Chemical cross-linking of Ia alloantigen alpha and beta chains with dimethyl 3,3'-dithiobispropionimidate.

We have examined the polypeptide chain composition of membrane-bound and detergent-solubilized Ia antigens using the chemical cross-linking reagent dimethyl 3,3'-dithiobispropionimidate (DTBP). Products of the I-E/C subregion of the major histocompatibility complex, which were solubilized from spleen cells with the detergent NP-40 and partially purified by affinity chromatography on lentil lectin-agarose, could be almost completely cross-linked by DTBP. Thus, the characteristic 33,000 m.v. (alpha) and 28,000 (beta) polypeptide chains seen on sodium dodecylsulfate polyacrylamide gels disappeared and a major new species of 60,000 m.w. appeared after cross-linking. When isolated and reduced with 2-mercaptoethanol, the 60,000 m.w. peak was found to be comprised to alpha and beta chains. Similar results were obtained when I-E/C, as well as I-A, alpha and beta chains were crosslinked on the cell surface. These data demonstrate that the alpha and beta chains of the Ia antigens exist primarily in the form of a dimer both in detergent solution and in situ.     

Structural comparison of I-A antigens produced by a cloned murine T suppressor cell line with B-Cell-Derived I-A

A cloned, antigen-specific T suppressor cell line derived from a CBA mouse expresses large amounts of I-A and I-E antigens. Comparative two-dimensional polyacrylamid gel electrophoresis of biosynthetically labeled I-A antigens immunoprecipitated with a variety of monoclonal I-Ak-specific antibodies suggested that alpha, beta and Ii polypeptide chains are identical with B-cell-derived I-A. Dimeric complexes formed by I-A chains derived from B or T suppressor cells were also similar with two major exceptions. Pulse-labeled T-cell-derived Ia antigen was complexed with two additional unknown components of about 31K. These components were not visible in pulse-chased (processed) materials. In addition, T suppressor-cell-derived I-A antigens did not contain S-S linked dimers consisting of processed alpha and beta chains, which are usually formed during solubilization of B cells. We consider the possibility that in T cells these chains are associated with other structures, thus preventing S-S linkage between alpha and beta chains.     

Hybrid Ia antigens: Genetic,serologic, and biochemical analyses

Ia specificities 22 and 23 were found to be determinants on hybrid Ia molecules, formed by the noncovalent binding of a 26,000–28,000 dalton beta polypeptide chain (A_e) coded by the I-A subregion and a 32,000–35,000 dalton alpha chain (E_α) coded by the I-E subregion. For expression of Ia. 23 the A_e chain, coded by the I-A subregion, must be derived from the H-2^d haplotype, while A^b, A^s, or A^k can provide the complementing beta chain for the expression of Ia. 22. For expression of Ia. 22 and Ia. 23, most Ia. 7 positive strains can provide the complementing alpha chain (E_α), with the one exception of B 10. PL (E^u), which is Ia. 7 positive but will not complement with A^d to express Ia. 23. Antisera were also produced against hybrid Ia antigens by immunizing with F₁ cells expressing Ia. 22 or Ia. 23 generated by transcomplementation. These antisera detect the same specificities as conventional anti-Ia. 22 and anti-Ia. 23 sera produced against cis-complementing Ia antigens. It is postulated that hybrid Ia determinants are involved in recognition and generation of immune response to antigens under dual Ir gene control. It is also suggested that there are 2 types of Ia specificities: (1) allotypic Ia specificities expressed on the alpha or beta chains (for example, Ia. 7 on the E_α chain) and (2) hybrid Ia specificities, which are unique interaction determinants formed by the association of alpha and beta chains (for example, Ia. 22 and Ia. 23). These interaction gene products may be involved in antigen recognition and presentation.     

Characterization of Ia antigens in mouse serum.

Sera obtained from normal B10.BR mice were shown to inhibit selectively a specific anti-Ia alloantiserum.Partial purification of the Ia antigenic activity was accomplished by isolation of the high density lipoproteins from these sera by fractional precipitation with sodium phosphotungstate and MgCL2. Both H-2.23 and Iak antigens present in this high density lipoprotein fraction were completely adsorbed by rabbit anit-rat beta2-microglobulin immunoadsorbents, whereas specific anti-H-2.23 immunoadsorbents removed only the H-2 activity. These data deomnstrate that Ia antigens, like H-2 antigens in the sera of B10.BR mice are associated with high density lipoproteins and further suggest that both H-2 and Ia antigens are associated with a beta2-microglobulin-like molecule.     

Isolation and characterization of murine Ia antigens

The isolation and characterization of Ia antigens from both lymphoid and nonlymphoid cells was attempted by SDS-polyacrylamide gel electrophoresis of radiolabeled, NP-40 solubilized, and anti-Ia precipitated lysates. The profiles obtained indicate that membrane proteins with a molecular weight of approximately 30,000 can be isolated from peripheral B but not from peripheral T cells. Ia antigens cannot be immunoprecipitated from cortisone-resistant thymocytes, total thymocytes, allogeneically activated T cells, Con A stimulated T cells, and anti-Ig immunoadsorbent purified T cells. Ia antigens seem to comprise only 1%–2% of labeled splenic intracellular and membrane-associated proteins. They differ from H-2 antigens and immunoglobulin H and L chains with respect to size and serological reactivity. Ia antigens cannot be found to be secreted from lymph node cells or splenocytes into the extracellular incubation media. Tissue distribution studies indicate that Ia antigens are present on macrophages, fetal liver cells, epidermal cells, and bone marrow cells. They have not been found on such tumor cells as myelomas, teratomas, and lymphocytic leukemias.     

Molecular architecture of a light-harvesting antenna. Structure of the 18 S core-rod subassembly of the Synechococcus 6301 phycobilisome

The 18 S subassembly particles obtained by partial dissociation of phycobilisomes from Synechococcus 6301 (Anacystis nidulans) strain AN 112 contain approximately one-half of the mass of the phycobilisome and include core-rod junctions (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086). The polypeptide composition of 18 S complexes, determined by analysis of uniformly 14C-labeled phycobilisomes, gave the following stoichiometry: 75K:27K:18.3K:alpha beta allophycocyanin monomer: alpha beta phycocyanin monomer of 1:2:1:5:6; where 75K, 27K, etc. represent polypeptides of 75, 27 kilodaltons, etc. The 18.3K polypeptide is a hitherto underscribed biliprotein bearing a single phycocyanobilin. The NH2-terminal sequence of this subunit was determined to be homologous to that of the beta subunit of allophycocyanin. Chromatography of products resulting from limited trypsin treatment of the 18 S complex led to the isolation of three subcomplexes: a mixture of (alpha beta)3 . 22K and (alpha beta)3 . 24K phycocyanin complexes, an (alpha beta)3 allophycocyanin trimer, and an (alpha beta)2 . 18.3K.40K.11K allophycocyanin-containing complex. The 22K and 24K components were products of the degradation of the 27K polypeptides, whereas the 40K and 11K components were derived from the 75K polypeptide. The subcomplexes accounted for the composition of the 18 S complex. Determination of the composition, stoichiometry, and spectroscopic properties of the subcomplexes has led to a model of the polypeptide arrangement within the 18 S complex and of the pathway of energy transfer among these polypeptides.     

Monoclonal antibodies identifying a novel T-Cell antigen and Ia antigens of human lymphocytes

We describe here two new monoclonal antibodies that react with surface antigens of human lymphocytes. Antibody 7.2 identified a determinant on the framework region of the human Ia antigen. It was cytotoxic for all cultured B-cell lines, normal B cells, and monocytes. The antibody was not cytotoxic for normal T cells or for established T leukemic cell lines. In immune precipitation assays, the 7.2 antibody reacted with a bimolecular complex of two chains that resolved in polyacrylamide gels as polypeptides with molecular weights of 29000 and 34000 daltons. These precipitation results were analogous to those achieved with a rabbit antiserum prepared against human Ia antigens. Antibody 9.3 identified a determinant on the framework region of a T-cell antigen. It was cytotoxic for 50–80% of peripheral T cells and for 20–50% of thymocytes. It was not cytotoxic for cultured B-cell lines, normal B cells, or monocytes. In immune precipitation assays, the 9.3 antibody reacted with a single polypeptide with a molecular weight of 44000 daltons. Due to the expression of this antigen on a limited subpopulation of human T cells, we have designated the antigen HuLyt-1.     

Monoclonal antibodies specific for Ia glycoproteins raised by immunization with activated T cells: possible role of T cellbound Ia antigens as targets of immunoregulatory T cells

Two monoclonal antibodies, Y-3P and Y-8P, specific for conventional mouse Ia glycoprotein antigens are described. Both were raised by repeated immunization of primed mice with activated T cells over a period of 1 yr, and both detect a new and broad public Ia specificity. Both of the antibodies react with I-A subregion-controlled A alpha:A beta complexes of all mouse strains apart from the responder strain (H-2d), as well as the equivalent of A alpha:A beta complexes in rats carrying seven independent haplotypes. These antibodies have great utility as almost universal Ia reagents. On the basis of these results, we propose that Ia antigens presented to the immune system bound to activated T cells are immunogenic, and may induce the production of Ia antibodies of novel and broad specificity. In addition, we propose that such bound Ia glycoproteins could be a target for immunoregulatory T cells, and could account for the specificity of suppression of graft-vs-host reactions and Ia-restricted helper T cells observed by others in F1 animals injected with parental T cells.     

Genetic mapping of the component chains of Ia antigens

The genes encoding the two polypeptide chains ( and) that comprise the murine Ia antigens were localized within distinct regions of the major histocompatibility complex (MHC). This was accomplished by correlating allelic forms of the and chains with the MHC congenic strains of mice from which they were isolated. Allelic forms of and chains were distinguished by their unique structural markers, such as isoelectric points, amino acid sequences or peptide maps. The results indicate that the structural genes for both the and chains of I-A subregion antigens are located within the K to I-A genetic interval. In contrast, the gene encoding the chain of I-E subregion antigens is located outside of theI-E subregion and within the K to I-B genetic interval. These findings may have important implications for analysis of observations that complementation by twoI-region genes is sometimes required for development of immune responses.     

Transverse membrane topography of the B875 light-harvesting polypeptides of wild-type Rhodobacter sphaeroides.

Purified B875 light-harvesting complex, chromatophores, and spheroplast-derived vesicles from wild-type Rhodobacter sphaeroides were treated with proteinase K or trypsin, and the alpha and beta polypeptides were analyzed by electrophoretic, immunochemical, and protein-sequencing methods. With the purified complex, proteinase K digested both polypeptides and completely eliminated the A875 peak. Trypsin digested the alpha polypeptide and reduced the A875 by 50%. Proteinase K cleaved the beta polypeptide of chromatophores and the alpha polypeptide of spheroplast-derived vesicles. Sequence analyses of polypeptides extracted from proteinase K-treated chromatophores revealed that the beta polypeptide was cleaved between amino acids 4 and 5 from the N terminus. The N terminus of the alpha polypeptide was intact. We concluded that the N terminus of the beta polypeptide is exposed on the cytoplasmic membrane surface, and the difference in the digestion patterns between the spheroplast-derived vesicles and chromatophores suggested that the C terminus of the alpha polypeptide is exposed on the periplasmic surface.     

HLA-D region antigen-associated invariant polypeptides as revealed by two-dimensional gel analysis. Glycosylation and structural inter-relationships

Two-dimensional polyacrylamide gel analyses of immunoprecipitates of HLA-D region antigens prepared from [35S]methionine-labeled B lymphoblastoid cells revealed a number of invariant polypeptides (Ii and theta) that co-precipitate with the alpha and beta polypeptides of the class II (Ia) antigens. The invariant polypeptides comprised at least three Ii spots of Mr = 31,000 (Ii1-Ii3) and a series of six theta spots of Mr = 34,000 (theta 1-theta 6). The structural inter-relationships of these polypeptides have been investigated. Tryptic peptide fingerprints showed that Ii and theta have closely related amino acid sequences. In contrast, the fingerprints of the HLA-DR alpha and beta polypeptides clearly differed from those of theta and Ii as well as from each other. Analyses of immunoprecipitates prepared from cells cultured in the presence of tunicamycin revealed the presence of two N-linked oligosaccharides on each invariant polypeptide and suggested that the more acidic theta polypeptides (theta 1 and theta 2) differed from the other invariant polypeptides by the presence of sialic acid on one or both N-linked oligosaccharides. Removal of sialic acid by neuraminidase simplified the pattern of theta spots into three distinct Ii-related polypeptides. Endo-beta-N-acetylglycosaminidase H digestion indicated that the individual theta polypeptides represent stages in carbohydrate processing whereby Ii with two N-linked immature oligosaccharides are converted initially to theta 6-theta 3 with one immature and one complex, but nonsialylated, oligosaccharide and finally to theta 2-theta 1 with two complex oligosaccharides. Digestion of the theta polypeptides with N-acetylgalactosamine oligosaccharidase indicated that the theta spots are also derived by O-glycosylation from the Ii polypeptides. This assignment is supported by results obtained using monensin to block glycosylation within the Golgi. At least three spots persisted after complete removal of the N- and O-linked oligosaccharides, suggesting the presence of a family of invariant polypeptides differing in amino acid sequence.     

Structural studies of the protein portion of the H-2-linked Ia glycoprotein antigens of the mouse: tryptic peptide comparison of products from the I-A and I-C subregions of B10-HTT.

Ia antigens from the I-A8 and I-Ck subregions of the B10.HTT (H-2t3) strain of mice were isolated by indirect immunoprecipitation of arginine-labeled, nonionic detergent-solubilized materials. After biochemical purification the electrophoretically homogeneous 28,000 dalton glycoprotein beta chains from the Ia precipitates were digested with trypsin and the resultant radiolabeled tryptic peptides were compared by analytical ion exchange chromatography. These comparisons reveal that the beta chains of Ia antigens from the A (I-A8) and C (I-Ck) subregions of B10.HTT share only two out of 12 to 14 of their arginine tryptic peptides. Thus these noncross-reactive Ia antigens are structurally quite diverse, and would possess sufficient structural variability to account for their lack of antigenic cross-reactivity.     

The structure-function relationship of I-A molecules: a biochemical analysis of I-A polypeptides from mutant antigen-presenting cells and evidence of preferential association of allelic forms

Chemically induced mutants of an I-Ak,d-expressing, antigen-presenting B cell-B lymphoma hybridoma have recently been generated by immunoselection in vitro with I-Ak-specific monoclonal antibodies, and were found to possess alterations in some of the I-Ak region-dependent functions. The mutants were categorized as alpha-polypeptide mutants or beta-polypeptide mutants on the basis of the patterns of reactivity with anti I-Ak alpha and anti I-Ak beta monoclonal antibodies. To delineate the structural alterations underlying the differences in serologic and functional properties of these mutants, I-A molecules from several of these mutant hybridomas were compared biochemically with wild type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic (HPLC) tryptic peptide analyses. These results suggest that the marked alterations in antibody reactivity and T cell-activating functions of the beta-polypeptide mutants G1, K2, and LD3, as well as the Ia alpha-polypeptide mutant JE50, may be due to very limited alterations in the Ia polypeptides. The functional deficiencies of the alpha-polypeptide mutant JE67 could be attributed to the change in net charge exhibited by its Ak alpha polypeptide. HPLC tryptic peptide analysis of I-A molecules isolated from the alpha-polypeptide mutant J4 indicates that the functional deficiencies exhibited by this mutant are due to a complete loss of expression of the Ak alpha polypeptide. The inability to detect significant amounts of Ad alpha Ak beta and Ak alpha Ad beta hybrid molecules in immunoprecipitates from some of these cell lines suggests that some hybrid molecules may be expressed at low levels due to preferential Ia polypeptide chain association. Together, these results indicate that most serologically defined epitopes are localized on either one or the other Ia polypeptide, whereas T cell-defined epitopes are determined by a combination of both Ia polypeptides. The results of these analyses also enable us to evaluate different immunoselection strategies for the most efficient production of mutants expressing limited alterations in Ia polypeptides.     

Cytofluorometric isolation of I937, an Ia antigen-bearing variant of the Ia-negative human monocytic cell line U937

Cells of the human monocyte cell line U937 are generally considered devoid of any Ia antigens on their surface. In analyzing U937 cells with a large panel of monoclonal anti-human Ia antibodies by flow cytometry, we detected a small number of cells that appeared to react with antibodies to HLA-DR and HLA-DS/DC molecules. These Ia-positive cells were isolated and were cloned, resulting in a human monocyte cell line that expresses high levels of Ia antigens. We analyzed these antigens by one- and two-dimensional polyacrylamide gel electrophoresis, after radiolabeling and immunoprecipitation. Three distinct Ia molecules, alpha 1 beta 1, alpha 1 beta 3 (HLA-DR-like), and alpha 2 beta 2 (HLA-DC/DS-like) are synthesized by I937 cells, alpha 1 beta 3 molecule being the predominant species. The Ia antigen-bearing human monocyte cell line is expected to be useful for studying events involved in antigen presentation.     

Comparison and partial characterization of guinea pig Ia alpha- and beta-chain oligosaccharides

The structures of the N-linked oligosaccharides of mature guinea pig Ia molecules were partially characterized by serial lectin affinity analysis. Those Ia antigens that are thought to be allelic products (Ia.3,5 and Ia.4,5) were found to bear identical oligosaccharides, whereas differences in glycopeptide distribution were found for Ia antigens known to be products of separate I subregions (Ia.2 and Ia.4,5). The two predominant oligosaccharides present on alpha-chains from all three Ia molecules were of the high mannnose type and the triantennary or tetraantennary complex type. Two structurally distinct beta-chains were isolated from Ia.3,5 and Ia.4,5 molecules; beta 1 bore primarily triantennary or tetraantennary complex oligosaccharides, and beta 2 had predominantly biantennary complex-type carbohydrate chains. The composition and distribution of the oligosaccharide moieties of guinea pig Ia molecules indicate that there are structural features shared among guinea pig, murine, and human Ia antigens.     

Lymphocytes express Ia antigens of foreign haplotype following treatment with neuraminidase

Evidence is presented which indicates that neuraminidase (NA) treatment of spleen cells both destroys old Ia antigens and reveals new Ia specificities which are not normally expressed by splenocytes. It was found that NA treatment unmasked alien I-A^k-like specificities on A.TH (I ^s ) spleen cells, and I^s-like antigens on A.TL (I ^k ) spleen cells. These conclusions were based on direct testing of NA-treated targets with a range of alloantisera and on cell-absorption experiments. Furthermore, the cellular distribution of NA-exposed antigens resembled that of convential Ia antigens, the new antigens being expressed on more than 90 percent of splenic B cells and a subpopulation of splenic T cells. However, although some of the antigens exposed by NA on A.TH cells appeared to resemble the Ia. 3 and 15 specificities, additional antigens were involved which did not correlate with any previously described Ia antigens.Sugar inhibition experiments demonstrated the NA-exposed antigens to be carbohydrate in nature, D-galactose being an effective inhibitor in these studies. The proportion of- and-linked D-galactose residues associated with the new antigens depended upon the target cell used and the anti-Ia serum tested. Furthermore, glycolipid extracts from lymphoid cells were shown to contain the NA-exposed antigens.Collectively, these results support the existence of carbohydrate-defined Ia antigens. The simplest interpretation of the findings is that NA clips off terminal sialic acid residues from carbohydrate-defined Ia antigens on the cell surface and exposes subterminal sugars which resemble antigens expressed by otherI-region haplotypes.     

Transplantation in miniature swine. V. Characterization of Ia antigens.

The complexity of the I region analog associated with the MHC of miniature swine has been probed by sequential antibody precipitation studies of Ia antigens. Treatment of solubilized lymphocyte preparations from MSLA homozygotes of the DD haplotype with excess AA anti-DD alloantiserum led to precipitation of only a portion of the total Ia antigens, as determined by secondary precipitation of the remaining material with CC anti-DD serum. The presumed I region of miniature swine must therefore code for more than one Ia antigen-bearing polypeptide chain. In addition, certain mouse alloantisera that had previously been shown to react with rat Ia antigens were tested for reactivity with swine Ia antigens. Anti-Iak mouse alloantisera precipitated Ia molecules from every swine extract tested, regardless of MHC type, precluding genetic mapping studies. However, sequential precipitation studies demonstrated that the cross-reactive mouse alloantisera reacted only with a subclass of swine Ia antigens, again suggesting genetic complexity of the pig Ia locus.     

Coordinate induction of Ia alpha, beta, and Ii mRNA in a macrophage cell line

We demonstrated a tightly coordinated timing in the appearance of mRNA for the four class II (Ia) MHC chains, A alpha, A beta, E alpha, and E beta, and the Ia-associated invariant chain in a murine macrophage cell line after the addition of immune interferon (IFN-gamma) or of IFN-gamma-containing supernatants from Con A-stimulated spleen cells. The marked increase in mRNA levels for these molecules at approximately 8 hr after IFN-gamma addition contrasts sharply with the earlier, more gradual kinetics observed for class I (H-2) and beta 2-microglobulin mRNA. The difference in kinetics of IFN-gamma induction of class I and class II mRNA suggests differential regulation of the expression of Ia and H-2 antigens. The long lag period preceding detection of Ia mRNA raises the possibility that IFN-gamma may not directly mediate the increase in mRNA expression, but may act through an additional cellular intermediate.     

Ontogeny of human Ia antigens

Indirect immunofluorescence (IIP) staining of tissues from human fetuses (ages ranging from 8 to 32 weeks of intrauterine life) with monoclonal antibodies (MoAb) to monomorphic determinants of Ia antigens and HLA-A,B,C antigens has shown that both types of antigens are already detectable in tissues of 8-week-old fetuses. Ia antigens and HLA-A,B,C antigens reach their almost-complete tissue distribution after 32 and 24 weeks of intrauterine life, respectively. The structure of Ia antigens synthesized by fetal thymus cells is similar to that of B-lymphoid cell-derived Ia antigens. Ia antigen-bearing thymic fetal cells can stimulate allogeneic lymphocytes in mixed lymphocyte reactions (MLRs). These reactions are blocked by monoclonal antibodies to monomorphic determinants of human Ia antigens and of HLA-A,B, antigens.