Murine antigen H-2.7: localization of its antigenic determinant to the Ss (C4) molecule

Murine blood group antigen H-2.7 is encoded by a locus mapping in the vicinity of the S locus which codes for the Ss antigen carried by the fourth component of the complement pathway (C4). Normal mouse serum of H-2.7-positive strains contains a substance which inhibits anti-H-2.7 hemagglutination. This substance cannot be removed by passage of the serum through an anti-Ss immunoabsorbent column indicating that the Ss and H-2.7 antigens are present on separate molecules or molecular fragments in the serum. In contrast, fresh plasma either does not contain the H-2.7-bearing substance at all or it contains it at a far lower concentration than normal serum, although it has a normal level of the Ss-antigen-bearing substance. However, the H-2.7-positive substance appears when the plasma is allowed to stand for several hours, or when it is dialyzed and treated subsequently in a manner favoring spontaneous degradation of complement components. Removal of the Ss substance from the fresh plasma prevents the appearance of the H-2.7 antigen at any time thereafter. These findings indicate that the Ss and H-2.7 antigens are carried by the same molecule or molecular complex. The intact molecule expresses only the Ss antigen; the H-2.7 antigen is either hidden or masked so that it is inaccessible or poorly accessible to H-2.7 antibodies. Degradation of these molecules results in the generation of two fragments, a large fragment carrying the Ss antigen and a smaller H-2.7-positive fragment. The data are consistent with the interpretation that the H-2.7 antigen is encoded by the S locus, and that it is carried by that portion of the C4 molecule split off during complement activation.     

Murine antigen H-2.7: Its genetics,tissue expression,and strain distribution

Reinvestigation of alloantisera containing antibodies to murine antigen H-2.7 revealed that the crucial recombinant, A.TFR1 (H-2 ^an1 ), which was reported to separate theH-2G locus from theSs-Slp loci, has ak-like instead off-like H-2.7 antigen. Therefore, the crossover position inH-2 ^an1 and the position of the G locus in theH-2 map are now uncertain. By using the hemagglutination-serum inhibition test, anti-H-2.7 reactive substance was found to be present in normal mouse serum in a strain-specific manner. Tissue distribution study by absorption analysis indicated that H-2.7 antigen is present, in addition to RBCs, on spleen and lymph node cells, but is absent on thymus cells. Thirty B10.W congenic lines were analysed for the presence of the H-2.7 antigen. Two lines (B 10.CHA2 and B 10.KPA44) were found to be H-2.7 positive by both direct hemagglutination and absorption tests.Abbreviations used in this paper ACT Ammonium chloride Tris-buffer - BSA Bovine serum albumin - HA Hemagglutination - HASI Hemagglutination serum inhibition - HBSS Hanks balanced salt solution - PBS Phosphate buffered saline - PVP Polyvinylpyrrolidone - RBCs Red blood cells     

A mouse plasma cell surface antigenic determinant (PLKB) recognized by xenoantiserum. Its relationship to the PC.1 antigenic determinant.

The specificities of the xenoantisera made against mouse myeloma cells have been compared to those recognized by alloantiserum by studying patterns of cytotoxicity on both normal and malignant plasma cells. Goat antiserum obtained by immunization with Balb/c mouse myeloma ADJ-PC-22A cells and purified by in vivo absorption could detect cell surface antigenic determinants present on plasma cells and on cells of liver, kidney, and brain (PLKB antigen), as we had previously reported for a similarly prepared rabbit antiserum. In spite of an apparent similarity between the tissue representation of the PLKB determinant and that of PC.1 antigenic determinants which were detected by DBA/2 anti-ADJ-PC-22A cell alloantiserum, the PLKB antigenic determinant is not identical with the PC.1 antigenic determinant, since the former is found on the tissues of PC.1-negative as well as PC.1-positive strains of mice. However, it was deduced that the PLKB antigenic determinant and the PC.1 antigenic determinant reside in close proximity on the cell surface or maybe even on the same molecule, since Fab fragments of antiserum against either PLKB or PC.1 blocked the cytotoxicity against both antigens. On the other hand, these Fab fragments did not inhibit the cytotoxicity of anti-H-2 antiserum, indicating that neither PLKB nor PC.1 antigenic determinants are in close proximity to H-2 antigens. Association of PLKB and PC.1 determinants was further supported by the finding that the loss of the PLKB determinant in a variant of myeloma MOPC-70A corresponds to the loss of PC.1 determinant on the same cells.     

Evidence for a fifth (G) region in theH-2 complex of the mouse

Antisera (B10.129×A)F₁ anti-P and (B10×A)F₁ anti-B10.P contain antibodies that define, in the PVP hemagglutination test, an antigen originally described as G or H-2.7. Of the independentH-2 haplotypes, the H-2.7 antigen is present inf, j, k, p, ands. In addition, the antisera also contain a weak cytotoxic antibody, distinct from anti-H-2.7. The cytotoxic antibody reacts with antigens controlled by theK orI regions. The hemagglutinating H-2.7 antibody does not have cytotoxic activity. The genetic determinant coding for antigen H-2.7 can be mapped into the chromosomal segment between theS andD regions. The H-2.7 antigen thus serves as a marker for a new region of theH-2 complex. The locus coding for antigen H-2.7 is designatedH-2 G and the correspondingH-2 regionG. The H-2.7 antigen has a tissue distribution distinct from that of the H-2 antigens controlled by theK orD regions. So far it could be detected primarily on erythrocytes.     

Murine antigen H-2.7: Phenotypic conversion of erythrocytes in radiation chimeras

By use of radiation chimeras produced between H-2.7⁺ and H-2.7^}- strains, A.SW and A.BY and B10.S(7R) and B10.S(9R), we demonstrate that the H-2.7 antigen can be passively attached to or detached from red blood cells. Thus, genetically H-2.7^}- red blood cells derived from H-2.7^}- bone marrow cells, gain H-2.7 antigen while maturing in the H-2.7⁺ host. Similarly, genetically H- 2.7⁺ red blood cells derived from H-2.7⁺ bone marrow cells become H-2.7^}- while maturing in H-2.7^– recipients. This behavior of the H-2.7 antigen is similar to that described for human Chido and Rodgers blood group antigens.Abbreviations used in this paper BMT bone marrow transfer - BSA bovine serum albumin - CT cytotoxicity test - HA hemagglutination - HBSS10 Hank's balanced salt solution containing 10% fetal calf serum - NMS normal mouse serum - PBS phosphate-buffered saline - PVP polyvinylpyrrolidone - RBCs red blood cells     

Secretion of a transplantation-related antigen

Analysis of mouse cDNA clones has led to the identification of a class I (H-2)-related gene that encodes a truncated transplantation-like antigen. Unlike the products of the class I genes (H-2K, H-2D, and H2-L), which are synthesized and displayed on the surface of all cells, the class I-related gene product is expressed only in liver cells and is secreted. The region of the secreted molecule corresponding to the extracellular domain of the membrane-bound class I antigens shows unusual amino acid substitutions at positions otherwise invariably conserved. There is also loss of a glycosylation site that is used in all class I antigens. Within the region corresponding to the transmembrane domain are multiple nonconservative substitutions of hydrophobic residues, alterations that render the encoded protein incapable of inserting into the plasma membrane. Toward the end of the same domain, the polypeptide chain terminates abruptly and thus lacks the intracellular domain present on all class I antigens. A candidate for this secreted molecule, detected using various heteroantisera against class I antigens, has been identified. A potential role for this serum protein in mediating active tolerance is discussed.     

Characterization of the Ss sialoglycoprotein and its antigens in Rhnull erythrocytes

The Ss sialoglycoprotein (glycophorin B) and its antigens in Rhnull erythrocytes, which lack the Rhesus blood group antigens, due to apparently silent (amorphic type) or independent suppressor (regulator type) genes, were investigated. The quantity of the molecule in amorphic and in regulator type red cell membranes was found to be decreased by about 60%-70%, as judged from sodium-dodecylsulfate polyacrylamide gel electrophoresis. The Ss glycoprotein content in the erythrocytes from heterozygotes (regulator type) was diminished to an extent of about 30%. Confirming and extending previous studies, the S, s, Ux, Uz and 'N' antigens were slightly weakened in Rhnull erythrocytes. The U and Duclos receptors were only slightly or not depressed in amorphic Rhnull cells, but almost absent from or not detectable in those of the regulator type. This demonstrates that an additional alteration, apart from the decreased Ss glycoprotein content of the membranes, accounts for the weakness of these receptors in regulator type cells. We propose the hypothesis that (a) protein(s) encoded by the Rhesus locus form(s) a complex with the Ss glycoprotein. Thus, it (they) might facilitate the incorporation of the Ss glycoprotein into the membrane and also contribute to the complete expression of the U and Duclos antigens in normal cells.     

Murine antigen H-2.7: In vitro phenotypic conversion of erythrocytes

The H-2.7 antigen in normal mouse serum can be passively adsorbed to H-2.7^– erythrocytes in 10 percent sucrose (low ionic strength) solution. This antigen can also be stripped off the H-2.7⁺ erythrocytes under the same conditions provided the H-2.7⁺normal serum is absent. The stripped red blood cells can regain the H-2.7 antigen upon reincubation with H-2.7⁺ normal serum. The attachment of the H-2.7 antigen to erythrocytes probably occurs via a specific receptor.Abbreviations used in this paper BSA bovine serum albumin - B10 C57BL/10Sn - HA hemagglutination - LIS low ionic strength solution - NMS normal mouse serum - PBS phosphate-buffered saline - PVP polyvinylpyrrolidone - RBCs red blood cells     

Inhibition of the induction of cytolytic T lymphocytes with alloantisera directed against H-2K and H-2D gene products.

Alloantisera directed at gene products of the H-2Kd or H-2Dd loci on the stimulator cell were shown to inhibit specifically the generation of cytolytic T lymphocytes to those antigens. Thus, masking the antigens of one H-2 locus on the stimulator cell inhibits the induction of CTL to products of that locus but does not inhibit the induction of CTL to antigens of another H-2 locus. Alloantisera inhibition of the induction of cytolytic T lymphocytes occurred with both normal and primed responder cells and also occurred when the stimulating antigens were on whole cells or purified plasma membrane. Absorption on the appropriate spleen cells removed the inhibitory capacity of these alloantisera.     

The genetic origin of minor histocompatibility antigens

The purpose of this study was to elucidate the genetic origin of minor histocompatibility (H) antigens. Toward this end common inbred mouse strains, distinct subspecies, and species of the subgenus Mus were examined for expression of various minor H antigens. These antigens were encoded by the classical minor H loci H-3 and H-4 or by newly identified minor H antigens detected as a consequence of mutation. Both minor H antigens that stimulate MHC class I-restricted cytotoxic T cells (Tc) and antigens that stimulate MHC class II-restricted helper T cells (Th) were monitored. The results suggested that strains of distinct ancestry commonly express identical or cross-reactive antigens. Moreover, a correlation between the lack of expression of minor H antigens and ancestral heritage was observed. To address whether the antigens found on unrelated strains were allelic with the sensitizing minor H antigens or a consequence of antigen cross-reactivity, classical genetic segregation analysis was carried out. Even in distinct subspecies and species, the minor H antigens always mapped to the site of the appropriate minor H locus. Together the results suggest: 1 minor H antigen sequences are evolutionarily stable in that their pace of antigenic change is slow enough to predate subspeciation and speciation; 2 the minor H antigens originated in the inbred strains as a consequence of a rare polymorphism or loss mutation carried in a founder mouse stock that caused the mouse to percieve the wild-type protein as foreign; 3 there is a remarkable lack of antigenic cross-reactivity between the defined minor H antigens and other products.     

Serological and genetic study of the ELA W21 specificity

Investigations on the W21 specificity showed that this antigen is expressed on lymphocytes and platelets but not on erythrocytes. The molecule carrying the antigen W21 moves in the cell membrane independently from ELA locus A and B encoded antigens, as observed in 'lysostripping'. The W21 specificity occurs with very different gene frequencies in various breeds. In informative families it segregates together with defined gene products of the MHC region. The data suggest strongly that the W21 specificity belongs to the ELA system as a class I gene product, but is governed by a separate locus than the known locus A and locus B allelic series.     

Mouse mitochondrial superoxide dismutase locus is on chromosome 17

The hamster × mouse hybridoma cell line GCL28 carries only one copy of mouse chromosome 17 but expresses H-2 antigens controlled by the major histocompatibility complex of the mouse. The cell line and clones derived from it were subjected to treatment with H-2-specific antisera and complement and a series of H-2-antigen-negative clones was produced. Typing of the clones for the mouse enzyme glyoxalase 1, which is encoded by an H-2-linked gene, revealed that the loss of H-2-antigen expression was accompanied by the loss of chromosome 17 in these clones. This suggestion was verified by karyotype analysis of selected clones. Typing of the clones and subclones for the mouse mitochondrial superoxide dismutase (SOD-2) indicated complete concordance between loss of chromosome 17 and loss of SOD-2 activity. This finding suggests that the locus controlling the expression of SOD-2 is located on chromosome 17. Since a similar locus in the human is linked to HLA, the human major histocompatibility complex, extensive homology must exist between the mouse and human MHC-bearing chromosomes.     

Lack of anti-TSTA antibodies in mice immunized with a methylcholanthrene-induced fibrosarcoma

Summary The 3-methylcholanthrene (MCA)-induced BALB/c (H-2^d) fibrosarcoma C-1 bears a strong tumor-specific transplantation antigen (TSTA) which, in previous studies, appeared to be distinct from H-2^k alien antigens expressed by this tumor. To see whether a syngeneic anti-C-1 serum obtained by multiple immunizations with C-1 tumor cells contained anti-TSTA-specific antibodies, in vitro cytotoxicity tests were performed. The syngeneic anti-C-1 serum had a high cytotoxic activity on C-1 cells, which allowed an absorption analysis to be carried out. Absorption of the serum with C3Hf, AKR, or B10.BR normal lymphoid cells (all sharing H-2^k antigens) reduced the cytotoxic activity on C-1 cells to 30%–50%. This residual activity could not be absorbed by FMR+ or G+ murine leukemias, by ecotropic endogenous virus obtained from SC-1 cells infected with the C-1 virus, by embryonic cells, or by normal BALB/c or C57BL/6 lymphoid cells. Conversely, the serum activity was abrogated by absorbing with the MCA-induced BALB/c fibrosarcomas C-1, ST2, C-3, GI-17, or CMS-1, and significantly lowered by the MCA-induced C3Hf fibrosarcoma C3H-7. A significant reduction of the anti-C-1 cytotoxicity was also obtained by absorbing with the two BALB/c fibrosarcomas teflon-9 and SCS (both lacking TSTA), by means of fresh newborn BALB/c or C3Hf muscle cells or of in vitro-cultured newborn BALB/c fibroblasts. These results suggest that, in spite of the strong transplantation immunity elicited by C-1 cells, antibodies to the individual TSTA of C-1 were undetectable in the syngeneic anti-C-1 serum obtained from animals highly resistant to the challenge of C-1 cells.     

Molecular heterogeneity of D-end products detected by anti-H-2.28 sera

Immunoprecipitation of NP-40 lysates of 125I-labeled lymph-node cells with different anti-H-2 sera and with anti-Qa-2 serum has shown that the BALB/cByA strain (H-2d, Qa-2-negative) expresses, besides H-2Ld, another molecule that is not detectable in the BALB/c-H-2dm2 strain. Electrophoresis in SDS polyacrylamide gels indicated that this molecule, provisionally designated Lq, has an apparent molecular weight of 41000 daltons, in contrast to approximately 49000 daltons for H-2Kd and H-2Ld, and 47000 daltons for H-2Dd molecules. The anti-Qa-2 serum precipitated from the Qa-2-positive strains BALB/cHeA but not from the Qa-2-negative strains BALB/cByA and BALB/c-H-2dm2 a protein that gave a very strong band corresponding to the molecular weight 41000 daltons in the gel electrophoresis. The biochemical characteristics of the Lq molecule are thus more similar to those of Qa-2 than of H-2 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.     

Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(alpha 1) and C1(alpha 2) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2Ld has been replaced by that of H-2Dd. Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2Dd replaced by that of H-2Ld) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2Ld and H-2Dd or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2Ld and H-2Dd to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2Ld substituted for that of H-2Dd), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2Ld or anti-H-2Dd N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of beta 2M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2Dd and anti-H-2Ld CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2Ld-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ia antigens: markers of specific T suppressors immune to H-2 complex antigens

Two antisera to Ia antigens, products of the H-2 complex I-Cd and I-JkEk subregions, respectively, have been obtained by immunisation of the F1 hybrids of recombinant strains of mice. These antisera are shown to display the 50 per cent cytotoxic effect in vitro in the presence of complement upon lymphocyte populations immune to the H-2 complex antigens and enriched for specific suppressor T cells (SSC) by fractionation on the monolayer of target cells. The specificity of anti-Ia cytotoxins is shown by the cross antibody absorption with T- and B-cells of mice originated from the recombinant H-2 haplotypes and bearing either particular I-Cd, I-Jk and I-Ek antigens, or their combinations. Anti-I-Cd cytotoxins are found to react with both B and T cells at a different rate, and the anti-I-JkEk serum contains two antibody types directed to I-Ek and I-Jk products, respectively, the latter being able to react preferently with T cells. Although both antisera do inactivate the in vitro SSC function in the presence of complement at a similar degree, the inactivating action of the anti-I-Cd serum, but not that of the anti-I-JkEk serum, occurs without complement. SSC are established to bear both Ia-antigens, I-J and I-C on the same cell, as demonstrated by the cross antibody absorption and variation of the H-2 origin of SSC. These two markers are suggested to function differently in the SSC immune to the H-2 antigens and the I-C antigen expression on the SSC surface is presumed to be required for their interaction with the inhibited responder T cells proliferating in MLC.     

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s.

The major histocompatibility complex (MHC) class I antigens contain a light chain, beta 2-microglobulin, non-covalently associated to the transmembrane heavy alpha-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with beta 2-microglobulin, and we recently found that activated C1s complement was capable of cleaving beta 2-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22,000 and 24,000 g/mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the alpha 2-and alpha 3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the alpha 1-and alpha 2-domains which represent the binding site for antigenic peptides.     

An erythrocyte and serum antigen with a specificity antithetical to the mouse C4 associated H-2.7 antigen

Repeated immunization of intra-H-2 recombinant strain A.TBR16 with lymphoid tissue from strain A.TBR13 produced an antiserum that agglutinated the erythrocytes from inbred strains of mice carrying the b, d, r, q, u, wr7, w13, w17, w19, and w21 haplotypes of the H-2 complex. The antiserum was negative with erythrocytes of strains carrying the haplotypes f, j, k, p, and s. This pattern of reactivity among fifteen H-2 haplotypes is unlike the strain distribution for any known H-2 erythrocyte antigen, and is exactly antithetical to the S region-controlled H-2.7 antigen. An examination of 12 intra-H-2 recombinant haplotypes mapped the genetic determinant controlling the new antigen to the IC or S regions of the H-2 complex. In addition, hemagglutination inhibition studies revealed the antigen was also expressed in serum and plasma. The serologic, genetic, and tissue distribution data suggest the gene controlling the newly defined antigen is allelic to the gene controlling the H-2.7 antigen.     

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.