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Using the polyoma virus-induced ascitic SEYF-a tumor, we evaluated isopycnic and velocity sedimentation gradients of Percoll as methods for separating tumor-seeking lymphocytes and tumor cells. It was established that the velocity sedimentation method is suitable for separation of small lymphocytes lodging within the SEYF-a tumor. This was confirmed by a serological analysis of the separated SEYF-a cell population. The results of this study strongly support our previously reported data demonstrating the in vivo coating of the tumor cells proper with potentially cytotoxic antibodies.

Murine B16 melanoma cells and metastasis variants of this tumor are resistant to NK activity mediated by normal splenocytes. The B16 cells are, however, sensitive to splenocyte-mediated cytostasis. Cytostasis was measured by a [125I]UDR incorporation-inhibition [(125I]UDR I-I) assay. The main characteristics of the [125I[UDR I-I assay and of the cells mediating it are as follows: The activity is mediated by splenocytes but not by thymocytes, it is not syngeneically restricted and it does not decrease with age. The presence of effector cells is required as splenocyte supernatants or supernatants of effector-target cell mixtures do not cause [125I]UDR I-I. The activity is probably mediated by at least 2 populations of non-phagocytic splenocytes. The first population adheres to plastic surfaces and to Sephadex G-10 columns while the other does not. The sensitivity to [125I]UDR I-I of the high metastasis B16 variant was similar to that of the low metastasis variant.

With the aid of an assay measuring complement-dependent cytotoxocity mediated by syngeneic antibodies, we performed a serological analysis of surface antigens of a polyoma-virus-induced murine tumor (SEYF-a). In vivo propagated SEYF-a ascites tumor cells expressed a specific membrane antigen in addition to various other cross-reacting antigens. Among these we could identify at least four separate specificities. Two of these were present on MuLV-induced lymphoma cells, the first on Moloney-virus-induced YAC cells and the second on Gross-virus-induced GHA cells. The third cross-reacting antigen was detected on EL-4 cells. At least one additional specificity was present on two methylcholantthrene-induced murine sarcomas. Normal syngeneic lymphoid cells were insensitive to cytotoxicity mediated by the anti-tumor antisera. Quantitative and perhaps also qualitative differences between that antigenic expression of in vivo propagated on cultured SEYF-a cells were indicated. These studies show that hyperimmune sera produced in syngeneic mice against transplanted tumors may contain a considerable number of antibody specificities, only some of which are specific for the tumor. Furthermore, the results also suggest that polyoma-virus-induced tumors may possess individually distinct antigenic specificities, over and above the known cross-reacting TSTA or TSSA type antigen.

Low pH eluates were prepared from trophoblasts derived from 8 term human placentas. A qualitative analysis for immunoglobulins revealed the presence of IgG, IgA, and IgM in these eluates. IgC-rich fractions were obtained by DEAE-cellulose chromatography of ammonium sulfate-concentrated eluates. These fractions were able to neutralize, in vitro, the catalytic activity of reverse transcriptases (RT) from several retroviruses. RT from baboon endogenous virus (BEV) seemed to be more susceptible to the neutralizing activity of some eluates. This was in contrast to RT from feline leukemia virus (FeLV) which were neutralized by eluates of leukocytes from chronic myelogenous leukemia. In contrast to previous and present results with purified IgG from leukemic leukocytes, the purified IgG from placenta eluates was incapable of RT neutralization. However, such purified IgG fractions inhibited mixed lymphocyte reactions.

Immunoglobulin (Ig) "coating" on different in vivo growing mouse tumors was investigated using several approaches. Radioiodine-labelled purified protein A from Staphylococcus aureus, a specific reagent for Fc of IgG, and/or iodinated purified antibodies against mouse immunoglobulins, were fixed by various in vivo-grown mouse tumor cells, but not by the corresponding in vitro-cultivated tumor cells. Removal of host macrophages from the in vivo tumor-cell preparations did not affect the fixation of anti-mouse Ig reagents by the tumor cells. After an initial lag period in vivo, the intensity of the Ig coating of tumor cells increased with time after tumor inoculation. Conversely, the detectable coating decreased rapidly as a function of time after ascites tumor cells were explanted in vitro at 37 degrees C. Iodoacetamide did partially block this 37 degree C in vitro-induced "uncoating process". Surface-bound Ig could also be released from in vivo-coated tumor cells treated at low pH in vitro. Analysis of the behavior of tumor-bound Ig indicated a composite pattern. At the membrane level, uncoating was best shown with in vitro incubation favorable to cellular metabolism; however IgG was detected equally well in the supernatants of the cells, whether they were incubated at 37 degrees C or at 4 degrees C, and so far has failed to display antibody activity towards uncoated tumor cells. This was in contrast to the case of IgG released from ascites tumor cells cultured at low pH in vitro: such eluted Ig, when neutralized, could rebind to the "same" uncoated tumor cells. Under favorable metabolic conditions, the fate of tumor-bound Ig might be one of internalization and/or of degradation.

Serum from young normal BALB/c mice was found to contain IgM antibodies able to mediate complement-dependent lysis of certain syngeneic or allogeneic tumor target cells. The titer of such naturally occurring antitumor antibodies ( NATA ) was found to increase with aging. A longitudinal serological study comparing the cytotoxicity potential of NATA from normal and from urethan-treated BALB/c mice was performed. It was found that urethan-treated mice that did not develop primary lung-adenomas within the duration of the experiment had significantly lower NATA titers, against one out of 4 target cells assayed, than urethan-treated animals that developed lung adenomas. This difference was evident in two independent experiments. The results suggested that the lower NATA activity of the urethan-treated mice that did not develop tumors existed even before exposure to the carcinogenic insult. This raises the possibility that certain populations could be segregated according to their natural antibody profile into those individuals which will develop primary tumors within a certain period if exposed to a subthreshold amount of carcinogen, and those which will not.

Previous results obtained in our laboratory suggested that natural antibodies reactive with L5178Y lymphoma cells play a role in the induction of lung tumors by the chemical carcinogen urethane. In order to characterize some of the naturally-occurring L5178Y reactive antibodies we prepared hybridomas that secreted natural monoclonal IgM antibodies reactive with L5178Y lymphoma cells. In the present study we characterized some of these antibodies and provided further proof as to their role in urethane carcinogenesis. One hybridoma secreted a cytotoxic antibody that reacted only with mouse lymphoma cell lines. Other non-cytotoxic monoclonal L5178Y-reactive antibodies showed various degrees of cross-reactivity with syngeneic, allogeneic and xenogeneic cells of normal or malignant origin. One of these antibodies reacted much better with activated T cells than with resting ones. Four groups of mice were treated with urethane. Three groups were injected twice a week during 5 months with different IgM preparations of natural monoclonal antibodies. The mice in the fourth group were not treated with IgM and served as controls. Five months after the urethane treatment the mice were sacrificed and the number of tumor foci in the lungs of each mouse was determined. The results show that the group treated with the cytotoxic monoclonal antibody 1.80 had a significant decrease, while the group treated with the IgM myeloma protein 104E had a significant increase in the number of tumor foci compared to urethane-treated mice that did not receive any IgM treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal IgM anti-erythrocyte autoantibody produced by a NZB-derived hybridoma has been found to react with trimethylammonium-containing compounds. Such compounds are able to prevent the lysis of bromelain-treated mouse erythrocytes (BrMRBC) by those autoantibodies. Using a column of insolubilized betaine hydrazine (BH) the monoclonal anti-erythrocyte antibody has been specifically retained. Elution of this antibody was accomplished by 0.15 M choline (Ch).

A spontaneous lymphoma (141) producing monoclonal IgM is established in NZB/NZW F(1) (B/W) mice who spontaneously develop an autoimmune disease. Idiotypic determinants of 141 IgM are present on the lymphoma cell surface as shown by indirect immunofluorescence and specific cytotoxicity with rabbit anti-idiotypic antiserum. Fluorescence and cytotoxicity are inhibited by 141 IgM but not by 104E IgM, a monoclonal IgM produced by a BALB/c plasmacytoma. Immunization of B/W mice with 141 IgM before transplantation of lymphoma 141 confers protective immunity. No such protection occurs after immunization with 104E IgM or other unrelated proteins. Protected mice contain spleen cells cytotoxic for 141 lymphoma cells. This cytotoxicity is blocked by incubation of spleen cells with 141 IgM but not with 104E IgM. Moreover, splenic lymphocytes from protected mice are stimulated to synthesize DNA by 141 IgM but not by 104E IgM. These results suggest that specific cellular immune responses to idiotypic determinants may participate in the observed protection against challenge with the corresponding B-cell tumor.

NIH 3T3 cells transformed in vitro with the c-H-ras oncogene were subcloned. The resulting subclones were assayed for in vivo tumorigenicity in nude and in immunocompetent mice. The response of two high tumorigenic and two low tumorigenic clones to mediators of natural immunity was analyzed. The clones did not differ in sensitivity to NK cell-mediated lysis. However, compared to low tumorigenic clones, the high tumorigenic ones had a down-regulated expression of a membrane determinant recognized by a certain monoclonal naturally occurring antibody. The determinants recognized by other monoclonal naturally occurring antibodies available in the laboratory were equally expressed on the high and low tumorigenic clones. The high tumorigenic cells showed an increased resistance to cytotoxicity mediated by lymphotoxin. These results suggest that naturally occurring antibodies and lymphotoxin may participate in controlling the tumorigenicity of transformed cells. The high tumorigenic clones but not the low tumorigenic ones contained a novel 3.5-kb ras mRNA.

In this study we demonstrated that cells lodging in tumors have the capacity to fix antibody or immune complexes in vivo and in vitro. Although some of the fixation is probably by host, at least in one system studied, tumor cells, per se, were found to exhibit immune complex fixation.

In this study we investigated the relationship between binding of anti-tumor antibodies by polyoma-virus-induced SEYF- a tumor cells and the expression of immune-complex receptors on these cells. It was shown that in vivo propagated cells became progressively coated with IgG. The increase in the IgG coating of SEYF-a cells, occurring during the second week of propagation, was directly correlated with an increase in the coating of cells with potentially cytotoxic anti-tumor antibodies. The activity of these antibodies was demonstrated by cell lysis following addition of exogenous complement. Cell populations propagated in vivo for longer periods (3 weeks or more) became less sensitive to exogenous complement although their IgG coating remained high, and although higher titers of anti-tumor antibodies could be eluted from them. This indicated that antibodies coating young tumor cell populations have the capacity to activate complement whereas those coating older tumor cell populations are incapable of complement activation. Previous findings that SEYF-a cell populations are able to bind unrelated immune complexes were confirmed in the present study. We also found that the capacity of these populations to bind such unrelated complexes decreased with propagation time in vivo. The involvement of anti-tumor antibodies in this phenomenon was indicated by the finding that such antibodies inhibited binding of unrelated immune complexes by young cells but not by old cells. Furthermore, treatments causing dissociation of Ig from the cell surface restored, to some extent, the capacity of anti-tumor antibodies to inhibit immune complex binding by old SEYF-a populations.

In this paper we present evidence that antibodies unrelated to the tumor cell can comprise part of the in vivo Ig surface coat of cells derived from the non-lymphoid murine tumor, TA3/St. This was shown by incubating TA3 cells originating from other OA or BSA preimmunized mice with radioiodinated 125I-OA and 131I BSA. Radioiodine-labeled 125I-OA specifically fixed to cells derived from TA3/St tumors originating from OA-preimmunized mice. On the other hand 131I-BSA was specifically fixed by cell populations from BSA-preimmunized mice. Incubation of these cells in vitro at 37 degrees C abolished the specific binding and antibody could subsequently be detected in the tissue culture medium. Radioiodine labeled purified soluble antibody-antigen complexes could also be bound to cells derived from freshly harvested TA3/St tumors but not to their in vitro propagated counterparts. Removal of phagocytic or adherent cells from these cell populations decreased the binding of the complexed antibody on freshly harvested TA3/St populations, but did not eliminate it. Inhibition of complexed antibody binding was obtained when TA3/St cells (an H-2a tumor) were pre-incubated with anti-H2a antiserum. Propagation of the tumor in an F1 hybrid (A X C57BL) in which host cells could be distinguished from tumor cells by using an anti H-2b antiserum showed that binding of the immune complex was mostly limited to host cells infiltrating into the tumor population.

The IgG fraction from rabbit antisera against a mouse plasmacytoma (RaPCT-IgG) was incubated, under acidic conditions, with lysosomal extracts (LE) derived from the corresponding tumors. The LE-treated IgG demonstrated a significant decrease in its complement-dependent cytotoxic potential when compared to RaPCT-IgG incubated without LE under the same conditions (control IgG). Ammonium sulfate (AS) precipitation of the control IgG and the LE-treated IgG preparations at 40% saturation of AS revealed that some of the IgG lost its precipitability at this AS concentration after LE treatment, but could be precipitated at 60% or 70% saturation. The LE-treated IgG and control IgG fractions that precipitated by 40% saturated AS showed similar gel filtration profiles. The LE-treated IgG fraction precipitating at 70% saturation of AS (but not at 40%) showed a different gel filtration profile. This fraction was separated into several subfractions, some with an apparent lower molecular weight. Some of these subfractions which still contained antigenic IgG lacked any expression of antigenic Fc, and were not cytotoxic to tumor cells. They could, however, rebind to tumor cells and block the complement-dependent cytotoxic activity of native RaPCT-IgG. Lysosomal enzymes of tumor origin seem, therefore, to convert cytotoxic anti-tumor antibodies into molecules devoid of Fc but still retaining their antigen binding activity. Such an activity, if occurring in vivo might enhance tumor growth.