Yosef H. Pilch

Detection of antibodies bound to tumor cell surface antigens with radioiodinated Staphylococcus aureus protein A (SPA)

SummaryStaphylococcal protein A (SPA) is known to bind to the Fc portion of certain subclasses of IgG. On the basis of this property, radioiodinated SPA (125I-SPA) was used to detect antibodies reacting with surface antigens of tumor cells. Target cells derived from an osteosarcoma growing in C3H/fHeJ mice and antiserum to this tumor prepared in female Hartley guineapigs were used to establish optimum conditions for the assay. Similar optimum conditions were also determined for human melanoma target cells. Target cells were plated at a concentration of either 3×105 cells per well or 1×105 cells per well in Microtest II plates, and allowed to form semiconfluent monolayers for 24–48 h respectively. Target cells thus prepared were treated with antiserum and then with 125I-SPA. A minimum of 30 min incubation time was found to be optimal for the antigen-antibody reaction. The quantity of 125I-SPA bound to antisera-treated target cells was found to depend on the time of incubation with 125I-SPA and on the concentration of SPA used. Longer incubation times and increasing concentrations of SPA resulted in greater amounts of 125I-SPA being bound to antiserum treated target cells. This assay was employed for the detection of antibodies in the sera of two melanoma patients and two colon carcinoma patients. The results of absorption analysis suggest that the antibody activity in the sera of the melanoma patients may be of four different specificities: (a) autoantibodies, (b) alloantibodies, (c) antibodies reacting with common, cross-reacting melanoma-associated antigens, and (d) antibodies reacting with unique antigens specific for autologous melanoma cells.

IMMUNE RNA IN TUMOR IMMUNOLOGY

Our laboratories have reported the mediation of specific antitumor immune reactions with immune RNA (I-RNA) in a number of model systems. The purpose of this present paper is to summarize results of studies which were designed to elucidate the mechanisms by which I-RNA converts lymphocytes in vitro to become “killer” cells that are cytotoxic for tumor target cells. We present the results of experiments which demonstrated that antitumor I-RNAmediated cytotoxic reactions in vitro that were directed specifically against tumor-associated antigens of human and animal tumor cells. Additionally, we report on the kinetics of synthesis of I-RNA in immunized animals and the isolation of immunologically active I-RNA fractions on sucrose density gradients. We also have shown that the antitumor activity of I-RNA is localized in the cytoplasm of lymphoid cells from immunized animals. Finally, we determined which subpopulation( s) of lymphoid cells participated in the cellular antitumor immune reactions mediated by I-RNA. By using a variety of commonly employed cell separation techniques, experiments were performed to determine the lymphoid cell of origin of I-RNA, and the effector cell responsible for the immunoreactivity mediated by I-RNA. T-cells appeared to play a major role both as the cell type which synthesized I-RNA, and as the effector cell which was acted upon by I-RNA. For studies in animal systems, we used both xenogeneic and syngeneic sources of I-RNA. With the former, xenogeneic I-RNA was extracted from guinea pigs immunized with tumors that had been chemically induced in C3H mice. These two tumors, designated MC-1 and BP-1, had individually distinct tumor-associated antigens, as denionstrated by in vivo transplantation experiments. Within ,his model, normal syngeneic C3H spleen cells were incubated with xenogeneic I-RNA and the resultant cytotoxicity for the C3H tumor target cells was measured in a classic lymphocytotoxicity test. For studies utilizing syngeneic I-RhA, we used inbred Fischer rats. The tumors used, MC3-R and BPI-R, were chemically induced. In this totally syngeneic system, the I-RNA

Production of cytotoxic antibody to a benz(a)pyrene-induced sarcoma in mice receiving xenogeneic antitumor immune rna.

SummaryGroups of normal C3Hf mice were injected subcutaneously and intraperitoneally over a 2-week period with various xenogeneic immune RNA (I-RNA) preparations. I-RNAs were extracted from the lymphoid organs of guinea pigs following immunization with normal C3Hf tissue cells, or tumor cells from a benz(a)pyrene-induced sarcoma (BP-1), a methylcholanthrene-induced sarcoma MC-1, or a spontaneous mammary carcinoma (SMT). In sera from mice injected with anti-BP-1 I-RNA, antibodies were detected which were specifically cytotoxic to BP-1 target cells, in vitro, but not to MC-1 target cells. Absorption with BP-1 sarcoma cells removed this cytotoxic activity while absorption with syngeneic spleen cells, or MC-1 sarcoma cells did not. Sera from mice receiving any of the other I-RNAs were not cytotoxic for either BP-1 or MC-1 target cells. Treatment of mice with BP-1 I-RNA may have induced the production of tumor-specific cytotoxic antibody, in vivo.

Conversion of human lymphocytes to antitumor immune reactivity by xenogeneic and allogeneic imune RNA detected by leukocyte-adherence inhibition tests

SummaryUsing leukocyte adherence inhibition (LAI) tests, we studied the activity of xenogeneic immune RNA (I-RNA) extracted from the spleen and lymph nodes of sheep after immunization with human breast carcinoma tissue or keyhole limpet hemocyanin (KLH) in inducing lymphocytes from normal healthy donors to mediate immune responses in vitro. Mononuclear cells isolated from venous blood of normal donors, depleted of monocytes and, in some experiments, separated into T cells and non-T cells, were incubated with and without anti-breast carcinoma I-RNA or anti-KLH I-RNA for 20 min at 37° C. Then, lymphocyte adherence was determined by a Coulter counter method in the presence of 3 M KCl extracts of breast carcinoma tissues, control tissue, or KLH. Following incubation with anti-breast carcinoma I-RNA, the adherence of lymphocytes from normal donors was found to be inhibited only in the presence of breast carcinoma extracts. Following incubation with anti-KLH I-RNA, lymphocyte adherence was inhibited only in the presence of KLH. The principal effector cells involved appeared to be T lymphocytes. I-RNA treatment with RNase, but not with DNase or pronase, completely abrogated the LAI responses. In a blind study utilizing coded samples of xenogeneic and allogeneic I-RNA of unknown origin, samples containing activity against breast cancer extracts were identified correctly by LAI.