Effect of Propionibacterium acnes on the cellular immune responses to tumor-specific antigens on malignant rat liver cells.

The effect of Propionibacterium acnes on the cellular immune responses to tumor-specific membrane antigens was investigated by microcytotoxicity assays (MA) and 51Cr release assays (CRA) with use of mesenteric lymph node cells (LNC) of syngeneic BD IV and BD VI rats. BD rat liver cell lines transformed in vitro by chemical carcinogens were used as target cells with tumor-specific antigens. By MA, the LNC from rats that were inoculated with malignant liver cells under the adjuvant effect of heat-killed P. acnes showed significant cytotoxic response to the target cells but not to nonmalignant liver cells. By CRA, these LNC did not show specific cytolysis to the malignant liver cells. Assays with various target cells derived from BD rat liver and inhibition tests with syngeneic and xenogeneic antisera against tumor-specific antigens on the malignant liver cells proved that LNC reacted with tumor-specific individual or tumor-specific cross-reacting antigens on the malignant liver cells. Cytotoxic responses against the malignant liver cells were not demonstrated even by MA with use of the LNC from rats inoculated with either the malignant liver cells or P. acnes alone. LNC from the rats inoculated with both nonmalignant liver cells and P. acnes were not cytotoxic to malignant or nonmalignant liver cell lines.     

Tumor-specific antigens on rat liver cells transformed in vitro by chemical carcinogens.

With the use of membrane immunofluorescence and xenogeneic antisera, tumor-specific membrane antigens were detected on rat epithelial-like liver cells transformed in vitro by chemical carcinogens. These antigens were not detected in 10-, 15-, and 19-day rat fetuses. Xenogeneic antisera were produced in rabbits by immunization of the rabbits with cultivated BD rat liver cells transformed by dimethylnitrosamine or N-methyl-N'-nitro-N-nitrosoguanidine. The specific antisera against tumor-associated antigen(s) were obtained by in vivo absorption in syngeneic male rats and by in vitro absorption with various cell lines. One tumor-specific individual antigen and two tumor-specific cross-reacting antigens were shown to be present on the surface of chemically and/or spontaneously transformed rat liver cell lines. They were not detected on liver and spleen cells of normal BD adult rats, on fetal liver cells, or on liver and intestinal carcinoma cells of Wistar rats. Sera from multiparous pregnant rats had no antibodies against these tumor antigens (although they reacted with fetal cells).     

Membrane immunofluorescence studies on cells producing rat C-type virus particles

The WF-l rat cell line was investigated for the presence of virus-associated cell membrane antigens similar to those found in the membranes of cells infected with murine leukemia viruses. These cells transform spontaneously in tissue culture from embryos derived from the Wistar-Furth ( W/Fu) strain of rats and produce large amounts of rat C-type virus particles. When sera from W/Fu rats bearing WF-l tumors were tested for antibodies directed against the WF-l cells by indirect membrane fluorescence, it was found that 20–35% of the cells reacted strongly. The sera did not react with normal W/Fu embryonic fibroblasts nor did absorption of a pooled positive serum with these cells remove antibodies reactive with the WF-l cell line. Interestingly, however, these sera also reacted with two other virus-positive cell lines (R-35 and RMTL-8) derived from mammary tumors originating in Sprague-Dawley rats but not with virus-negative cell lines. Absorption experiments verified that the R-35 and RMTL-8 cells contained cross-reacting antigens with the WF-l cells. These results suggested that (a) virus-associated antigens were expressed in the membranes of cells infected with rat C-type particles and (b) C-type virus particles found in cell lines originating from different Sprague-Dawley rats as well as from the Wistar-Furth strain were antigenically closely related if not identical.     

Tumour antigens and alloantigens. I. Relation of a rat tumour-specific antigen with normal alloantigens of the host strain.

It has been shown that tumour-specific antigen from a chemically-induced rat hepatoma is capable of binding to immunoadsorbent columns made from the Ig fractions of antisera raised in allogeneic animals against a variety of normal tissues. This reactivity is observed with antisera directed against normal syngeneic liver, spleen and lymph-node cells but it is not detected in normal sera, F, hybrid sera or an antiserum directed against syngeneic erythrocytes. This phenomenon is specific since soluble extracts of normal syngeneic liver, but not of normal altogeneic liver, are capable of inhibiting the binding of hepatoma-specific antigen to an immunoadsorbent column of Ig from an allogeneic anti-normal-liver antiserum. Syngeneic and allogeneic rats were immunized with immune complexes made from xenogeneic sera, alloantisera or syngeneic immune serum and soluble extracts of normal tissue and affinity-chromatography-purified hepatoma-specific antigen. The resulting sera were examined by means of the membrane immunofluorescence test for reactivity against a panel of syngeneic and allogeneic cells. Xeno-antisera directed against normal syngeneic tissue precipitated with purified hepatoma antigen and this material generated tumour-specific antibody following immunization into syngeneic rats. The phenomenon of cross-reactivity of one chemically-induced rat hepatoma-specific antigen with normal syngeneic alloantigens is not a unique phenomenon as preliminary results indicate that two other immunologically distinct hepatomas show similar characteristics.     

Characterization of the surface proteins of SV40-transformed mouse and human cells: absence of SV40-specific proteins

The proteins of a number of SV40- and spontaneously transformed mouse and human cell lines were compared in an effort to identify a surface protein which would correspond to the SV40 tumor-specific transplantation antigen (TSTA). Analysis of the one- and two-dimensional electrophoretic patterns of 35S-methionine-labelled total proteins and 125I-labelled surface proteins of several of these cell lines failed to reveal the presence of proteins specific to transformation by SV40. Antisera were prepared against SV40- and spontaneously transformed mouse cells in syngeneic mice. In serological assays, these antisera reacted with surface antigens common to both SV40- and spontaneously transformed mouse cell lines. Electrophoretic analysis of the 125I-surface-labelled proteins which these antisera immunoprecipitated from extracts of SV40- and spontaneously transformed mouse and human cells identified a set of common surface proteins with apparent molecular weights of 15, 46, 50, 72, 77, 105, 150 and 230kdal. No SV40-specific surface proteins were detected. Two of the transformed cell surface proteins (105 and 150kdal) were present as well in membrane fractions of 35S-methionine-labelled primary mouse kidney cultures. The proteins of the primary cultures could not be iodinated by lactoperoxidase suggesting that these proteins were present at a "cryptic" location at the surface of normal cells. We were not able to obtain serological or immunochemical evidence for the presence of SV40 large T-antigen at the surface of any of the SV40-transformed cell lines tested using either hamster anti-SV40 tumor sera, a rabbit antiserum against SDS-denatured gel-purified large T-antigen or antisera against SV40-transformed mouse cells. In conjunction with the report that large T-antigen released from disrupted SV40-transformed cells will bind to cell surfaces (Lange-Mutschler and Henning, 1982), we consider the possibility that the specific rejection of SV40-induced tumors by sensitized animals is the result of immunological reactions against both common transformation-related surface antigens and SV40 T-antigen from disrupted cells that has bound to the surface of other tumor cells.     

Studies on murine sarcoma virus: II. Detection of group-specific antigens by immunofluorescence

The group-specific (GS) antigen of murine tumor viruses was demonstrated by immunofluorescence in mouse cells recently infected by mouse sarcoma virus, strain Moloney (MSV-M), with the serum of rats carrying long-transplanted MSV-M tumors. GS antigen was detected 15 h post-infection and was also present in various mouse and rat cell lines chronically infected with murine tumor viruses. The antigen was strictly localized in the cytoplasm of infected cells and was also found in mouse and rat cells chronically infected by members of the two major subgroups of murine tumor viruses. Further, the sera employed were shown to contain exclusively GS antibodies and the tumors used for immunization were found by several techniques to be free of virus envelope (V) antigens after a given number of passages in vivo. V antigens were visible only at the cell membrane and the time course of appearance of both GS and V antigens in recently infected cells was parallel. In contrast, GS antigen was not observed in two hamster tumor lines transformed by MSV-M.     

Detection and isolation of tumour-specific antigen associated with a spontaneously arising rat mammary carcinoma

Cell surface expressed tumour-specific antigen was detected in one (Sp4) of eight spontaneously arising rat mammary carcinomas by membrane immnofluorescence tests with viable tumour cells in suspensioni and sera from syngeneic rats immunized against each tumour. These findings are consistent with the frequency of expression of significant tumour antigenicity as defined by transplantation methods. Also the tumour antigen detected by membrane immunofluorescence tests in mammary carcinoma Sp4 showed the individual characteristics as the tumour-specific transplantation antigens, and no evidence was obtained of weak common antigens in these tumours. Tumour-specific antigen in subcellular fractions of mammary carcinomas Sp4 was assayed by its capacity to absorb antibody from tumour-immune sera. By this criterion, it was established that plasma membrane fractionis retained tumour-specific antigen although recoveries were low, amounting to less than 5% of cell-surface experssed antigen. In comparison, almost total recovery was obtained of alloontigen experessed on mammary carcinoma Sp4 cells. Plasma membrane fractioins from mommary carcinoma Sp4 lacked the capacity to induce tumour rejectino reactions in syngeneic rats although a weak humoral antibody response was obtained. These findings contrast with the more consistent immune response elicted by heavily irradiated mammary carcinoa Sp4 cells.     

Tumour antigens and alloantigens. II. Lack of association of rat hepatoma-D23-specific antigen with beta 2 microglobulin.

It has previously been shown that the tumour-specific antigen of the chemically-induced rat hepatoma D23 has determinants recognised by alloantisera raised against normal syngeneic liver and spleen. However, no reactions were observed with alloantisera raised against syngeneic erythrocytes suggesting that the alloantigeneic determinant responsible for this cross-reactivity is not a major serologically-defined histocompatibility antigen. This concept has been further examined using a defined turkey anti-rat beta 2 microglobulin antiserum. This antiserum failed to block the binding of syngeneic antihepatoma D23 sera to hepatoma D23 target cells as assessed using membrane immunofluorescence and complement-dependant 51Cr release tests. Furthermore, immune precipitates formed from soluble tumour extracts containing hepatoma D23 specific antigen with turkey anti-rat beta 2 microglobulin failed to generate a tumour-specific antibody response in syngeneic rats althouth a cross-reactive antiserum was produced following immunization of allogeneic rats with the immune precipitate.     

New antigens in cells transformed by the SV40 virus. II. Evidence of their cytoplasmic localization in a cell line (TSV5 CL2) of hamster fibroblasts transformed by SV40

A clone of hamster fibroblasts transformed by SV40 virus (TSV₅ CL₂) induced antibodies in hamsters against new antigenic constituents not present in the untransformed fibroblasts. The antibodies in the immune sera against histocompatibility antigens and those against nuclear T antigen were absorbed. The absorbed sera contained antibodies which agglutinated the transformed cells. The sera contained also precipitating antibodies against new antigens present in a crude extract of TSV₅ CL₂. These antibodies were directed to common new antigens present in SV40-transformed fibroblasts from different species. Immunofluorescence and the “indirect enzyme-labelled antibody technique” were used for the cellular localization of the antigens corresponding to the precipitating antibodies. Both techniques showed that these antigens were localized in the cytoplasm, mostly in the microsome-ribosomal fractions, much less on the cell membrane and not at all in the nucleus.     

Tk, a new colon tumor-associated antigen resulting from altered O-glycosylation

Erythrocyte polyagglutination antigens T and Tn are truncated O-glycan chains that are also carcinoma-associated antigens. We investigated whether Tk polyagglutination antigen could similarly be a carcinoma-associated marker and a target of immunotherapy. Monoclonal antibody LM389 was raised against Tk erythrocytes and tested by immunohistochemistry. LM389 strongly reacted with 48% human colorectal carcinomas. Labeling of normal tissues was visible on epithelial cells, mainly digestive, but was confined at a supranuclear level. Expression of the antigen on cloned human carcinoma cells correlated with sialosyl-Tn expression. O-Sialoglycoprotein endopeptidase treatment revealed that on carcinomas and cell lines, the epitope was present on O-glycans. Antibody specificity was determined using synthetic carbohydrates. Direct binding and inhibition studies indicated that LM389 best ligands were terminated by two branched N-acetylglucosamine units. Screening of murine cellular cell lines with LM389 allowed development of an experimental model with Tk-positive and -negative cells in syngeneic BDIX rats. Vaccination of rats with Tk erythrocytes provided a protection against growth of rat Tk-positive, but not of Tk-negative, tumor cells in association with the development of antibodies. Taken together, the results indicate that Tk polyagglutination antigen is a new colorectal carcinoma-associated antigen, absent from the normal cell surface, resulting from alteration of O-glycans biosynthesis and with potential as a target of immunotherapy.     

Cellular and humoral immunity against human malignant melanoma

The humoral and cellular immunological responses of melanoma patients against tumor-associated antigens were studied by means of the indirect membrane immunofluorescence test and a microassay for cell-mediated cytotoxicity. Melanoma target cells obtained from 22 different surgical specimens were used. A total of 15 different sera were tested in 27 fluorescence assays. Humoral antibodies were found in all the 13 autochthonous sera tested: eight out of ten sera cross-reacted with one or two allogeneic melanoma cells. The 31 microassays performed with lymphocytes from 16 patients showed that effector cells specifically killed the melanoma target cells in seven out of 12 autochthonous tests and in 14 out of 19 allogeneic tests. No cytotoxicity was found when melanoma-patient lymphocytes were tested against control cells or when control lymphocytes were tested against melanoma cells. The observed cross-reactivity pattern indicates that more than one tumor-specific antigen may be present in melanoma cells.     

Tumor-specific T cell lines: capacity to proliferate and produce interleukin 2 in response to various forms of tumor antigens.

Anti-tumor proliferative T cell lines were established from cultures of lymph node cells from BALB/c mice immunized to syngeneic CSA1M fibrosarcoma with the CSA1M tumor cell membrane. The cultures were maintained throughout in the absence of exogenous interleukin 2 (IL2). Cell surface phenotypes of all T cell lines established were Thy-1+, Ig-, L3T4+ and Lyt-2-. Their proliferation was induced in a tumor antigen dose-dependent fashion and a tumor antigen-specific way. Such proliferative responses were inhibited by the addition to cultures of anti-class II H-2d (anti-I-Ad) or anti-L3T4 but not of anti-class I H-2d or anti-Lyt-2 monoclonal antibody. None of the T cell lines exhibited any cytotoxic T lymphocyte activity but they all produced IL2 upon stimulation with CSA1M tumor antigens, indicating that they represent helper-type T cell (Th) lines. The activation of these tumor-specific Th lines was induced with either CSA1M tumor cells themselves, or their membrane or detergent-solubilized fraction depending on the presence of antigen-presenting cells (APC). Most importantly, activation was also inducible by membranous tumor antigen-pulsed APC, which were capable of producing potent anti-tumor protective immunity when administered in vivo into syngeneic BALB/c mice. These results indicate that the tumor-specific Th lines established here can be activated with various forms of tumor antigens for their expression of helper function. Since Th lines of this type have not been described previously, our Th lines provide an intriguing tool for investigating the cellular and molecular mechanisms by which tumor-specific Th recognize tumor antigens.     

Tumour-specific complement-dependent serum cytotoxicity against a chemically induced rat hepatoma.

A short-term 51Cr release test was used for the detection of complement-dependent cytolytic activity of syngeneic serum for transplanted aminoazo dye-induced rat hepatoma cells in suspension. Serum samples from rats bearing intraperitoneal implants of one hepatoma (hepatoma D23) were specifically cytotoxic for hepatoma D23 target cells, although this activity was not detected in sera from donors bearing subcutaneous tumour grafts. Other sera containing demonstrable IgG antibodies reactive in membrane immunofluorescence tests with individually distinct tumour-specific antigens or tumour-associated embryonic antigens were not cytotoxic for hepatoma D23 cells; and even though serum from donors carrying intraperitoneal tumour grafts contained tumour-specific IgG antibody, the complement-dependent reactivity was confined to the 19s region of fractionated tumour-bearer serum. These findings are discussed in relation to the development of humoral responses in the tumour-bearing host and with regard to the significance of the availability of an objective and reproducible assay for measuring humoral responses directed against the tumour-specific antigens associated with chemically induced rat tumours.     

Tumour-related antigen specificities associated with 3-methylcholanthrene-treated rat embryo cells.

Rat embryo cells were treated in vitro for 18 h with 10 micrograms/ml 3-methylcholanthrene (MCA). Syngenetic male rats were immunized with several inocula of treated cells to prepare antisera which were screened for membrane immunofluorescence reactivity against panels of established chemically-induced syngeneic rat tumours. Three separate antiserum pools raised against MCA-treated cells reacted with certain chemically-induced tumours, whereas antisera to control (DMSO-treated) cells were completely negative. The reactions observed were reproducible and highly specific for particular target tumours. Absorption studies indicated that each antiserum contained antibodies to several different antigens, present on different tumours. Antiserum prepared against extranuclear membrane from MCA-treated cells, rather than intact MCA-treated cells, was negative. This suggests that the antibody responses were directed against antigens arising subsequently to MCA treatment and injection into syngeneic hosts. It is postulated that carcinomgen treatment results in the acquisition of multiple neoantigens among a treated cell population, which represent an early change in a sequence of events leading to malignant transformation.     

Studies on a gross-virus-induced lymphoma in the rat. II. The role of cell-membrane-associated and serum P30 antigen in the antibody and cell-mediated response.

The internal viral protein, p30-1, has been identified by cytotoxicity tests using xenogeneic sera, on MuLV-G-induced rat lymphoma cells. In this system internal virion antigens, and in particular p30, are cytotoxic target antigens for syngeneic antibody and cell-mediated cytotoxicity; p30 is also present in the serum of rats with progressively growing lymphomas, and is the principal antigen responsible for the inhibition of cell-mediated cytotoxicity produced by these sera.     

A carcinofetal antigen located on the membrane of cells from rat intestinal carcinoma in culture.

Heteroantisera were produced in rabbits by immunization with cultivated cells from rat intestinal carcinomas. The sera were made specific by in vivo absorption in syngeneic rats. On immunofluorescence, these sera recognized a membrane-associated antigen common to five different intestinal carcinomas and to fetal intestine. The antigen was not detected in noncancerous adult intestine, nonintestinal fetal tissues, or three nonintestinal tumor cell lines.     

Expression by human neuroblastoma cells of an antigen recognized by naturally occurring mouse anti-brain autoantibody.

A number of human tumor cell lines of both neural and nonneural origin have been assayed for the expression of an interspecies brain antigen (mouse brain antigen 2), detected by naturally occurring antibodies in normal mouse sera. These experiments indicate that mouse brain antigen 2 is present on four human neuroblastoma cell lines but not on other human tumor cell lines tested, including four glial cell lines and a retinoblastoma. These findings demonstrate the value of naturally occurring antibodies in normal mouse sera for the detection and serological classification of human tumor antigens and indicate that considerable caution should be used in attempts to distinguish tumor-specific and tissue-specific antigen expression on human neuroblastoma cells.     

Detection of tumor antigens by syngeneic antiserum on in vitro-transformed tracheal epithelial cell line 2-10-1

Sera obtained from rats showing transplantation immunity to syngeneic, malignant epithelial cell line, 2-10-1, were used to identify antigens associated with transformation in vitro. The 2-10-1 cell line was derived from exposure of tracheal explants, in vitro, to the carcinogen MNNG. Tumorigenic passages of the cell line were shown to have common antigenic determinants, shared by independently transformed malignant tracheal epithelial cell lines, as well as antigenic determinants that appear to be limited to the immunizing cell line, 2-10-1. Care was taken to ensure that antibodies were not produced against viral antigens or non-specifically absorbed serum components. Early passages of the 2-10-1 cell line were not tumorigenic in athymic BALB/c (nu/nu) mice, but became malignant with serial passage in vitro. Antigenic specificities recognized by 2-10-1 immune sera were also present on early-passage 2-10-1 cells that had not as yet acquired the malignant phenotype. Such antigen expression must be an early event in neoplastic development.     

Tumour-associated transplantation antigen in sera of rats with large RSV-induced sarcomas.

A factor inhibiting tumour growth in syngeneic hosts was found in the sera of inbred Lewis rats carrying Rous sarcoma virus-induced tumour (RSL). The findings presented here suggest that the serum factor is a tumour-associated transplantation antigen (TATA) shed from the neoplasm into the circulation. All the tumour bearers' sera tested with RSL cells were negative in indirect membrane immunofluorescence;however, on passive transfer into syngeneic rats, they protected the animals against the growth of an RSL tumour inoculum. A similar protective effect was also observed after injection of TATA prepared from RSL cell membranes by solubilization with potassium cholate. When incorporated into Freund's adjuvant, tumour-bearers' sera immunized the animals against a subsequent RSL sarcoma graft. Sera collected from immunosuppressed rats bearing large sarcomas which presumably contain neither tumour-specific antibody nor antigen-antibody complexes, transferred inhibition of tumour growth to syngeneic hosts. Intact immunological reactivity of recipients was a necessary prerequisite for the protective effect of sera, since the passive transfer of an inhibitory serum to immunosuppressed rats did not inhibit tumour growth. We assume that the TATA present in tumour-bearers' serum is released from the growing neoplasm as a result of either cell death or membrane metabolic turnover.     

Specificity of serum from rodents immune to Moloney C-type virus-induced tumours.

When cells are infected by C-type viruses such as Moloney sarcoma/leukaemia virus, new antigens appear on the cell membrane. Mice and rats will respond immunologically to the antigen(s). It was uncertain whether the antigens were related to the viral structural proteins or to non-virion, tumour-specific surface antigens (TSSA) or both. In an 125I-antiglobulin binding assay, Moloney virus completely blocked the binding of mouse and rat sera to virus shedding target cells, thus suggesting that mice and rats recognise only viral proteins. Mice responded to type-specific and rats mainly to group-specific determinants on the virus. Individual Moloney viral proteins were prepared using the guanidine HCl method and were used to block the binding of the rat anti-Moloney immune serum to Moloney virus shedding target cells. By this method, it was demonstrated that the rat serum contains specificities for the viral proteins gp70 and p30, but it was not possible to detect any antibodies directed towards non-virion or TSSA-like molecules.