Identification of transformation-related antigen by monoclonal antibody on Swiss 3T3 cells induced by transfection with murine cultured colon 36 tumor DNA

The cell surface antigen associated with the transformed state of cells that could grow in an anchorage-independent manner was analyzed by use of techniques of DNA transfection and hybridomas secreting the monoclonal antibody (MoAb). Spleen cells of C57BL/6 mice immunized with a highly tumorigenic, chemically induced murine cultured colon 36 tumor (C-C36) of BALB/c origin were hybridized with NS-1, a hypoxanthine phosphoribosyltransferase-deficient myeloma line of BALB/c mice. Screening of hybridomas revealed an antibody that reacted with C-C36 and transformed Swiss 3T3 cells growing in soft agar after transfection of 3T3 cells with C-C36 DNA. The hybridomas that did not react with nontransformed 3T3 and the less tumorigenic BALB/c hemangioendothelioma line D10 were then selected. An MoAb was designated "#71295." This MoAb immunoprecipitated the antigen that consisted of 65,000- and 14,000-molecular-weight components with soluble C-C36 membrane antigens. It also reacted with 2 other chemically induced syngeneic colon tumor lines, cultured colon 26 tumor line and cultured colon 51 tumor line, and with fibrosarcoma Meth A. However, #71295 was not found in NS-1, D14, and BALB/c normal thymus, liver, colon, and kidney tissues. In addition, this MoAb could not inhibit the anchorage-independent growth of C-C36 and transformed 3T3 cells. These results suggest that although the molecule defined by #71295 might not be associated with the anchorage independence of cell growth, it could be a newly expressed determinant on the cell surface that is related to the events of cell transformation.     

Alien histocompatibility determinants on the cell surface of sarcomas induced by methylcholanthrene. I. In vivo studies.

Groups of BALB/c mice were immunized to normal tissues (skin and/or liver plus kidney) of C3Hf, C57Bl/6, DBA/2 and AKR strains and challenged with either of two syngeneic 3-methylcholanthrene-induced immunogenic sarcomas, ST2 and TZ15, or with a "spontaneous" non-immunogenic BALB/c sarcoma, B2. It was found that anti-C3Hf and anti-DBA/2 immune mice were significantly protected against the growth of ST2, whereas anti-AKR immune mice rejected TZ15; no protection was elicited by immunizing with normal tissues of any strain against B2, which lacked individual tumor-associated transplantation antigens (TATA). The reciprocal experiment, i.e. the immunization of BALB/c mice with tumor cells and challenge with skin grafts of different strains, was also carried out with ST2 and TZ15. Accelerated rejection of all the various allogeneic skins was observed in anti-ST2 immune mice and of AKR and C3Hf skin in anti-TZ15 immune animals. In addition the Winn test demonstrated that lymph-node cells of BALB/c mice immune to C3Hf or DBA/2 tissues were specifically inhibitory for ST2, and that lymph-node cells immune to AKR tissues protected against TZ15. In a further experiment both ST2 and TZ15 tumors were left to grow in (C3Hf X BALB/c)F1, (C57Bl/6 X BALB/c)F1, (BALB/c X DBA/2)F1 and (BALB/c X AKR)F1 mice; the tumors were then excised and the "immune" mice challenged with the related tumor to measure their immune response in comparison with that elicited by the same procedure in BALB/c mice. ST2 was highly immunogenic in syngeneic BALB/c mice and in all the hybrid combinations except (C3Hf X BALB/c)F1 mice, where it completely lost its immunogenicity; TZ15 showed a certain loss of immunogenic strength in (BALB/c X AKR)F1 hybrids. It was concluded that TATA of ST2 contain antigenic determinants expressed on the normal cells of C3Hf and DBA/2 strains, and that TATA of TZ15 are likely to share antigens with AKR normal tissues.     

Kinetics of responses to tumor antigens and mouse mammary tumor virus in BALB/cCrgl and BALB/cfC3H mice

Spleen cells from BALB/cCrgl mice responded to murine mammary tumor virus (MuMTV) in cell-mediated immune assays at higher levels than did the spleen cells from the syngeneic BALB/cfC3H mice. Implantation in BALB/cCrgl with a chemically induced mammary tumor and in BALB/cfCrgl mice with spontaneous mammary tumors (SMT) arising in the same strain resulted in sensitization of these animals to the antigens of their tumors. Reactivities peaked 3 weeks after transplantation, whereas no positive reactions could be detected when tumors reached maximum size. A kinetic study with the use of MuMTV antigen(s) showed that the responses of lymphocytes from BALB/cfC3H with SMT followed the same pattern as that obtained with tumor antigens, which indicated that this might be a de novo sensitization. In sharp contrast, a steady type of response to MuMTV was observed with the spleen cells from BALB/cCrgl mice; i.e., the levels of responsiveness to MuMTV did not significantly vary at any stage of tumor development. In vivo studies explored the possible relevance of the in vitro cell-mediated immunity to the host defenses. MuMTV-expressing mammary tumor cells were implanted in BALB/cCrgl and BALB/cfC3H mice. The total incidence of tumors was significantly reduced and a delay occurred in their time of appearance in BALB/cCrgl mice in relation to BALB/cfC3H animals. Thus the in vitro reactivity to MuMTV antigen(s) in BALB/cCrgl mice was found to be coincidental with a degree of protection against the development of MuMTV-expressing mammary tumors.     

Sequential involvement of two distinct CD4+ regulatory T cells during the course of transplantable tumor growth and protection from 3-methylcholanthrene-induced tumorigenesis by CD25-depletion.

The involvement of two phenotypically different regulatory T cells in different stages of tumor growth was investigated. Treatment of BALB/c mice with anti-CD25 monoclonal antibody (mAb) (PC61), but not anti-CD4 mAb (GK1.5) before RL male 1 or Meth A inoculation caused tumor rejection. On the other hand, treatment of BALB/c mice with anti-CD4 mAb (GK1.5) but not anti-CD25 mAb (PC61) on day 6 after inoculation of the same tumors caused rejection. The findings suggest that CD4+CD25+ T cells downregulated the rejection response in the early stage of tumor growth. On the other hand, putative CD4+CD25- T cells downregulated the tumor rejection response in the late stage. Both CD4+CD25+ and putative CD4+CD25- T cells appeared to inhibit the efficient generation of cytotoxic T lymphocytes (CTL). The present study also demonstrated that the treatment of BALB/c mice with anti-CD25 mAb (PC61) at 4 or 6 weeks after 3-methylcholanthrene (3-MC) inoculation retarded tumor occurrence and prolonged survival.     

Common tumor rejection antigens in methylcholanthrene-induced squamous cell carcinomas of mice detected by tumor protection and a radioisotopic footpad assay.

Seven transplantable lines of squamous cell carcinoma of the skin initiated in BALB/c mice by skin painting with methycholanthrene were systematically tested for cross-reactivity of their tumor rejection antigens in a 7 X 7 matrix. As determined by decreased tumor frequency after tumor cell challenge, each line was immunogenic against and/or immunosensitive to at least one and usually more than one of the other lines. A radioisotopic footpad assay for delayed hypersensitivity against viable tumor cells confirmed the cross-reactivity shown by tumor rejection. More than two antigens appeared to be present in the lines. Tests for C-type viruses were positive in all tumors; those for polyoma virus were negative. Whether the uniform presence of C-type viruses can account for the number and variety of antigens found, or whether the tumor rejection antigens are independent of virus expression, remains an open question. The finding of cross-reacting tumor rejection antigens in methylcholanthrene-induced squamous cell carcinomas encourages prospects for the development of more broadly applicable immunodiagnostic and immunotherapeutic reagents.     

Augmentation of tumor immunity against syngeneic tumors in mice by beta-carotene

The effect of beta-carotene on tumor immunity was examined with the use of a syngeneic murine tumor system. Oral administration of beta-carotene (120 micrograms/mouse/day) for 9 days from day 1 to the BALB/c mice inoculated sc with 10(7) syngeneic BALB/c Meth A fibrosarcoma cells (Meth A) led to a remarkable rejection against rechallenged Meth A implanted sc on day 10. The growth of Meth 1 fibrosarcoma (Meth 1), another syngeneic tumor of BALB/c origin, as a rechallenge tumor was unaffected by treatment with beta-carotene, thereby suggesting that beta-carotene may augment tumor rejection specific to tumor-specific antigens. Winn assay revealed that the suppressive effect on tumor growth of immune lymph node cells obtained from Meth A-inoculated beta-carotene-treated mice on day 12 was enhanced dose dependently. Primary effector cells responsible for the augmented rejection are Thy-1-positive, Lyt-1-negative, and Lyt-2-positive lymphocytes, presumably cytotoxic T-lymphocytes.     

Sequential Involvement of Two Distinct CD4+ Regulatory T Cells during the Course of Transplantable Tumor Growth and Protection from 3–Methylcholanthrene-induced Tumorigenesis by CD25–depletion

The involvement of two phenotypically different regulatory T cells in different stages of tumor growth was investigated. Treatment of BALB/c mice with anti-CD25 monoclonal antibody (mAb) (PC61), but not anti-CD4 mAb (GK1.5) before RL male 1 or Meth A inoculation caused tumor rejection. On the other hand, treatment of BALB/c mice with anti-CD4 mAb (GK1.5) but not anti-CD25 mAb (PC61) on day 6 after inoculation of the same tumors caused rejection. The findings suggest that CD4⁺CD25⁺ T cells downregulated the rejection response in the early stage of tumor growth. On the other hand, putative CD4⁺CD25⁻ T cells downregulated the tumor rejection response in the late stage. Both CD4⁺CD25⁺ and putative CD4⁺CD25-T cells appeared to inhibit the efficient generation of cytotoxic T lymphocytes (CTL). The present study also demonstrated that the treatment of BALB/c mice with anti-CD25 mAb (PC61) at 4 or 6 weeks after 3–methylcholanthrene (3–MC) inoculation retarded tumor occurrence and prolonged survival.     

Adoptive transfer enables tumor rejection targeted against a self-antigen without the induction of autoimmunity

We have previously described a tumor model in which the influenza hemagglutinin protein (HA) expressed on the BALB/c-derived MT901 tumor line serves as an immunization-dependent tumor rejection antigen in normal syngeneic mice. Although the HA antigen in this model is clearly foreign to normal BALB/c mice, many tumor antigens recognized by T cells in humans and mice are nonmutated antigens that are expressed on normal tissues as well as on the tumor cells, thereby raising issues of self-tolerance and autoimmunity in attempts to use such antigens therapeutically. To examine these issues, we have applied our tumor model to syngeneic mice that broadly express HA at low levels as a "self"-transgene. Unlike the situation in normal BALB/c mice, immunization of HA-transgenic mice did not result in tumor protection nor did it generate cytotoxic T cell or IgG responses against the HA self-antigen. However, if immunization of HA-transgenic mice was preceded by adoptive transfer of spleen and lymph node cells from normal untreated BALB/c mice, then HA-specific tumor protective immune responses were generated. Despite the self-nature of the HA antigen, no obvious manifestations of autoimmunity were observed. The immunity established in the transgenic mice was notably different from that observed in normal mice in that it was considerably more transient and required CD4 T cells for both successful immunization and subsequent tumor protection, qualities that were not associated with the immunity established in normal BALB/c mice. Collectively our results suggest that transferred cells can be transiently and selectively directed against a tumor-expressed self-antigen before returning to a tolerant state.     

Antigenic diversity of tumors chemically induced within the progeny of a single cell

To explain why the antigens of tumors induced by oncogenic hydrocarbons are individually distinct, it has been suggested that the carcinogen induces a mutation-like hereditary alteration expressed in the cell membrane. An alternative explanation recently proposed is that the tumor antigens pre-exist in the normal precursor cells. Since each precursor cell contains different antigens, these are not in sufficient quantity to be recognized by the immune mechanism until clonal amplification occurs in the tumor. In order to distinguish between these propositions, low tumorigenic BALB/c 3T3 cells were first cloned in vitro and the progeny cells were then neoplastically transformed by methylcholanthrene in diffusion chambers in the abdomen of mice. The antigens of the tumors induced were still individually distinct, even though they all had been derived from the same original cell. It is concluded that the antigenic diversity of chemically induced tumors cannot be explained solely on the basis of cloning of pre-existing variants.     

Immunologic studies with mouse mammary tumor viruses: comparative studies with spontaneous mammary tumor cell vaccines from five inbred mouse strains

Vaccines were prepared from cells of primary spontaneous mammary tumors (MT) of RIII/Imr, GR/Imr, C3H/Imr, A/Imr, and C3HfC57BL/Imr mice. Each vaccine was used to immunize "murine mammary tumor virus (MuMTV)-free" BALB/c/Imr and C57BL/Imr mice before challenge with infectious RIII-MuMTV, GR-MuMTV, C3H-MuMTV, and A-MuMTV. RIII-MT and C3H-MT cells protected mice against tumorigenesis by all four MuMTV's; GR-MT cells protected against all but the RIII-MuMTV challenge; A-MT cells were ineffective against challenge by all four MuMTV's and significantly enhanced development of A-MuMTV-induced tumors. The activity of the C3H-MT cells was not seen when C3HfC57BL cells, free of the standard C3H-MuMTV, were used. The same cell vaccines were inoculated into the five donor strains of mice in attempts to prevent spontaneous MT in these high-incidence strains, which are naturally infected with MuMTV at birth. None of the vaccines prevented tumorigenesis in these mouse strains although delays in the appearance of tumors were observed in RIII and GR mice inoculated with either isologous MT cells or C3Hf-MT cells. The role of viral antigens acting alone or in concert with cellular antigens is discussed.     

Fusion of plasmacytoma and host cells in vivo: selection of proliferating and nonproliferating cultures

The double-mutant cell line 4T00.1 is derived from a plasmacytoma of a BALB/c mouse and is resistant to 6-thioguanine and ouabain. These cells were inoculated into (BALB/c X C57BL)F1 mice by different routes--sc, ip, intrasplenically, and intrathymically. The degree of tumorigenicity and pattern of tumor development were site-dependent. Intrasplenic inoculation of 10(3)-10(4) 4T00.1 cells resulted in development of large omental tumors accompanied by marked ascites. Tenfold to a thousandfold more 4T00.1 cells were required to obtain tumors by other routes. From all solid tumors and ascites and from various organs of tumor-bearing mice, which were explanted into culture in double selective medium containing hypoxanthine, aminopterin, and thymidine plus ouabain (in which only hybrids between 4T00.1 and normal cells can survive), proliferating and nonproliferating cultures were obtained. Of the 14 proliferating cultures, 9 proved to be hybrids by chromosome and H-2 isoantigen analyses and were tumorigenic when 10(6) cells were inoculated sc into syngeneic F1 mice.     

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.     

Specific sensitization of lymphocytes to tumor antigens by Co-cultivation with peritoneal cells exposed to such antigens

Peritoneal cells from BALB/c mice were seeded into 50-mm plastic Petri dishes and exposed either to antigen extracts prepared from individual BALB/c sarcomas having unique tumor-specific antigens, to similar extracts prepared from 14- to 18-day-old mouse embryos, or to culture medium only. Lymphoid cells from BALB/c lymph nodes were then added to the peritioneal cells. Following 4-5 days of co-cultivation, the lymphoid cells were harvested and tested in a 30 h microcytotoxicity assay for reactivity against tumor cells carrying the respective sensitizing antigens as well as against control cells which consisted of either tumor cells lacking the sensitizing antigens or normal skin fibroblasts. By this approach sensitization to individually unique tumor antigens, as well as to antigens shared by mouse embryos and mouse sarcoma cells, was achieved.     

A unique mucin immunoenhancing peptide with antitumor properties

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5' sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.     

Systemic targeting of CpG-ODN to the tumor microenvironment with anti-neu-CpG hybrid molecule and T regulatory cell depletion induces memory responses in BALB-neuT tolerant mice

We have shown that neu transgenic mice are immunotolerant and that immunizations with dendritic cells (DC) pulsed with neu-derived antigens were not able to control tumor growth in these animals. We tested whether, by modulating the tumor microenvironment with Toll-like receptor ligands, it could be possible to induce the activation of antitumor responses in neu mice. Our results indicate that only intratumoral (i.t.) injections of CpG-ODN induce an antitumor response in neu mice. To target the CpG-ODN to the tumor site anywhere within the body, we chemically conjugated an anti-Her-2/neu monoclonal antibody (mAb) with CpG-ODN. The anti-neu-CpG hybrid molecule retained its ability to bind to Her-2/neu(+) tumors, activate DCs, and induce antitumor responses. Our results indicated that injections of anti-neu-CpG induced the rejection of primary tumors in 100% of BALB/c mice and only in approximately 30% of BALB-neuT mice. After challenging the BALB/c and BALB-neuT mice, we observed that BALB/c mice developed a protective memory response; in contrast, BALB-neuT mice succumbed to the challenge. After injections of anti-neu-CpG, T regulatory cells (T-reg) were drastically reduced at the tumor site, but a large number were still present in the lymphoid organs. When BALB-neuT mice were treated with anti-neu-CpG plus anti-GITR mAb, but not with anti-CD25 mAb, 100% of the BALB-neuT mice rejected the primary tumor and developed a protective memory response indicating the critical role of T-regs in regulating the repertoire against self antigens. Taken together, these results indicate that CpG-ODN-targeted therapy and depletion of T-regs optimally activate a primary response and generate a protective memory response against self-tumor antigens.     

Progression of a Weakly Tumorigenic Mouse Fibrosarcoma at the Site of Early Phase of Inflammation Caused by Plastic Plates

To elucidate tumor progression-enhancing factor(s), we examined the effects of host inflammation and host immunological status on in vivo tumor progression. One × 10⁴ cells of QR clones (QR-32, -20 and -18), regressor tumor clones of 3-methylcholanthrene-induced fibrosarcoma, were unable to grow when injected s.c. into C57BL/6 mice in cell suspension form. However, QR clones grew and were lethal when s.c. implanted, attached to plastic plates. Furthermore, the tumor lines (QRpP) obtained from the tumors which had arisen from the plate-attached QR-32 clone cells no longer required plastic plates for their growth in normal mice, and had acquired stable malignant phenotypes. Although QR-32 cells became lethal when injected at the site of plastic plate implantation 1, 5 and 10 days before tumor injection, few tumors developed when plastic plates had been implanted 20 or 30 days before tumor injection. We established culture clones from the tumors arising in normal mice and mice immunosuppressed by irradiation. Clones derived from the tumors which had arisen in normal mice after implantation with plastic plates were lethal when re-implanted in normal mice (71%). On the other hand, clones derived from the tumors that arose in irradiated mice with or without plastic plates were lethal in only a few normal mice, when re-implanted (20 and 8%, respectively). These results indicate that QR clone cell progression is enhanced by the early phase of inflammation at the site of plastic plate implantation and that the progression-enhancing activity of co-implantation with a plastic plate is inhibited by previous whole-body irradiation of hosts.     

Xenogenization of a mouse lung carcinoma (3LL) by transfection with an allogeneic class I major histocompatibility complex gene (H-2Ld)

We investigated the tumorigenicity and immunogenicity of tumor cells transfected with an allogeneic class I major histocompatibility complex gene. A single clone (3LL/3) from a Lewis lung carcinoma in the C57BL/6 strain (H-2b) was cotransfected with a BALB/c genomic clone containing an H-2Ld gene and a bacterial neo gene conferring resistance to G418. Three Ld-positive, three Ld-negative, and two Neor clones were selected by means of a 125I-protein A binding assay using an anti-H-2Ld monoclonal antibody. The antigenic expression of the H-2Ld gene products was only 20-40% on the Ld-positive clones compared with Meth-A tumor cells of BALB/c mice. The 50% lethal tumor dose of these clones in C57BL/6 mice was 5.6 X 10(6) in the Ld-positive clones, but only 1.3 X 10(5) in the 3LL/3 parent clone, 1.2 X 10(5) in the Neor clones, and 2.2 X 10(5) in the Ld-negative clones. The tumorigenicity of the Ld-positive clones was, therefore, reduced to less than 1/40 of that of the parent tumor cells. The decreased tumorigenicity of the Ld-positive clones was abrogated in mice irradiated with 600 rads. After inoculation and spontaneous regression of the viable Ld-positive clone cells, the mice acquired transplantation resistance against the challenge of a parental 3LL/3 tumor. However, the immunogenicity variation between Ld-positive, Ld-negative, Neor, and 3LL/3 parent clones showed no statistical difference. These results indicate that tumor cells transfected with an allogeneic class I H-2 gene can express an H-2 foreign antigen, can regress in syngeneic hosts, and can induce antitumor transplantation resistance against the original tumors, although they are not able to enhance their immunogenicity.     

Loss of marrow allograft resistance in mice with transplanted methylcholanthrene-induced sarcomas.

To determine if the effector cells responsible for allogeneic marrow stem cell rejections were suppressed in mice with tumors, C57BL/6 (B6) mice were inoculated with 3-methylcholanthrene (MCA)-induced sarcoma cells. When the tumor reached 2.0--2.5 cm in diameter, these mice and control B6 and (BALB/c times A)F1 (CAF1) uninoculated animals were irradiated and given BALB/c marrow cells in the first of a two-step "stem cell rescue" experiment. Four days later, spleen cells of the primary hosts were reinoculated into irradiated CAF1 secondary hosts compatible with BALB/c marrow cells and immunized against B6 antigens. Splenic uptake (percent) of 125I-5-iodo-2'-deoxyuridine 5 days after spleen cell regrafting was used as a measure of cell proliferation and reflected growth of the stem cells in the primary hosts. BALB/c stem cells grew as well in B6 mice with tumors as in CAF1 primary hosts but were rejected by B6 controls. Seeding efficiency of BALB/c stem cells 6 hours after infusion of marrow cells and growth of syngeneic B6 stem cells were enhanced twofold in spleens of tumor-bearing B6 mice. To exclude the possibility that enhanced seeding resulted in greater survival of allogeneic stem cells, more DBA/2 marrow cells were infused into control B6 primary hosts than into tumor-bearing B6 and control DBA/2 mice. Control B6 mice resisted growth of even 7.5 times 10(6) DBA/2 marrow cells, whereas B6 tumor bearers allowed growth of 2.5 times 10(6) cells. No "suppressor cells" capable of inhibiting marrow cell allograft reactions were detected in spleens of tumor-bearing mice. Thus transplanted syngeneic MCA-induced sarcomas abrogated the ability of mice to reject allogeneic marrow stem cells.     

Neutrophils are required for 3-methylcholanthrene-initiated, butylated hydroxytoluene-promoted lung carcinogenesis

Multiple studies have shown a link between chronic inflammation and lung tumorigenesis. Inbred mouse strains vary in their susceptibility to methylcholanthrene (MCA)‐initiated butylated hydroxytoluene (BHT)‐promoted lung carcinogenesis. In the present study we investigated whether neutrophils play a role in strain dependent differences in susceptibility to lung tumor promotion. We observed a significant elevation in homeostatic levels of neutrophils in the lungs of tumor‐susceptible BALB/cByJ (BALB) mice compared to tumor‐resistant C57BL/6J (B6) mice. Additionally, BHT treatment further elevated neutrophil numbers as well as neutrophil chemoattractant keratinocyte‐derived cytokine (KC)/chemokine (C‐X‐C motif) ligand 1 (Cxcl1) levels in BALB lung airways. Lung CD11c+ cells were a major source of KC expression and depletion of neutrophils in BALB mice resulted in a 71% decrease in tumor multiplicity. However, tumor multiplicity did not depend on the presence of T cells, despite the accumulation of T cells following BHT treatment. These data demonstrate that neutrophils are essential to promote tumor growth in the MCA/BHT two‐step lung carcinogenesis model. © 2011 Wiley Periodicals, Inc.     

Antitumor properties of an anti-idiotypic monoclonal antibody in relation to N-glycolyl-containing gangliosides

We examined the antitumor effects of 1E10 monoclonal antibody, an anti-idiotypic IgG to an IgM monoclonal antibody, named P3, that reacts specifically with N-glycolyl-containing gangliosides and also recognizes antigens in human breast and melanoma tumors. Two murine tumor cell lines positive for the P3 antibody, F3II mammary carcinoma (BALB/c) and B16 melanoma (C57BL/6), were employed. In BALB/c mice, vaccination with several i.p. doses at 14-day intervals of 50 microgram of 1E10 coupled to keyhole limpet hemocyanin in Freund's adjuvant, significantly reduced s.c. tumor growth of F3II carcinoma cells and the number of spontaneous lung metastases. Also, the effect of 1E10 as a biological response modifier on tumor lung colonization was evaluated in C57BL/6 mice injected i.v. with B16 melanoma cells. Interestingly, i.v. administration of 10 microgram of uncoupled 1E10 antibody, 10-14 days after inoculation of B16 cells, dramatically reduced the number of experimental metastases in comparison with lungs from mice treated with an irrelevant IgG. The present data suggest that this 'non-internal image' anti-idiotypic monoclonal antibody may activate more than one mechanism of antitumor response against melanoma and mammary tumor cells.