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.     

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.     

Persistence of the immunoprotective effects of leukemia X fibroblast hybrid cells toward leukemia in histocompatible mice

Hybrids of ASL-1 murine leukemia cells and LM(TK-) cells, a cultured line of mouse fibroblast origin, stimulate partial immunity toward ASL-1 cells in (A/J X C3H/HeJ)F1 mice (F1 mice). Such mice ordinarily exhibit no resistance to the malignant proliferation of ASL-1 cells. Unprotected animals invariably die within 14-18 days after receiving as few as 200 ASL-1 cells. The hybrid cells, the mice used in the experimental studies and the leukemia cells used for challenge all share the same histocompatibility antigens. ASL-1 cells are H-2a; LM(TK-) cells are H-2k, both ASL-1 X LM(TK-) hybrid cells and A/J X C3H/HeJ)F1 mice are H-2a/k. The long-term persistence of the immunoprotective properties of the hybrid cells toward murine leukemia was investigated by using cells that had been in continuous culture for approx. 36 months. (A/J X C3H/HeJ)F1 mice injected previously with hybrid cells in continuous culture and then challenged with up to 10(7) ASL-1 cells survived longer (p less than 0.001) than mice who had not received hybrid cells previously. Some mice challenged with lesser number of ASL-1 cells survived indefinitely (greater than 200 days). The median survival time of F1 mice injected simultaneously with 10(7) hybrid cells and 200 or 2000 ASL-1 cells was significantly (p less than 0.001) prolonged as well, although the differences between experimental and control groups are less pronounced than if the hybrid cells were injected before challenge with ASL-1 cells. The hybrid cells like those freshly prepared continue to be rejected by histocompatible precipients. In no instance has there been evidence of a progressively growing tumor of hybrid cells in immunocompetent F1 mice. Hybrid cells like those investigated previously do form rapidly growing metastasizing tumors in immunodeficient nu/nu (BALB/c) or X-irradiated (550 R) F1 mice. The cells possess approx. 70 chromosomes (reduced from 85) including chromosomes identified as having originated in each parental source. Like (A/J X C3H/HeJ)F1 animals, they continue to express both H-2a and H-2k antigenic determinants.     

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.     

Expression of alien H-2 specificities of a chemically induced BALB/c fibrosarcoma.

The presence of alien histocompatibility antigens on the cell surface of the 3-methylcholanthrene-induced BALB/c (H-2d) fibrosarcoma C-1, was investigated by serological and transplantation studied. Absorption experiments with monospecific alloantisera showed that C-1 cells expressed their original private (H-2.4 and 31) and public (H-2.3, 8, 28, and 35) specificities. C-1 cells were also able to absorb monospecific antisera directed to the private specificity H-2.23 of the H-2k haplotype, as well as antisera to the public specificities H-2.1, 5, 11 11 and 25 (H-2k and in part H-2q, H-2a and H-2b haplotypes), which are absent from H-2d normal cells. Conversely, other alien specificities (H-2.2, 17, 30, 32, and 33) were not detected on C-1 cells. The C-1 cells were also unable to absorb the activity of an anti-Ia serum (1-28) directed to 1a.1, 2 and 19 (lak) specificities. Transplantation studies showed that resistance against the challenge of C-1 cells could be induced in syngeneic BALB/c mice by preimmunization with normal tissues from C3Hf and AKR (H-2k), A (H-2a) and C57BL/6J (H-2b) strains (expressing all or some of the extra H-2 antigens of the tumor) whereas no protection was obtained with DBA/2 (H-2d) or with W/Fu rat tissues. The anti-tumor activity could be passively transferred by BALB/c lymphoid cells immune to normal C3Hf, AKR, A, and NIH (H-2q) tissues, but no protection was achieved with lymphoid cells immune to DBA/2 or to W/Fu normal rat tissues. These data indicate that foreign H-2 antigens are expressed on C-1 tumor and that they might function as tumor-associated transplantation antigen which was shown to be present and individually distinct on this sarcoma by appropriate in vivo tests.     

Antitumor immunity induced by hybrid tumor cells: comparison between hybrids and the parental tumor

The ability of hybrid tumor cells to induce antitumor immunity has been evaluated in the line 1 alveolar cell carcinoma (L1) model of BALB/c mice. Hybrid tumor cells were produced by fusing freshly dissociated L1 cells isolated from in vivo tumors with the hypoxanthine:aminopterin:thymidine-sensitive cell line, GM 347, derived from C3H mice. Each hybrid was characterized by DNA content and expression of H-2 antigens using a fluorescence-activated cell sorter. Irradiated L1 cells in the presence or absence of Corynebacterium parvum were capable of immunizing BALB/c mice against a challenge of live L1 cells, provided the challenge dose was small (50% lethal dose was between 6 X 10(4) and 1.2 X 10(5) L1 cells). Testing of five hybrid clones and 1 uncloned hybrid line for their immunizing ability demonstrated a range in immunizing ability with none showing a statistically significant improvement in survival (p less than 0.0018) when compared to untreated controls. However, one hybrid clone, MoHb-L1-C2, was selected in which the survival of mice immunized with it compared to controls had a p value of 0.0255. A tumor (labeled L1/A) which grew in one of the mice immunized with this clone was removed and hybridized with GM 347 to yield a second set of hybrids. Both this variant of L1 cells and a hybrid clone made from it (MoHb-L1A-C18) were capable of immunizing mice against a challenge of live L1/A (p values of 0.0000 and 0.0028, respectively, when compared to controls). However, L1 cells were not able to immunize effectively against L1/A, and MoHb-L1A-C18 did not immunize against L1. This suggests that L1/A is a subpopulation of L1 cells with a different antigenic composition. The limited success of MoHb-L1A-C18 against L1/A is thought to be due to the narrower range of antigenic specificities in L1/A and the ability of MoHb-L1A-C18 to represent an important antigenic subpopulation of L1/A.     

Effects on syngeneic tumors of F1 lymphocytes sensitized in vitro by parental stimulator cells

F1 lymphocytes stimulated in vitro by parental cells were evaluated for cytotoxicity against semisyngeneic tumors and lymphoblasts. B6AF1 (H-2a,b) spleen cells were placed in culture with C57BL/6 (H-2b) or B10.A (H-2a) cells and 6 days later were assayed for cell-mediated cytotoxicity in vitro; also subcutaneous, intraperitoneal, and intrapulmonary tumor neutralization experiments were performed. F1 lymphocytes sensitized by parental cells showed high levels of cytotoxicity to the tumor cells of the parental haplotype but no lysis of parental blastoid cells. Tumor cells from irrelevant haplotypes were also lysed. The cells mediating in this type of killing were characterized phenotypically as Thy-1.2- and Lyt-2.2-positive. In a subcutaneous tumor neutralization test (Winn assay), marked suppression of EL-4 lymphoma (H-2b) and B16 melanoma (H-2b) was observed. Likewise, tumor control was seen in an intraperitoneal tumor model. These studies show that F1 versus parental sensitization can be used to lyse tumor in vitro and in vivo and should be explored as an immunotherapeutic tool.     

Alien H-2 antigens on a chemically induced fibrosarcoma: further evidence in crude membrane and soluble extracts of the tumor

Crude membranes (CM) were obtained from in vivo subcutaneous nodules of the methylcholanthrene-induced BALB/c fibrosarcoma C-1 by forcing tumor fragments through a mechanical press and subsequent differential centrifugation. This immunogenic tumor has been previously shown to express both H-2d and extra H-2k-like antigens. Original H-2d and alien H-2k antigenic activities were present in CM C-1 as judged by the specific inhibition of the C'-dependent cytotoxicity of monospecific H-2 alloantisera on normal 51Cr-labelled lymphoid cells. Both K- and D-end private H-2d antigens (31 and 4), and H-2d public antigens 8, 29, 35 were detected in CM C-1. In addition, the alien H-2Kk.23 private specificity and the public H-2k.1, 5, and 25 were also found in CM C-1. A weak but reproducible activity attributable to the Dk private antigen 32 was also revealed in this material. A hierarchy in the expression of both H-2d and H-2k specificities was evident in CM C-1 which paralleled, although with an overall lower antigenic activity, those of two other BALB/c (H-2D) FIBROSARCOMAS AND OF A C3Hf (H-2k) lymphoma, respectively. CM from normal BALB/c and C3Hf spleens, while expressing higher amounts of all the tested H-2 antigens, displayed a hierarchy of the different specificities similar to that of neoplastic tissues. Crude soluble (CS) material was obtained from CM C-1 by deoxycholate treatment and was tested in the inhibition assay for the presence of H-2d and alien H-2k antigens. Only specificities with the highest expression in CM were found in CS, i.e. H-2.4 and 29 for H-2d and H-2.25 for H-2k. Both CM and CS from C-1, but not from another control BALB/c sarcoma, were able to significantly inhibit the activity of an oligospecific serum to the Kk-coded antigens.     

In vitro and in vivo expression of original and foreign H-2 antigens and of the tumor-associated transplantation antigen of a murine fibrosarcoma

We have previously shown that the methyl-cholanthrene-induced BALB/c fibrosarcoma C-1 syngeneically transplanted in vivo expressed, in addition to its original H-2^d and tumor-associated transplantation antigens, also the foreign H-2K^k 23, 1, 5, 11, 25 specificities. In the present study we have investigated the expression and the immunogenicity of these antigens on an in vitro line of C-1 tumor. The binding of C57BL/6J or C3Hf anti-BALB/c sera or that of monospecific alloantisera to H-2^d specificities on plated C-1 cells (evaluated by the indirect isotopic ¹²⁵I-antiglobulin assay) showed that the expression of H-2^d antigens was very low or undetectable on the tumor kept in vitro. Absorption by the in vivo and in vitro maintained C-1 cells of the complement-dependent cytotoxicity on BALB/c normal lymphoid cells of the C75BL/6J anti-BALB/c serum confirmed the lower expression of H-2^d antigens on C-1 kept in vitro as compared to the in vivo transplanted tumor. The H-2^k antigens found on C-1 were also examined by the above methods using BALB/c anti-C3Hf, anti-AKR, anti-C57BL/6J polyspecific sera and also monospecific alloantisera directed to H-2^k antigens. The C-1 cultured cells displayed a significant reduction of H-2^k antigens as compared to the in vivo C-1 tumor, the antigen 5 being undetectable on the in vitro cells. Allogeneic and syngeneic sera raised against in vitro or in vivo C-1 cells were tested on either BALB/c or C3Hf normal lymph-node cells by the complement-dependent cytotoxicity assay. A significantly higher titer of cytotoxic antibodies to H-2^d and to H-2^k was obtained in the allogeneic and syngeneic sera respectively, after immunization with the in vivo C-1 cells as compared to sera raised against the in vitro tumor line. Transplantation studies showed that both in vitro and in vivo C-1 lines possess the same tumor-associated transplantation antigen whose expression was non-significantly reduced on the cultured cells.     

Mouse strain (STS/A) resistant to mammary tumor induction by hypophysial isografts

Implantation of hypophysial isografts does not lead to induction of mammary tumors in all strains of mice lacking the exogenous murine mammary tumor virus. While O20, C3Hf, and BALB/c females are highly susceptible and C57BL and TSI females are of intermediate susceptibility, the STS females appear to be nearly totally resistant. The resistance of the STS strain is not due to failure of prolactin production by the hypophysial isografts and may therefore be due to a genetically controlled mechanism at target cell level. Neither resistance, i.e., low incidence of mammary tumors (2% in STS), nor susceptibility, i.e., high incidence at low age (93% at 349 days in C3Hf; 83% at 360 days in BALB/c), is dominant. F1 hybrids of strain STS and the two strains C3Hf and BALB/c show high incidences (STS X C3Hf F1, 90%; STS X BALB/c F1, 60%), but the age at which tumors appear (476 and 604 days, respectively) is much higher, suggesting that more than one gene is involved in this type of hormonal carcinogenesis of the mammary gland in mice.     

Development and characterization of the BALB/cNIV mouse strain

The strain BALB/cNIV/Crgl was developed by infecting BALB/c/Crgl mice with mouse mammary tumor virus from C3Hf mice. A BALB/c normal mammary duct was transplanted into the gland-free fat pad of a hormone-stimulated female C3Hf X BALB/c F1 mouse. A hyperplastic alveolar nodule was found in the BALB/c ductal outgrowth and was transplanted into another hybrid gland-free fat pad. The resultant hyperplastic alveolar outgrowth was finally transplanted to female BALB/c mice. The hyperplastic alveolar outgrowth contained an exogenous, infectious mouse mammary tumor virus named the nodule-inducing virus, which was thought to be derived from the endogenous low oncogenic mouse mammary tumor virus found in C3Hf mice. The hyperplastic alveolar outgrowth-bearing BALB/c mice were inbred for four generations, and one family was selected as the strain BALB/cNIV/Crgl. It was found that (a) the mouse mammary tumor virus found in the BALB/cNIV strain was milk transmitted, but not transmitted by infected males; (b) the BALB/cNIV breeding females had a low tumor incidence (40%) and a longer latent period (14 months) than did female BALB/cfC3H mice (92% at 8 months); (c) the BALB/cNIV nodule outgrowths had low tumor-producing capabilities (50%) and longer latent periods (13.4 months) than did nodule outgrowths derived from female BALB/cfC3H mice (100% at 7.7 months).     

Hybrid resistance to BALB/c plasmacytomas: F1 hybrid anti-MPC-11 immunological responses correlated with resistance to tumor challenge

BALB/cJ X C57BL/10Sn F1 (hereafter called B10F1) hybrids resist challenge with the BALB/c plasmacytoma, MPC-11, by a radiation-sensitive, silica-insensitive mechanism, whereas BALB/cJ X BALB.B F1 (hereafter called BALB.BF1) hybrids are as susceptible to MPC-11 as are homozygous BALB/c mice themselves. To investigate the mechanism of resistance, we have compared anti-MPC-11 immune responses by these F1 hybrids both before and at various times after tumor challenge. Resistance is not determined by natural killer cell reactivity inasmuch as neither hybrid harbors splenic natural killer cells with lytic activity directed against MPC-11. Nor is it determined by antibody-dependent cell-mediated cytotoxicity since neither hybrid produces an appropriate anti-MPC-11 antibody. Spleen cells and lymph node cells from both hybrids are capable of generating high levels of anti-MPC-11 cytotoxic T-lymphocyte activity in both primary and secondary mixed-lymphocyte tumor cell cultures. Such cytotoxic T-lymphocytes protect susceptible hybrids from tumor growth in Winn assays. The susceptible but not the resistant hybrids lose the ability to generate high levels of cytotoxic T-lymphocytes activity in spleen mixed lymphocyte tumor cell cultures by 28 days, and in lymph node mixed-lymphocyte tumor cell cultures by 14 days postchallenge. The reduction in spleen cell reactivity is due to suppression mainly by adherent cells and can be abrogated by pretreatment of the susceptible hybrids with a low dose of Cytoxan 2 days before challenge. This pretreatment does not, however, protect the mice. They develop tumor at the same rate and die at the same time as do controls. Both the late appearance of suppression and the lack of effect on survival of its ablation suggest it to be a concomitant of tumor growth rather than its cause. Resistance to tumor growth in this model system may reflect an enhanced ability of the resistant hybrid to deliver effector cells to the site of tumor implantation.     

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.     

Mechanisms of the potentiation of specific antitumor immunity by intratumor injection of Corynebacterium parvum

Meth A fibrosarcoma-bearing BALB/c mice given intratumoral injections of 0.5 mg of Corynebacterium parvum showed a highly and tumor-specific transplantation antigen specifically potentiated concomitant immunity to a subsequent tumor challenge. This potentiated antitumor immunity could be locally transferred in the Winn assay to normal recipients with whole draining lymph node cells from the tumor-bearing mice, but the potentiated effect disappeared when adherent cells were removed from these cells. Moreover, the potentiated cytostatic effect on tumor cells was detected in the peritoneal macrophages but not in the nonadherent draining lymph node cells in in vitro tests. On the other hand, nonadherent draining lymph node cells from the tumor-bearing mice, when mixed with C. parvum-induced macrophages, exhibited a specifically potentiated antitumor effect. In addition, this effect was completely abolished by treatment of the draining lymph node cells with anti-Thy-1 and complement. Thus, the potentiated antitumor effect following intratumor injection of C. parvum may be ascribed to the collaboration of specifically sensitized T-lymphocytes with C. parvum-activated macrophages.     

Tumor immunity to murine plasma cell tumors. III. Detection of common and unique tumor-associated antigens on BALB/c, C3H, and NZB plasmacytomas by in vivo and in vitro induction of tumor-immune responses.

Tumor-associated antigens (TAA) were demonstrated on plasmacytomas derived from BALB/c, NZB, and C3H mouse strains, by in vivo and in vitro techniques. By immunizing the appropriate F1 hybrid mice with these tumors, it was possible to show that all the plasmacytomas expressed cross-reactive tumor-associated transplantation antigens. When cytotoxic lymphocytes (CL) were induced in vitro by the coculturing of syngeneic or F1 hybrid spleen cells with irradiated plasmacytoma cells, "shared" and "unique" plasmacytoma TAA were demonstrable. This was accomplished by inducing CL in vitro against one syngeneic plasmacytoma and assaying for lytic activity on a range of 51Cr-labeled BALB/c, NZB and C3H plasmacytoma cells in vitro. In a second in vitro assay, unlabeled plasmacytoma cells were tested for their ability to inhibit the lysis of a particular 51Cr-labeled plasmacytoma, with the use of CL induced in vitro against it. The possibility that these TAA were "self" antigens was excluded by demonstrating in the inhibition assay that they were not present on T lymphomas and spleen cells of the same strain, and that CL "autosensitized" in vitro could not significantly lyse 51Cr-labeled plasmacytoma cells in vitro. From both in vivo and in vitro studies of immunity to these tumors, it was concluded that any one plasmacytoma line possesses multiple TAA of both shared and unique types.     

The collaboration of H-2 identical bone marrow cells in in vivo antitumor activity of immune mouse splenocytes

Antitumor activity of Meth A-immune BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by a Winn test in CD-1(nu/nu) or BALB/c(nu/nu) chimeric mice which were reconstituted with the bone marrow cells of various strains of mice. It was found that Meth A-Im-SPL (L3T4+ T cells) neutralized the tumor in collaboration with H-2 compatible bone marrow or bone marrow-derived cells.     

Studies of genetic transmission of mammary tumour virus by C3Hf mice.

By radioimmunoassay (RIA) mammary tumour virus (MTV) antigens were detected in individual milk samples of C3Hf mice, (female BALB/c X male C3Hf)F1 mice and (female C3Hf X male BALB/c)F1 mice; milk samples of BALB/c mice were negative. In the segregating backcross I population, female BALB/c X male (female BALB/c X male C3Hf) viral antigens were found in the milk of 93 out of 169 mice (55%). In the Bc II population (daughters of Bc I mothers and BALB/c fathers) two groups were distinguished. In the first group, derived from positive Bc I mothers, 55 out of 110 mice (50%) had detectable levels of viral antigens in the milk. In the second group, progeny of negative Bc I mothers, 1 mouse out of 47 was positive. These data are consistent with the assumption that one dominant gene is responsible for the presence of viral antigens in the milk of C3Hf mice. This gene (Mtv-1) seems to be linked with the albino locus situated on chromosome 7; the recombination percentage was about 29. In the first experiment with Bc I mice a significant difference was found between the tumour ages of the mice with virus-positive milk and of the mice with virus-negative milk: all mice (18) with viral antigens in the milk developed mammary tumours at an age ranging from 7 to 18 months, whereas in only 7 out of 16 mice with virus-negative milk were mammary tumours found before the age of 21 months. Viral antigens were detectable (by RIA) in the tumours of mice of both subgroups; however, the amounts (mU/mg tumour) were significantly lower in the tumours derived from mice with virus-negative milk. Although MTV-L of C3Hf mothers could be transmitted to BALB/c mice by foster-nursing, viral antigens could not be detected in milk samples collected prior to the third lactation period; thus an influence on the data of extrachromosomally transmitted MTV-L is unlikely.     

Cross-reactions between tumor cells and allogeneic normal tissues. inhibition of a syngeneic lymphoma outgrowth in h-2 and non-h-2 alloimmune balb/c mice

To test whether alloimmunization with H-2 or/and non-H-2 different normal tissues may increase the immunity to syngeneic tumors, groups of BALB/c (H-2^d) mice were immunized with a series of allogeneic lymphoid cells and then challenged i.p. with syngeneic lymphoma cells. The outgrowth of otherwise lethal doses of the Moloney virus-induced lymphoma YC8 and of its clones was inhibited in BALB/c mice immune to DBA/2 (H-2^d), C3Hf (H-2^k), C3H.SW (H-2^b), C3H.OH (H-2^o2) and to B10 background tissues but not in mice immunized to A/He, BALB.K (H-2^k) or BALB.B (H-2^b) normal tissues. Anti-YC8 effect was also induced by immunizing BALB/c recipients with a pool of five different allogeneic cell lines which included C3Hf, C57BL/6J (H-2^b), N:NIH (H-2^q), B10.M (H-2^f), and DBA/2 lymphoid cells. No growth inhibition of other BALB/c lymphomas induced by Moloney virus (LSTRA), X-rays (RL♂I) or urethane (UR-I) was evident in alloimmune mice. In vivo transfer of growth inhibition of YC8 was obtained with BALB/c anti-B10.D2 peritoneal exudate cells in a Winn assay. The ability of these alloimmune lymphoid cells to delay significantly the survival time of BALB/c mice injected with the mixture of immune cell and YC8 cells was abrogated by anti-Thy 1.2 plus C' treatment. In addition, nu/nu BALB/c mice were unable to develop resistance to YC8 outgrowth after alloimmunization. The results of this study show that: (1) syngeneic growth of a lymphoma can be prevented by alloimmunization with normal cells; (2) this cross-reaction involved non-H-2 antigens; (3) the phenomenon appeared to be mediated by T cells.     

Bioactivity of male (sperm transmitted) mouse mammary tumor virus as influenced by donor, recipient and age at treatment.

The sperm collected from mammary tumor virus (MTV)-carrying mice (C3H, BALB/cfC3H, BALB/cfRIII and RIII) was separately tested for mammary tumor-inducing activity in (BALB/c x C3Hf)F1, (BALB/c x RIIIf) F1, and BALB/c female recipients by i.p., injection of 0.1 ml of the sperm at 1-2 weeks or at 3 months of age. A total of 551 recipents was observed, including control mice. The results may be summarized as follow: 1) mammary tumor incidence in experiments with or without histocompatibility between sperm donor and recipient is the same; 2) bioactivity is related to the type of MTV (C3H, RIII) and to the type of recipient, not to the sperm donor; 3) the activity of RIII MTV released in the sperm appears to be less influenced by the age of recipients than is that of C3H MTV; 4) BALB/c recipients are more susceptible to C3H than to RIII sperm-released MTV; 5) (BALB/c x RIIIf) F1 hybrids are resistant to sperm-released MTV, especially to C3H MTV infection, and show a 34% incidence of late spontneous lymphomas inherited by the RIIf male parent; 6) (BALB/c x C3Hf) F1 hybrids are susceptible to both C3H and RIII sperm-released MTV and show a 30% incidence of late spontaneous mammary tumors due to genetic transmission of MTV by the C3H male parent.     

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.