Characterization of "H-2K,D-like structures" on MM2 x mouse L cell hybrids. II. An "H-2Kd-like" structure on C3H-derived tumor

Anti-MM2 serum, which had been prepared by immunizing C3H/He mice with syngeneic MM2 mouse mammary ascites tumor, immunoprecipitated "H-2K,D-like" molecules on DBA/2 and C3H.H-2 degrees lymph node cells as well as on somatic cell hybrids between MM2 tumor and mouse L cells. Preclearing of lysates from C3H.H-2 degrees lymph node cells with anti-H-2.31 serum removed all "H-2K,D-like" molecules reactive with anti-MM2 serum, indicating that the molecules detected by anti-MM2 serum are H-2Kd antigens. The anti-H-2.31 serum detected an "H-2K,D-like structure" on the hybrid cells and absorption of the anti-H-2.31 serum with the hybrid cells deprived the serum of anti-H-2Kd reactivity. The hybrid cells could induce antibodies against the H-2Kd antigen in C3H/He mice. These results indicate that on the hybrid cells, whose parental cells were both derived from C3H mice, there is an "H-2K,D-like structure" that has the H-2Kd private specificity. Absorption of anti-MM2 serum with EL4 cells did not affect the capacity of the serum to detect the H-2Kd antigen on C3H.H-2 degrees lymph node cells, indicating that the "H-2Kd-like structure" is distinct from "H-2K,D-like structure A" which was previously reported. Nine isozymes were examined and MM2 cells, mouse L cells, and the hybrids were found to have the same isozyme markers as those of the C3H/He mouse.     

Structure of tumor antigen on hybrid cells between mouse mammary ascites tumor and mouse fibroblast L cells.

Somatic cell hybrids between mouse fibroblast L cells and MM2 mouse mammary ascites tumor grown in BALB/c mice were isolated and the structures of tumor-associated surface antigen of the hybrid cells, and parental MM2 and mouse L cells were investigated by the methods of radioiodination of membrane proteins, immunoprecipitation with a specific antiserum against tumor-associated surface antigens of MM2 tumor (anti-MM2 serum), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two molecules of 105,000 and 76,000 daltons were detected on the MM2 cell surface, but no MM2 tumor antigen was detected on the mouse L cells. On the hybrids between these two kinds of cells, in addition to the two MM2 tumor antigens, molecules of 48,000-51,000 and 12,000 daltons were observed. On Sendai-virus-infected mouse L cells only a molecule of 68,000 daltons was detected by the anti-MM2 serum, and furthermore this molecule was also detected by normal mouse serum, indicating that antibodies against Sendai virus were contaminating in both the anti-MM2 and normal mouse sera used, and thus the molecules detected on the hybrid cells were distinguishable from possible viral components of Sendai virus on the hybrid cells. The results indicate that somatic cell hybrids between mouse L cells and MM2 tumor grown in BALB/c mice expressed on their cell surface the molecules that were not exposed on either parent cell. The experiments comparing newly detected molecules with the H-2 antigen suggested that they were similar to H-2 in their electrophoretic pattern.     

Antigenic modulation in vitro. III. Failure to modulate H-2 antigens on several mouse tumors.

The capacity of various malignant and normal mouse cells to acquire resistance to lysis by guinea pig complement during exposure to H-2 antisera in vitro at 37 degrees C (antigenic modulation) was examined. All tumors tested, including cell lines of the TL+ leukemias RADA1, ASL1, and RLmale1, the TL- leukemia EL 4, myelomas MOPC-70A and S194, and the sarcoma Meth A, failed to modulate when incubated with multispecific or monospecific H-2 antisera up to 24 hours, even though under comparable conditions thymus-leukemia (TL) antigens and surface IgG molecules modulated within several hours. Indirect sensitization of RADA1 leukemia cells with H-2 antisera followed by antiserum against mouse IgG also failed to induce H-2 antigen modulation. Normal peritoneal cells from certain mouse strains were partially modulated with H-2D-specific or H-2K-specific and monospecific antisera within several hours, but normal thymus and lymph node cells did not modulate. Modulation of peritoneal cells occurred without a complete loss of sensitizing H-2 antibody from the cell surface and required a cobra venom factor-sensitive activity that could be restored by human complement component C3. Modulation of TL antigens in vitro had previously been shown to have similar characteristics.     

H-2Db gene transfer into highly metastatic D122 cells results in tumor rejection in allogeneic recipients, but does not affect metastasis in syngeneic recipients. Implications for mechanisms of allorejection.

Highly metastatic, weakly immunogenic Lewis lung carcinoma clones express very low levels of H-2Kb and moderate levels of H-2Db class-I major histocompatibility complex antigens. These cells metastasize spontaneously in mice with C57BL/6 genetic background possessing the H-2Db locus, and grow as local tumors across allogeneic barriers. Transfection of the H-2Db genes into the highly metastatic clone D122 did not alter the growth or metastatic capacity of these cells in syngeneic mice. However, these cells were rejected in allogeneic mice. Transfection of the H-2Kd or H-2Kk genes into D122 elicited a CTL population that cross-reacted with cells bearing native H-2Db antigens. These data suggest that overlapping allo-CTL populations are induced by a native alloantigen and by alloantigen peptides presented through self class-I molecules.     

Immuno-selection in vivo of H-2D phenotypic variants from a metastatic clone of sarcoma cells results in cell lines of altered metastatic competence

To find out whether manipulation of H-2 expression on metastatic cells could alter their metastatic properties, we immunoselected in vivo H-2 antigen variants from a metastatic clone of the T10 sarcoma [originating in a (C57BL/6J X C3H.eB)FI mouse] and tested their metastatic capacity. The unselected metastatic cells (IE7) were previously found to express H-2Db and H-2Dk antigens, but they did not express the H-2K antigens of either parental haplotype. Transplantation of IE7 cells into C57BL/6J irradiated mice resulted in loss of H-2Dk expression and a reduction in H-2Db antigen density. Further transplantation of these cells into non-irradiated C57BL/6J mice led to a total loss of H-2 expression. The cells concomitantly lost their metastatic potency. Immunoselection of IE7 cells in C3H.eB irradiated and non-irradiated mice resulted in cells which were H-2Dk-positive but H-2Db-negative. Cells of these selected variants not only retained their metastatic potential, but in fact were far more metastatic than the unselected IE7 cells. Thus, changes in H-2 expression on tumor cells may alter their metastatic potential. In the case of T10 cells, H-2Dk expression seems to be directly involved in their metastatic capacity.     

Abolishment of metastasis formation by murine tumor cells transfected with "foreign" H-2K genes

High metastatic, low immunogenic Lewis lung carcinoma clones express low levels of H-2Kb major histocompatibility complex antigens. These cells metastasize spontaneously in mice with C57BL/6 genetic background possessing the H-2Db locus. Transfection of different H-2K genes abrogates metastasis in H-2K, H-2D compatible mice and in C57BL/6 recipients. The transfected cells are potent inducers of H-2K-restricted and alloreactive cytotoxic lymphocytes that kill H-2K-positive cells and cross-react with parental nontransfected cells.     

Detection of H-2Dd and H-2Kd molecules in purified Rauscher leukemia virus

Evidence is presented that an H-2 antigenic activity is associated with Rauscher murine leukemia virions grown in vitro. Purified Rauscher MuLV grown in fibroblasts of BALB/c (H-2d), C57BL/6 (H-2b) or (BALB/c X C57BL/6)F1 (H-2d/b) were used to absorb the activity of anti-Dd, anti-Kd, anti-Db or anti-Kb antisera tested against H-2-related targets by cellular radioimmunoassay. The results show that Dd and Kd activities were associated with the virions grown in H-2d or H-2d/b fibroblasts. No H-2b antigenic activity was detected in the virions grown in C57BL/6 or F1 fibroblasts. Immunoprecipitation of surface-labelled spleen cells revealed that Rauscher MuLV grown in BALB/c or F1 fibroblasts inhibited the precipitation of the 48 000 dalton peaks characteristic of the Dd and Kd molecules whereas the precipitation of H-2b molecules was not modified by Rauscher MuLV grown in the different cell lines. Class II molecules (Ia) were not detected in Rauscher virions. Comparison of the absorbing activities of intact and disrupted viruses suggests that the H-2 activity was localized at the viral surface. Taken together, these results confirm the possible non-random association of H-2 molecules to type-C viral particles whatever the nature of this association and its possible cellular or extracellular origin. However, the H-2 antigens found in the virions being a non-restricting element of anti-viral cytolytic T lymphocytes in the same system, these results give no particular support to the "altered self" hypothesis.     

Immunological studies on mouse mammary tumors. VI. Further characterization of a mammary tumor antigen and its distribution in lymphatic cells of allogeneic mice

A new lymphatic cell antigen of some laboratory mice which also occurs as a tumor-specific antigen of ascitic mammary tumors of C3H mice was discovered. This antigen is not present in normal cells of C3H/He mice and is therefore capable of eliciting tumor-specific immune reactions in C3H/He mice carrying that particular tumor, and also appears as an alloantigen in many of the 24 strains of laboratory mice examined, such as C57Bl/6, AKR, C58, 129, C57L, H-2H, 101, RF, SJL/J and Swiss mice. The name “MM antigen” seems appropriate for this cross-reacting antigen detected on the surface of lymphatic cells. The antigen is regulated by a dominant gene independent of other known alloantigens. A designation of the locus MMa was given for this newly discovered antigen. The similarity and difference between this antigen and TL antigen are discussed.     

The differential expression of H-2K versus H-2D antigens,distinguishing high- metastatic from low- metastatic clones,is correlated with the immunogenic properties of the tumor cells

Two clones of the 3LL Lewis lung carcinoma, a low-metastatic clone A9 and a high-metastatic clone D122, were studied for MHC expression and immunogenic properties. Using monoclonal antibodies, we demonstrated that the A9 clone expresses both the H-2K^b and the H-2D^b, whereas the D122 expresses only the H-2D^b, and lacks the expression of the H-2K^b encoded molecules. Cells of the low-metastatic clone A9 grew progressively in syngeneic (C57BL/6J) or in F₁ mice, but were rejected in allogeneic recipients. The high-metastatic D122 grew progressively in all mouse strains tested, yet metastases were formed only in syngeneic recipients. When H-2 recombinant mice were used, the A9 again manifested a significantly greater immunogenic potency than the metastatic D122, which grew in all 4 recombinants tested. Metastases, however, were formed in B10HTG and to a lesser extent in B10A(4R), thus indicating that metastasis formation is restricted by both C57BL background and H-2D^b sub region. We subsequently tested whether the higher immunogenicity of the H-2K^b-positive A9 cells is expressed also in syngeneic mice, to examine whether this could account for its low metastatic phenotype. We found that immunization by A9 cells significantly inhibited the growth of a subsequent A9 graft and even of D122, yet D122 did not retard the growth of secondary D122 or A9 cells. The increased immunogenic effect was expressed also in the generation of syngeneic cytotoxic lymphocytes by A9 but not by D122 cells. We suggest that expression of H-2K molecules on the 3LL clones, immunogenicity and the metastatic phenotype are causally related in this system.     

Oncogenicity of friend-virus-infected cells: Determination of origin of spleen colonies by the H-2 antigens as genetic markers

Spleen cells from mice infected with Friend leukemia virus (FLV) inoculated by the intravenous route give rise to macroscopically visible colonies in the spleens of normal F₁ histocompatible hybrid hosts. A study of H-2 antigens as generic markers for identification of strains of origin of cells constituting the spleen colonies was undertaken. The standard cytotoxic test was demonstrated to be suitable for characterizing the H-2 antigens present on the surface of spleen cells from normal or FLV-leukemic parents or F₁ hybrid mice. Individual colonies dissected out of the spleen of (C3H × C57BL/6) F₁ recipients (H-2^k/H-2^b), 10 days after the intravenous graft of FLV-infected spleen cells of C3H origin (H-2^k), were all sensitive to anti-C57BL/6 antibodies. In the same way, colonies obtained from the spleens of (DBA/2 × C57BL/10) F₁ recipients (H-2^d/H-2^b) grafted with DBA/2 leukemic spleen cells (H-2^d) were all sensitive to both anti-H-2^b and anti-H-2^d antibodies. These results directly prove that the main cell population constituing a spleen colony arises from the recipient. The authors conclude that the spleen colonies do not result from the neoplastic proliferation of injected donor cells but rather from the multiplication of host cells transformed by Friend virus produced by the grafted cells.     

Cessation of autonomous proliferation of mouse lymphoma EL4 by fusion with a T cell line

Benzanthracene-induced C57BL/6 (H-2b) mouse T-cell lymphoma EL4 (a thymidine kinase-deficient cell line) was fused by using polyethylene glycol with an Mlsa (Mls for minor lymphocyte stimulatory) antigen-dependent T cell line, which was designated G4 and had been derived from a C3H/He mouse (H-2k), and the fused cells were cultured in HAT medium. Although no growing cells appeared in most of these fusions, we consistently obtained growth-arrested H-2Kb-positive cells from the fused cell populations by the panning method. The cells were tetraploid and were able to proliferate in response to Mlsa antigen. Three H-2Kb-positive clones, isolated by limiting dilution from three different fusions, were shown to be EL4 x G4 hybrids, because (1) they had both H-2k and H-2b antigens; (2) each of the clones had one submetacentric chromosome which was a marker chromosome of EL4, and they were tetraploid with modal chromosome numbers of 74, 78, and 79, respectively; (3) they had 4 isozymes of both parental cells. These results indicate that EL4 lymphoma cells cease to proliferate when fused with T cell line G4. The malignant phenotype of lymphoma EL4 is thus suppressed at the level of cell transformation by the introduction of the G4 cell genome.     

Detection of a unique antigen on radiation leukemia virus-induced leukemia B6RV2

Radiation leukemia virus-induced leukemia of a male C57BL/6 mouse, B6RV2, is immunogenic to female BALB/c X C57BL/6 F1 mice. In these mice, B6RV2 tumors regressed after initial growth, and after tumor regression the mice were resistant to repeated inocula of up to 10(8) B6RV2 cells. Serum from these mice reacted with B6RV2 in mixed hemadsorption or protein A assays, and absorption analysis indicated that the antigen was restricted to B6RV2; it could not be detected in normal thymocytes or spleen concanavalin A blasts from different inbred strains, nor in 16 C57BL/6 or BALB/c leukemias. Spleen cells from mice in which the tumor had regressed were cytotoxic to B6RV2 after in vitro stimulation with B6RV2, as shown by 51-chromium release assay. This cytotoxicity was eliminated by pretreatment of the cells with anti-Thy-1.2, anti-Lyt-2.2, anti-Lyt-3.2, and complement, indicating that the effector cells were T-cells. The specificity of T-cell killing of B6RV2 was examined by competitive inhibition assays with unlabeled cells; only B6RV2 inhibited killing, while eight other C57BL/6 leukemias did not inhibit. Thus, the antigen on B6RV2 defined serologically and by cytotoxic T-cells is a unique antigen. However, it was not revealed by antibody-blocking test whether the unique determinant defined serologically was related to that recognized by T-cells; B6RV2 antiserum did not block lytic activity in the absence of added complement, irrespective of whether the target cells were untreated or anti-H-2b-treated B6RV2. H-2Kb antisera, but not H-2Db antisera, blocked lysis. This indicated that the H-2Kb molecule was exclusively involved in recognition of B6RV2 by cytotoxic T-cell.     

Effects of H-2Kb gene on expression of melanoma-associated antigen and lectin-binding sites on BL6 melanoma cells

An H-2Kb negative BL6 melanoma clone (BL6-8) was transfected with plasmids containing either the class I H-2Kb or class II H-2IAk gene in combination with the neor gene. The effects of the transfected genes on the expression of the melanoma-associated antigen (MAA) recognized by the monoclonal antibodies MM2-9B6 and MM2-3C6 and the cell surface carbohydrates recognized by 15 different lectins were studied. The original H-2Kb- clone or clones transfected with neor or class II H-2IAk genes expressed high levels of MAA and very low levels of soybean agglutinin (SBA), Griffonia simplicifolia I-B4 (GSIB4), and peanut agglutinin (PNA) lectin-binding sites. In contrast, clones that expressed high levels of the transfected H-2Kb gene completely lost the expression of MAA. In addition, these clones were characterized by the appearance of high levels of expression of the sugars specifically reacting with SBA, GSIB4, and PNA lectins. When the original BL6-8 clone was transfected with the H-2Kd gene, 25 clones subsequently isolated had relatively low expression of the transfected H-2Kd gene but high expression of the endogenous H-2Kb gene accompanied by an alteration in expression of the MAA and lectin binding identical with patterns common for H-2Kb+ melanoma cells. These changes were not due to the transfection, plasmid construction, or place of insertion, since similar phenotypic characteristics were found in H-2Kb+ but not H-2Kb- clones isolated from the N-methyl-N'-nitro-N-nitrosoguanidine-treated BL6T2 or parental BL6 melanoma lines. In total, 73 BL6 melanoma clones were investigated and all of the 41 H-2Kb+ clones displayed loss of MAA and appearance of SBA, GSIB4, and PNA-binding sugars. None of the 32 H-2Kb- clones showed these changes. This study indicates that the class I H-2Kb gene product might alter several phenotypic properties of BL6 melanoma cells. The mechanisms of these changes remain unknown. We consider that these effects of the class I H-2Kb gene are indirect, involving interactions with the B-tropic ecotropic retrovirus specific for melanomas of C57BL/6 mice origin.     

Characterization of interleukin 2-dependent cytotoxic T-cell clones: specificity, cell surface phenotype, and susceptibility to blocking by Lyt antisera

Cytotoxic T-cells were derived from the peritoneal cavity of a C57BL/6 mouse immunized with BALB/c sarcoma Meth A and from the spleens of BALB/c x C57BL/6 F1 (hereafter called CB6F1) mice immunized with BALB/c leukemia RL male 1. The cells were cultured in interleukin 2 and cloned by limiting dilution, and their specificity was determined by direct tests and competitive inhibition assays. C57BL/6 anti-Meth A effector cells recognized H-2Dd determinants. CB6F1 anti-RL male 1 effector cells recognized a unique cell surface antigen of leukemia RL male 1. The specificity was maintained in long-term culture. The cell surface phenotype of the cloned effector cell lines as determined by absorption analysis was Thy-1.2+, Lyt-1.2+, 2.2+, and 3.2+. Cytotoxicity was blocked at the target cell level by antisera against H-2Dd, but not H-2Dk or H-2b, and at the effector cell level by antisera against Lyt-2.2 and 3.2, but not Lyt-1.2, Ly-5.1 or Thy-1.2.     

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.     

Resistance to NK and metastatic potential of fibrosarcoma cells is associated with products encoded by the H-2D region.

We have used the murine 3-methylcholanthrene induced T10 fibrosarcoma tumor cell system originating in (C3II/en x C57BL/6)F1 mice (H-2b x H-2k) to elucidate the possible correlation between metastatic potential, expression of individual H-2 antigens and susceptibility to NK cells. Transfection of the non metastatic and NK sensitive IC9 cells (Db+, Kk, Kb, Kk-) with the H-2Dk gene, altered the metastatic phenotype of the parental cells, yet had no effect on the susceptibility of these tumor cells to lysis by NK and did not elicit a specific CTL response in syngeneic hosts. Variants of the metastatic and NK resistant IE7 clone (Db+, Kk-, Kb-, Kk-), lacking H-2Dk, were selected by treatment with monoclonal anti H-2Dk antibodies and complement. These variants were sensitive to NK and poorly or non metastatic. Transfection of Dk negative variants with the H-2Dk gene, resulted in the isolation of several clones which expressed a wide range of metastatic phenotypes but maintained sensitivity to NK. In addition, by cloning the cDNA of the H-2Dk gene of the metastatic T10-IE7 variant cells and analyzing its nucleotide sequence, we found four single nucleotide changes. Two of them are not expected to alter the encoded amino acids, whereas the others should result in two amino acid substitutions in the alpha-2 domain of the class I H-2Kd protein product. These changes might account, at least partially, for the failure of the transfection of H-2Dk to restore resistance to NK.(ABSTRACT TRUNCATED AT 250 WORDS)     

H-2 control of the cytotoxic antibody response against a newly defined MuLV-related cell-surface antigen: G(B10.A)

H-2-congeneic C57BL mice with milk transmission of B-tropic murine leukemia virus (V+ mice) have a much higher lymphoma incidence than the same strains without milk-transmitted virus (V- mice). Gene(s) within the major histocompatibility complex (H-2) influence virus titers, lymphoma incidence, lymphoma type and the anti-MuLV envelope antibody response. In this paper, we report that the prevalence of cytotoxic antibodies to virus-induced lymphomas is also regulated by the H-2 complex. Milk transmission of MuLV resulted in the formation of cytotoxic antibodies against primary virus-induced C57BL lymphomas. These antibodies detect an antigen that is also present on the RADAI tumor-cell line, and on normal spleen cells of young adult B10.A (H-2a) mice of both V+ and V- sublines, but not on spleen cells of young adult B10 (H-2a) mice of either subline. These cytotoxic antibodies were detected in the sera of B10V+ and B10.A(5R)V+ animals, but not in the sera of B10.AV+ mice. This indicates that the prevalence of these antibodies is controlled by a gene in the K- and/or I-A region of the H-2 complex. The presence of these cytotoxic antibodies in serum is recessively inherited. The specificity of the cytotoxic antibodies was investigated with a standard panel of transplantable tumor-cell lines. Of these, only the RADAI cells expressed the target antigen in direct cytotoxicity tests and by absorption. The ability of B10V+ sera to lyse the B10.AV+ and RADAI tumor cells is ascribed to antibody activity against a new MuLV-related cell-surface protein: G(B10.A). Immunochemical analysis and absorption experiments with different types of purified MuLV and MuLV-infected cell lines indicate that the cytotoxic antibodies belong to low-avidity IgM antibodies that are directed to MuLV.     

Antitumor effect of actinomycin D entrapped in liposomes bearing subunits of tumor-specific monoclonal immunoglobulin M antibody

Sonicated liposomes containing actinomycin D in the membranes were chemically coated with the subunits of monoclonal immunoglobulin M (IgM) antibody against a mouse mammary tumor-associated antigen (MM antigen) and examined for their in vitro and in vivo antitumor effects against MM46 (MM+) and MM48 (MM-) tumors of C3H/He mouse origin. The antibody-bearing, actinomycin D-containing liposomes (chemoimmunoliposomes) were selectively bound to MM+ tumor cells and showed much more in vitro cytotoxicity against the tumor cells than that shown by free actinomycin D. The in vivo antitumor effect of the chemoimmunoliposomes was tested on the mammary tumor cells (5 X 10(4) to 5 X 10(6) transplanted i.p. into syngeneic mice. A single i.p. injection of the chemoimmunoliposomes containing 0.3, 0.5, or 1 microgram of actinomycin D into MM46 tumor-bearing mice resulted in the cure of some mice and a prolonged survival time in the rest of the mice as compared to results in controls. In this test, free actinomycin D, anti-MM IgM antibody, and bovine serum albumin-coated liposomes containing actinomycin D were marginally effective or ineffective. To examine a systemic antitumor effect of chemoimmunoliposomes, mice were inoculated with MM46 tumor cells and then treated with a single i.v. injection of liposomes 4 days later. If the mice were pretreated with an i.v. injection of unmodified multilamellar liposomes, an injection of the chemoimmunoliposomes containing 1 microgram of actinomycin D resulted in a significant inhibition of tumor growth. Both free actinomycin D and bovine serum albumin-coated liposomes containing actinomycin D were ineffective against the s.c. tumor. These results indicate that an antitumor drug entrapped in the membranes of small sonicated liposomes bearing antitumor monoclonal antibodies can be delivered to antigenic tumor cells and exert more efficient antitumor activity than does the free drug.     

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.     

Immunological studies on mouse mammary tumors. II. Characterization of tumor-specific antibodies against mouse mammary tumors

After injection of isografted mammary tumor MM2 sensitized with rabbit antiserum, resistance was induced in C3H/He mice after repeated challenges with MM2. The serum taken from these mice was found to be cytotoxic against MM2 cells. The serum also inhibited the outgrowth of transplant of primary tissue culture cells of spontaneous mammary tumor of C3H/He mice. A series of transplanted mammary tumors recently converted into ascitic form in our laboratory (MM3, MM4-1, MM4-2, MM4-3, MM6, MM8, MM9, MM11, MM12 and MM13), and Ehrlich ascites tumor were found to be susceptible to this serum, as tested by trypan blue uptake in vitro. Outgrowth of these tumors was also inhibited when tumor premixed with this serum was injected. No cytotoxic effect was observed against normal mouse mammary gland cells of a C3H/He mouse. Sera obtained from hyperimmunized syngeneic C3H/He mice were able to fix complement with MM2 tumors. They were partially inactivated by heating at 56° C and by treatment with 2-mercaptoethanol. After gel filtration through Sephadex G-200, complement fixing and cytotoxic activity were found in both 19S and 7S fractions. The 7S fractions, after DEAE cellulose column chromatography, gave a precipititin line at the IgG position in immunoelectrophoresis. From the above evidence, it is concluded that the target cells of the cytotoxic factors of this serum are primary cultured cells or isografted cells of mammary tumors of C3H/He mice. The cytotoxic factors present in the serum are considered to be antibodies against isografts of mammary tumors in C3H/He mice.