complement-dependent lysis was observed after trypsin treatment of D23cc cells. Absorption of cytotoxic antibodies with D23cc cells showed no or very low antigen expression on the surface of these cells. They did, however, contain the antigen(s), since 3 M KCl extracts of the D23cc cells could inhibit the complement-dependent cytotoxicity of D23sol TBS against D23sol cells. From these data it was concluded that the susceptibility of hepatoma cells for antibody-mediated complement lysis is not only correlated with antigen expression, as was the case with the in-vitro-cultivated cells, but is also dependent on increased lytic susceptibility after trypsin treatment of the cells.