Expression of embryonic and MHC antigens in heterokaryons of murine embryonal carcinoma and human melanoma cells

Mouse embryonal carcinoma cells were fused with human melanoma cells or with cytoplasts of these cells. The expression of embryonic and major histocompatibility complex (MHC) antigens was studied in single heterokaryons and cybrids in the population after fusion. Recognition of heterokaryons by differential staining of mouse and human nuclei was combined with indirect immunofluorescent staining of specific membrane antigens. Complete suppression of embryonic antigen expression was found in heterokaryons within 2 days after fusion. Cybrids, formed by fusion of embryonal carcinoma cells with melanoma cytoplasts, showed a transient decrease in the expression of embryonic antigens. The expression of human MHC antigens, both class I (HLA-A, B, C) and class II (HLA-DR), was only slightly influenced in heterokaryons. No activation of mouse MHC antigens was found. The results indicate that melanoma cells contain trans-acting factors exerting a negative control on the expression of embryonic antigens. In contrast the continued expression of human MHC antigens in heterokaryons suggests that embryonal carcinoma cells either are devoid of or contain only a very limited amount of trans-acting factors controlling the expression of MHC antigens.     

Expression of MHC antigens by mouse thymic dendritic cells.

Thymic epithelial cells express MHC antigens in several different patterns. I-A is present throughout the thymic cortex on dendritic cells. The remainder of the I region and H-2K/D are expressed on dendritic cells apparently only variably in the cortex (at least in some haplotypes). All MHC antigens tested are present in the medulla on epithelial cells; expression on medullary lymphocytes cannot be evaluated. Monoclonal anti-MHC antibodies confirm these results. The significance of these findings to T cell maturation is discussed.     

Expression of interferon genes in murine macrophages: Possible role of endogenous interferon in the modulation of cell differentiation

The expression of interferon (IFN)- gene was studied in mouse peritoneal macrophages (PM) harvested from normal mice (lps ⁿ ) or LPS-hyporesponsive mice (lps ^d ). A strong direct correlation between the LPS response of PM and their capacity to expressing basal levels of IFN was found. The results suggest that the constitutive expression of IFN- gene can play an important role not only in the resistance to viral infection but also in the modulation of cell differentiation.     

Expression of CD1 and class I MHC antigens by human thymocytes

The acquisition of surface class I MHC molecules is associated with the maturation of thymocytes. Here, surface expression of class I MHC and CD1, which represents a family of MHC-related molecules, was analyzed on various human immature and mature thymocyte subpopulations. Class I expression was inversely related to the expression of CD1. The majority of CD4+ CD8+ cortical type thymocytes expressed low levels of class I MHC Ag, the previously described CD4+ CD8+ thymocyte subpopulation with low CD8 expression exhibited intermediate levels of class I MHC, whereas most of the single positive CD4 and CD8 thymocytes displayed high levels of class I MHC. Biochemical comparison of CD1 and class I showed that thymic class I molecules were post-translationally modified by phosphorylation, whereas CD1 was not phosphorylated. Furthermore, our studies suggested that in addition to CD1/CD8 complexes, thymocytes bear CD8/class I complexes. Chemical cross-linking and peptide mapping studies clearly identified the CD8-associated protein on thymic clones as the class I MHC molecule.     

Expression of ia antigens by murine keratinizing epithelial langerhans cells

Immunofluorescent and immunoelectron-microscopic staining methods were utilized to investigate the localization of Ia antigens in murine keratinizing epithelia. Approximately 3-5% of epidermal cells were shown to be Ia positive. Only dendritic Langerhans cells in the interfollicular epidermis and outer root sheaths were found to express Ia antigens. These Ia determinants were shown to be controlled by both theI- A andI- EC subregions of theH-2 complex. The results were confirmed by identifying positively stained cells containing Langerhans cell granules at the ultrastructural level. No staining was noted on the surface of keratinocytes, melanocytes, or immigrant lymphocytes. The results presented are in close agreement with those previously reported for Ia-bearing Langerhans cells in human and guinea pig epidermis.     

Localization of type I interferon in murine trophoblast and decidua during decidual formation.

Mouse trophoblast and decidua were examined by means of immunohistochemistry to define the localization of type I interferon. The decidua were stained for type I interferon at the time of implantation. The strong reaction was first observed in the primary decidual zone on day 5 and subsequently in the secondary decidual zone on day 6. After day 10, the decidua basalis and decidua capsularis showed a strong reaction. At the one-cell stage, embryos were weakly labelled, but a positive reaction was recognized in compacted morulae. Blastocysts on days 3 and 4 were positive in trophoblast and inner cell mass and a strong reaction was observed in the primitive endoderm on day 4. The visceral endoderm on day 5 and the trophoblast on day 6 were positive. After day 10, the trophoblast giant cells, labyrinth, visceral yolk sac and fetal blood cells gave a positive reaction. This study is the first demonstration of type I interferon localization in situ in mouse trophoblast and decidua during decidual formation.     

Immunochemical analysis of the modulation of human melanoma-associated antigens by DNA recombinant immune interferon

By utilizing the human melanoma cell line Colo 38, a panel of monoclonal antibodies, and a combination of serologic and immunochemical assays, the effect of recombinant immune interferon (IFN-gamma) on the synthesis, expression, and shedding of a cytoplasmic melanoma-associated antigen (MAA) and of the membrane-bound high m.w. MAA (HMW-MAA), 115K MAA, and 100K MAA has been investigated. IFN-gamma increased the synthesis and shedding of the cytoplasmic MAA, but reduced the synthesis and cell surface expression of the HMW-MAA and of the 100K MAA. The cell surface expression of the 115K MAA on IFN-gamma-treated melanoma cells was reduced, although its synthesis was not markedly changed. The effects were dose-dependent and were related to the incubation time of cells with IFN-gamma. Among the three membrane-bound MAA analyzed, the 100K MAA was the most susceptible to modulation by IFN-gamma. The effects of IFN-gamma preparations are not mediated by contaminants in IFN-gamma preparations because removal of IFN-gamma by affinity chromatography on anti-IFN-gamma monoclonal antibodies abolished its modulating activity. The effects of IFN-gamma on the cytoplasmic MAA are similar to those of leukocyte and fibroblast interferons, whereas those on the membrane-bound MAA are significantly different. The potential implications of the marked changes in the antigenic profile of melanoma cells treated with IFN-gamma are discussed in view of the changes in the immunogenicity of IFN-gamma-treated melanoma cells.     

Induction and functional characterization of class II MHC (Ia) antigens on murine keratinocytes

To induce Ia molecules on the surface of murine keratinocytes (KC), healthy mice were treated daily with i.p. injections of rIFN-gamma at a dose of 50,000 U/day for 6 days. This resulted in strong Ia expression by KC as determined by immunofluorescence of epidermal sheets or cell suspensions with anti-class II mAb. To obtain a population of Ia-bearing KC devoid of Langerhans cells, a method of depleting Langerhans cells from such suspensions was developed. Although Ia+ KC were unable to stimulate allogeneic T cells in a primary epidermal cell-lymphocyte reaction (less than 5% control), they did induce a proliferative response in an allospecific T cell line. Ia+ KC were unable to present native peptide molecules to class II restricted, Ag-specific T cell hybridomas. However, Ia+ KC were able to present a peptide fragment of pigeon cytochrome c to a hybridoma, suggesting that although these cells cannot process native protein Ag, they can present antigenic peptides. Ia+ (but not Ia-) KC also served as targets for class II restricted cytolytic T cell clones. These data indicate that the Ia expressed by KC is a functional molecule, and that Ia+ KC can participate in some immunologic reactions.     

Expression of MHC class I, MHC class II, and cancer germline antigens in neuroblastoma

Background: Neuroblastoma is the most common solid extracranial tumor in childhood, still with poor survival rates for metastatic disease. Neuroblastoma cells are of neuroectodermal origin and express a number of cancer germline (CG) antigens. These CG antigens may represent a potential target for immunotherapy such as peptide-based vaccination strategies. Objective: The purpose of this study was to analyze the presence of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 on an mRNA and protein level and to determine the expression of MHC class I and MHC class II antigens within the same tumor specimens. Methods: A total of 68 tumors were available for RT-PCR, and 19/68 tumors were available for immunohistochemical (IHC) analysis of MAGE-A1, MAGE-A3/A6, and NY-ESO-1. In parallel, the same tumors were stained with a panel of antibodies for MHC class I and MHC class II molecules. Results: Screening of 68 tumor specimens by RT-PCR revealed expression of MAGE-A1 in 44%, MAGE-A3/A6 in 21%, and NY-ESO-1 in 28% of cases. Immunohistochemistry for CG antigens of selected tumors showed good agreement between protein and gene expression. However, staining revealed a heterogeneous expression of CG antigens. None of the selected tumors showed MHC class I or MHC class II expression. Conclusions: mRNA expression of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 is congruent with the protein expression as determined by immunohistochemistry. The heterogeneous CG-antigen expression and the lack of MHC class I and II molecules may have implications for T-cell–mediated immunotherapy in neuroblastoma.     

Expression of murine Ia antigens during embryonic development.

An immunochemical analysis of the kinetics of appearance of Ia antigens during embryonic development was performed. Ia antigens first appear on the surface of embryonic cells 11 days postconception and their expression between days 11 and 16 of gestation is confined to the fetal liver. Ia antigen synthesis by fetal liver cells is detectable at day 14. Ia seems to precede Ig as a surface marker of embryonic liver cells, since Ig cannot be detected until day 16 of gestation. H-2 antigens may be immunoprecipitated from day 10 whole embryo cells. F9 primitive teratocarcinoma cells are Ia negative and H-2 negative.     

Immunosuppressive properties of murine trophoblast

The modification of the immunological response by murine trophoblast cells of different sources was investigated using the mixed lymphocyte reaction (MLR) and the cell mediated lympholysis (CML) test. MLR between C57BL (H-2b) stimulator splenocytes (mitomycin C treated in the unidirectional MLR) and BALB/c (H-2d) responder lymph node cells were markedly suppressed by trophoblast of ectoplacental cone (EPC) and placental origin. The same in vitro effect was observed with supernatants (SN) of trophoblast cells and with supernatants of blastocysts. Addition of anti-progesterone serum (APS), anti-testosterone serum (RAT), and anti-immunoglobulin serum (RAHIg) in serial dilutions to the trophoblast-MLR system revealed that the immunosuppressive effect of trophoblast giant cells and trophoblast giant cell culture supernatants can be abolished with APS. Identical results were obtained with APS added to immunosuppressive doses of progesterone. CML between C57BL responder lymph node cells and mitomycin C-treated BALB/c stimulator spleen cells was also markedly suppressed when trophoblast of EPC origin was added. A similar suppression of cytotoxic T-cell induction was seen when progesterone was added to the system. The immunosuppressive action of trophoblast as detected in vitro is likely to play an important role in the maintainance of pregnancy by protecting the semiallogeneic conceptus against immune aggression by the maternal immune system.     

Expression of MHC class II antigens in human lung cancer cells.

Surgical specimens of lung cancers were examined immunopathologically for the expression of major histocompatibility complex class II (MHC-II) antigens in the tumor cells and their relationship to the lymphocytic infiltration. A lymphocytic infiltrate was frequently observed in the tumor tissue, though its intensity differed among the various histological types. MHC-II antigens were often demonstrated in tumors with a lymphocytic infiltrate. They were detected predominantly in the cytoplasm of tumor cells and to a lesser extent on the cell membranes. The emergence of the MHC-II-positive tumor cells was closely related to a local infiltration by lymphocytes including interferon-gamma (IFN-gamma)-producing T-cells. On the basis of the histological findings, an in vitro experiment was carried out. Four types of lung cancer cells were incubated with recombinant IFN-gamma in order to induce MHC-II antigens. MHC-II antigens (HLA-DR as well as HLA-DQ and HLA-DP antigens) were elicited in three cancer cell lines depending on the concentration of IFN-gamma. Immunoelectron microscopic study revealed that they were expressed on the surface of the cell membrane, though to a lesser extent than in the cytoplasm. It was considered that MHC-II antigens could be induced in some tumor cells in the immunological environment where IFN-gamma was secreted from T-cells and concentrated locally.     

Oncofoetal antigens of human trophoblast.

Rabbits immunized with human trophoblast cell membranes produced antibodies that were detected, by immunofluorescence, to react with normal human tissues, and, by complement-mediated cytotoxicity, with several transformed human cell lines. Absorption with trophoblast abolished all of these reactions, whereas multiple absorptions with lymphocytes, liver or kidney failed to remove reactivity with either trophoblast or certain transformed cells. To further identify the antigens responsible for these antibodies, rabbits were immunized with a chromatographed fraction of deoxycholate-solubilzed membranes prepared from KCl-extracted, ultracentrifuge-prepared trophoblast microvilli. The resultant IgG antibody reacted specifically with syncytiotrophoblastic membranes in sections of human placentae, in addition to recognizing the membranes of viable Chang liver, AV3, HEp-2, Sw/156 (kidney) and Sw/527 (breast) cells. That normal tissues, baboon or monkey placentae, and HeLa or Daudi cell lines did not react with this antibody, indicates the presence of species- and organ-specific antigens in human trophoblast, as well as the existence of trophoblast cross-reactive antigens on some transformed cells. The selective localization of these antigens at the interface of the materno-foetal graft suggests that they function biologically in the host-parasite relation of human pregnancy; their appearance on many transformed cells implies a similar function in the host-parasite relation of some human cancers.     

Immune interferon modulation of in vitro murine anti-human T cell monoclonal antibody-mediated cytotoxicity

Limited antitumor effects have been achieved in clinical trials with murine monoclonal antibody T101, perhaps because of its limited ability to effect complement-mediated or cell-mediated cytotoxicity. We explored the effects of recombinant immune interferon on T101-mediated cytotoxicity in vitro. Interferon failed to enhance expression of the antigen detected by T101 on target cells, but it did increase Fc receptor binding of T101 and other IgG2A and IgG3 murine proteins, but not IgG1 or IgG2B. Preincubation of U937, HL60, and human mononuclear cells with 100 U of immune interferon for 48 hr, while T101 was preincubated with various T cell line targets or human CLL cells at 4 degrees C for 30 min before combining effectors and targets for 4 hr at 37 degrees C, resulted in cytotoxicity of 18 to 44% of maximum. Cytotoxicity in the absence of interferon or T101 was less than 5%. Unfortunately, rapid modulation of antigen-antibody when T101 was preincubated with targets at 37 degrees C prevented any increase in cytotoxicity under those conditions. We conclude that immune interferon can augment T101-mediated cytotoxicity in vitro, but it is unlikely that it would enhance T101-mediated cytotoxicity via complement or cell-mediated mechanisms in vivo.     

Expression of membrane activation antigens on murine B lymphocytes stimulated with lipopolysaccharide

The expression of two membrane glycoproteins, RL388 antigen and transferrin receptor (TfR), was examined on murine B cells stimulated with lipopolysaccharide (LPS) in vitro. Immunofluorescent staining with monoclonal antibodies and flow cytofluorometric analysis were used to monitor the expression of these markers as a function of the time in culture, the state of membrane Ia antigen expression, the position in cell cycle, and the degree of B-cell differentiation. Freshly explanted splenic B cells expressed low levels of RL388 antigen and TfR. Following LPS stimulation, increased expression of RL388 antigen was detectable by 8 to 12 hr of culture, a time span characterized by increased Ia antigen expression, blast transformation, and G0 to G1 phase transition. The increased expression of TfR was apparent later and correlated with entry into late G1 phase and the onset of S phase. LPS-stimulated cell cultures treated with actinomycin D (G0/G1 block) exhibited increased expression of Ia antigen, but neither RL388 antigen nor TfR, whereas hydroxyurea treatment (G1/S block) allowed expression of all three markers. These results indicate that hyperexpression of RL388 antigen and TfR occurs during G1 phase and that these events are subsequent to Ia antigen hyperexpression. Finally, B cells in late G1 through M phase of the cell cycle simultaneously express high levels of RL388 antigen and TfR. These findings suggest that the expression patterns of RL388 antigen and TfR might be useful parameters for defining compartments of the murine B-cell cycle.     

Phagocytic processing of antigens for presentation by MHC molecules

Phagocytosis plays a major role in the defence of higher organisms against microbial infection not only by allowing ingested microbes to be destroyed by microbicidal mechanisms, but also by providing the basis for processing of their antigens to forms that generate immune responses. This article examines the role of the phagolysosome in antigen processing, and discusses the contributions of both MHC class II and MHC class I molecules to the presentation of antigens derived from phagocytosed material.     

Transplantation antigen expression on murine trophoblast detection by induction of specific alloimmunity

Cell-mediated immune responses to murine embryonic trophoblast cells were investigated using lymphocyte trophoblast cultures (LTC) and cell-mediated lympholysis (CML). Spleen cells from CBA (H-2^k) or C57BL/6 (H-2^b) mice hyperimmunized with 3.5-day-old Balb/c (H-2^d) blastocysts did not undergo DNA synthesis after in vitro exposure to Balb/c blastocyst outgrowths nor were cytotoxic lymphocytes (CTL) generated against H-2^d alloantigens. Splenocytes from Balb/c mice presensitized with semiallogeneic (Balb/c female × C57BL/6 male) trophoblast cells derived from 17- to 20-day placental tissue expressed a weak proliferative response in the presence of semiallogeneic placental trophoblast and produced a moderate number of CTL against H-2^b (paternal strain) alloantigens when compared to mixed lymphocyte cultures (MLC) between Balb/c responder and semiallogeneic (stimulator) spleen cells. CTL were also generated in vitro after splenocytes from Balb/c mice hyperimmunized with semiallogeneic spleen cells were restimulated in vitro with placental trophoblast cells. These studies showing that early-stage trophoblast cells fail to evoke transplantation immunity and placental trophoblast is capable of generating alloimmunity only after combined in vivo hyperimmunization with in vitro restimulation suggest that these trophoblast cells are poorly immunogenic due in part to the relatively weak functional expression of major transplantation antigens.