A2 Molecules Research Articles

Comparative structural analysis of HLA-A3 antigens distinguishable by cytotoxic T lymphocytes: variant E1.

Abstract Influenza-specific cytotoxic T cells restricted by HLA-A3 recognize differences between HLA-A3 antigens that are serologically indistinguishable. To examine whether this differential recognition had a structural basis, we have compared the structures of HLA-A3 molecules from Epstein Barr virus-transformed peripheral blood lymphocytes of two individuals, E1 and M17. M17 was representative of the majority of HLA-A3-bearing individuals, whereas E1 was a variant distinguished by cytotoxic T cells. Peptide map comparisons revealed a small number of differences when particular amino acids were used to radiolabel the A3 molecules. Sequence analysis and comparison of the results with the prototypic HLA-A3 sequence localized the variability to a tryptic peptide spanning residues 147-157. To obtain the complete amino acid sequence for the E1 and M17 A3 molecules in the 147-157 region, the CNBr fragments beginning at residue 139 were isolated and sequenced. Two amino acid differences were detected between the HLA-A3 molecule of the CTL-defined variant E1 and that of M17. At position 152, Glu in donor M17 had changed to Va1 in donor E1, and at position 156, Leu in donor M17 had changed to Gln in donor E1. The finding that A3 molecules from E1 are altered in the region between residues 147 and 157 is consistent with studies on HLA-A2 variants and Kb mutants showing that this region of class I molecules is important for CTL recognition but not for recognition by serologic reagents.

Allospecific T cell recognition of HLA-A2 antigens: evidence for group-specific and subgroup-specific epitopes.

Interleukin 2-dependent alloreactive cytotoxic T cell lines, with activity predominantly directed against the HLA-A2 antigen, have been generated in vitro by stimulating blood mononuclear cells from donors nonimmune to the Epstein-Barr (EB) virus with appropriate numbers of EB virus-transformed B cells from A2-homozygous individuals. Such effector cells were tested against a panel of EB virus-transformed target cell lines all expressing the serologically defined A2 antigen but typed into "common A2" and "variant A2" subgroups on the basis of their recognition by A2-restricted EB virus-specific cytotoxic T cells. "Variant A2" responder cells cocultivated with "common A2"-bearing stimulators gave rise to effector T cell lines which recognized only the "common A2"-bearing subgroup of targets. By contrast, responder cells from A2-negative donors stimulated with "common A2"-bearing cells produced effector T cell lines in which the strong lysis of "common A2"-bearing targets was accompanied by a lower, but still significant, lysis directed against all targets within the "variant A2" subgroup. In both cases, lysis of the target cells was blocked equally well by the anti-A2-specific monoclonal antibody MA2.1 as by the monoclonal antibody W6/32 specific for HLA-A, -B, and -C determinants. This suggests that HLA-A2 molecules possess at least two distinct sets of epitopes capable of inducing alloreactive T cell cytotoxicity: first, epitopes probably associated with T cell-restricting sites, which generate subgroup-specific responses, and second, epitopes shared by all A2 molecules, and perhaps associated with serologically defined sites, which generate "pan A2" group-specific responses.

Clonal diversity and homology of latent and nominal group a immunoglobulin allotypes in the rabbit

Latent V H immunoglobulin allotypes are expressed unexpectedly and transiently at low concn in the serum of rabbits. Latent group a1 molecules in sera from rabbit colonies in Philadelphia (U.S.A.) and Birmingham (U.K.) were examined for a1 specificity and clonal diversity using reference nominal allotypic reagents and isoelectric focusing (IEF) autoradiography. Latent a1 molecules from rabbits of both colonies had diverse spectrotypic patterns in the pI range 5.5–8.3, as identified with 125I-labelled affinity-purified specificity-tested, anti-nominal a1 antibody. Comparisons of spectrotypes between nominal a1 antigen and latent a1 focused molecules revealed a marked correspondence in banding over the pI range. Reference anti-nominal a1 antibodies could be absorbed out substantially by the IgG fraction of serum from two rabbits containing latent a1 molecules; in a reciprocal fashion absorption with nominal a1 molecules reduced the binding of focused latent a1. The latent a1 molecules from both U.S.- and U.K.-bred rabbits displayed strikingly similar IEF spectra and their antigenic similarities were confirmed by similar absorption capacities of the reference anti-a1 serum. When sequential serum samples from one (U.S.) latent a1 rabbit were compared by IEF, some bands, e.g. those between pH 7.75 and 8.3, appeared to fluctuate in their presence, whereas others, e.g. between pH 5.3 and 7.4, were expressed continuously. We can conclude that latent a1 molecules are clonally complex and some are consistently produced in small amounts. As they also show antigenic similarity, if not identity with nominal a1, we believe that they are probably the product of the same gene (or genes) with an equivalent capacity to be associated with specificity-determining genes even though the level of synthetic activity is lower and possibly governed differently. Anti-a1 antibody was raised in a rabbit in which latent a1 allotype had been previously detected. This antibody was of low avidity and, while inhibitable on RIA by nominal a1 it was not inhibitable by the donor's latent a1 or by a second latent a1 of the same (partially inbred) U.K. colony, but was inhibitable by a latent a1serum from the Philadelphia, U.S.A. colony. This result suggests that a1 molecules are the products of more than one gene.

Mutant HLA-A2 antigens as restricting elements for virus-specific cytotoxic T cells.

Mutants of the EB virus-transformed cell line T5-1 (HLA-A1, 2; B8, 27), bearing well-characterized alterations in HLA-A2 antigen expression and unable to bind the HLA-A2-specific monoclonal antibody BB7.2, have been tested for their susceptibility to EB virus-specific cytolysis using effector T-cell preparations functionally restricted through relevant HLA antigens. Initial experiments first confirmed that the parent line T5-1 was susceptible to cytolysis by both "common" A2-restricted and B27-restricted effector cells. While those T5-1 mutants with little or no surface A2 expression were not lysed by A2-restricted effectors, those targets with quantitatively normal expression of mutant A2 molecules were as susceptible to A2-restricted lysis as the parent line itself. In contrast, all the T5-1 mutant lines were susceptible to B27-restricted cytolysis. The results demonstrate that experimentally induced mutations of HLA-A2 antigen structure, affecting a serologically defined site on the molecule, can occur without altering that same molecule's expression of the T cell-restricting determinant(s). Such experimentally induced mutations are quite different from the naturally occurring "variant" A2 antigens which are present within the serologically defined A2 antigen group and which show changes at the T cell-restricting site.

Delineation of immunologically and biochemically distinct HLA-A2 antigens.

Cytotoxic T cell (CTL) recognition of influenza virus in conjunction with HLA-A2 was examined in a population study. Virus-infected target cells from three unrelated A2-positive donors were not lysed by virus-immune CTL from any donor matched only for A2. The A2 antigens of these three donors were indistinguishable from the A2 antigens of other A2-positive donors as assessed by extensive serologic analyses; however, isoelectric focusing (IEF) of A2 molecules from these three donors demonstrated that their A2 heavy polypeptide chains are structurally distinct from those of "normal" A2-positive donors. To date 11% of all A2-positive donors tested exhibited a "variant" A2-associated CTL restriction antigen, and IEF of A2 heavy chains from all "variant" A2-positive cells revealed structural differences in each of these polypeptides. These results suggest there may be considerably greater polymorphism of HLA-A gene products than has been revealed by current serologic techniques.

Virus-immune cytotoxic T cells recognize structural differences between serologically indistinguishable HLA-A2 molecules

The self-specificity of human influenza virus-immune cytotoxic T cells has been analyzed in order to identify the relationship between the self-determinants which they recognize and the serologically defined HLA-A and -B antigenic determinants. Virus-immune T cells were generated in vitro by culture of normal adult peripheral blood lymphocytes with A/HK influenza virus. Virus-immune effectors from HLA-A2 positive donors were tested on panels of virus-infected target cells from donors who were either HLA-mismatched or matched only for the HLA-A2 specificity. Virus-immune T cells from 11/11 A2-positive donors lysed all 2A-matched virus-infected target cells (and no HLA-mismatched targets), except that each of these effector cell populations consistently failed to lyse the virus-infected target cells from ove A2-positive donor (designated M7). Although the A2 antigen of donor M7 could also be distinguished from the A2 antigen of other donors by alloimmune cytotoxic T cells, no differences in the A2 antigen of donor M7 could be defined by extensive serological analyses. Results of iscelectric focusing of A2 molecules from three individuals plus M7 demonstrated that the M7 A2 heavy polypeptide chain is structurally distinct. These results indicate that: 1) there is a strong but incomplete associatione between a self antigen recognized by virus-immune T cells and the serologically defined HLA-A2 specificity; and 2) there may be at least two structurally and functionally distinct epitopes on the same A2 molecule: one is the serologically defined HLA-A2 antigenic determinant; the other is the self determinant recognized by T cells on HLA-A2 molecules.

Rabbit allotype a locus subspecificities of homogeneous anti-streptococcal antibodies.

Rabbits were immunized with group A-variant (Av) streptococcal vaccine to elicit antisera containing antibodies of "restricted heterogeneity" specific for Av polysaccharide. Antibodies that migrated as single bands in polyacrylamide gel isoelectric focusing were isolated on agarose block electrophoresis and preparative isoelectric focusing. These homogeneous antibodies were analyzed for content of the a locus markers by inhibition of allotype-anti-allotype radioimmunoassays. Both single-band preparations and preparations containing 2--5 bands were deficient in content of the a locus markers. These analyses were strongly influenced by the different anti-allotype antisera used. Single-band preparations, deficient in a 2 or a 3 markers, were further used to produce anti-allotype antisera, and these sera were assayed for reaction with pooled a2 or a3 molecules. The results, in contrast to the a1 allotype, failed to demonstrate common determinants on either a 2 or a 3 molecules indicating that each a allotype consists of a set of different Ig molecules. The data suggest different evolutionary pathways of the a 1, as opposed to the a 2 and a 3 molecules.

Structure and thermodynamic properties of the complexes between phospholipase A2 and lipid micelles

The interaction between porcine pancreatic phospholipase A2 and a homogeneous population of micelles of the subtrate analogue n-hexadecylphosphorylcholine containing 155 lipid monomers was studied by light scattering, equilibrium gel filtration, and isothermal calorimetry. From the detergent/protein molar ratio and the equivalent "molecular weight" of the resulting complex it is concluded that insertion of the enzyme into the detergent micelle results in a protein--detergent complex containing two phospholipase A2 molecules and 80 lipid monomers at 25 degrees C. The affinity constants and complex composition have been determined at different temperatures, allowing calculation of the thermodynamic parameters of the binding process. It is concluded that the interaction of phospholipase A2 with micellar lipids is predominantly hydrophobic.

Cross‐Reaction of a Minor Variant of the al Allotypic Specificity with Anti‐a2 Antibodies

Certain samples of hare IgG can combine with cross-linked anti-a2 antisera prepared in the a3/a3 rabbit. This cross-reaction permitted the isolation, on hare IgG immunoadsorbent, of anti-a2 cross-reacting antibodies (anti-a2(Lv) antibodies). The binding of labeled rabbit a2 IgG to insolubilized anti-a2(Lv) antibodies is inhibited by a1 IgG, demonstrating a cross-reactivity with a2. The percentage of a1 IgG cross-reacting with anti-a2 antiserum (a1(2) IgG) is about 0.5% of total a1 IgG. The a1(2) molecules represent another variant (or set of variants) of the a1 specificity. Demonstration of this variant of a1 IgG brings to seven the minimum number of described a1 variants, and its low concentration among a1 IgG (0.5%) is in favor of a larger number of variants of allotypic specificities. Arguments in favor of common ancestor genes for allotypy in lagomorphs are given.

Are “hidden” genes for immunoglobulin allotypes of rabbit heavy and light chains expressed upon hyper‐immunization?

AbstractThe question of whether genes for some Ig‐allotypic forms may be “hidden” in the genome was investigated by hyperimmunization of 36 allotypically pedigreed rabbits with a vaccine of Micrococcus luteus (Micrococcus lysodeikticus). Since Strosberg et al. (J. Immunol 1974.113: 1313) have reported that such hyperimmunization elicited expression of a third “allele” at two different loci (a2 and b6 in a rabbit whose original phenotype was ala3b4b5), we wished to determine whether their immunization protocol would reproducibly elicit expression of “hidden” allotypes. None of the 36 immunized rabbits developed elevated expression of an unexpected allotype during the 7‐38 weeks of immunization and bleeding. However, we did observe elevated expression of unusual variable regions. In particular, a cross‐reaction between some VH region gene products produced by al rabbits, and the majority of VH products of a2 rabbits was detected with anti‐a2 antisera made in a3a3 but not in a1a1 or a1a3 rabbits. There is a small percentage of Ig molecules in a1a1 rabbits whose V regions contain an antigenic determinant also present on most or all a2 molecules of rabbits. A similar determinant is also found on Ig of related genera (Lepus and Sylvilagus) Expression of this population was elevated during immunization of an a1a3 rabbit with Micrococcus. In immunodiffusion, a reaction of partial identity was observed. In hemagglutination inhibition, the reaction of a3 anti‐a2 with homologous a2 was not inhibitable by the cross‐reacting component. Anti‐a2 antisera made in a3 rabbits could be depleted of cross‐reactive antibody by absorption with insolubilized sera from normal al rabbits but not by insolubilized a3 sera. Radioautography of isoelectric focusing plates overlayed with a3 anti‐a2 antibodies which had been affinity‐purified and radiolabeled, revealed distinct spectrotypes in sera from al rabbits that carried the cross‐reacting VH determinants.

Electron microscopy of negatively stained jackbean urease at three levels of quaternary structure, and comparison with hydrodynamic studies.

Electron microscopy, with sodium phosphotungstate as negative stain, has been carried out on purified jackbean urease prepared at three levels of quaternary structure: (a) A1 urease, Mr = 240 000, S20,W = 11.5 S (b) alpha urease, Mr = 480 000, S20,w = 18.3 S (c) polymers of alpha urease above the tetramer stage. The compatibility of the images from level to level leaves no doubt that the enzyme itself is being visualized, and the following geometry is suggested by electron microscopy: A1 molecules are cyclic trimers, which pair up in eclipsed position across a 1-nm cleft to form the hexameric alpha, which displays D3 (or 32) symmetry of a trigonal prism. Polymers consist of alpha molecules aligned with their clefts coplanar and an angle of 120 degrees between each triplet of 3-fold axes. These features correspond reasonably well with sedimentation and electrophoretic studies of the solvated enzyme, which have indicated a hemispherical A1, a spherical alpha, and string-of-beads polymers. Sedimentation constants of the urease polymers up through the pentamer level were found to be compatible with the rosette, straight-chain, and zig-zag forms seen in the electron microscope, and with the suggested protomer arrangement in A1 and alpha urease.

Evidence for quasi-silent germline genes coding for phylogenetically ancient determinants of the rabbit a locus allotypes.

Anti-a2 sera raised in a3 rabbits are shown to detect determinants on a2 molecules which are different from those detected by anti-a2 sera raised in a1 animals. The former determinants are occasionally observed at a low level in rabbits of the a1 allotype and at a high level in sera of Leporidae of different genera. The two types of anti-a2 sera are shown to compete for the same sterical region of the a2 molecules. All homogeneous a2 molecules which have been tested show both types of determinants.

Electrostatic binding in the first-row AH and A2diatomic molecules

The charge migration that converts two overlapping spherical atoms into a bound molecule produces attractive Hellmann-Feynman fields at the two nuclear positions, offsetting the repulsive field that each nucleus encounters on penetrating inside the charge cloud of its neighbour. The magnitudes of these fields correlate positively with the molecular dissociation energies. The contribution of the σ valence orbitals to the field at a first-row nucleus varies regularly from binding in lithium to anti-binding in fluorine. The electrostatic effect of these orbitals is weakened by reversed polarization inside the 2s nodal sphere and is further opposed by a tiny but effective exchange polarization of the core. The major binding role in the first row thus falls to the π orbitals, which, like the σ orbital in hydrogen, have no radial node to inhibit a uniformly binding forward polarization. The net electrostatic interaction of two spherical atoms, derived with neglect of antisymmetry, is always binding. Especially in the heavier first-row A2 molecules, this classical Coulomb attraction provides more of the binding energy than either charge accrual on the bond axis, which is opposed by the exclusion principle, or contraction towards the nuclei, which is characteristic of hydrogen but hardly occurs in first-row atoms.

Expression of the a, x, and y Variable Region Genes of Heavy Chains among IgG, IgM, and IgA Molecules of Normal and a Locus Allotype-Suppressed Rabbits

Abstract The contribution of the VH subgroup genes, a, x and y, to the formation of individual IgG molecules has been studied in normal and a locus allotype-suppressed rabbits. Using anti-x32, anti-y33 and antisera to the a locus allotypes (anti-a1, anti-a2 and anti-a3), we used indirect precipitation of 125I-labeled IgG or Fab of specific allotype or inhibition of precipitation for the quantitative analyses. In normal rabbits homozygous with respect to the a locus, about 70 to 90% of the total IgG molecules possess the a locus allotypic specificities and most or all of the remaining molecules, depending on the genotype, could be accounted for as x32 or y33 molecules. Additional IgG molecules with unknown allotypic specificities for the VH region were found in two normal a3 rabbits and in an a1 allotype-suppressed rabbit; these molecules are probably the products of allelic alternatives to x32 and/or y33. Allotype-suppression of a locus molecules was compensated for by an increase in the number of molecules synthesized by closely linked VH subgroup genes, x and y. A similar ratio of the a2, x32, and y33 molecules was observed among Ig fractions with different net electrical charges. In a partially a2-suppressed rabbit, the results of indirect precipitation and sequential precipitation indicated that no two of the allotypic specificities, a2, x32, or y33, are located on the same IgG molecule or on the same Fab fragment. In homozygotes, a locus suppression was found for IgM and IgA as well as for IgG. The expression of a selected VH gene in a cell synthesizing Ig molecules results from linked gene exclusion apparently independent of CH gene selection.

Distribution of Allelic Allotypes among γG-Immunoglobulins and Purified Anti-Hapten Antibodies from the Same Rabbit

Summary Antibodies were prepared to the two haptenic groups 2,4-dinitrophenyl (DNP) and p-azobenzenearsonate (R-azo) in 17 rabbits heterozygous at either or both the a or b allotypic specificity loci. By qualitative Ouchterlony test, every purified antibody preparation contained the same allotypic specificities as that of the immune serum from which it had been isolated. By quantitative Oudin analysis in agar gel tubes, the quantitative variation of allotypes among the antibody preparations of individual rabbits was considerable. Among five b4/b5 heterozygous rabbits, the concentration of b4 and b5 molecules in the purified anti-DNP population was not consistently different from that in the bulk γG-immunoglobulins from which they were isolated (P > 0.20). On the other hand, among the same five b4:b5 heterozygotes as well as two more, the concentration of b4 molecules was consistently greater (P < 0.05) and of b5 molecules consistently less (P < 0.01) in the purified anti-R-azo population than in the bulk γG-immunoglobulins from which they were isolated. Furthermore, among four a1/a3 heterozygous rabbits, the concentration of a1 molecules was consistently less (P < 0.01) and of a3 molecules consistently more (P < 0.09) in the anti-DNP population than in the bulk γG-immunoglobulins from which they were isolated. While these results suggest that antibodies of a given specificity are selectively formed from certain allotypes, confidence in the biologic significance of the present results must be restrained until additional experimental conditions outlined in this article are satisfied.