Enhanced expression of class I major histocompatibility complex gene (Dk) products on immunogenic variants of a spontaneous murine carcinoma

Both immunogenic and nonimmunogenic variant clones were isolated from a recently obtained spontaneous murine adenocarcinoma after treatment (xenogenization) with either the mutagen ethyl methanesulfonate or the DNA hypomethylating agent, and "gene activator," 5-azacytidine. Clonal analysis of the untreated tumor population confirmed that immunogenic variants arose as a consequence of the xenogenization protocol. At a dose of 10(6) cells per mouse, nonimmunogenic variants, like the parental tumor line, grew progressively in normal syngeneic recipients. In contrast, immunogenic variants were rejected in normal syngeneic mice and grew progressively only in T-cell-deficient nude mice. Serologic analysis of the respective clonal variants revealed that immunogenic variants expressed substantially elevated (fourfold to tenfold) levels of class I H-2Dk antigen relative to parental or nonimmunogenic cell lines. Two variants exhibiting marginal immunogenicity expressed high and low levels of major histocompatibility complex (MHC) antigen, respectively suggesting that elevated MHC expression, although possibly a contributing factor, did not account for the immunogenic phenotype in all cases. Finally, the immunogenic phenotype of two variants decayed with time in culture. Clones in the process of reversion lost their elevated Dk gene expression and became progressively more tumorigenic in normal syngeneic mice. Together, these data are consistent with a hypothesis that elevated MHC expression can contribute to the immunogenic phenotype of originally low MHC-expressing tumors and that the reduced level of MHC observed in certain clinical cancers may have significant implications with regard to immunologic aspects of the tumor-host relationship.     

Failure of expression of class I major histocompatibility antigens to alter tumor immunogenicity of a spontaneous murine carcinoma

We have shown previously that clonal immunogenic variants of murine mammary adenocarcinoma 10.1 can be isolated after treatment in vitro with the DNA-hypomethylating agent 5-azacytidine (5-aza). Such immunogenic variants frequently express elevated class I major histocompatibility complex antigens relative to the level of expression in the parent tumor and are rejected in syngeneic mice by a T-cell-dependent process. To ascertain whether elevated immunogenicity is a function of increased class I antigen expression, we isolated high class I antigen expressors from 5-aza-treated 10.1 cells by using the fluorescence-activated cell sorter. Clonal variants displaying any increase in class I antigen expression were more efficient stimulators of allo-class I antigen-specific cytolytic T-cell precursors. However, these variants displayed unaltered tumorigenicity in immunocompetent syngeneic mice. Thus, phenotypic changes other than, or in addition to, elevated class I antigen expression cause the reduced tumorigenicity of immunogenic clones of 10.1 cells isolated after 5-aza treatment.     

Reduced tumorigenicity of murine tumor cells secreting gamma-interferon is due to nonspecific host responses and is unrelated to class I major histocompatibility complex expression

Spontaneous SP1 murine adenocarcinoma cells transfected with the murine gamma-interferon (IFN-gamma) gene expressed IFN-gamma (SP1/IFN-gamma) failed to grow in syngeneic hosts and grew in nude mice. The rejection of SP1/IFN-gamma cells was related to the amount of IFN-gamma produced and appeared to be mediated primarily by nonspecific cellular mechanisms, although some role for T-cells in the afferent arm of this response is possible. SP1 cells are H2-Kk negative but express class I antigens when producing IFN-gamma. However, class I major histocompatibility complex (MHC) expression, while likely necessary, was insufficient in itself to prevent tumor growth since secretion of greater than 64 units/ml IFN-gamma was needed to inhibit tumorigenicity while only 8 units/ml IFN-gamma could induce class I antigens. Similar results were obtained with the murine colon carcinoma CT-26, a tumor that constitutively expresses class I MHC antigens, further supporting the contention that class I MHC expression is not essential for the rejection response induced by IFN-gamma. The failure of SP1/IFN-gamma cells to protect against a challenge with parent SP1 cells argues that factors other than IFN-gamma production or class I MHC expression are needed to induce a protective response against weakly or nonimmunogenic tumor cells.     

Major histocompatibility complex class I and unique antigen expression by murine tumors that escaped from CD8+ T-cell-dependent surveillance

The rejection of murine UV-induced skin cancers by normal mice is a striking example of powerful immune surveillance of the normal host against malignant cells. In this study, we show that UV-induced regressor tumors regularly grew progressively and killed mice that were depleted of CD8+ T-cells. Depletion of CD4+ T-cells had no effect, suggesting that CD8+ but not CD4+ T-cells were required for this immune surveillance. To determine whether change in major histocompatibility complex (MHC) class I expression was a frequent event that caused low immunogenicity of tumors or facilitated escape from immune destruction, recently isolated murine tumors of varying degrees of immunogenicity, including highly immunogenic UV-induced regressor, less immunogenic UV-induced progressor, and poorly immunogenic spontaneous progressor tumors, were compared. There was no correlation between the ability of a tumor to grow progressively in a normal immunocompetent host and the level of constitutive class I expression or the level of expression induced in vitro by gamma interferon. (Only 1 of more than 20 progressor tumors analyzed showed complete loss of a MHC class I molecule.) Some progressor variants showed loss of a unique tumor-specific cytotoxic T-lymphocyte-defined antigen, consistent with earlier evidence of antigen loss providing a mechanism for tumor escape. However, most of the host-selected progressor variants retained both MHC class I antigens and the unique tumor antigens that we could detect with cytotoxic T-lymphocyte clones, suggesting that mechanisms other than loss of MHC class I or of the unique target antigen may be involved in escape of some tumors from a highly effective CD8-dependent host surveillance.     

Metastatic capacity of cloned T10 sarcoma cells that differ in H-2 expression: inverse relationship to their immunogenic potency

Clones of the T10 sarcoma, originated in a (H-2b X H-2k)F1 mouse, differ in their metastatic competence, correlated with differences in the expression of antigens of the two parental haplotypes. In fact, the metastatic phenotype is determined by the H-2Dk antigen. Whether the different major histocompatibility complex gene products control the metastatic phenotype via their different immunogenic properties was tested. The involvement of the immune system in controlling the development of metastases was inferred from experiments in which nonmetastatic T10 cloned cells were found to produce both experimental and spontaneous metastases in syngeneic immune-suppressed mice. After the testing of T-cell-mediated immune responses, metastatic T10 cloned cells, which expressed the H-2Db and H-2Dk antigens, were nonimmunogenic in their syngeneic hosts, whereas nonmetastatic T10 cloned cells, which expressed predominantly the H-2Db antigens, evoked a strong T-cell response. H-2Db and H-2Dk antigens expressed on T10 cells appeared to differ in their immunogenicity. This was further supported by the observation that whereas a good antibody response was elicited by H-2Db antigens expressed on T10 cells, only a low anti-H-2Dk antibody was produced. The different T10 cloned cells were not susceptible to natural killer (NK) activity in an in vitro assay, yet in vivo studies suggested the participation of NK activity in controlling T10 metastasis. In animals with depressed NK activity, metastases were generated even by nonmetastatic clones, whereas in animals in which NK activity was elevated, even metastatic clones failed to generate metastases. Both T-cell-mediated immune responses, probably restricted by the H-2D products and NK reactivity, appeared to participate in controlling the development of metastases by T10 cells.     

Clonal variation in tumorigenicity of L1210 lymphoma cells: nontumorigenic variants with an enhanced expression of tumor-associated antigen and Ia antigens

Clonal variations in the tumorigenicity and in the expressions of tumor-associated antigens (TAA) as well as normal cell surface antigens were studied using clones of a highly tumorigenic DBA/2 lymphoma, L1210, which were isolated by limiting dilution in vitro. The majority of the clones were highly tumorigenic (tum+) in normal syngeneic mice, as was the parent L1210. The rest were nontumorigenic (tum-) in such mice; these clones, however, were tumorigenic in host mice that had been immunosuppressed by irradiation with 450 rads. Moreover, these tum- variants were shown to have an ability to elicit, in syngeneic mice, strong host resistance specifically directed against challenge with the parent L1210 and tum+ cloned cells and an ability to generate an in vitro primary syngeneic cytotoxic T-cell response against L1210 clones, indicating an enhanced immunogenicity in tum- variants. The expression of TAA by tumor clones was defined by determining the reactivity of monoclonal antibody, raised in syngeneic mice against an immunogenic L1210 subline, L1210/GZL. Marked clonal variation in the expression of monoclonal antibody-defined TAA was demonstrated, while no significant variation was seen in the H-2Dd expression. There was an inverse relationship between the TAA expression and the tumorigenicity. Furthermore, the enhanced expression of the TAA and the increased immunogenicity was associated with the I-Ad expression on the tum- variants. The unique characteristics of the tumor variants were very stable and heritable although occasional revertant phenotypes were detected on some clones. The results suggest that the tumor variants bearing distinct immunological properties exist in the parent L1210 line and carry a potential to modulate host immune responses directed against tumor cells.     

Immune response to somatic cell hybrids between ultraviolet radiation-induced regressor and spontaneous progressor C3H mouse tumor cells

We used somatic cell hybridization to determine whether the regressor phenotype exhibited by UV-induced murine tumors was dominant or recessive and whether this technique could confer immunogenic properties on nonimmunogenic syngeneic tumors. We transfected a highly antigenic UV-induced C3H mouse tumor cell line (UV-2240) with the plasmid pSV2-neo and selected G418-resistant clones. The resulting cell line was fused with a spontaneously transformed nonimmunogenic C3H progressor tumor cell line (SF-2T) that had been selected previously for resistance to 3.0 mM ouabain. These two cell lines were fused by a brief exposure to polyethylene glycol and heterokaryons isolated by growth in medium containing both G418 and ouabain. Hybrid cell lines established from individual colonies and from pools of colonies were tested for tumorigenicity in normal C3H and athymic nude mice. The results indicated that all the hybrid cell lines tested were highly antigenic in that they were completely rejected when transplanted into normal syngeneic mice but grew progressively in nude mice. Furthermore, immunization of C3H mice with the hybrid cell lines induced protective immunity against challenge with the immunizing tumor and generated cross-protective immunity against challenge with the regressor parental cell line but not against challenge with the progressor parental cell line. These results demonstrate that the regressor phenotype of the UV-2240 tumor is dominant in nature and that the immune response induced by somatic cell hybrids is uniquely directed against the dominant tumor-specific transplantation antigens expressed on the regressor tumor. This implies that introduction of tumor-specific transplantation antigens from an immunogenic tumor into a nonimmunogenic tumor, although sufficient to confer immunogenic properties to the hybrid, is insufficient to induce cross-protective transplantation immunity against the nonimmunogenic tumor.     

MHC class I genes controlling the metastatic phenotype of tumor cells.

Metastatic clones of murine tumors that manifest impaired expression of class I MHC antigens do not induce an antitumor CTL activity. Transfection of H-2Kb genes into D122 carcinoma and B16 melanoma clones converted these cells to low metastatic immunogenic clones that can be used to protect in vivo against metastases of parental clones. Amplification of the protective effect can be achieved by combination of syngeneic and allogeneic MHC class I genes. Studying the mechanisms involved in MHC class I suppression in tumor cells, we found that changes in H-2 promoter activity were the cause of low expression. Proteins that might be involved were demonstrated by migration retardation methods. The involvement of the fos-jun complex in regulation of MHC expression is discussed.     

Association in the expression of Kirsten-ras oncogene and the major histocompatibility complex class I antigens in fibrosarcoma tumor cell variants exhibiting different metastatic capabilities

The metastatic properties of the methylcholanthrene-induced T-10 sarcoma tumor variants which originated in C3H x C57Bl/6 F1 mice are correlated with the relative expression of class I major histocompatibility complex antigens. Both the nonmetastatic and the highly metastatic clones were found to lack the H-2K region-controlled H-2Kb and H-2Kk antigens. However, the nonmetastatic clones express only the H-2Db molecule whereas the metastatic clones express both the H-2Db and the H-2Dk molecules. Transfection of the highly metastatic lines with cloned H-2K genes (Kb, Kk) reduced their tumorigenicity and abolished the formation of metastasis in syngeneic mice, while the transfection of the nonmetastatic lines with cloned H-2Dk genes resulted in shifting the cells to the metastatic phenotype. The present study is aimed to investigate the expression of protooncogenes in the T-10 fibrosarcoma lines that exhibit distinct metastatic properties in correlation with the expressed H-2 antigens. The major oncogene which showed differential expression in the T-10 clones is Ki-ras. The amounts of specific Ki-ras messenger RNA and the Ki-ras Mr 21,000 protein are expressed in elevated levels in the H-2Dk-negative nonmetastatic clones in comparison with a low level of expression in the H-2Dk-positive highly metastatic clones. Expression of H-2K antigens following transfection with cloned H-2K genes had no effect on the expressed Ki-ras oncogene in the T-10 clones. However, transfection of the nonmetastatic cells with the cloned H-2Dk gene resulted in shifting of the cells to a highly metastatic phenotype and in reduction of the expressed c-Ki-ras oncogene.     

Association between MHC class I antigen expression and malignancy of murine T lymphoma variants

Tumor cell variants were derived from an AKR T-cell lymphoma cell line (BW5147, H-2k haplotype). These variants differed in their malignant potential and in their membrane expression of class I MHC antigens. High tumorigenic and spontaneous metastatic capacity was found to be predominantly associated with a decrease of H-2Kk class I major histocompatibility complex (MHC) antigen expression. In contrast, high experimental metastatic capacity correlated strongly with an increased H-2Dk antigen expression. The in vitro invasive potential and the LFA-1 expression of the BW variants showed no correlation with the differential MHC antigen expression and the differential metastatic and tumorigenic capacity of the BW variants. Furthermore, the susceptibility of the BW 5147 variants to TNF and NK-mediated cytotoxicity was not related to the differential metastatic potential and the expression of the class I MHC antigens.     

MHC class I-deficient metastatic tumor variants immunoselected by T lymphocytes originate from the coordinated downregulation of APM components

Previous reports from our group indicated that the MHC class I phenotype of metastatic lung colonies produced by a mouse fibrosarcoma tumor clone (B9) were, depending on the immune status of the host, MHC class I negative in immunocompetent mice and MHC class I positive in immunodeficient athymic nude/nude mice. Now we report the identification of the molecular alterations responsible for the changes of MHC class I molecules in both situations. Metastatic nodes were analyzed for the mRNA level of H-2 class I and beta2-microglobulin genes, and several gene components of the major histocompatibility complex (MHC) class I antigen-processing machinery (APM). These included the genes coding for the low-molecular-weight proteins LMP2, LMP7, LMP10, the transporter associated with antigen processing (TAP-1, TAP-2), and calnexin, calreticulin, tapasin, PA-28-alpha, PA-28-beta, ERP-59 and ER-60. Analyses with RT-PCR showed that TAP-1, TAP2, LMP-2, LMP7, LMP10, tapasin and calnexin mRNA specific for these genes was absent in metastases produced in immunocompetent mice. In contrast, similar techniques with mRNA preparations obtained from metastatic nodes from immunodeficient mice showed that the mRNA expression level of these genes was highly positive. Interestingly, the MHC class I-positive or negative phenotypes of the metastatic colonies correlated with in vivo immunogenicity. H-2 positive metastasis grew more slowly than the H-2 negative ones when injected intrafootpat in syngeneic immunocompetent animals and were finally rejected. These results provide evidence of the role of T cells in immune surveillance against tumors and identify a mechanism targeted by antitumor T lymphocytes to generate MHC class I-negative tumor escape variants.     

Antigen loss variants of a murine renal cell carcinoma: Implications for tumor vaccination

Vaccination with tumour cells genetically modified to support induction of an immune response either by production of cytokines or expression of co-stimulatory molecules provides a promising therapeutic approach. We have evaluated the efficiency of tumour vaccination using RENCA cells, a renal cell carcinoma of the BALB/c strain, which were stably transfected with MHC class II, B7.1 or both. Tumour growth after vaccination with MHC class II and/or B7.1 transfected RENCA cells was extremely variable, with protection close to 100% after vaccination with some clones and no effect of vaccination with others. To unravel the underlying mechanism, untransfected RENCA cells were cloned, and individual clones were tested for immunogenicity; that cloned RENCA cells varied considerably in immunogenicity. Whereas all clones displayed comparable growth rates in nude mice, some grew very slowly in immunocompetent syngenetic hosts. Vaccination with rapidly growing clones was ineffective and, importantly, this feature remained unaltered by vaccination with MHC class II and/or B7.1 transfected clones. Instead, 8 of 10 mice rejected the parental line after immunisation with a pool of MHC class II and B7.1 transfected clones. Finally, by cloning RENCA cells, we obtained one highly immunogenic clone (P2). Vaccination with this clone led to an individual-specific response, which indicates that during the cloning procedure a new strongly immunogenic entity must have arisen. Taken together, our results indicate that vaccination with MHC II and/or B7.1 transfected tumour cells induces an efficient immune response, but only if the tumour is weakly immunogenic. Since tumours may be composed of clones displaying different antigenicities, it is mandatory to use bulk cell populations for transfection and vaccination. Int. J. Cancer 77:114–122, 1998.© 1998 Wiley-Liss, Inc.     

Differences in the immunogenicity of latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus genomes derived from LMP1-positive and -negative nasopharyngeal carcinoma

We have previously shown that an EBV-encoded latent membrane protein 1 (LMP1) gene derived from a nude mouse-propagated nasopharyngeal carcinoma (NPC) tumor and expressed in nonimmunogenic murine mammary carcinoma S6C cells failed to convey immunogenicity (rejectability) in syngeneic mice, whereas the corresponding B-cell derived LMP1 gene made the mice highly immunogenic. This raised the question of whether LMPL-expressing NPCs have been selected for low immunogenicity at the viral gene expression level. If so, LMP1-negative tumors that carry highly methylated LMP1 regulatory sequences may not have been exposed to a similar immunoselection. In the present study, we have compared LMP1 genes derived from two LMP1-positive NPCs and two LMP1-negative NPCs. All four genes were expressed in S6C cells in parallel with the previously tested isolates from a B-cell (B95-8)-derived and a nude mouse-propagated NPC (Cao)-derived gene. As in the previous study, we have found that the B-cell-derived LMP1 isolate was highly immunogenic. LMP1-positive tumor-derived isolates were poorly immunogenic, whereas the isolates from the LMP1-negative NPC tumor had intermediate immunogenicity. Sequence data revealed that LMP1 genes from LMP1-expressing NPC had 16 amino acid substitutions, whereas LMP1 from non-LMP1-expressing NPC had only 9 amino acid changes in the coding region. Three of the changes were at shared sites, but with different modifications. The fact that the gene from non-LMP1-expressing NPC mutated at a low frequency but was more immunogenic than the LMP1 gene derived from LMP1-expressing NPC, which was highly mutated but less immunogenic, favors the idea that LMP1-positive tumors escape immunosurveillance in immunocompetent hosts by either a selective down-regulation of LMP1 expression, methylation in the LMP1 promoter sequence, or mutation of LMP1 in LMP1-expressing samples.     

Effects of mutagens on the immunogenicity of tumor cells: comparison of cell surface changes induced by 5-azacytidine and N-methyl-N'-nitro-N-nitrosoguanidine

The reduced tumorigenicity of mutagenized cells appears to be due to the induction or alteration of cellular determinants leading to a more effective immune recognition. We previously reported that clones of the murine T-lymphoma Eb obtained after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment (Eb-MNNG) show a moderate to strong decrease in tumorigenicity and in addition altered expression of several cell surface proteins. We report here that treatment of Eb cells with 5-azacytidine (5-Aza) induces cell surface changes that are very similar to those seen in MNNG clones; likewise, Eb-5-Aza clones were decreased in tumorigenicity and increased in immunogenicity. Regressor mice that had been inoculated with Eb-5-Aza or Eb-MNNG cells could be shown to be protected against a subsequent challenge of Eb cells but not against the syngeneic mastocytoma P 815. Analysis of cytosine methylation of Eb-MNNG and Eb-5-Aza clones by high pressure liquid chromatography revealed decreased levels of methylcytosine content in several clones. Because of these similarities and because both drugs are DNA hypomethylating and gene inducing agents we assume that hypomethylation is involved in causing amplified protein expression. The possible role of amplified proteins in increased immunogenicity is discussed.     

Expression of H-2 antigens and inducibility of antitumor immune responses in various tumor cell clones established from methylcholanthrene-induced fibrosarcomas

A large number of fibrosarcoma cell lines was established in vitro from a tumor mass induced freshly by inoculating 3-methylcholanthrene (MCA) subcutaneously (sc) into C3H/HeN mice, and more than five clones were isolated from each cell line by the limiting dilution technique. The present study investigated a) qualitative and quantitative comparison of the immunogenicity [tumor-associated transplantation antigen (TATA) activity] of different tumor clones and b) the relationship between such immunogenicity and the expression of H-2 class I antigens. When TATA were compared between different clones from the same tumor, these TATA were revealed to be cross-reactive to each other. On the other hand, the comparison of TATA between clones from different tumors demonstrated the existence of individually unique TATA in these clones. In addition to qualitative heterogeneity of TATA from different tumors, the magnitude of immunogenicity was also heterogeneous in the individual clones established. Whether or not such quantitative heterogeneity of immunogenic strength was related to the expression of H-2 (class I) antigens was examined by flow microfluorometry studies using anti-H-2k antibodies. The results demonstrated that there was no correlation between TATA activity capable of inducing in vivo tumor resistance and the expression of H-2 antigens. This contrasted with parallelism between the expression of H-2 antigens and inducibility of cytotoxic T lymphocytes (CTL) or lysability of tumor cell clones by CTL. These results are discussed in the context of the cellular mechanism of tumor cell eradication in vivo and the regulation of cell surface H-2 expression in vitro and in vivo.     

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)     

Immunological characteristics of immunogenic variants induced in the MCA-F murine fibrosarcoma using 1-methyl-3-nitro-1-nitrosoguanidine, 5-aza-2'-deoxycytidine, or ultraviolet radiation

The purpose of this study was to compare the frequency of generation and in vivo cross-reactivity of highly immunogenic (Imm+) clones induced in a single parental murine fibrosarcoma cell line MCA-F by 4 weekly treatments with either UV-B radiation, 1-methyl-3-nitro-1-nitrosoguanidine, or 5-aza-2'-deoxycytidine. These agents are believed to induce Imm+ variants by different mechanisms. The frequency of Imm+ variant generation was similar for the three different protocols, suggesting that the frequency of Imm+ generation was related more closely to the cell line than the inducing agent used. The strength of the immunogenic phenotype, however, was better correlated to the agent used, since 1-methyl-3-nitro-1-nitrosoguanidine yielded clones with the strongest immunogenicities. Three of four UV-B-induced Imm+ clones grew preferentially in chronically UV-irradiated syngeneic mice, a phenotype associated with UV-induced skin tumors. Cross-reactivity was tested with two Imm+ clones from each treatment group in a modified immunoprotection assay that selectively engendered antivariant, but not antiparental, immunity. Under these conditions each clone, except one, protected against itself. The clones displayed a complex pattern of cross-protection. Intervariant cross-protection was sensitive to the challenge dose, suggesting possible differences in the strengths of the cross-reacting immunities. Conversely, parental cross-protection was observed only with high immunizing multiplicities of Imm+ cells. The clones expressed the Imm+ phenotype in both C3H/HeN and C3H/HeJ mice, suggesting that expression of mammary tumor virus antigens did not account for the strong antitumor immune response. We also investigated whether the level of major histocompatibility complex class 1 or class 2 expression and immunogenic phenotype were correlated. Flow cytofluorography using haplotype-specific anti-Kk and anti-Dk monoclonal antibodies did not reveal a consistent difference in the constitutive or gamma-interferon-induced class 1 expression by Imm+ clones. However, we did observe a significant increase in the constitutive expression of IAk by most of the Imm+ variant clones. Together, these data demonstrate that in this system Imm+ variants engendered by a variety of mechanisms can express a range of cross-reactive tumor rejection neoantigens, independent of parental tumor antigens or major histocompatibility complex antigen expression.     

Immunoresistant metastatic tumor variants can re-express their tumor antigen after treatment with DNA methylation-inhibiting agents

Immuno-escaping variants which arise during metastasis of ESb lymphoma cells in syngeneic DBA/2 mice have been shown to exhibit selective resistance to lysis by ESb-specific cytotoxic T-lymphocytes (CTL). The immuno-resistant variants present no changes in the expression of H-2Kd molecules which appear to be the restricting elements for ESb-specific CTL. We now show that treatment of clonal immuno-resistant ESb variant cells with MNNG or 5'azacytidine can restore the sensitivity to tumor-specific CTL lysis in a high percentage of cloned progenitor cells. The acquisition of susceptibility to lysis by these clones is most likely due to re-expression of ESb-type tumor antigens because such cells regain the capacity to compete with original 51Cr-labelled ESb cells for lysis by ESb-specific CTL, and regain the capacity to induce ESb-specific CTL in vivo. Our data suggest that the immuno-resistant variants are not cellular mutants but rather gene regulatory variants. This could explain: their high frequency of occurrence during metastasis; the relative stability of the variant phenotype; and the reversibility observed after the use of DNA-demethylating and gene-activating drugs like 5'-azacytidine or MNNG.     

Induction in a murine tumor of immunogenic tumor variants by transfection with a foreign gene

Transfection of the undifferentiated murine colon carcinoma line CT-26 with the gene coding for the hemagglutination antigen (HA) of influenza virus resulted in the generation of highly immunogenic tumor cells. CT-26 cells transfected with HA not only failed to grow in syngeneic mice but also protected normal animals against a challenge with otherwise lethal doses of parental nontransfected cells. The immunogenicity of HA-transfected cells appeared to correlate with surface HA expression in that tumorigenic clones of HA-transfected CT-26 cells expressed little HA, while immunogenic clones were high expressers of HA. Irradiation of immunogenic HA clones did not abrogate their immunogenicity. These observations demonstrate that immune recognition of a poorly immunogenic tumor can be produced by immunization with tumor cells expressing a defined, foreign cell surface antigen.