Resistance to 402AX teratocarcinoma involves immunity to minor histocompatibility antigens

The 402AX teratocarcinoma is a 12/J-derived mouse major histocompatibility complex (MHC) antigen negative tumor that is induced to express H-2^b class I antigens during rejection. Resistance to 402AX by MHC allogeneic and syngeneic mice is immunologically mediated and involves the recognition of tumor-associated antigens (TAA) in the context of induced MHC class I antigens. The current studies were undertaken to define the 402AX TAAs. Reconstitution of irradiated susceptible hosts (129/J) with 402AX-primed resistant spleen cells (C57BL/6) results in acute graft-versus-host disease, suggesting that tumor-primed C57BL/6 splenocytes are reactive to tumor genotype (129/J) minor histocompatibility (Hm) antigens. C57BL/6 anti-129/J effector cells, although not directly cytotoxic for 402AX cells, are specifically cold target inhibited by 402AX cells. Genetically susceptible hosts (C3H.SW) immunized to 129/J Hm antigens by skin grafting become resistant to an i.p. challenge of 402AX cells. These results suggest that 129/J Hm antigens may be the TAAs recognized during genetically controlled rejection of the 402AX teratocarcinoma.     

402AX teratocarcinoma MHC class I antigen expression is regulated in vivo by Lyt 1, Lyt 2, and L3T4 expressing splenic T cells

The murine 402AX teratocarcinoma is a MHC class I antigen negative tumor of 129 strain origin. Host resistance to the 402AX tumor is genetically controlled. When passed intraperitoneally in genetically resistant mice, the tumor cells are induced to express MHC Class I antigens of the 129 genotype. When passed in genetically susceptible mice, the tumor cells remain MHC class I antigen negative. Earlier studies have demonstrated that resistance to the tumor and regulation of tumor cell MHC class I antigen expression are under the control of the host's immune system. The present studies indicate that splenic Lyt 1-, Lyt 2-, and L3T4-expressing cells regulate tumor cell MHC class I antigen expression, and that these cells require a genetically resistant host environment in which to differentiate. Splenic T cells primed to the 402AX tumor and transferred into genetically susceptible 129 mice give rise to GVHD, suggesting that immunity to the tumor involves reactivity to 129 minor histocompatibility antigens.     

Teratocarcinoma cell MHC antigen expression is regulated in vitro by a soluble noninterferon factor

The 402AX murine teratocarcinoma is a spontaneous testicular tumor of 129 (H-2b) origin which does not express MHC encoded antigens. Rejection of this tumor is immunologically mediated and the tumor cells are induced in vivo to synthesize H-2b antigens when passaged in genetically resistant host mice. The present studies demonstrate that serum from tumor primed genetically resistant host mice can induce tumor cell MHC antigen expression in vitro as measured by indirect immunofluorescence using monoclonal antibodies. The inducing factor is specific for 402AX tumor cells and is not interferon as shown by the lack of response of the 402AX tumor to gamma interferon, and the absence of significant interferon activity in inducer serum. These studies demonstrate another factor independent of interferon that can induce MHC class I antigen expression on tumor cells.     

H-2Kb antigen expression has no effect on natural killer susceptibility and tumorigenicity of a murine hepatoma

Recent reports suggested a correlation between decreased expression of tumor cell MHC class I Ag and increased susceptibility to NK cells. These studies led to the hypothesis that tumor cells displaying reduced levels of MHC class I Ag have reduced tumorigenicity in vivo because they are eliminated from the host by endogenous NK cells. The present studies use the murine hepatoma BW7756 and a spontaneous H-2Kb loss variant, Hepa-1, to test this hypothesis. The parental BW7756 tumor is highly malignant in syngeneic C57L/J hosts while Hepa-1 cells do not give rise to tumors, suggesting that the loss of H-2Kb Ag expression correlates with decreased tumorigenicity and NK susceptibility. Hepa-1 cells were therefore transfected with an H-2Kb gene to generate H-2Kb Ag expressing clones. The resulting clones were tested for tumorigenicity. Syngeneic or NK-deficient C57BL/6-beige/beige mice challenged with Hepa-1 or the H-2Kb transfectants rejected the cells, suggesting that reexpression of H-2Kb Ag does not restore tumorigenicity and that NK cells are not involved in Hepa-1 rejection. In vitro H-2Kb Ag-negative and -positive Hepa-1 cells are equally susceptible to tilorone-boosted NK cells, indicating that MHC class I Ag expression also does not affect in vitro NK susceptibility. Tumor challenged athymic nude and sublethally irradiated syngeneic mice develop tumors demonstrating that T cells are probably responsible for rejection of the Hepa-1 tumor, and that H-2Kb Ag expression has no effect on rejection. Inasmuch as the expression of H-2Kb Ag on Hepa-1 cells does not effect tumorigenicity or in vitro NK susceptibility, the previously reported association between reduced MHC class I Ag levels and increased NK susceptibility is not universally applicable.     

Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.     

Mouse Hepa-1 tumor is rejected by H-2Db-restricted CTL despite decreased MHC class I antigen expression

It has recently been hypothesized that tumor cells with reduced levels of MHC class I antigens are more susceptible to NK-mediated lysis and are rejected by NK cells, whereas tumor cells with normal levels of class I are rejected by tumor-specific CTL. We have tested this hypothesis using a mouse hepatoma system. The Hepa-1 tumor is a spontaneous H-2Kb loss variant that arose from the BW7756 tumor, when BW7756 was adapted to growth in culture. Our studies have shown that despite the loss of H-2Kb antigen, Hepa-1 is not more susceptible to NK lysis than its H-2Kb-transfected variants. These studies also suggested that NK cells were not responsible for rejection of the Hepa-1 tumor. The Hepa-1 tumor, therefore, appears to contradict the hypothesized linkage of MHC levels and NK susceptibility. Because NK cells are not involved in immunity to this tumor, we have sought to identify the effector cell responsible for Hepa-1 rejection. Cytotoxic T lymphocyte assays demonstrate that in vitro, Hepa-1 cells are lysed by Hepa-1-specific H-2Db-restricted CD4-CD8+ T lymphocytes. Footpad assays demonstrate that in vivo, Hepa-1 rejection requires CD4+CD8- and CD4-CD8+ Hepa-1-primed splenocytes. These results indicate that immunity to Hepa-1 is T cell mediated. Hepa-1 is therefore an example of an unusual tumor in that down-regulation of MHC class I antigen expression is associated with increased CTL susceptibility.     

Transplantation of cultured thymic fragments. VI. H-2 recombinant donors

Athymic (nude) mice were transplanted with cultured thymic fragments from syngeneic, allogeneic, and partially allogeneic (recombinant) mice. Lymphocyte proliferation and cytotoxicity in vitro were measured to assess immunologic reconstitution. Transplanted nude mice were immunocompetent whether donor and recipient were disparate for class I, class II, or both H-2 gene types. Furthermore, allotolerance for thymic H-2 class I antigens was achieved independently of class II antigen allotolerance. Class I antigen tolerance was not broken during lymphocyte responses to unrelated alloantigens, ruling out insufficient help as the tolerance mechanism. Splenocytes, isolated from nude mice transplanted with fully allogeneic or syngeneic thymic fragments and stimulated in vitro with trinitrophenyl-modified cells, displayed H-2-restricted, hapten-specific cytotoxicity. Cytotoxic cells from allotolerant mice were restricted to either host or thymic H-2 antigens, depending on the stimulating cell haplotype. Response levels for thymic and host trinitrophenyl-modified cells were comparable. We have shown that allogeneic thymic epithelium transplanted into adult nude mice can induce allotolerance to class I and II H-2 antigens equally, and permits T lymphocyte interaction with cells bearing thymic donor or host H-2 antigens. Our results are consistent with a model wherein T lymphocyte self-receptors retain their genomic repertoire but can be selectively mutated or expanded by appropriate H-2 antigen presentation by the thymus.     

Altered donor and recipient Ly49+ NK cell subsets in allogeneic H-2d --> H-2b and H-2b --> H-2d bone marrow chimeras

NK cells reject non-self hematopoietic bone marrow (BM) grafts via Ly49 receptor-mediated MHC class I-specific recognition and calibration of receptor expression levels. In this paper we investigated how Ly49+ subset frequencies were regulated dependent on MHC class I expression. The development of donor and host Ly49A+ (recognizes H-2Dd and H-2Dk ligands) and Ly49C/I+ (Ly49CBALB/c recognizes H-2Kb, H-2Kd, and H-2Dd, and Ly49CB6 recognizes only H-2Kb) NK cell frequencies were monitored for 120 days in murine-mixed allogeneic BM chimeras. C57BL/6 (H-2b) BM was transplanted into BALB/c (H-2d) mice and vice versa. Peripheral NK cell populations were examined every 5 days. Chimerism was found to be stable with 80-90% donor NK cells. In contrast to syngeneic controls reexpressing pretransplant patterns, donor and host NK cells revealed new and mainly reduced subset frequencies 55 days after allogeneic transplantation. Recipient NK cells acquired these later than donor NK cells. In H-2d --> H-2b chimeras Ly49A+, Ly49C/I+, and Ly49A+/Ly49C/I+ proportions were mainly diminished upon interaction with cognate ligands. Also in H-2b --> H-2d chimeras, Ly49A+ and Ly49A+/Ly49C/I+ subsets were reduced, but there was a transient normalization of Ly49C/I+ proportions in the noncognate host. After 120 days all subsets were reduced. Therefore, down-regulation of developing Ly49A+ and Ly49C/I+ chimeric NK cell frequencies by cognate ligands within 7-8 wk after BM transplantation may be important for successful engraftment.     

Rejection of allogeneic tumor is not determined by host responses to MHC class I molecules and is mediated by CD4-CD8+ T lymphocytes that are not lytic for the tumor

In previous studies, the murine SaI (A/J derived, KkDd) sarcoma was transfected with the allogeneic MHC class I H-2Kb gene, and expressed high levels of H-2Kb antigen. Contrary to expectations, the tumor cells expressing the alloantigen (SKB3.1M tumor cells) were not rejected by autologous A/J mice. Because these results contradict the laws of transplantation immunology, the present studies were undertaken to examine the immunogenicity of SKB3.1M and SaI cells in allogeneic hosts. Similar to SKB3.1M, SaI cells are lethal in some allogeneic strains, despite tumor-host MHC class I incompatibilities. Tumor challenges of SKB3.1M and SaI cells, however induce MHC class I-specific antibodies and CTL in both tumor-resistant and -susceptible hosts. Although the tumors induce specific CTL, tumor cells are not lysed in vitro by these CTL, suggesting that the tumor cells are resistant to CTL-mediated lysis. Since growth of these tumors does not follow the classical rules of allograft transplantation, and because the tumor is not susceptible to CTL-mediated lysis, we have used Winn assays to identify the effector lymphocyte(s) responsible for SaI rejection. Depletion studies demonstrate that the effector cell is a CD4-CD8+ T lymphocyte. Collectively these studies suggest that the host's response to MHC class I alloantigens of SKB3.1M and SaI cells does not determine tumor rejection, and that effector cells other than classically defined CTL, but with the CD4-CD8+ phenotype, can mediate tumor-specific immunity.     

Tumor rejection mediated by transfection with allogeneic class I histocompatibility gene

Non-self class I histocompatibility Ag can act as strong alloantigens and be recognized as distinct targets by CTL. To study the possibility of using allograft rejection to generate tumor-specific immunity, we have introduced an allogeneic class I histocompatibility gene, the H-2Kb gene, into a k haplotype tumor, K36.16, by DNA-mediated gene transfer. The K36.16 tumor grows readily and does not confer protective immunity in AKR mice. A total of 37 H-2Kb-transfected K36.16 clones (Kb/K36.16) was isolated and studied individually. The Kb/K36.16 clones were found to differ significantly in the amount of the exogenous H-2Kb antigens expressed on their cell surface. Moreover, as a result of the transfection, the level of expression of the endogenous H-2Dk Ag was also altered when compared to that of the parental K36.16 tumor cells. All the Kb/K36.16 clones that were positive for the H-2Kb Ag were rejected by the semisyngeneic AKR mice. Moreover, some of these Kb/K36.16 clones were also rejected by syngeneic (AKR x C57BL/10)F1 mice. In consequence of immunization with the Kb/K36.16 clones, the AKR and F1 mice were able to survive a subsequent challenge of the wild-type, unmodified, parental K36.16 tumor cells. More importantly, some of these Kb/K36.16 clones demonstrated an active and specific immunotherapeutic effect, and they were able to eradicate the growth of the parental K36.16 tumor cells in AKR mice. This observation therefore reinforces the feasibility of using DNA-mediated gene transfer as a molecular approach to abrogate tumor growth.     

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.     

NK-mediated elimination of mutant lymphocytes that have lost expression of MHC class I molecules

Mutant cells generated in vivo can be eliminated when mutated gene products are presented as altered MHC/peptide complexes and recognized by T cells. Diminished expression of MHC/peptide complexes enables mutant cells to escape recognition by T cells. In the present study, we tested the hypothesis that mutant lymphocytes lacking expression of MHC class I molecules are eliminated by autologous NK cells. In H-2b/k F1 mice, the frequency of H-2Kb-negative T cells was higher than that of H-2Kk-negative T cells. The frequency of H-2K-deficient T cells increased transiently after total body irradiation. During recovery from irradiation, H-2Kk-negative T cells disappeared more rapidly than H-2Kb-negative T cells. The disappearance of H-2K-deficient T cells was inhibited by administration of Ab against asialo-GM1. H-2Kk-negative T cells showed higher sensitivity to autologous NK cells in vitro than H-2Kb/k heterozygous or H-2Kb-negative T cells. Adding syngeneic NK cells to in vitro cultures prevented emergence of mutant cells lacking H-2Kk expression but had little effect on the emergence of mutant cells lacking H-2Kb expression. Results in the H-2b/k F1 strain correspond with the sensitivity of parental H-2-homozygous cells in models of marrow graft rejection. In H-2b/d F1 mice, there was no significant difference between the frequencies of H-2Kb-negative and H-2Kd-negative T cells, although the frequencies of mutant cells were different after radiation exposure among the strains examined. H-2b/d F1 mice also showed rapid disappearance of the mutant T cells after irradiation, and administration of Ab against asialo-GM1 inhibited the disappearance of H-2K-deficient T cells in H-2b/d F1 mice. Our results provide direct evidence that autologous NK cells eliminate mutant cell populations that have lost expression of self-MHC class I molecules.     

Alloantigenic sites on class I major histocompatibility complex antigens: 61-69 region in the first domain of the H-2Kb molecule induces specific antibody and T cell responses

The most polymorphic residues in the first domain of class I major histocompatibility complex (MHC) molecules are in the 61-69 region. We have chosen the H-2Kb molecule for determining the role of this region in the induction of alloimmune responses. A synthetic peptide, Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly corresponding to this region was synthesized. T cells enriched from the lymph nodes of allostrain mice that were previously primed with H-2Kb containing cells or with the synthetic peptide in complete Freund's adjuvant undergo extensive in vitro proliferation in response to the synthetic (61-69)H-2Kb peptide. The response was dependent on the presentation of the (61-69)H-2Kb peptide by the syngeneic antigen-presenting cells and was blocked by anti-class II MHC monoclonal antibodies. This peptide fragment of class I MHC molecule activates only helper/inducer type T cells that are involved in the primary responses but not the effector cytotoxic T cells. When coupled to a carrier protein, (61-69)H-2Kb peptide induced antibodies in allostrain mice that bind to intact H-2Kb molecule. No antibodies or T cell responses could be induced in syngeneic H-2b mice. The antigenic site on the H-2Kb molecule recognized by two H-2Kb-specific monoclonal antibodies B8 X 3 X 24 and Y-25 was also mapped in the 61-69 region by direct binding to the synthetic peptide. Therefore the 61-69 region on the H-2Kb molecule represents the first defined sequence on a class I molecule that is directly involved in the induction of alloimmune responses.     

Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.     

Cell-mediated lympholysis responses against autologous cells modified with haptenic sulfhydryl reagents. IV. Self-determinants recognized by wild-type anti-H-2Kb and H-2Db-restricted cytotoxic T cells specific for sulfhydryl and amino-reactive haptens are absent in certain H-2 mutant strains

Virus-specific H-2-restricted cytotoxic T cells (CTL) have been found to discriminate between wild-type and mutant class I molecules. The only results reported concerning a hapten-self model, however, indicate that TNP-specific CTL do not discriminate between wild-type and mutant self determinants (7). In the present study, hapten-specific CTL generated against N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine-modified syngeneic cells (AED-self) were used to determine whether a hapten that is known to react with different cell surface sites than TNP can induce CTL that distinguish mutant H-2K and D molecules from those of wild type. The findings of this study indicate that H-2Kb-AED-self cytotoxic effector cells can discriminate between self-determinants of H-2Kb wild-type and the H-2bm1 and H-2bm11 mutants, but not between wild-type and the H-2bm6 and H-2bm9 mutants. H-2Db-AED-self effector cells were also found to discriminate between self-determinants of H-2Db wild-type and the H-2bm13 and H-2bm14 mutants. Furthermore, cold target competition experiments indicated that the bm1 and bm11 Kb products also lack some determinants recognized by anti-wild-type Kb TNP-specific CTL. These findings provide the first demonstration that hapten-self-specific effectors can detect alterations in H-2 mutant class I molecules. The results in the present report also support the hypothesis that haptens do not have to derivatize H-2 molecules in order to form antigens recognized by H-2-restricted CTL. These findings are discussed with respect to the involvement of self-determinants on MHC and non-MHC cell surface molecules.     

Co-expression of immunogenic determinants by the same cellular immunogen is required for the optimum immunotherapeutic benefit in mice with melanoma

 Tumor-associated T cell epitopes are recognized by T cells in the context of determinants specified by class I loci. Since the rejection of foreign histocompatibility antigens is known to enhance tumor immunity, immunization with a cellular vaccine that combined the expression of both syngeneic and allogeneic class I determinants could have important immunological advantages over a vaccine that expressed either syngeneic or allogeneic determinants alone. To investigate this question in a mouse melanoma model system, we tested the immunotherapeutic properties of B16 melanoma × LM fibroblast hybrid cells in C57BL/6J mice with melanoma. Like C57BL/6J mice, B16 cells expressed H-2K^b class I determinants and (antibody-defined) melanoma-associated antigens. LM cells, of C3H mouse origin, formed H-2K^k determinants along with B7.1, a co-stimulatory molecule that can activate T cells. The B16 × LM hybrid cells co-expressed H-2K^b and H-2K^k class I determinants, B7.1 and the melanoma-associated antigens. C57BL/6J mice with melanoma, immunized with the semi-allogeneic hybrid cells, developed CD8-mediated melanoma immunity and survived significantly (P<0.005) longer than mice with melanoma immunized with a mixture of the parental cell types. The failure of melanoma immunity to develop in mice injected with the mixture of parental cells indicated that co-expression of the immunogenic determinants by the same cellular immunogen was necessary for an optimum immunotherapeutic effect. Augmented immunity to melanoma in mice immunized with the semi-allogeneic hybrid cells points toward an analogous form of therapy for patients with melanoma. Received: 19 May 1997 / Accepted: 23 July 1997     

Failure or success in the restoration of virus-specific cytotoxic T lymphocyte response defects by dendritic cells

C57BL/6 (B6, H-2b) mice are CTL responders to both Sendai virus and Moloney leukemia virus. In the former response the H-2Kb class I MHC molecule is used as CTL restriction element, in the latter response the H-2Db molecule. B6 dendritic cells (DC) are superior in the presentation of Sendai virus Ag to CTL in comparison with B6 normal spleen cells. Con A blasts have even less capacity to present viral Ag than NSC, and LPS blasts show an intermediate capacity to present viral Ag. H-2Kb mutant bm1 mice do not generate a CTL response to Sendai virus, but respond to Moloney leukemia virus, as demonstrated by undetectable CTL precursors to Sendai virus and a normal CTL precursor frequency to Moloney virus. Compared to B6 mice, other H-2Kb mutant mice show decreased Sendai virus-specific CTL precursor frequencies in a hierarchy reflecting the response in bulk culture. The Sendai virus-specific CTL response defect of bm1 mice was not restored by highly potent Sendai virus-infected DC as APC for in vivo priming and/or in vitro restimulation. In mirror image to H-2Kb mutant bm1 mice, H-2Db mutant bm14 mice do not generate a CTL response to Moloney virus, but respond normally to Sendai virus. This specific CTL response defect was restored by syngeneic Moloney virus-infected DC for in vitro restimulation. This response was Kb restricted indicating that the Dbm14 molecule remained largely defective and that a dormant Kb repertoire was aroused after optimal Ag presentation by DC. In conclusion, DC very effectively present viral Ag to CTL. However, their capacity to restore MHC class I determined specific CTL response defects probably requires at least some ability of a particular MHC class I/virus combination to associate and thus form an immunogenic complex.     

The unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not due to a lack of H-2 antigen expression.

It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.