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.     

Suppressor T cell recognition of major and minor histocompatibility alloantigens: selected suppression of MHC-directed responses by minor alloantigen Ts

The induction of suppression by i.v. administered alloantigens in the murine host was analyzed as a model of the possible effects of blood transfusion on transplant survival. The results indicated that suppressor T cells (Ts) specific for minor histocompatibility alloantigens could be readily induced by the i.v. presentation of minor alloantigen-disparate spleen cells. In contrast, similar priming with cells differing solely at the H-2 major histocompatibility complex stimulated only positive T cell immunity, with no evidence of suppression. The induction of H-2 directed Ts activity could be accomplished only by i.v. priming with major plus minor incompatible donor cells, suggesting that suppressor cell recognition of minor alloantigens may have facilitated the generation of Ts against H-2-encoded major transplantation antigens. A role for minor histocompatibility antigens in the regulation of H-2-specific immunity at the effector level was also indicated. Ts induced by i.v. pretreatment with minor antigen-disparate donor cells not only suppressed the delayed-type hypersensitivity (DTH) response to the relevant minor alloantigens, but also inhibited DTH against unrelated H-2 alloantigens introduced during subsequent intradermal immunization. Suppression of H-2-directed T cell reactivity was specific in that the presence of the Ts-inducing minor alloantigens was also required and occurred only when the minor and unrelated major alloantigens were presented within the same inoculum, if not on the same cell surface. The capacity of Lyt-2+Ts or Ts-derived suppressive factors specific for one set of cell surface molecules to modulate responses to an unrelated group of surface antigens does not appear to represent a general phenomenon, because similar suppression of immunity to unrelated tumor-specific transplantation antigens by minor-specific Ts was not observed. These results are discussed with respect to the possible mechanism of H-2-directed suppression and the role of the I region in Ts recognition of antigen.     

Flow microfluorometric analysis of H-2L expression

The cell surface expression of H-2L, a major transplantation antigen, was compared by flow microfluorometry to the expression of products of H-2K and H-2D loci, using monoclonal antibodies. By this methodology, the ontogeny and tissue distribution of Ld antigens were found to be indistinguishable from those of the K and D antigens. In a reciprocal blocking assay, using fluorescein-labeled test reagents, it was shown that monoclonals anti-H-2.65 and anti-H-2.64 did not inhibit the binding of each other. These results suggest that the alloantigenic determinants H-2.64 and H-2.65 are located at distinct sites on Ld molecules. Quantitative comparisons using the fluorescein-labeled monoclonal reagents indicated that Ld molecules are expressed at 2- to 3-fold lower levels on the cell surface compared with K and D molecules. These findings give new credence to a "3-locus" model for the major histocompatibility complex of man and mouse, where H-2L and HLA-C share several homologies that are unique and distinguish them from the other histocompatibility loci.     

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.     

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.     

Specific T-cell-mediated killing of autologous lung tumour cells

The phenomenon that strong syngeneic T-cell-mediated cytotoxicity is observed if killer, stimulator, and target cells share H-2 histocompatibility antigens is called H-2 restriction. Here a syngeneic model system making use of hapten-coupled stimulator and target cells is used to explore whether H-2 restriction is absolute or not. Using TNP-coupled spleen or tumor cells as stimulator or target cells in syngeneic and allogeneic situations, it is shown that neither the induction step nor the effector step of TNP-dependent killing is H-2 restricted. By varying the experimental assay conditions more or less H-2-restricted, TNP-dependent killing can be observed. For instance, suboptimal coupling of TNP to targets may result in H-2-restricted killing. Similarly, the use of spleen cell targets as opposed to spleen blast cells or tumor cells may result in H-2-restricted lysis. In contrast optimal coupling of TNP to sensitive target cells and coupling of TNP to cells with certain H-2 haplotypes may lead to significant TNP-dependent killing which is not H-2 restricted. Since hapten-coupled cells lacking H-2 are neither stimulators nor targets these results suggest that the T-cell receptor recognizes TNP-modified H-2 antigens simply as nonself-H-2. Thus hapten coupling of syngeneic cells appears to lead to a histocompatibility antigen change similar to the situation in an allogeneic cytotoxic reaction. Experiments are presented which support this view showing that TNP-coupled and uncoupled syngeneic or allogeneic stimulator and target cells cross-react. For instance allogeneic sensitization may lead to killing on TNP-coupled targets syngeneic to the effector cells and TNP-coupled stimulator cells syngeneic to the effector cells may induce killing on uncoupled syngeneic targets. TNP-dependent cytotoxicity can therefore be envisaged as a kind of allogeneic reactivity due to modification of H-2 antigens by the TNP coupling. This conclusion may have bearing on other model systems in which syngeneic killing appears to be H-2 restricted. In support of this possibility it is shown that allogeneic sensitization may lead to priming of memory cells able to respond to minor histocompatibility antigens.     

Variable expression of histocompatibility antigens on tumor cells

Conclusions During the last decade the H-2 major histocompatibility complex (MHC) has become the focal point of cellular immunology, with emphasis on its role in regulating the magnitude and specificity of T cell responses. The studies reviewed in this paper add to the growing realization that the MHC may also determine the antigenicity and immunogenicity of tumor-associated antigens. It is imperative that appropriate assays be developed to analyse human tumors for variations in histocompatibility antigen expression and to determine the exact genetic and molecular mechanisms involved. A thorough understanding of the variable expression of histocompatibility antigens on tumor cells should have far-reaching consequences for the concept of immunesurveillance and for the design of effective immunotherapeutic procedures.     

Does the major histocompatibility complex serve as a specific receptor for Semliki Forest virus?

Murine F9 and PCC4 teratoma cells do not express H-2 major transplantation antigens according to virus-specific T-lymphocyte cytotoxic or serological assays. However, such cells can be infected with and readily replicate many types of viruses (coxsackie B 3, mouse hepatitis, Sindbis, Semliki Forest [SFV], lymphocytic choriomeningitis, Pichinde, vesicular stomatitis, herpes simplex type 1) to the same extent as do murine F12 teratoma cells and mouse embryo fibroblasts, all of which express the H-2 determinants. In contrast, F9 and PCC4 cells are not productively infected with murine cytomegalovirus, whereas F12 and mouse embryo fibroblast cells are. In addition to replicating in H-2-negative murine teratoma cells, SFV replicates in H-2-negative murine lymphoblastoid cells. The ability of SFV to infect cells without H-2 antigens and then to effect viral antigenic expression in the cells' cytoplasm and on their surface with similar kinetics and in equivalent amounts as cells with H-2 antigens indicates that the H-2 receptor is not needed for SFV infection. Daudi cells, which lack HLA antigens, block the replication of SFV. This occurs at some point after receptor binding, as demonstrated by diminished viral mRNA. In addition, a possible membrane defect precludes viral exit in Daudi cells transfected with SFV infectious RNA. These results indicate that a cell's possession of H-2 antigens is not a requirement for SFV infection and that major histocompatibility complex antigens are not specific receptors for this virus.     

The Chinese hamster gene map. Assignment of four genes (DTS, PGM2, 6PGD, EnoI) to chromosome 2

Mouse morulae and blastocysts express cell surface antigens that fortuitously cross-react with antisera to human chorionic gonadotropin (hCG). In the present study, the cell surface and cytoplasmic expression of these antigens was followed in mouse unfertilized oocytes, different stages of preimplantation embryos and in early post-implantation embryos cultured from blastocysts. In addition to their known stage-dependent cell surface expression on morulae and blastocysts, these antigens (1) were already present in the cytoplasm of mature unfertilized oocytes and pre-morula stages of embryos; (2) remained expressed as cell surface antigens on cells of the inner cell mass (ICM), but not on the surface of trophectodermal cells with further blastocyst development although (3) they persisted as cytoplasmic antigens in trophectodermal cells. In addition, these antigens were also detectable by antiserum to the alpha subunit of hCG.     

Inverse Relationship of Polyoma Tumour Specific Cell Surface Antigen to H-2 Histocompatibility Antigens

NEOPLASTIC transformation is known to be associated with changes in the strength of normal cellular antigens, but the effect can be either an increase or a decrease. In the former category are Forssman antigens in guinea-pig hepatoma¹ and SV40 transformed cells²; HL-A antigens in leukaemic cells³; and “G” antigen in human tumour cells⁴. On the other hand, the intensity of the expression of mouse H-2 histocompatibility antigens is decreased in TL(+) leukaemia⁵ and methylcholanthrene (MCA)-induced tumours⁶. We set out to tell whether the expression of histocompatibility antigens was also affected by transformation with an oncogenic virus and have found that in tumours induced by polyoma virus, the quantity of H-2 antigens varied inversely with the amount of tumour-specific cell surface antigen.     

Anti-MHC immunity detected prior to intentional alloimmunization

Cell fusion was performed between spleen cells from young BALB/cBy (H-2 ^d) mice which have never been immunized and SP2/0 mouse plasmacytoma cells. A monoclonal H-2-specific cytotoxic IgM antibody was obtained (By-1) which detected a new public biregional H-2 specificity, H-2.m210. The mcAb By-1 reacted strongly with H-2K^d, D^d, and H-2^s antigens, gave weak cross-reactions with H-2K^k, D^q, H-2^r, and H-2^v antigens and was negative with H-2^b, H-2^f, H-2^p, and H-2L^d antigens. A polymorphic reaction pattern was also observed on a panel of lymphocytes from B 10.W strains. The intriguing finding on this reaction pattern was the reactivity on H-2^d cells, including the syngeneic BALB/cBy and truly autologous cells. As shown by capping and immunoprecipitation experiments on H-2^d cells and by studies on H-2^d-transfected mouse L cells, the target molecules for McAb By-1 were H-2K^d and H-2D^d molecules. The BALB/cBy mouse, from whose spleen cells the McAb By-1 was obtained, survived after the fusion experiment, and serum was examined for the presence of cytotoxic H-2-specific antibodies during the rest of its life. At the time of the fusion, no autoreactive serum antibodies were found, but about 4 months later, we found in the serum of this mouse autoreactive H-2-specific cytotoxic IgM antibodies. The serum antibodies followed the same reaction pattern as that of the McAb By-1. As far as we know, this is the first report of autoreactive H-2-specific antibodies in serum of a mouse which has never been immunized and of the first natural autoreactive H-2-specific monoclonal antibody.Abbreviations McAb monoclonal antibody - MHC major histocompatibility complex - H-2 major histocompatibility complex of mice - CTLs cytotoxic T cells - FMF flow microfluorometry - FITC fluorescein isothiocyanate - LPS lipopolysaccharide W.E. coli 0111:134 - PBS phosphate-buffered saline - Iodogen 1,3,4,6,-tetrachloro-3,6-diphenylglycoluril - GAMIg goat-antimouse immunoglobulin - Staph-A Staphylococcus aureus Cowan I     

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     

Membrane-reconstituted, purified H-2Kb specifically inhibits T-cell-target cell conjugate formation

We report the use of a sensitive microassay to detect purified H-2Kb antigens which have been functionally reconstituted into membrane vesicles of defined composition. The histocompatibility antigens have been purified by monoclonal antibody affinity chromatography. The assay utilizes inhibition of specific conjugate formation between allogeneically primed (H-2d anti- H-2b) cytotoxic T cells and H-2b target cells by the membrane-reconstituted H-2Kb antigens. Cytoskeletal proteins were added to the H-2Kb (and control H-2k) antigens. Sucrose density fractionation of reconstituted vesicles and Pronase E cleavage studies suggested that the cytoskeletal proteins aided in the incorporation and vectorial orientation of the antigens into large, cholesterol-containing membrane vesicles. As little as 6 ng purified H-2Kb plus 28 ng cytoskeletal proteins in vesicles of defined lipid composition (0.28, 0.25, 0.47 mol fraction cholesterol, dimyristoylphosphatidylcholine, and dipalmitoylphosphatidylcholine, respectively) inhibited specific conjugate formation to 50% of the maximum inhibition observed. This inhibition was shown to be specific in two ways: (i) the same H-2Kb-containing vesicles did not inhibit nonspecific conjugate formation, and (ii) control vesicles containing the same amounts of lipid, cytoskeletal proteins, and purified H-2k proteins inhibited conjugate formation but only at significantly higher H-2k concentrations, indicating the specificity of the response with the vesicles containing H-2Kb.     

The influence of major histocompatibility complex class I antigens on tumor growth and metastasis

The work described here demonstrates the importance of major histocompatibility complex class I antigens for the control of tumor growth and metastasis by the host's immune system. In certain murine tumor cells which have lost expression of H-2 class I antigens, a de novo expression of H-2 can be achieved by transfection with syngeneic class I genes. In contrast to the parental cells the transfected tumors do not grow any more in syngeneic mice, or in other cases they do not form metastases. The studies suggest that the de novo expression of the H-2 antigens renders the tumors highly immunogenic and leads to effective recognition of a tumor-associated antigen in conjunction with the transfected H-2 antigen. These conclusions were confirmed in other tumor systems. For example, separation of a heterogeneous tumor into clones expressing high or low amounts of H-2 showed that only the tumor cell with low H-2 grew well in syngeneic mice, whereas the H-2 high tumor clones were rejected. In other studies in vitro induction by IFN-gamma of H-2 antigen on H-2 negative tumors led to reduced tumor growth in vivo which was due to the increased immunogenicity. About 10% of human tumors are also low or defective for HLA class I expression and often these tumors appear to be more malignant. The class I negative tumors could either have arisen from class I low or negative tissues or are HLA loss variants which escaped the attack of the immune system. Altogether, our studies and the data of other laboratories demonstrate the important role of class I antigens for anti-tumor immunity and they suggest that modulation of class I expression by gene transfection or by induction with soluble mediators could be a useful tool for the manipulation of tumor immunity.     

T-cell and antibody typing of a mouse population segregating for Sxr and H-2 haplotype

During investigation of the frequency of recombination of the testis determining gene, Tdy, and the minor histocompatibility antigen gene Hya on the Sxr segment in an outbred mouse stock, we identified two fertile males, one XY and the other XYSxr, which typed H-2k positive using the H-2b anti-H-2k monoclonal antibody HB50, but whose cells failed either to stimulate H-Y specific H-2k restricted T-cell clones, or to be killed by anti-H-2k or anti-H-2k restricted H-Y specific cytotoxic T cells. We investigated these two mice and their existing relatives, using H-2 and H-Y typing methods. The progeny of their test matings with H-2b homozygous C57BL/6 females were also investigated. The results indicate that the transmission of the Hya gene on the Y chromosomes from both mice, and the additional Hya gene on the Sxr segment of the carrier male, allowed for the expression of the H-Y antigen and its detection in the presence of an H-2 haplotype for which we had H-2 restricted H-Y specific typing cells (H-2b and H-2k). Furthermore, we identified the haplotype of the two original males as expressed in the H-2 homozygous and heterozygous F2 progeny as H-2q and discovered an unexpected cross reactivity of the monoclonal anti KkDk antibody HB13 with half the cells of H-2q homozygotes, but not qb heterozygotes.     

Loss of reactivity of a myeloma tumor with H-2 compatible thymus-derived lymphocytes.

It was previously shown that MOPC-315-EL, a subline of the BALB/c myeloma tumor MOPC-315, reversibly alters its reactivity with T cells that recognize H-2^d, 2,4,6-trinitrophenyl, and minor histocompatibility antigens. This report demonstrates (a) that CTLs directed against vesicular stomatitis virus and Sendai virus are unable to recognize viral antigens associated with the unreactive tumor cell, and (b) that incorporation of Sendai virus antigens into the same membrane as the H-2 gene products is required for effective recognition by cytotoxic T lymphocytes.     

Monoclonal antibodies directed against major histocompatibility complex antigens bind to the surface of Treponema pallidum isolated from infected rabbits or humans

Evidence is presented for the association of class I major histocompatibility complex (MHC) antigens with the surface of Treponema pallidum during infection. A monoclonal antibody (IgG2a) directed against a murine H-2Kb epitope of public specificity reacted with the cell surface of T. pallidum, as assayed by the binding of protein A-colloidal gold in immunoelectron microscopy. Monoclonal antibodies directed against class I rabbit MHC antigens also reacted in immunofluorescence assays with material on the surface of rabbit-cultivated T. pallidum. In addition, impression smears of human syphilitic genital ulcers that were darkfield-positive for the presence of spirochetes were tested in immunofluorescence assays with monoclonal antibodies directed against human MHC antigens; antibody directed against HLA-ABC (class I) was reactive whereas antibody directed against HLA-DR (class II) was nonreactive. Results of the study suggest that the association of host-derived class I MHC antigens or molecular mimicry may play a role in T. pallidum evasion of host immune defenses.     

Biochemical evidence for expression of a semi-allogeneic, H-2 antigen by a murine adenocarcinoma

LT-85 is an alveolegenic adenocarcinoma induced in mutant C3HfB/HeN (C3Hf) mice. This tumor, however, grows preferentially in allogeneic, wild-type C3H/HeN (C3H) mice. The tumor-associated transplantation antigen has been mapped to the K end of the major histocompatibility complex. H-2K antigens were isolated from detergent extracts of LT-85 cells by immunoprecipitation with monoclonal antibody. The tryptic peptides of these antigens were compared, by using high-pressure liquid chromatography, with the tryptic peptides of H-2K antigens isolated from syngeneic mutant C3Hf and ancestral wild-type C3H spleen cells. We found that the H-2K antigens of the LT-85 tumor cells were very similar to, but distinct from, those present on syngeneic C3Hf lymphoid cells. We also found, however, that the H-2K antigens of LT-85 tumor cells were clearly different from the H-2K antigens of allogeneic C3H spleen cells. The H-2K antigens of LT-85 cells are therefore foreign to syngeneic C3Hf cells, but do not represent expression by the tumor cells of the allogeneic H-2K antigens expressed by normal C3H cells. Furthermore, the nature of the differences observed between the H-2K antigens of LT-85 cells and C3Hf and C3H spleen cells strongly suggests that the structure of the H-2K molecule of LT-85 cells is identical in some regions to the H-2K molecule of C3Hf cells, and in other regions to the H-2K molecule of C3H cells.