Tumorigenicity and H-2 expression of papillomavirus-transformed mouse cell lines

Summary Tumorigenicity in immunocompetent syngeneic mice and H-2 class I antigen expression of BPV1-transformed mouse cell lines had no correlation. H-2 expression was examined using monoclonal anti-(H-2K^b) and anti-(H-2D^b) antibodies in immunofluorescence staining for flow cytometry analysis and by determining the sensitivity of the cells to cytolysis by allostimulated spleen cells. Nontumorigenic cell lines were as resistant as tumorigenic cell lines to natural killer activity. The results indicate that in our model defence by natural killer cells is not a decisive factor. The results also show that instead of or in addition to H-2 class I antigens other factors (e. g. the presence or absence of virus-specific antigens) are important in determining the tumorigenicity of BPV1-transformed cell lines.     

Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer

Among the more than 20 H-2-like genes in the BALB/c mouse genome, there are two classical transplantation antigens (H-2Dd and H-2Ld) encoded at the D-end of the major histocompatibility complex. Here we report the identification of a bacteriophage clone that encodes H-2Dd. The H-2Dd gene was identified by nucleotide sequence analysis and by characterization of the new H-2 antigen expressed when the cloned gene was introduced into mouse L cells by DNA-mediated gene transfer. The previously identified H-2Ld gene was then compared with the H-2Dd gene. The two genes appear to have the same general structure, and for the 854 nucleotides that have been compared, the two genes are 89% homologous. The H-2Ld and H-2Dd antigens expressed on mouse L cells after DNA-mediated gene transfer were examined by immunologic criteria. The stably transformed cell lines express apparently normal levels of H-2Dd and H-2Ld on the cell surface as measured by quantitative immunofluorescence by using monoclonal anti-H-2 antibodies. They synthesize H-2Dd and H-2Ld at normal rates as determined by endogenous labeling and immunoprecipitation of cell extracts. They evoke a strong specific serologic response when used to immunize C3H mice. The newly expressed antigens are able to serve as targets for alloreactive T cells. These cloned genes provide good substrates for examining the evolution of two closely linked H-2 antigen genes. Comparison of the structures of these genes provides clues to the basis for the differential expression of these antigens and their different biologic functions.     

Generation of H-2-reactive T cell lines that bear the 5936 idiotype(s)

The present experiments showed 1) that it was possible to produce mouse T cell lines against MHC determinants with a relatively high success rate by stimulation of purified T cells with allogeneic cells in the presence of irradiated syngeneic spleen cells; 2) that these lines could be led to react against selected H-2 specificities; 3) that only T cell lines established from Ig-1b allotype mice contained 5936-Id+ T cells (5936-Idiotypes are defined by an antiserum against B6 anti-CBA IgG produced in rabbit no 5936, which was tolerant to mouse gamma-globulin); and 4) that antigenic determinants coded by IAk genes induce the 5936-Idiotype(s). The latter data are in accordance with the 5936-idiotype characteristics of primary MLC T blasts. All T cell lines contained both specific MLC-responding cells and cytolytic cells. However, studies on the functional capacity of 5936-Id+ T cells from both primary MLC and the T cell lines showed that neither MLC-responding cells nor cytolytic cells directed against H-2Kk, IAk, or H-2Dk were 5936-Id+. Thus, 5936-Id+ T cells may be regulator cells induced by IAk antigens.     

Antibody-induced redistribution of normal and tumor associated surface antigens

Redistribution (capping) of normal and tumor-associated surface antigens was studied on murine and human cells by the indirect membrane immunofluorescence (MIF) technique. The capping of H-2 isoantigens was compared on normal mouse T-lymphocytes and on YAC cells, a Moloney leukemia virus (MLV) induced lymphoma. H-2 and Moloney virus induced cell surface antigen (MCSA) capping was compared on three YAC lines with different MCSA concentrations. H-2 and tumor-associated surface antigen capping was compared on two polyoma induced sarcoma lines and five methylcholanthrene induced sarcoma lines. In the human system, IgM-capping was compared on normal lymphocytes and on the Burkitt lymphoma derived Daudi line. Capping of HL-A and the Epstein-Barr virus (EBV) determined membrane antigen (MA) was compared on the Burkitt lymphoma derived line Maku and on EBV-superinfected Daudi cells. H-2 antigens on normal murine cells capped more promptly and on a larger fraction of the cell population on the various tumor cells. Surface associated IgM showed a better capping on normal lymphocytes than on Daudi cells. All tumor associated antigens except MCSA, showed good capping. MCSA was almost completely refractory to capping. Increasing concentrations of MCSA appeared to inhibit the capping of H-2 on the YAC sublines with different concentrations of MCSA. The polyoma induced ascites sarcoma (SEWA) did not cap either with regard to H-2 or the polyoma determined surface antigen.     

The occurrence and distribution of H-2 antigens on mouse intestinal epithelial cells.

This report describes an immunoferritin labeling study of mouse H-2 histocompatibility antigens on epithelial cells dissociated from the small intestine by EDTA and trypsin. Before cell dissociation, the intestine was prefixed in paraformaldehyde or periodate-lysine-paraformaldehyde in order to preserve the shape of the cells and to immobilize H-2 antigens in their native positions. The results demonstrated the presence of H-2 antigens on the lateral and basal cell membranes at about the same high density that was observed at the surface of mouse monocytes. No H-2 antigens could be detected at the apical surface of dissociated or undissociated epithelial cells. It is unlikely that the fuzzy coat masked H-2 antigens at the apical surface because it was essentially absent from the apical membranes of dissociated cells. These observations extend our knowledge of the cellular distribution of transplantation antigens, and provide further evidence of a discontinuity in the expression of membrane components at the junctional complex of epithelial cells.     

Time course study of H-2 and other antigen expression by hybrids of a myeloma cell line with inflammatory macrophages

The hybrids (the CANS lines) between inflammatory macrophages from C57BL/6N (B6) mice (H-2^b) and BALB/c mouse (H-2^d)-derived myeloma cell line NS1 in the early period after cell fusion showed no macrophage functions. However, most of the hybrids expressed these functions after prolonged cultivation accompanied with chromosome loss. In contrast, the hybrids initially displaying myeloma functions ( light chain production) lost this function when they exhibited macrophage functions. We studied the expression of cell-surface antigens in these hybrids and found that hybrids in the early period after cell fusion codominantly expressed both parental cell H-2 antigens (H-2K^b, H-2K^d, and H-2D^d) but not the H-2D^b antigen. On the other hand, aged hybrids strongly expressed the H-2 d antigen but lacked the H-2K^b antigen. Alternatively, these aged hybrids with macrophage functions expressed antigen(s) as detected with antiaged CANS-196 cell sera and asialo GM1 antigen, both of which were thought to be found exclusively on macrophages. Thus, the expression of cell-surface antigens in these hybrids was greatly altered after cell fusion.     

Characterization of Ia8 antigen, thy-1.2 antigen, complement receptors, and virus production in a group of murine virus-induced leukemia cell lines.

Ia8, a cell membrane antigen controlled by gene(s) located in the I region of the H-2 complex, was found on 9 of 26 murine leukemia cell lines. Iaddition, 3 of the 9 Ia8-bearing lines had a membrane receptor for antigen-antibody-complement complexes. Six of 26 lines bore the Thy-1.2 antigen. Ia8 and Thy-1.2 antigens were mutually exclusive on the cell lines studied. The strain of virus used to induce the leukemia, the H-2 type of the cells, and the techniques of leukemia cell propagation all appeared to influence the antigenic characteristics of the cell lines obtained. Production of infectious murine leukemia virus in vitro and expression of leukemia virus-induced membrane antigens did not appear to correlate with the presence of I8 or Thy-1.2 antigens or with the H-2 type of the cells.     

Independence of H-2 and viral antigens on the cell surface and absence of H-2 antigens on murine leukemia virus and mouse mammary tumor virus particles

By indirect immunoelectron microscopy we tested for the presence of H-2 antigens on murine mammary tumor virus (MMTV) and murine leukemia virus (MuLV) particles. The association of H-2 antigens and viral antigens on the virus-infected cell surface was investigated with antibody-induced redistribution. Mammary tumor cells and leukemia cell lines with different H-2 genotypes and carrying different MuMTV or MuLV were used. No H-2 antigens could be demonstrated on the envelope of MMTV and MuLV particles, even after the permeabilization of their envelopes with saponin. On the surface of virus-infected cells antibody-induced patching or capping of the viral antigens did not result in copatching or cocapping of the H-2 antigens. In the reciprocal tests no co-redistribution of viral antigens with H-2 antigens was seen. Our experiments failed to show any physical association between H-2 antigens and MMTV or MuLV antigens on the cell surface.Abbreviations used in this paper MMTV mammary tumor virus - MuLV murine leukemia virus - MHC major histocompatibility complex - IEM immunelectron microscopy     

Analysis of the negative allogeneic effect using B cells and helper T cell lines as an indicator system: B cells identified as target cells for suppression

The target cells for H-2b T lymphocytes mediating a negative allogeneic effect in vitro were analyzed by using carrier-specific helper T cell lines of H-2b, H-2d, or F1 origin and hapten-primed T-depleted spleen cells also expressing one or both of these haplotypes. The helper T cell lines were shown to be carrier specific and H-2b or H-2d restricted. Most importantly, the lines derived from H-2b homozygous mice were devoid of alloreactivity against H-2d and vice versa. Titration of naive H-2b T lymphocytes to the indicator cultures resulted in suppression of the secondary anti-DNP response of the indicator cells whenever the B cells expressed H-2d antigens. The lack of suppression observed in mixtures in which only the helper T cell lines expressed H-2d antigens was not reversed by the increased addition of naive H-2bxd cells, indicating that an insufficient amount of H-2d antigens present on the low number of helper T cells used did not account for this finding. Moreover, the polyclonal plaque-forming cell responses of F1 spleen cells to LPS were also suppressed by naive parental T cells. From these findings it is concluded that the suppressor T cells directly recognize and inhibit allogeneic B cells without the involvement of helper T cells. In addition, it was shown that the suppression of secondary anti-hapten responses by naive allogeneic T cells is blocked by monoclonal anti-Lyt-2 antibody added at the onset of culture. Addition late in culture had no effect, pointing to a functional role of the Lyt-2-bearing structure at an early stage of the suppressive events resulting in the negative allogeneic effect.     

Inhibition of tumor growth mediated by lymphocytes sensitized in vitro to a syngeneic murine teratocarcinoma 402AX

TerC, a cell line derived from a strain 129 teratocarcinoma 402AX, was used to sensitize syngeneic 129 (H-2bc) splenic lymphocytes in vitro. The effector cells generated inhibited in vitro growth of TerC as measured by an 125I-IUDR ost-labeling technique. It was also shown, with a modified Winn assay, that the sensitized cells were effective in preventing TerC growth in vivo. The effector lymphocyte was nonadherent to nylon wool was sensitive to anti-Thy-1.2 + C, and was phenotypically Ly 1-2+. The anti-TerC effector T lymphocytes were not functional in a 51Cr-release assay. However, this failure to lyse appears not to be due to some intrinsic membrane resistance since both BCG and ConA-activated killers were able to lyse TerC. The TerC-sensitized lymphocytes displayed no H-2 restriction and were able to growth inhibit in vitro a wide range of tumorigenic cell lines, e.g., P815 (H-2d), EL-4 (H-2b),Sal (H-2a), and BALB/c (H-2d) 3T12. Mouse blastocyst cell lines were also inhibited. BALB/c 3T3 and mouse fibroblast cell strains were not growth inhibited. Thus, it appears that oncofetal antigens expressed on TerC are capable of initiating a cell-mediated response and that these antigenic specificities are shared by many transformed cell lines.     

Anti-major histocompatibility complex immunity detected prior to intentional alloimmunization. II. Monoclonal H-2-specific antibodies obtained from an unimmunized C57BL/KaLwRij (H-2b) mouse

A monoclonal 'natural' anti-H-2 IgM antibody produced by a hybridoma cell line OL-3.17 (H-2 m. 209) is described. The OL-3.17 monoclonal antibody was obtained by hybridization of spleen B cells from an unimmunized C57BL/Ka (H-2b) mouse in the serum of which simultaneously an IgM kappa paraprotein of high concentration and a natural H-2-specific antibody of high titer was detected. The monoclonal antibody OL-3.17 reacted strongly with H-2d and H-2s and weakly with H-2k,q,r lymphocytes, thereby detecting a hitherto unknown H-2 public determinant. The target molecules for OL-3.17 cocapped with class-I H-2 antigens, but immunoprecipitation of H-2 antigens was not achieved. This is the first monoclonal H-2-specific antibody obtained from a mouse without intentional immunization and, with high probability, was derived from a B-cell clone which produced natural H-2-specific antibodies detectable in the serum of the original mouse.     

Differential phosphorylation of murine class I major histocompatibility antigens

Recent studies have shown that the H-2K and H-2D transplantation antigens are expressed differentially in different tissues of mouse. Our previous investigations also established that in thioglycolate-stimulated peritoneal macrophages the H-2D^k antigen exists in distinct cell surface and intracellular forms. These two forms are glycosylated differently. In this report, we have found that (1) H-2D^k antigen is phosphorylated whereas H-2K^k antigen is not, and (2) only the cell surface form of H-2D^k antigen is phosphorylated in thioglycolate-stimulated macrophages derived from C3H/Heha mice. This differential phosphorylation of H-2 antigens will provide a model system for further studies on the molecular mechanism and function of phosphrrylation of H-2 antigens.     

Viral-restricted cytolytic T lymphocyte recognition of hybrid human-murine class I histocompatibility antigens

Hybrid human-murine major histocompatibility antigens have been constructed and expressed on the surface of both human RD and murine L cell lines after DNA mediated gene transfer. These antigens linked the polymorphic domains (alpha 1 and alpha 2) of H-2Kb and the carboxy-terminal domains (alpha 3, transmembrane, and intracellular) of HLA-A2. Previously we demonstrated that these antigens were serologically intact and were recognized by allospecific cytolytic T lymphocytes. However, the cell lines expressing the hybrid antigen were less well lysed than the native H-2Kb expressing cell lines. In this study, we extend these observations and demonstrate that virally restricted cytolytic T lymphocytes specific for vesicular stomatitis virus and for Sendai virus can recognize cell lines expressing the hybrid antigen, whether expressed on murine (L cell) or human (RD cell) lines. Furthermore, the data show a profound influence by the carboxy-terminal domains upon the polymorphic T-cell restricting epitopes.     

Modulation of natural cytotoxicity by alloantibodies: III. Augmentation of natural killer activity by alloantisera directed against K,D, and I regions of the murine H-2 complex

Natural killer activity of mouse spleen cells toward a human myeloid leukemia cell line, K562, can be enhanced by alloantisera directed against individual antigens in the H-2 region. By using a panel of 13 antisera (8 directed against antigens in the K and D regions and 5 directed against antigens in the I region) and four strains of mice (C57BL/6J, CBA, DBA/2, and A/J) it was found that certain antisera would stimulate target cell lysis by spleen cells only if the antisera had specificity for antigens which were a part of the haplotype represented on the spleen NK effector cells. Anti Ia antisera could stimulate the anti K562 NK activity of nude mouse spleen cells which lack mature T cells. Depletion of B cells and macrophages from nude spleen cells, by passing through a nylon-wool column also did not abolish the effect of anti-Ia antiserum. It appears likely therefore that the anti-Ia antibodies exert this effect directly on NK cells and that Ia antigens may be expressed on NK cells. Since the antisera directed against different antigens in H-2 complex irrespective of subregion specificity (K, D, or I) stimulated the NK activity of mouse spleen cells, the phenomenon offered an interesting method for testing the presence of a given alloantigen on mouse spleen cells. Log-dose response curves for the augmentation of lysis induced by appropriate alloantisera were linear over a dilution range of 1:320 to 1:5120. By using the dose-response curves, potency ratios of two preparations of antisera (directed against antigen 33 of the K region) could be successfully determined. Besides the K562 cell line, many human lymphoblastoid cell lines could also be used as target cells in this assay system.     

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.     

Localization of H-2Kk in developing mouse teeth using monoclonal antibody

The in situ distribution of H-2 antigens during mouse tooth morphogenesis was investigated using monoclonal antibodies to H-2Kk and indirect immunofluorescent techniques. H-2 antigens were detected in the basement membrane region of fetal molars; they were absent from both the epithelial and dental mesenchyme. H-2 antigens were not found in newborn and 4-day-old mouse molars.     

Association of mouse adenovirus-induced cell surface antigen(s) with histocompatibility antigens

The topological relationship on the mouse adenovirus (M-Ad)-infected cell surface between virus-induced specific cell surface(s) antigens and serologically defined major histocompatibility antigens (H-2) was analyzed by the cap formation technique. Rhodamine-isothiocyanate (RITC)-labeled anti-S serum failed to stain the surface of virus-infected lymphoid cells which were pretreated with anti-H-2 serum and fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulin serum (anti-M-Ig) to cap the appropriate H-2 antigens. Conversely, the capping of the S antigens by pretreatment with anti-S followed by FITC anti-M-Ig serum induced cocapping of H-2 antigens. The β₂ microglobulins (β₂m) were also shown to be cocapped with S antigens by anti-β₂m or by anti-S serum. The S antigens, however, did not cocap with mouse-immunoglobulins or Thyl. 2 antigens on virus-infected B or T lymphocytes, respectively. To further elucidate the molecular relationship between S and H-2 antigens, radio-iodinated virus-infected cells were solubilized with Nonidet P40 (NP40) and S antigens were precipitated with anti-S serum. When the precipitates were analysed with sodium dodecyl sulfate Polyacrylamide gel electrophoresis, two major peaks were seen at positions of molecules of about 45,000 and 12,000 daltons both of which corresponded with molecules which were observed when NP40 extracts of virus-infected or uninfected cells were precipitated with anti-H-2 serum. Sequential immunoprecipitation analysis of infected cell extracts showed that S antigens were coprecipitated with either H-2K or H-2D antigens. These results suggest that the S antigens are somehow associated with H-2K or H-2D antigens separately.     

Immunocytochemical localization of class I H-2 histocompatibility antigens of mouse liver

The subcellular location of class I H-2 histocompatibility antigens was determined for mouse liver using immunocytochemical techniques and correlated with information determined by cell fractionation and analysis in situ. Surface antigens first were localized by standard procedures involving surface labeling with ferritin-labeled antibody. This approach could not be used for internal membranes either in situ or in fractions since the antigens are not expressed at the cytoplasmic surface. For this purpose, thin sections of tissues embedded in Lowicryl were analyzed and quantitated. The in situ analysis confirmed the presence of H-2 antigens on internal membrane compartments as well as on the cell surface and helped rule out the possibility that distributions based on analyses by immunoprecipitation of fractions of internal membranes were influenced greatly by plasma membrane contamination. Quantitation was provided by immunoprecipitation of H-2 antigens from radioiodinated or metabolically labeled isolated and highly purified cell fractions. The findings establish the presence of class I H-2 histocompatibility antigens in endoplasmic reticulum, Golgi apparatus and plasma membrane in the approximate ratios of 1:3:7. No class I H-2 histocompatibility antigens could be detected in mitochondria, salt extracts of isolated membranes or NP-40-insoluble membrane material.