Synthesis of H-2 antigens by preimplantation mouse embryos

An enzyme-linked immunosorbent assay (ELISA), which utilized anti-H-2 monoclonal antibody, was used to detect H-2 antigens on preimplantation mouse embryos. All embryonic stages studied, including unfertilized eggs and 1-cell, 2-cell, 8-cell, and blastocyst-stage embryos, showed the presence of H-2 antigens. To prove that the H-2 antigens were not cytophilically adsorbed to the embryos, blastocysts were treated with papain to strip off the H-2 antigens, and then the embryos were further incubated to allow the H-2 antigens to regenerate. After a 3-h incubation time, 60% of the H-2 antigens on the embryos had reappeared, proving that the H-2 antigens were synthesized by the embryos themselves.     

Immunofluorescence study of H-2 antigens in early mouse embryogenesis

The time of appearance and degree of expression were studied for antigens of the main locus of histocompatibility of the mouse embryos by indirect immunofluorescence. H-2 antigens appeared on the 5.5 day of embryogenesis on the cells of still undifferentiated rudiments of embryonic endoderm and ectoderm. By the 8th day of embryogenesis, rather intensice fluorescence of the cells of amnion, yolk sac and embryonic ectoderm was observed suggesting a marked expression of H-2 antigens during this period. The cells of trophoblast gave practically no positive reaction with anti-H-2-serum.     

H-2 histocompatibility antigens of subcellular membranes of mouse liver

Purified fractions of plasma membrane, Golgi apparatus, rough endoplasmic reticulum vesicles, nuclear envelope, and mitochondria were isolated from mouse liver and the distribution of H-2 histocompatibility antigens determined by indirect radioimmunoassay before and after membrane disruptive treatments. Fractions enriched in plasma membrane (surface membrane) revealed H-2 antigens in highest concentration; disruptive treatments were not necessary to reveal H-2 antigens with surface membranes. In contrast, internal membranes did not possess H-2 antigens which were accessible to antibody. Golgi apparatus fractions or some component of these fractions (e.g. secretory vesicles) possessed the antigens but in a latent form where accessibility was provided by simple rupture of the membrane vesicles. With endoplasmic reticulum, detergent solubilization of the membranes was required before H-2 antigen could be detected. Nuclear envelope preparations contained little or no demonstrable H-2 activity. These results were confirmed by several techniques including immunoprecipitation of labelled solubilized membrane components with anti-H-2 serum and subsequent analysis in SDS-PAGE.     

Blocking action of the soluble H-2 antigens and H-2 antibody isolated by various methods and mouse immune lymphocytes]

The treatment of tumour and lymphoid cells of mice with 3M KC1 solution having high ionic strength, nonionic detergent and by subsequent freeze-thawing resulted in obtaining serologically active H-2 antigen preparation capable of specifically blocking the cytotoxicity of H-2 antisera. The antigenic activity of the preparations thus obtained depended on the source from which they were isolated (spleen cells and their membrane fragments proved to be the best source), on the degree of maturity of tumor cells and the degree of purification of the preparation, as well as on the methods of solubilization. The blocking action of soluble H-2 antigens on the cytotoxicity of immune lymphocytes depended on the method used for isslating these antigens. The interaction of immune lymphocytes and H-2 antisera with soluble antigens was probably effected by different mechanisms.     

Immunogenetic analysis of microsomal and non-microsomal lipoproteins from normal and malignannt mouse tissues for histocompatibility-2 (H-2) antigens

The H-2 antigenic properties of lipoprotein fractions from malignant (L-5178Y leukemia) and normal (spleen, thymus, liver, kidney) mouse tissues have been studied by serological and immunological tests, and the results compared to the previously described activities of these fractions in homograft-sensitization tests. Although, in general, the relative activities in the different assays parallel each other some notable exceptions were found. The non-microsomal lipoproteins from leukemic tissue, inactive in homograft-sensitization tests, did elicit H-2 antibody. Also, the liver microsomal lipoproteins, which are inactive in homograft-sensitization tests in amounts 400 × the minimal effective doses of spleen preparations, exhibited, in in vitro agglutinin-inhibition tests, approximately one-fourth the H-2 activity of the latter. Other findings of note include the high antibody-eliciting potency of the spleen and leukemia microsomal lipoproteins (15 μg protein was sufficient to initiate primary immunization and 1 μg protein to cause an anamnestic response); and the quantitive identity of H-2 antigen activity of the microsomal lipoproteins from spleen and thymus.     

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.     

Dimethyl sulfoxide induces expression of H-2 antigens on mouse lung carcinoma cells

The addition of dimethyl sulfoxide (DMSO) to cultures of line 1 carcinoma cells can increase the surface expression of H-2K and H-2D antigens at least 100-fold from barely detectable initial levels, as determined by using specific monoclonal antibodies and flow cytometry. H-2 values stabilize approximately 1 wk after exposure to maximally inducing concentrations of DMSO (3% vol) at densities found on normal spleen cells. Increased expression of H-2 antigens is not the result of cell selection, it is specific in that expression of an unrelated surface protein decreases, and it is associated with increased synthesis of these antigens as measured by incorporation of [35S]methionine. Additional DMSO-induced changes in the growth, cycling, lectin binding, and antigenic properties of line 1 cells are consistent with increased cell maturation. All changes are reversed when DMSO is removed. This system may facilitate study of products associated with differentiation that influence tumor cell malignancy.     

Immunohistological and flow cytometric analysis of glycosphingolipid expression in mouse lymphoid tissues.

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb(3)Cer), globoside (Gb(4)Cer), gangliotriaosylceramide (Gg(3)Cer), gangliotetraosylceramide (Gg(4)Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb(4)Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg(4)Cer, and Gb(4)Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment.     

Studies on mouse autoreactive cells: I. Role of H-2 antigens in mouse autologous rosette formation

A minority (1–2%) of normal mouse lymphoid cells bind autologous erythrocytes and form rosettes. In this study we examined the antigenic specificity involved in the formation of such rosettes. A significant difference in the incidence of rosettes formed, respectively, with autologous and allogeneic mouse erythrocytes is found. Moreover, preincubation of lymphoid cells with low concentrations of syngeneic erythrocytic ghosts causes significant competitive inhibition of subsequent rosette formation. Allogeneic ghosts obtained from nonrelated or from congenic resistant strains of mice do not display this inhibitory effect under the same conditions. It is thus suggested that mouse autologous rosette-forming cells bear receptors for syngeneic H-2 antigens that are involved in the binding of autologous erythrocytes. More precisely, compatibility between lymphocyte and erythrocyte restricted to K or D only is sufficient to ensure a level of rosettes similar to that obtained when complete identity occurs for K, I, and D regions.     

Lateral diffusion and patch formation of H-2Kk antigens on mouse spleen lymphocytes

We have studied the diffusion and aggregation of H-2Kk antigens labeled with a fluorescent anti-H-2Kk monoclonal antibody (IgG) on mouse splenic lymphocytes, employing fluorescence photobleaching recovery and fluorescence microscopy. The H-2Kk antigens were initially distributed homogeneously on all lymphocytes. Upon antibody binding, sub-micron patches were formed on 50-60% of the cells. A lateral diffusion coefficient, D, of 7.1 X 10(-10) cm2/s and a mobile fraction of 0.73 were found for H-2Kk antigens on diffusely-labeled cells, while these antigens were immobile (D less than or equal to 5 X 10(-12) cm2/s) on patched cells. The patched and nonpatched sub-populations did not correspond to B- and T-lymphocytes. Subjection to low temperature or treatment with NaN3 or cytoskeleton-disrupting drugs did not affect the diffusion or patching of H-2Kk, indicating no involvement of metabolic energy or drug-sensitive cytoskeletal components. These findings could be related to the interactions of H-2 antigens on the cell surface, and to the different susceptibilities of various cells to lysis by cytotoxic T-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     

Interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. II. Levels of expression of H-2K, H-2D, and H-2L in different mouse strains

The numbers of MHC class I molecules expressed by spleen cells from various mouse strains were determined by using MHC-specific monoclonal antibodies and a radioactive binding assay. Although small differences were found to exist in some cases, our general conclusion is that different mice of the same strain, congenic mice of different haplotypes, and syngeneic mice of varying background all express similar numbers of class I antigens. B10.A mice (8 to 10 wk old), for example, express 5.3 X 10(4) Kk molecules/cell, 5.4 X 10(4) Dd molecules/cell, and 2.2 X 10(4) Ld molecules/cell. Some of the differences observed in class I antigen expression included: 1) the level of Kk expression increased to a small but significant extent with age in B10.A mice; 2) female B10.A mice expressed slightly higher amounts of Kk than male mice; and 3) B10.A(2R) and B10.A(4R) recombinant strains expressed elevated levels of K-end antigens and slightly decreased levels of D-end antigens when compared with the unrecombinant B10.A strain. In several strains, F1 mice express approximately 50% as many copies of each parental antigen as do the homozygous parents. B10 mice, which are negative for the L antigen, nevertheless express the same total number of D-end molecules as do B10.A mice. The data suggest that the levels of expression of MHC class I molecules are controlled by at least two factors: gene dosage and another factor(s) that gives rise to the small variations in class I antigen expression seen with age, sex, and strain, and to the low expression of Ld relative to Dd and Kk.     

Demonstration of H-2 antigens on preimplantation mouse embryos using conventional antisera and monoclonal antibody

Mouse embryos at the 2-cell, 8-cell, and blastocyst stages of development were examined for the presence of H-2 antigens by immunoperoxidase labeling and transmission electron microscopy. Conventional antisera made in congenic mouse strains were used to study embryos of four different haplotypes: b, a, k, and d. Blastocysts showed uniform heavy labeling of all cells of the trophectoderm, 8-cell embryos showed lighter labeling of only some of the cells, and 2-cell embryos showed no labeling. Similar results were found for all four haplotypes studied. In addition, monoclonal antibody 11-4.1 (anti-Kk) was reacted with homologous (H-2k) and heterologous (H-2b) blastocysts. Positive results with the monoclonal antibody corroborates the concept that H-2 antigens are expressed on early mouse embryos.     

The cellular mechanism of maintenance of neonatally induced tolerance to H-2 class I antigens

We used limiting dilution analysis protocols to investigate the mechanism by which in vitro cytotoxic T lymphocyte (CTL) hyporeactivity is maintained in adult mice that had been neonatally tolerized to major histocompatibility complex-encoded antigens. Class I molecules, presented on donor cells having an H-2 K or D region haplotype difference from recipients, readily induce tolerogen-specific CTL hyporeactivity. All attempts to identify any in vitro effects of active suppressive cells operative in the maintenance of this hyporeactivity have been unsuccessful. We conclude that this cytotoxic deficiency is the consequence of in vivo mediated clonal inactivation of the precursors of tolerogen-specific CTL. A presentation and evaluation of the assumptions inherent in this conclusion are made. In contrast to class I molecules, class II molecules, presented on donor cells having an H-2 I region haplotype difference from recipients, are unable to induce tolerogen-specific CTL hyporeactivity, even when injected neonatally at high doses. This inability of class II molecules to induce CTL tolerance parallels the considerable difficulty of inducing helper T lymphocyte tolerance to class II molecules.