In vitro detection of cell-mediated immunity to individual tumor-specific antigens of chemically induced BALB/c fibrosarcomas

In this study we have analyzed the in vitro cell-mediated cytotoxicity of immune peritoneal exudate cells (PEC), elicited in syngeneic mice against the MCA-induced, TSTA-bearing BALB/c fibrosarcomas CA-2, GI-17 and C-3. The 4 h ⁵¹Cr-release assay showed the immune PEC effectors to be specifically cytotoxic to fibrosarcoma used for the immunization, but not to other syngeneic MCA-induced tumors or normal fibroblasts. Cold target inhibition experiments on CA-2 cells confirmed the specificity of the reaction. When PEC, lymph-node and spleen cells from BALB/c anti-CA-2 mice were compared for anti-tumor activity, only PEC were found to kill tumor cells significantly. PEC effectors did not have a significant level of NK or NC activities since they were unable to destroy YAC-1 target and the PEC-mediated anti-tumor activity was not inhibited by unlabelted YAC-1 or WEHI-164 tumor cells. PEC anti-CA-2 were analyzed for the expression of T-cell markers by anti-Thy 1.2, anti-Ly 1.2 and Ly 2.2 monoclonal antibodies. Anti-tumor specific effector cells were identified as mature T cells since they were not adherent to plastic and showed Thy l.2⁺, Ly 1.2^? and Ly 2.2⁺ phenotypes. In addition, anti-H-2K^d but not anti-H-2D^d alloantiserum added to target cells, blocked CA-2 tumor lysis, thus supporting the conclusion that the T-cell response against TSTA is H-2 restricted.     

Transplantable mouse tumor line induced by injection of SV40-transformed mouse kidney cells

A transplantable tumor of inbred mice was obtained by inoculating BALB/c mice subcutaneously with SV40-transformed mouse kidney (mKS-A) cells. Tumors were produced by mKS-A cells in the 71st cell culture passage, but not by cells in the 26th passage. The tumor line has been serially passed in BALB/c mice 14 times. In vitro cell culture lines were derived from tumors after 1, 2, 8, 10 and 12 passages in mice. The tumors, as well as the In vitro tumor cell lines, contained SV40 T-antigen, and sera from the tumor-bearing mice contained antibodies to the SV40 T-antigen. SV40 was rescued from the In vitro tumor cell lines after fusion with green monkey kidney (CV-1) cells in the presence of UV-irradiated Sendai virus. The In vitro tumor cell lines derived from mouse passages 8, 10 and 12 were used as SV40 virus; 2) SV40-transformed cell lines; 3) primary mouse (BALB/c or Yale Swiss) kidney cells, or 4) primary mouse (BALB/c or Yale Swiss) embryo cells. These results showed that the tumor line and the In vitro tumor cell lines have the transplantation antigen.     

Merkel cell polyomavirus (MCV) T-antigen seroreactivity,MCV DNA in eyebrow hairs,and squamous cell carcinoma

Background

The role of Merkel cell polyomavirus (MCV) infection in the etiology of non-melanoma skin cancers, other than Merkel cell carcinoma, is unclear. Previously, we reported a significant association between seropositivity to MCV capsid antigen and MCV DNA-positive cutaneous squamous cell carcinoma (SCC). Here we present associations between SCC and seroreactivity to MCV T-antigen (T-Ag) oncoprotein, as well as MCV DNA detected in eyebrow hairs.

Findings

A clinic-based case–control study, including 171 SCC cases and 300 controls without skin cancer, was conducted at Moffitt Cancer Center in Tampa, Florida. Multiplex assays were used to measure serum antibodies against MCV small and large T-Ag and MCV DNA in both eyebrow hairs and SCC tumors (n?=?144). Odds ratios (ORs) and 95 % confidence intervals (CIs) were estimated using logistic regression to evaluate the associations between MCV and SCC. No significant association was observed between seroreactivity to MCV full-length large or small T-Ag and SCC, overall [OR_{large T-Ag}?=?0.99 (0.48-2.08), OR_{small T-Ag}?=?0.31 (0.06–1.62)] or when comparing tumor MCV DNA-positive cases to controls [OR_{large T-Ag}?=?1.06 (0.38–2.93)]. Only presence of MCV DNA in eyebrow hairs was significantly associated with MCV DNA-positive SCC [OR?=?4.05 (2.01–8.18)].

Conclusion

MCV infection is unlikely to play a direct role in SCC.

     

Expression of alien minor histocompatibility antigens distinct from tumor-specific transplantation antigen on a murine fibrosarcoma

The fibrosarcoma ST2, induced by 3-methylcholan-threne in BALB/c (H-2^d) mice, also expressed alien histocompatibility antigens of the C3Hf and B10 background not encoded by the MHC. To examine the relationship between these alien, minor antigens and the tumor-specific transplantation antigen (TSTA) of the tumor, in vivo immunogenicity test were performed in BALB/c mice and in hybrids between BALB/c and C3Hf (H-2^k), C3H.OH (H-2⁰²), C3H.SW (H-2^b), BALB.K (H-2^k), B10.BR (H-2^k), and B10.D2 (H-2^d) mice. A significant loss of TSTA immunogenicity was found in (BALB/c X C3Hf) and in (BALB/c X C3H.OH)F₁ animals and, to a lesser extent, in (BALB/c X C3H.SW)F₁ mice as compared to the immunogenicity of the tumor in BALB/c mice. Immunogenicity tests with ST2 in BALB/c X (BALB/c X C3Hf) or in BALB/c X (BALB/c X B10.D2) backcross mice, respectively, revealed that half of the BALB/c X (BALB/c X C3Hf) and 97% of the BALB/c X (BALB/c X B10.D2) animals were able to mount an immune response to ST2. To see whether the loss of TSTA immunogenicity in (BALB/c X C3Hf) was due to common determinants shared between TSTA and alien non-H-2 C3Hf antigens or to a genetically linked low responsiveness to TSTA introduced by C3Hf and C3H.OH strains, BALB/c mice were immunized with normal normal tissues of some BALB/c X (BALB/c X C3Hf) back-cross, anti-ST2 resistant mice. Normal tissues of anti-ST2 resistant, dd and dk typed backcrosses were able to immunize BALB/c mice against a challenge of an otherwise lethal dose of ST2 cells. Some but not all BALB/c X (BALB/c X B10.D2) anti-ST2 resistant donors had tissues able to immunize BALB/c hosts against the ST2 growth. Since resistance to tumor growth and expression of minor “alien” antigens shared with the tumor segregate independently, we concluded that alien, minor C3Hf and B10 antigens of the BALB/c sarcoma ST2 are distinct from the TSTA of this tumor.     

Restoration of specific immunity against SV40 tumor-specific transplantation antigen to lymphoid cells from tumor-bearing mice.

Specific cell-mediated immunity to SV40 tumor-specific transplantation antigen (TSTA) in BALB/c mice undergoing progressive tumorigenesis by syngeneic SV40-transformed cells (VLM) was investigated in vivo using a tumor-cell neutralization test. Specific cellular reactivity to SV40 TSTA was not detected in BALB/c mice bearing large tumors (10-15 mm mean diameter) but was demonstrable after tumor excision. Specific cytotoxic reactivity against syngeneic SV40-transformed cells in vivo could be restored to lymphoid cells from VLM tumor-bearing mice either by culturing the lymphoid cells in vitro or by treating them with papain or trypsin. Enzyme-treated lymphoid cells from MCA tumor-bearing BALB/c mice had no cytotoxic reactivity against VLM cells. These studies suggest that tumor-bearing hosts possess lymphocytes which are sensitized to the TSTA of the tumor but that the reactivity of these lymphocytes is blocked.     

Immune response of athymic nude mice to papovavirus SV40 tumor-associated antigens.

The requirement of T cell functions in the induction of immune response to SV40-specific transplantation rejection antigen and intranuclear tumor antigen was studied using athymic nude mice. The results obtained indicate that virus-immunized athymic nude mice were unable to reject SV40 tumor cell challenge, and sensitized lymphocytes capable of inhibiting tumor growth in vivo could not be demonstrated in the spleens of virus-immunized mice. Athymic nude mice bearing tumor induced by virus-free SV40-transformed BALB/c cells failed to develop antibodies to intranuclear T antigen. Athymic nude mice also failed to respond to viral antigens. Thus it can be concluded that T cell functions are required in the induction of cellular immune response to SV40-specific transplantation rejection antigen and in humoral immune response to SV40-specific T antigen and virion antigen.     

Naturally occurring cell-mediated cytotoxicity against simian virus 40-transformed 3T3 murine tumor cells

The tumorigenicity and host protective mechanisms induced by simian virus 40 (SV40)-transformed 3T3 cells (SV403T3) were evaluated in syngeneic BALB/c mice. Tumors were regularly produced by sc inoculation of SV403T3 cells; the incidence, latent period, and survival were proportional to the size of the initial inoculum. With the use of an in vitro 18-hour 51Cr cytotoxicity assay, spleen cells from normal mice showed a dose-related killing activity against the SV403T3 cells. At an effector cell-to-target cell ratio of 200:1, the average lysis was 56 +/- 6%. This reaction appeared specific for the virally transformed targets; the mean lysis of parent 3T3 cells was 23 +/- 5%. Effectors were resistant to anti-theta serum and not removed by adherence to plastic or nylon wool. Tissue distribution studies indicated that these effectors were present in high concentrations in spleen, bone marrow, lymph nodes, and peritoneal cavity. Low levels of activity were associated with cells from the thymus. In the present studies specific T-cell cytotoxicity against the SV403T3 cells could not be demonstrated. Animals challenged with nonviable SV403T3 cells prior to tumor cell inoculation did not show increased in vivo resistance. In parallel, the in vitro cytotoxicity of animals inoculated with SV403T3 tumor cells showed no heightened cell killing compared to the cytotoxicity of normal controls.     

Isolation of a tumor-associated transplantation antigen (TATA) from an SV40-induced sarcoma. Resemblance to the TATA of chemically induced neoplasms

A tumor rejection antigen (TATA), obtained from the cytosol of a BALB/c mKSA sarcoma induced by SV40 virus, has been partially purified. This partially purified antigen is strikingly immunogenic against mKSA, providing more than 90% inhibition of growth at levels of 10-30 ?g. This antigen preparation does not protect against challenge with another SV40-induced BALB/c sarcoma VLM which, however, shares a group-specific TATA with mKSA. The antigen also does not immunize against challenge with the methylcholanthrene-induced sarcomas of BALB/c mice, Meth A, CI-4, CII-7 and CII-10, each of which has its own unique TATA. Binding assays, using ELISA, failed to detect any SV40 antigen in the antigen preparation despite the fact that the large T antigen of SV40 (or fragments of it) constitutes the immunodominant TATA of mKSA.     

Induction of graft vs. tumor effect in a murine model of mammary adenocarcinoma

We have attempted to induce immune-mediated graft-vs.-tumor (GVT) effects against solid tumors, using a murine model of mammary adenocarcinoma derived from BALB/c(H-2^d) mice. A cell line (4TI) isolated from this tumor model was highly tumorigenic in syngeneic (BALB/c) or haplo-identical (BALB/c × C57B1/6)F₁ mice (F₁), was only partially tumorigenic in an H-2^d congenic strain of mice (DBA/2) and was non-tumorigenic in a major histocompatible (MHC)-unrelated (H-2^b) strain of mice (C57B1/6). 4T1 cells express class I MHC antigens and adhesion molecules but do not express MHC class II antigens or B7-I co-stimulatory molecules. Female BALB/c (H-2^d) or F₁ (H-2^d/b) mice were reconstituted with male bone marrow (BM) cells derived from minor histocompatible (MiHC)-mismatched DBA (H-2^d) donors or with MHC-mismatched C57B1/6 (H-2^b) BM cells, respectively, 24 hr following lethal total body irradiation. Recipient mice carrying MiHC- or MHC-mismatched donor cells were inoculated with 4T1 cells 2–3 months following BM reconstitution. Chimeras reconstituted with allogeneic donor cells that were MiHC- or MHC-incompatible with tumor cells were able to down-regulate the development of the primary tumour expressing host-type MHC alloantigens. Tumor size in BM chimeras across MiHC or MHC antigens was significantly smaller than tumor size observed in normal BALB/c or F₁ controls. The GVT effect might be of help in improving immunotherapy for solid tumors in humans. Int. J. Cancer, 71:59–63, 1997. © 1997 Wiley-Liss, Inc.     

Induction of simian virus 40 (SV40) transplantation immunity in mice by SV40-transformed cells of various species.

Specific tumor rejection was obtained with the use of simian virus 40 (SV40)-transformed cells from several species including man, rat, ape, sheep, and hamster. Growth of the syngeneic sarcoma mKSA in BALB/c mice was strikingly inhibited following a single immunization with as few as 10(3) intact, viable cells. Non-SV40-transformed cells did not induce tumor rejection activity nor did SV40-transformed lines induce immunity against the 3-methylcholanthrene-induced sarcoma Meth A, syngeneic with BALB/c mice. A close relationship existed between the tumor rejection antigen, the tumor-specific transplantation antigen (TSTA) located on the plasma membrane, and the intranuclear tumor antigen (T-ag). Both were associated with the DNA sequence of the early region of the SV40 genome, and TSTA activity was found in the nucleus. However, we did not observe a close parallelism between T-ag activity and TSTA. Neverthesless, the results strongly suggested that TSTA, like T-ag, was encoded by the virus.     

Genetic control of in vivo immunity to tumor-specific transplantation antigens of chemically induced murine fibrosarcomas.

A possible genetic control of the in vivo immunity to tumor-specific transplantation antigen (TSTA) of two methylcholanthrene-induced BALB/c fibro-sarcomas, C-1 and ST2, was studied in F₁ compatible hosts. Both tumors, which have been previously shown to share their TSTA, lost their immunogenicity in (BALB/c×C3Hf)F₁ and C-1 also in (BALB/c×BALB.K)F₁ mice; the immunogenicity of both sarcomas remained unchanged in other hybrid combinations of BALB/c with H-2^b or H-2^a animals. For C-1 this was true either for a primary immunity to low doses (10⁴ and 5 × 10⁴) of tumor cells or for the secondary immune response measured as a capacity of preimmunized animals to resist a challenge of 10⁵ cells. Since (BALB/c×B10.BR) or (BALB/c×AKR)F₁ hybrids (both H-2^d × H-2^k) were able to reject C-1 cells as strongly as BALB/c mice, the proof of a possible genetic influence involving dominant genes coding for low responsiveness to C-1 or to ST2 and linked to H-2^k haplotype was sought in a back-cross experiment. The primary and secondary immune response to C-1 and the secondary immunity to ST2 were evaluated in BALB/c × (BALB/c × C3Hf) back-cross mice. Results for both tumors were compatible with the presence of an H-2 dominant gene (or cluster of genes), whose products suppress immunity to TSTA; the linkage of low anti-tumor responsiveness with H-2^k was demonstrated for both C-1 and ST2. Further evidence for this linkage was found in the study of immunity to C-1 in (BALB/c×C3Hf)×(BALB/c×C3Hf) F₂ animals. An attempt to map gene(s) governing the low immune response to C-1 and ST2 was performed by studying the anti-tumor immunity of BALB/c, (BALB/c×A), (BALB/c×A.TL), (BALB/c×A.AL), (BALB/c×B10.A), [BALB/c×B10.A(4R) and (BALB/c×C3H.OH]F₁ mice. It was found that K^k and D^k regions are not necessary for the low immunity to C-1 to be expressed, therefore mapping the relevant gene(s) in the I^k region. The low immune response of (BALB/c×C3H.OH)F₁ mice to C-1, however, suggested that D^k genes are also important in this phenomenon. For ST2, only BALB/c×C3H.OH animals showed a low anti-tumor response thus mapping genes coding for a suppression of anti-ST2 immunity in the D^k region. Since (BALB/c×B10.A) but not (BALB/c × A)F₁ mice (both hybrids having an identical H-2^d × H-2^a genotype but different non-H-2 backgrounds) were able to develop an anti-C-1 immunity, an effect of non-H-2 factors which may counteract the suppressive activity of MHC genes is implied. The possible mechanims of this genetic control are discussed.     

Cell interactions in adoptive immune rejection of a syngeneic tumor

Studies of adoptive tumor neutralization (the Winn test) in BALB/c mice rendered immune to the Simian Virus 40-induced tumor mKSA indicate that: a) the reaction is extremely specific; b) there is a very close correlation between immunity detected by the Winn test and the ability of the intact animal to reject tumor; and c) immunity reflected by the ⁵¹chromium lymphocytotoxicity assay (CRA) and microcytotoxicity test (MCI) correlate poorly with in vivo resistance to tumor challenge and the Winn test. Investigation of the contribution of the immune donor lymphocytes to the reaction demonstrated that immune thymus-dependent lymphocytes (T cells) were essential, but other donor cell populations were not. Proliferation of immune donor cells was not required. Recipient T cells do not participate in the reaction. We propose that donor T cells do not attack the tumor directly, but must interact with recipient cells to generate an effector response. Some of the parameters of the postulated cooperative interaction are discussed.     

Immunogenicity, antigenicity and mechanisms of tumor rejection of mineral-oil-induced plasmacytomas in syngeneic BALB/c mice

Transplantation immunity studies were conducted to determine whether mineral-oil-induced plasmacytomas of syngeneic BALB/c mice possess tumor-associated transplantation antigens (TATAs). In a series of experiments to determine whether TATA could be detected, it was established that the optimum immunization regimen against TATA was obtained by immunizing syngeneic hosts with an intradermal inoculation of viable plasmacytoma cells, followed by therapeutic drug-induced tumor regression with aniline mustard. This immunization regimen induced transplantation immunity not only to the homologous tumor employed for immunization, but was effective in demonstrating some cross-protection when mice were rechallenged with other plasmacytomas. Thus, definite cross-reactivity of antigens was observed between some plasmacytomas, but others shown to possess TATA did not share antigens. These, therefore, may possess distinct TATA (s). Further, studies with Winn assays employing cells from BALB/c mice immune to plasmacytoma cells support the idea that cell-mediated immune functions are responsible for syngeneic plasmacytoma rejection. Specificity studies in subsequent Winn assays demonstrated that the kill was directed against plasmacytoma cells and not against TATA (s) of an MLV-induced Moloney leukemia and SV40 virus-induced tumor cells. The nature of the TATA (s) detected is not known, but they probably represent new “cell-associated” antigens acquired during malignant transformation. Finally, the selective removal of the thymus-dependent cell component of the immune lymphocyte population with anti-theta serum and complement eliminated the tumor-neutralizing properties of the immune cells in the Winn assay.     

Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer

Despite studies demonstrating that inhibition of cyclooxygenase‐2 (COX‐2)‐derived prostaglandin E₂ (PGE₂) has significant chemotherapeutic benefits in vitro and in vivo, inhibition of COX enzymes is associated with serious gastrointestinal and cardiovascular side effects, limiting the clinical utility of these drugs. PGE₂ signals through four different receptors (EP1–EP4) and targeting individual receptor(s) may avoid these side effects, while retaining significant anticancer benefits. Here, we show that targeted inhibition of the EP1 receptor in the tumor cells and the tumor microenvironment resulted in the significant inhibition of tumor growth in vivo. Both dietary administration and direct injection of the EP1 receptor‐specific antagonist, ONO‐8713, effectively reduced the growth of established CT26 tumors in BALB/c mice, with suppression of the EP1 receptor in the tumor cells alone less effective in reducing tumor growth. This antitumor effect was associated with reduced Fas ligand expression and attenuated tumor‐induced immune suppression. In particular, tumor infiltration by CD4⁺CD25⁺Foxp3⁺ regulatory T cells was decreased, whereas the cytotoxic activity of isolated splenocytes against CT26 cells was increased. F4/80⁺ macrophage infiltration was also decreased; however, there was no change in macrophage phenotype. These findings suggest that the EP1 receptor represents a potential target for the treatment of colon cancer.     

Studies of lymphocyte stimulation by intact tumor-cell and solubilized tumor antigen.

BALB/c mice were rendered immune to syngeneic SV40-induced sarcoma by subcutaneous injection of mKSA-TU5 tissue-culture adapted cells. Spleen cells from immune mice were examined for tumor-cell neutralization in the Winn assay as well as in in vitro lymphocyte stimulation assays. A microculture (200 mul) lymphocyte stimulation (LS) assay utilizing immune spleen cells was employed with mixed lymphocyte/tumor-cell cultures (MLTC) and the papain crude soluble (CS) extracts from mKSA-TU5 cells. Specificity in the LS assay was determined using spleen cells from mice immune to other syngeneic tumors and by soluble antigenic preparation of normal BALB/c spleen cells. The Winn assay studies demonstrated that spleen cells from mKSA-sensitized mice neutralized mKSA tumor cells and this was corroborated by their resistance to direct tumor challenge. Positive lymphocyte transformation responses in MLTC were observed when mKSA-TU5-sensitized spleen cells were mixed with mitocycin-C-treated intact tumor cells or when papain-solubilized antigens of mKSA cells were employed, but not with non-immune spleen cells or with a soluble antigen from normal cells. Papain-solubilized antigen preparations employed in in vitro assays also immunized against challenge with mKSA tumor cells. Specificity of these lymphocyte transformation reactions was demonstrated with non-sensitized lymphoid cells or lymphoid cells from mice sensitized with a syngeneic Kirsten virus-induced respond. Thus, mKSA tumor surface antigens were recognized on intact tumor cells or with microgram quantities of papain-solubilized extracts from these tumor cells. We believe the lymphocyte stimulation assay affords a method for demonstrating the presence of tumor-specific antigen and for monitoring further purification procedures.     

Differences in the capacity of simian virus 40 (SV40) tumor antigens on cells, membranes and in soluble form to induce transplantation immunity in hamsters and mice.

The immunogenicity of the SV40 tumor-specific transplantation antigen (TSTA) on cells, cell particulates and solubilized membranes was studied in mice and in Syrian hamsters. Immunizations were done with various concentrations of tissue-culture-passaged, non-virus-releasing transformed cells, purified cell membranes and in some cases purified nuclei and papain-solubilized membranes obtained from several species, including the mouse, hamster, man, and sheep. All transformed cell lines were T-antigen-positive. The immunosensitive mKSA line of BALB/c mice and the immunosensitive SV34 cell line of the hamster were used for tumor challenge. All materials, regardless of source and of type of preparation, were strikingly immunogenic in the mouse but only SV40 virus and SV34 (hamster) cells provided protection against tumor cell challenge in the hamster. Also, in a limited study, BKV-transformed hamster cells and purified cell membranes and JCV-transformed hamster cells were found to be immunogenic by the tumor rejection assay in the mouse but not in the hamster. SV40 immunization did not protect the hamster against BKV- and JCV-transformed hamster cells. These results are discussed in terms of possible different specificities resident on the TSTA molecule.     

Specific depression of the antitumor cellular immune response with autologous tumor homogenate.

A momogenate of an SV40-transformed firbosarcoma of BALB/c mice (E4 tumor) injected i.p. into E4, tumor-immune syngeneic mice specifically depressed their cell-mediated immune responses to autologous tumor cells, as measured by a radioisotopic foot pad assay. The fraction of the tumor homogenate that brought about this depression was present in the high-speed supernatant and pellet of a 3 M KCl extract of the tumor. The specificity of the depression was shown in three ways: (a) the serum of E4 tumor-immune mice, but not of normal mice, given injections of E4 tumor homogenate 24 hr previously, suppressed antitumor immunity in vitro, as measured by the release of 51Cr from labeled E4 tumor cells incubated with spleen cells from tumor-immune animals; (b) the i.p. inoculation of E4 tumor homogenate did not alter the cellular immune response of tuberculin-sensitized mice to tuberculin; and (c) the i.p. injection of a homogenate of antigenically unrelated tumor did not depress the cellular immune response of E4 tumor-immune mice to E4 tumor cells.     

Transplantation analyses of the immunogenicity of epidermal tumors generated in murine skin two-stage carcinogenesis protocols

SSIN mice are very sensitive to tumor promoters in two-stage skin carcinogenesis protocols. It was recently reported that SSIN mice have fewer CD8⁺ T-cells than other strains of mice and develop a weaker cytotoxic T-cell response upon challenge with an allogeneic tumor transplant. The significance of this muted immune response to processes involved in two-stage carcinogenesis depends on the immunogenicity of the tumors generated in such protocols. Although they have low CD8⁺ T-cell contents, SSIN rejected a variety of subcutaneously transplanted allogeneic murine tumors. Analyses of the growth of primary papillomas derived from 7,12-dimethylbenz[a]anthracene–initiated/12-O-tetradecanoylphorbol-13-acetate–promoted SSIN mice and then subcutaneously transplanted into triple-deficient (bg-nu-xid), athymic nude and immune-competent and immunosuppressed SSIN mice revealed that few tumors took and tumor takes were not markedly influenced by the immumological status of the transplant recipient. Two tumor cell lines (RS1 and RS2) were derived from the transplantation studies and could be passaged in normal SSIN mice (H-2^q haplotype). Both tumors were squamous cell carcinomas (SCCs) by the second in vivo passage and were rejected in allogeneic mice (BALB/c) but grew in FVB/N mice, a strain having the H-2^q haplotype. Transplantation studies revealed that prior exposure to RS1 and RS2 did not prime SSIN mice to reject a subsequent tumor challenge. Three primary SCC tumors derived from SSIN mice in a two-stage carcinogenesis protocol also grew when subcutaneously transplanted in SSIN mice and could be serially passaged. Consequently, the epidermal SCCs that develop in two-stage carcinogenesis protocols appear to be nonimmunogenic. Mol. Carcinog. 20:48–57, 1997. © 1997 Wiley-Liss, Inc.     

Further studies on tumor-producing activity in adult by embryonic tissue cultures exposed to a virus and grafted before manifestation of morphologic transformation

Experiments were carried out to test the possibility of tumour production in adult recipients of different mouse strains by syngeneic embryonic tissue cultures exposed to, and not transformed by several oncogenic viruses. Tumour-producing effect of embryonic tissue cultures exposed to C-Z strain of RSV was observed in 100% of A, C3H-H2^p, AKR, C57BL/Sn, CC57W and in a small proportion of CC57BR mice, but not in B10.D2 and BALB/c mice. However, introduction of infected embryonic tissue cultures, originating from the resistant strains of mice, into F₁ hybrids between the opposite strains resulted in tumour production. It was demonstrated that resistance to tumour production of the three strains mentioned above was not mediated by the strong immune reaction against the cells introduced. Tumour-producing effect by infected but untransformed cells was also seen with the C-Z strain in hamsters, with the S-R strain of RSV in mice, and with the 5D strain in hamsters but not in mice. Attempts to produce tumours in mice by the introduction of syngeneic embryonic tissue culture exposed to SV₄₀ and neurotropic strain (WSN) of influenza A viruses were negative.     

Immunogenic properties of a soluble tumor rejection antigen (TSTA) from a Simian virus 40-induced sarcoma.

Tumor rejection antigen (TSTA) has been prepared in soluble form and in good yield from the tissue-culture-adapted SV40-induced sarcoma, mKSA, without resorting to the usual extraction procedures of proteolysis, 3 M KCI extraction or treatment with detergents. Chromatography on a Biogel A.5m column of the high-speed supernatant material yielded a fraction, fraction 5, that contained the most active TSTA (and T antigen). This activity was purified in relation to the tumor-rejection activity of the cell-bound TSTA on intact cells or on membranes; a single immunization in the 1 μg to 10 μg protein range strikingly inhibited the growth of the syngeneic neoplasm, mKSA, in tumor-cell challenges at 5×10″ (500×TD₅₀), The soluble TSTA was immunologically specific and extremely stable. Inhibition of growth was not achieved against Meth A, another BALB/c syngeneic sarcoma, and preparations frozen for 6 months at ?20°C maintained their strong specific immunogenicity. The immune deviations of the host that have been reported following inoculations of solubilized (extracted) tumor antigens or crude extracts were not observed in this study using our soluble TSTA. We found no evidence of antigen “overloading;” i.e. an optimal protective effect in a restricted dose range of antigen or with a particular size of tumor-cell challenge; nor did we observe facilitated tumor growth or inhibition of cell-mediated immune responses under a variety of conditions.