Fetal antigens as tumor growth enhancing factors in Yoshida's tumor rats

Malignant neoplastic cells have been shown to have some antigenic features identical to those of embryonic cells. Since several antigens are likely to be shared by both embryonic cells and neoplastic tissue, we tried to understand the meaning of the appearance of such antigens and the type of effect that the immunization with embryonic antigens would have on the survival of Yoshida's tumor rats. Wistar rats were immunized with fetal antigens by fetal cells (1.5 x 10(6)) suspended in 0.5 ml of Hanks solution plus an equal volume of Freund adjuvant, were injected in hind footpads, i.p. and i.m., respectively, for active immunization. Rabbit antigen sera were used for passive immunization. All animals presented ascites and tumor growth. Animals immunized by means of fetal cell antigens showed a mean survival rate after neoplastic transplant of 14 days. Animals that received rabbit immune serum showed a mean survival rate after neoplastic transplant of 17 days. The immunization by means of fetal antigens elicited a scanty effect on the survival of Yoshida's tumor transplanted rats. It can be concluded that antibodies, which are able to cross react with neoplastic cells, do not have cytotoxic effect and do not interfere with the survival of the neoplastic transplanted animals. Therefore, fetal antigens are likely able to carry out an immunosuppressive action. The fact that they appear on neoplastic cells could be seen as a metabolic modification effect or as a growth enhancing factor.     

Further studies on the structural requirements of agents for immunotherapy of the guinea pig line-10 tumor

Summary We made a comparative study of the in vivo binding of immunoglobulins (Ig) to a polyoma virus-induced ascitic tumor propagated in syngeneic or allogeneic mice. The Ig coat was found to appear more rapidly and to be denser in H 2-incompatible than in H 2-compatible mice. This suggests that antibodies were fixed specifically on strong normal transplantation antigens (H-2) recognized as non-self by allogeneic mice. Experiments with mice in which immunosuppression had been achieved by means of X-irradiation confirmed that the Ig fixed on SEWA cells are actively bound antibodies. The only mice that could fix Ig on tumor cells were those that had been specifically immunized against cell surface antigens shared by SEWA cells before irradiation, while mice hyperimmunized against nonrelated antigens could not.In partial fulfilment of doctorate thesis requirements     

Influence of different routes of anti-tumor immunization: alternative induction of tumor immunity and tumor enhancement.

Chickens and quails were immunized in parallel either i.v. or intramuscularly (i.m.) with lectin column-purified antigens from chick embryo cells that were transformed in vitro by avain sarcoma virus (ASV). After five to six injections, immunity of the animals was tested by challenge with ASV into the wing webs. Whereas tumor growth was inhibited after i.v. immunization with respect to incidence rate and time of tumor appearance, tumor growth was enhanced after i.m. injection. Animals that were injected with normal cell antigens served as controls. Spleen cells from only those animals that were immunized i.v. exerted a cytotoxic effect in vitro against ASV-transformed cells, whereas spleen cells from i.m. injected animals, in contrast, suppressed such cytotoxicity. The search for serum blocking or arming factors suggested that sera from i.m. injected animals block cellular cytotoxicity whereas sera from i.v. immunized animals render normal spleen cells cytotoxic (arming effect). The use of viruses from different subgroups and of antigens from gp85-lacking ASV-transformed cells indicates that immune effects were obtained against tumor cell surface antigens that differ from the antigen that is involved in virus neutralization (s-gp85).     

Antigen-mediated macrophage adherence inhibition

Antigen-mediated macrophage adherence inhibition (MAI) was studied in inbred rats immunized with various transplantation, tumour-specific and protein antigens. A macrophage-rich suspension of peritoneal cells (PC) was obtained from the peritoneal cavity of immunized and control animals by washing. The adherence to glass of PC was specifically inhibited by the addition of the antigens used for sensitization of PC donors or by related (cross-reacting) antigens but not with unrelated antigens. The MAI seems to be due to the direct interaction of the respective antigen with a corresponding PC receptor and not due to the humoral factor released from immune lymphocytes of PC population upon contact with the specific antigen.     

Cytolytic antibodies to murine lymphomas elicited by immunization with allogenic and syngenic tumors

C57BL/6 mice immunized with allogenic L#2 tumor cells or syngenic EL-4 tumor cells produced lytic antibodies directed against cell-surface antigens present on EL-4 tumor cells but absent from normal C57BL/6 tissues. Such immunized C57BL mice also exhibited prolonged survival after challenge with syngenic EL-4 tumor cells.     

Liposome encapsulated tumor-associated antigens elicited humoral and cellular immune responses in mice bearing tumor

Chemically induced tumors in mice provide a system to investigate tumor-associated antigens (TAA). The cell surface glycoprotein antigens on such tumor cells have been identified as suitable targets for immune attack. The induction of immune responses against (TAA) in N-nitrosodiethylamine (DEN) exposed mice has been examined. In order to present antigens to the immune system, the liposome was used as vehicle to deliver the TAA. Liposomal-TAA formulation, elicited both humoral and the cellular immune responses, when administered intramuscularly in DEN-exposed mice. Presence of circulatory antibodies against TAA and the induction of cellular responses in immunized mice were monitored using ELISA and in vitro cell proliferation assay of lymphocytes respectively. Specificity of antibody against TAA in immune sera was analysed using immunoblotting technique. Based on these results, it is proposed that the liposome encapsulated TAA may successfully be used to induce humoral and cellular immune responses against tumor.     

Isolation and degranulation of mucosal mast cells from the small intestine of parasitized sheep.

The isolation of mucosal mast cells and globule leucocytes from the small intestine of sheep immunized with Trichostrongylus colubriformis is described. Sheep mast cell protease was released from these cells in a dose-dependent fashion after incubation with soluble protein from T. colubriformis larvae. Release also occurred with other T. colubriformis antigens whereas non-parasite antigens at comparable protein concentrations evinced only a minimal response. Mucosal mast cells prepared from worm-free sheep also produced a similar minimal response. This is the first report describing the release of sheep mast cell protease from isolated sheep intestinal mucosal mast cells after addition of specific parasite antigens.     

Potentiation of the in vitro cytotoxic response to syngeneic lymphoma cells by soluble products of tuberculin-sensitive lymphoid cells stimulated with PPD

Spleen cells from rats immunized with the syngeneic (C58NT)D Gross virus induced lymphoma have previously been shown to differentiate into cytotoxic effector cells following restimulation with tumor cells in vitro. Previous work has also demonstrated that the addition of PPD-primed syngeneic spleen cells and PPD to cultures of (C58NT)D-primed spleen cells will potentiate the in vitro cytotoxic response to tumor antigens. In the studies presented here, the potentiating effect was found to be mediated by a soluble factor(s) released by nonadherent cells from BCG-primed rats. The release of this immunopotentiating factor(IPF) required the presence of PPD and varied with the concentration of PPD added. IPF was produced by BCG-primed spleen, lymph node, and thymus cells. Maximal production of IPF in PPD-stimulated cultures was obtained after 6–12 hr of incubation. Supernatants obtained after 30 hr of incubation lacked apparent IPF activity when tested initially, but activity was recovered after mild heat treatment. Recovery of IPF activity after heat exposure is best explained by the presence of a heat-labile inhibitor. IPF itself is stable to heat treatment to 56 °C for 40 min. IPF was also shown to be capable of enhancing immune responses to histocompatibility antigens in vitro.     

Stimulation of lymphoid cells from normal and immune mice by syngeneic BALB/c plasma cell tumors.

Lymphoid cells from normal and immunized BALB/c mice could be stimulated in vitro by syngeneic PCT contrasted with an absence of response to a number of other tumors. Maximal responses of normal cells to PCT were found to occur 5 days after the initiation of the cultures at an optimal responding:stimulation cell ratio of 1:2. MLTI activity of normal cells could not be blocked or enhanced by PCT myeloma protein products indicating that MLTI reactivity was directed against non-idiotypec cell surface determinants. Lymphoid cells from immunized mice demonstrated increased MLTI responses to cells of the immunizing tumor but not to other PCT, indicating that the post-immunization MLTI responses were primarily to individual rather than shared tumor cell surface antigens. Activity of both normal and immunized spleen cells was found to involve thymus-derived lymphocytes. The persistence of residual MLTI activity after treatment with anti-theta serum and complement, however, implicated participation of non-theta antigen-bearing cells in MLTI reactivity. From these data, we conclude that lymphoid cells from un-immunized mice are capable of T cell-dependent reactivity to syngeneic PCT-associated antigens and that elevations in these reactivities after immunization may reflect specific cellular immune responses.     

Specific MIF release by rat lymphocytes following incubation with syngeneic anti-tumor “Immune” RNA

Other investigators have previously shown that normal nonimmune lymphoid cells, after incubation with “Immune” RNA, will release MIF when these cells are incubated with the specific antigen used to immunize the RNA donor. This conversion can be detected with the macrophage migration inhibition assay. These observations have been confirmed in a system involving the transfer of immune response to tumor associated antigens with syngeneic “Immune” RNA. Syngeneic “Immune” RNA was extracted from the spleens of Fischer 344/N rats bearing growing transplants of one or another of two syngeneic chemically induced sarcomas. Normal, nonimmune Fischer 344/N spleen cells were incubated with these RNA preparations. When these RNA-incubated spleen cells were exposed to solubilized antigens from that particular tumor used to immunize the RNA donor, MIF was released. RNAse treatment of the “Immune” RNA abrogated the response, while DNAse or pronase treatment did not.     

In vitro hemolysis of autologous erythrocytes caused by immune murine spleen cells and spherules of the fungus Coccidioides immitis.

This communication describes an in vitro system wherein mouse erythrocytes are lysed in the presence of spherules of the fungus Coccidioides immitis and spleen cells from syngeneic mice immunized with a variety of antigens. The antigens include: tobacco mosaic virus in complete Freund's adjuvant (CFA), CFA alone, separate components of CFA, sheep erythrocytes, and allogeneic tumor. Spleen cells from mice sublethally infected with C. immitis are also capable of participating in this response. The lytic phenomenon, which does not require complement, is dependent upon the number of spleen cells per culture, the number of spherules per culture, the time of culture incubation, the amount of antigen injected into the animal and the time after immunization at which spleen cells are recovered. Live spherules or spherules killed with heat, with dimethylsulfoxide, or with formalin were effective participants, together with immune spleen cells, in the lytic reaction.     

Soluble factors produced during an immune response regulate Ia antigen expression by murine adenocarcinoma and fibrosarcoma cells

LT-85 is an alveologenic adenocarcinoma of C3Hf/HeN mice. Comparisons of the in vitro and in vivo surface properties of these cells revealed that under normal conditions, they expressed I-A and I-E antigens iv vivo only. By using clonally derived cells, it was established that this phenomenon was not due to the selection of an Ia antigen-positive tumor cell subpopulation, but resulted from phenotypic conversion of Ia antigen-negative tumor cells. These tumor cells and 1053 cells (a fibrosarcoma of C3H/HeN MTV- mice) could, however, be induced to express I-A, I-E, and much higher levels of H-2 antigens in vitro by co-culturing them with spleen cells from LT-85 tumor-bearing C3H/HeN MTV- mice. In vitro induction of Ia and H-2 antigens did not result from contaminating splenocytes or from antigen transfer, because splenocytes from BALB/c (H-2d) mice immunized with A/J (H-2k/d) cells were able to induce the expression of Iak antigens by both tumor cell lines. It was found that this phenomenon was neither H-2-restricted nor antigen-specific. The results clearly indicated, however, that an immune response was required to generate phenotypic conversion of the tumor cells, both in vivo and in vitro. It was further found that soluble, rather than cellular, factors produced during an immune response induced the expression of Ia antigens by LT-85 and 1053 tumor cells. In contrast to what has been reported about the induction of Ia antigens on macrophages and normal epithelial and endothelial cells, the induction of Ia antigens on LT-85 and 1053 cells did not appear to require T cells, and did not involve gamma-interferon. These findings demonstrate that some tumor cells are capable of altering their MHC antigen phenotype in response to factors produced during an immune response in vivo or in vitro. Because of the involvement of Ia antigens in several aspects of immune phenomena, the ability of tumor cells to differentially express Ia antigens in response to environmental factors may have profound effects on host-tumor interactions. Furthermore, the differences seen in the phenotypes of tumor cells grown in vitro and in vivo suggest that in vitro methodologies of tumor cell characterization may not present a complete picture of the natural state of the tumor cell surface.     

诱导免疫应答的肿瘤抗原

肿瘤抗原可以诱导机体的免疫应答,是肿瘤的免疫治疗中多肽疫苗的分子基础,近十年来发展起来的肿瘤疫苗筛选方法,利用肿瘤抗原特异性T细胞或抗体识别肿瘤抗原,为临床肿瘤免疫治疗提供了大量备选抗原分子。文中总结了肿瘤抗原的种类,及迄今几乎所有被证明的含有T细胞识别表位的抗原分子及其血清学反应性,为临床肿瘤疫苗的选择提供了依据。     

Suppression of the guinea-pig line-10 hepatocarcinoma by a factor(s) released from line-10 cells

Summary A factor or factors released from line-10 hepatocarcinoma cells during mixing in a fluid medium suppressed tumor growth when injected intradermally (ID) with viable line-10 cells to strain-2 guinea-pigs. No suppressive factor was released from other syngeneic normal adult, fetal, or tumor cells. The mechanism behind the tumor suppression was not determined, although antigens derived from line-10 cells were demonstrated in media containing the suppressive factor(s).     

Co-expression of immunogenic determinants by the same cellular immunogen is required for the optimum immunotherapeutic benefit in mice with melanoma

 Tumor-associated T cell epitopes are recognized by T cells in the context of determinants specified by class I loci. Since the rejection of foreign histocompatibility antigens is known to enhance tumor immunity, immunization with a cellular vaccine that combined the expression of both syngeneic and allogeneic class I determinants could have important immunological advantages over a vaccine that expressed either syngeneic or allogeneic determinants alone. To investigate this question in a mouse melanoma model system, we tested the immunotherapeutic properties of B16 melanoma × LM fibroblast hybrid cells in C57BL/6J mice with melanoma. Like C57BL/6J mice, B16 cells expressed H-2K^b class I determinants and (antibody-defined) melanoma-associated antigens. LM cells, of C3H mouse origin, formed H-2K^k determinants along with B7.1, a co-stimulatory molecule that can activate T cells. The B16 × LM hybrid cells co-expressed H-2K^b and H-2K^k class I determinants, B7.1 and the melanoma-associated antigens. C57BL/6J mice with melanoma, immunized with the semi-allogeneic hybrid cells, developed CD8-mediated melanoma immunity and survived significantly (P<0.005) longer than mice with melanoma immunized with a mixture of the parental cell types. The failure of melanoma immunity to develop in mice injected with the mixture of parental cells indicated that co-expression of the immunogenic determinants by the same cellular immunogen was necessary for an optimum immunotherapeutic effect. Augmented immunity to melanoma in mice immunized with the semi-allogeneic hybrid cells points toward an analogous form of therapy for patients with melanoma. Received: 19 May 1997 / Accepted: 23 July 1997     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

Identification of a colon-specific antigen (CSA) in normal and neoplastic tissues.

An antigen has been isolated from a human signet-ring cell carcinoma serially growing in hamsters, GW-39, by saline, PCA, or phenol extraction, and has been found immunologically identical to a similarly extracted substance in normal human or hamster colon. No other hamster or human tissues or cells were found to contain this antigen, for which reason we have termed it colon-specific antigen, or CSA. CSA has been found to be distinct from the major blood group-specific antigens and from othercolon tumor-associated antigens, such as CEA, CCA-II, and CCA-III. It thus seems that a colon organ-specific antigen can be synthesized by this particular human tumor system. Hamsters immunized with CSA could reject cheek pouch grafts of GW-39 tumors, and tumor rejection by these animals correlated with their anti-CSA antibody titers. Preliminary characterization of CSA suggested that it is a glycoprotein on the cell surface having a molecular size of 30,000 to 50,000 daltons. It is proposed that CSA may play a role in the diagnosis of mucin-producing adenocarcinoma of the colon and in ulcerative colitis.     

Stimulation of mouse migration inhibitory factor (MIF) production form MSV-immune lymphocytes by soluble tumor-associated antigen: requirement for histocompatible macrophages.

Spleen cells from C57BL/6 mice immunized with murine sarcoma virus (MSV) are capable of producing migration inhibition factor (MIF) in response to stimulation with a specific tumor-associated antigen prepared by solubilization with 3 M KCL. We have previously demonstrated that this response is T cell-dependent. Further investigations into the effector cells involved in the production of MIF have revealed that spleen cells from mice immunized with MSV cannot produce MIF when stimulated with tumor extract if the population has been previously depleted of macrophages. However, the response can be restored by adding nonimmune syngeneic macrophages but not by allogeneic macrophages. The inability of allogeneic macrophages to provide this function was not due to their increased suppressor activity since in mixing experiments they did not interfere with the ability of immune spleen cells to produce MIF. Furthermore, they were not defective since they could supply this "cooperative function" to appropriate F1 mice. The results indicate that macrophages are required for stimulation of MIF by soluble tumor antigens and that for efficient interaction the macrophages and lymphocytes must share some genetic similarities.     

Prevention of transplantable tumors by adoptive transfer of spleen cells from immunized rats.

Fischer 344 rats were specifically hyperimmunized with allogeneic, nonvirus-producing [Kirsten murine sarcoma virus (KiMSV)] or syngeneic, virus-producing [KiMSV (Rasheed)] rat tumors. Spleen cells taken from these rats adoptively transferred protection against a 100 to 1,000 X rat tumor dose50 cell challenge with several different transplantable rat tumors. Protection was obtained with spleen cells after removal of adherent cells and macrophages but not peritoneal cells. The spleen cells were not directly cytotoxic but required more than 3 days residence in the recipient before protecting the recipient against challenge. No protection against tumor cell challenge was observed when spleen cells were lethally x-ray irradiated before injection into nontreated rats. Spleen cells taken from rats immunized with normal histocompatibility antigens did not protect in this test system.     

The spontaneous release of a high-molecular-weight aggregate containing immunoglobulin G from the surface of Ehrlich ascites tumor cells

The spontaneous release of tumor cell antigens from the cell surface into the circulation has been proposed as a mechanism whereby tumors may escape the immune response of the host. In this study we have found that Ehrlich ascites tumor cells after removal from the host (mouse) spontaneously release significant amounts of cell surface components during incubation for 1 h in cold isotonic buffer. Immunodiffusion studies revealed that immunoglobulin G (IgG) and a complement component (C3) are included in this spontaneously released material. These surface-bound humoral immune components are apparently released in the form of a high-molecular-weight aggregate (cell coat particle) as shown by ultracentrifugation and ultrafiltration experiments. Precipitation of IgG from the cell coat particle preparation with antibodies directed against mouse IgG followed by detergent gel electrophoresis of the immune precipitate revealed five major bands in addition to the heavy and light chains of IgG. These results suggest that host IgG is tightly bound to several other components at the cell surface, perhaps in the form of immune complexes. IgG is localized on the tumor cell surface in a highly heterogeneous pattern with the appearance of patches and caps in some cells as shown by immuno-fluorescence analysis. The possibility that humoral immune components bind to the tumor cell surface and result in the shedding of high-molecular-weight aggregates of cell surface antigens into extracellular fluids is discussed.