Susan E. Cullen

Structural and Serological Properties of Murine Ia Alloantigens

The gene products of the immune-response (/) region of the mouse major histocompatibility complex (MHC) were first isolated and described through the use of alloantisera raised between mouse strains differing in the / (and S) region, but not in the K or D regions of the MHC (Hauptfeld et al. 1973, David et al. 1973). The number of combinations for which the requirement of H-2K and H-2D identity and /-region difference can be fulfilled is relatively small, but rather widespread crossreactivity between la antigens of different haplotypes enabled the early definition of eight alloantigenic specificities in these few combinations (David et al. 1973, 1974, Hauptfeld et al. 1973, David & Shreffler 1974). It was also shown that antibodies to la alloantigens were present in alloantisera raised in strain combinations which differed in both H-2 and / regions, and that such alloantibodies could be analyzed on the basis of crossreactivity with haplotypes which did not share the H-2 alloantigenic specificity with the immunizing haplotype (Gotze et al. 1973, Sachs & Cone 1973). Further serological analysis of the system (Hess & Davies 1974, Staines et al. 1974, Sachs et al. 1975a, Colombani et al. 1976, David et al. 1976a) has raised the number of defined la alloantigenic specificities to about 20, and these are nimibered according to the guidelines set down at the 1975 la Workshop (Sachs et al. 1975b). Subdivision of the / genetic region on the basis of functional (immune-response gene) traits which reassort in recombinant haplotypes has been accompanied by the observation that Ia-antigenic

Chemical characteristics of Ia antigens

The discovery that maj or histocompatibility complex (MHC)-linked genes regulate the ability of an animal to respond to an immunologic challenge [4, 35] stimulated intense investigation into the mechanisms by which these immune response (Ir) genes exert their influence. As part of the investigation, an effort was made to identify and analyse products of Ir genes. Since it was possible to reciprocally immunize animals which differed virtually only by the genetic region (the immune response or I region) containing the Ir genes, the use of serological techniques to detect Ir gene products seemed an appropriate starting point [17,/8, 25, 28, 46]. The antisera raised by these reciprocal immunizations reacted primarily with lymphocyte cell surface antigens which were termed I region associated or Ia antigens. While it is not yet evident whether the Ia antigens are in fact determined by the Ir genes themselves, or by genes closely linked to the Ir genes, it is clear that these antigens play a critical role in the regulation of the immune response. Evidence for this contention was derived from several in vivo and in vitro functional studies. Ia antigens have been shown to be necessary for successful collaboration of immunocompetent cells [32, 44]. In addition anti-Ia antisera can inhibit antigen-elicited genetically controlled immune responses as measured by in vitro T cell proliferation [54, 59], can inhibit the induction of mixed lymphocyte reactions (MLR) [27, 37], can inhibit the induction of in vitro antibody responses [24, 40] and can react with antigen specific helper and suppressor factors derived from T cells [38, 65]. Thus, though identity of Ia antigens and Ir gene products has not been established, the Ia antigens have nonetheless been subjected to extensive biochemical analysis in an effort to elucidate the physiology of these antigens, and their relationship to Ir genes. This review will summarize the information presently available regarding the structure and chemistry of the Ia antigens in mice and guinea pigs.

Serological analysis of H-2 mutants

Serological absorption experiments showed that mutant B6.M505 (H-2bd) lacks an H-2K end specificity present in the parental B6 strain. It has a molecular weight of 45,000 dalton, indicating it is an H-2 antigen. It is a private specificity ofKb strains, and has been designated H-2.62.

Ir associated murine alloantigens. Serological and chemical definition of Ia specificities associated with the H 2(b) haplotype

Strains bearing recombinant H-2 haplotypes with known positions of recombination have been used to demonstrate antibodies reacting with Ia antigens associated with the H-2b haplotype in several H-2 antisera. Two new Ia specificities, Ia.8 and Ia.9, both determined by genes in the Ir-1A subregion, have thus been defined, and the strain distribution of these specificities has been determined. Immunoprecipitation of radio-labeled membrane antigens has been used to characterize Ia.9 and to distinguish it chemically from Ia.7 in a membrane preparation from a strain possessing both specificities.

Identity of molecules bearing murine ia alloantigenic specificities ia.7 And ia.22: Evidence for a single type of i-e molecule in homozygotes.

In homozygous mice bearingI regions derived from haplotypek, only a single type of Ia molecule bearing the alloantigenic specificities Ia.7 and Ia.22 was found using techniques of sequential immune precipitation and tryptic peptide analysis. As suggested at the fourth Ir Gene Workshop (Sachs 1978), Ia.7 is considered here to be an antigenic determinant associated with I-E-subregionencoded molecules, i. e., it is excluded from theI−C subregion. TheI−C subregion is currently defined mainly by functional traits. It is now known that the I-E molecules are composed of an α chain encoded in theI−E subregion, and aβ chain encoded in theI−A subregion. Since theI−C subregion is not involved with the determination of these Ia molecules, and since in homozygotes there is apparently only a single type of molecule bearing both specificities Ia.7 and Ia.22, the term “I-E/C” molecule should probably be dropped in favor of the simpler designation I-E.

USE OF IMMUNE PRECIPITATION TO IDENTIFY AND SUGGEST MAPPING POSITION FOR NEW Ia SPECIFICITIES

Publisher Summary The la alloantigens are associated with the immune response (I) region by the immunization of strains differing in the I region but not in the K or D regions of MHC. Strain pairs that differ at I alone are few, and other criteria for the definition of la specificities have been used. Anti-la sera are cytotoxic for a subpopulation of lymphocytes. Antisera with or without anti-H-2 antibodies can be identified as anti-la sera by capacity to bind Ia molecules from solubilized antigen preparations. The use of these methods to define Ia specificities had been demonstrated in several laboratories. This chapter discusses the use of immune precipitation to identify and suggest the mapping position for new Ia specificities— Ia.13 and Ia.W21.

THE CHEMISTRY OF MOUSE AND GUINEA PIG Ia ANTIGENS

Ia antigens of mice and guinea pigs are shown to be molecules of ∼58,000 daltons comprised of two glycoprotein chains of 33,000 and 25,000 daltons which are linked by disulfide bonds. One Ia antigen (murine Ia.7) was shown to retain its antigenic activity after digestion by glycosidases but not after pronase digestion, indicating the antigenic activity resides in the protein portion of the molecule. Sequential precipitation was utilized as a means of genetic mapping of Ia specificities. Two Ia determinants encoded within one I subregion were found on one molecule, while two Ia determinants encoded within different subregions were found on different molecules. This technique was employed to map Ia determinants to a subregion when no recombinants informative for a given specificity were available for analysis. Differences in Ia antigens isolated from guinea pig B cells, T cells and macrophages were demonstrated and were shown to be related to differences in carbohydrate structure or possibly content. These differences may have implications for cell interaction.

Structural characterization of murine ia antigen n-linked oligosaccharides and localization of specific structures to two unique α-chain glycosylation sites*

The sequence of N-linked oligosaccharides of differentially glycosylated murine I-Ak alpha-(alpha 2- and alpha 3-) and beta-chains was determined. I-Ak beta-chains predominantly bear a biantennary complex oligosaccharide with a core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. The I-Ak alpha-chain has two N-linked glycosylation sites at Asn-82 and Asn-122. When Lubrol-insoluble alpha 3-chains are examined they are found to bear high-mannose oligosaccharides of either the Man9GlcNAc2 or Man8GlcNAc2 type at both sites. When Lubrol-soluble alpha 2-chains are examined, in about 85% of the molecules the Asn-82 site bears a biantennary complex oligosaccharide with core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. Interestingly, the Asn-122 site bears a variety of structures. In about 50% of the molecules, the structure at Asn-122 is a biantennary complex oligosaccharide without core fucose and with the peripheral sequence SA----Gal----GlcNAc----Man. In addition, it can bear other complex structures which we did not define further. The apparently restricted addition of fucose to the oligosaccharide at the alpha-Asn-82 site, even when both alpha-sites bear biantennary complex structures with the same peripheral sequence, is a feature unique to this system. The unusual variety of structures present at the alpha-Asn-122 site may indicate differential processing in different cell types.

Disulfide structure of murine ia alloantigens.

The tryptic (T) and T insoluble chymotryptic (TIC) peptide maps from 35S-cysteine (Cys) labeled, disulfide-linked I-Ak dimer were compared to those from 35S-Cys labeled I-Ak alpha and beta chains which were not covalently linked. These comparisons indicated that the alpha and beta chains found in the covalent I-Ak dimer were not a specialized subset of I-A alpha and beta chains. Furthermore, these data, along with the knowledge that alkylation of spleen cells prior to and during detergent solubilization prevents the formation of disulfide-linked I-Ak dimer, indicate that covalent dimer formation is an inefficient and artifactual process. Comparison of the T and TIC peptide maps of reduced and nonreduced 35S-Cys labeled I-Ak alpha and beta chains suggests that the I-Ak alpha chain contains one intrachain disulfide bond, whereas the I-Ak beta chain contains two intrachain disulfide bonds. Examination of the T and TIC peptide maps of the reduced and nonreduced 35S-Cys labeled I-Ak dimer identifies the Cys-containing peptides which are involved in the formation of the artifactual I-Ak dimer interchain (alpha-beta) disulfide bond. Comparison of 35S-Cys labeled I-Ak and I-Ek alpha and beta chains by T and TIC peptide mapping reveals considerably more homology between the two alpha-chains and between the two beta-chains than is observed using other 3H-amino acid precursors, thus indicating that the I-Ak and I-Ek alloantigens are homologous in their amino acid sequences adjacent to the Cys resides. The reasons for the inability to induce formation of interchain (alpha-beta) disulfide bonds in I-Ek molecules are discussed.

INABILITY TO DETECT Ia ANTIGENS IN MOUSE SERA

Abstract: Inhibition assays with alloantisera failed to reveal presence of Ia antigens in mice serum. Most mouse sera at high concentrations inhibit the anti-Ia sera non-specifically. Immunoprecipitation assays with rabbit anti-mouse sera failed to detect Ia molecules on spleen cells. The antigenic specificity detected in serum and purportedly of oligosaccharide in nature is probably not the defined Ia specificities.