Structural relatedness of distinct determinants recognized by monoclonal antibody TP25.99 on beta 2-microglobulin-associated and beta 2-microglobulin-free HLA class I heavy chains

The association of HLA class I heavy chains with beta2-microglobulin (beta2m) changes their antigenic profile. As a result, Abs react with either beta2m-free or beta2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both beta2m-associated and beta2m-free HLA class I heavy chains. The reactivity with beta2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194-198 in the alpha3 domain. The reactivity with beta2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239-242, 245, and 246 in the alpha3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs.     

Human beta 2-microglobulin specifically enhances cell-surface expression of HLA class I molecules in transfected murine cells

Sequential transfections of P815 murine mastocytoma cells with class I gene encoding either HLA-Cw3, HLA-A3, or HLA-B7 H chain and subsequently with a human beta 2-microglobulin gene were performed to evaluate the relative efficiency of human and murine beta 2-microglobulins in promoting the cell-surface expression of HLA-class I molecules. A 6-, 11-, and 40-fold specific enhancement of the cell-surface expression of HLA-Cw3, HLA-A3, and HLA-B7 molecules, respectively, was observed in cells co-transfected with human beta 2-microglobulin gene. This effect was attributed to a more efficient association of HLA H chains with human than with murine beta 2-microglobulin, which apparently allowed a more rapid transport of the HLA molecules from the endoplasmic reticulum to the Golgi apparatus.     

Association with beta 2-microglobulin controls the expression of transfected human class I genes

The genes encoding HLA-B27K and HLA-B27W were transfected into murine recipient cells. A monoclonal antibody HC-10, directed against free B-locus heavy chain, was the only reagent capable of efficiently detecting the HLA-B27 heavy chains in detergent lysates. These heavy chains were devoid of sialic acid. Trace amounts of HLA-B27 could be isolated with the anti-HLA-A,-B antibody W6/32, which reacts with the heavy chain beta 2-microglobulin complex. In marked contrast, HLA-A2 and -B7 genes, when transfected, yielded easily detectable amounts of antigen precipitable with W6/32, which carried the usual complement of sialic acids. Because the alpha 3 domains of HLA-B27 and HLA-B7 and the more COOH-terminal portions are identical in amino acid sequence, structural elements in the polymorphic alpha 1 and alpha 2 domains must control association of heavy chain with beta 2-microglobulin. Introduction of a human beta 2-microglobulin gene into L cells transfected with the HLA-B27 gene rescued the expression of HLA-B27 at the cell surface, as evidenced by reactivity with W6/32, surface staining, and the presence of sialic acid on the heavy chain.     

Peptide binding to HLA-A2 and HLA-B27 isolated from Escherichia coli. Reconstitution of HLA-A2 and HLA-B27 heavy chain/beta 2-microglobulin complexes requires specific peptides.

The specificity of peptide binding by human leukocyte antigen (HLA) class I molecules was investigated in a cell-free direct-binding assay. Peptides were assessed for binding to HLA-A2 and HLA-B27 by measuring the formation of heterotrimeric HLA complexes that consisted of iodinated beta 2-microglobulin, HLA heavy chain fragments isolated from the Escherichia coli cytoplasm, and peptide. In this system, no detectable HLA heavy chain-beta 2-microglobulin complexes were formed unless appropriate peptides were intentionally added to the reconstitution solution. Analysis with monoclonal antibodies demonstrated that these heterotrimeric complexes were correctly folded. Five nonhomologous peptides, known to form complexes with HLA-A2 or HLA-B27 from T-cell functional studies, were tested for their capacity to bind to HLA-A2 and HLA-B27 using the reconstitution assay. Four of the peptides bound to the appropriate class I molecule only. One peptide and some (but not all) substitution analogs of it bound to both HLA-A2 and HLA-B27. The effect of peptide length on binding to HLA-B27 was studied, and it was found that the optimal length was 9 or 10 amino acid residues; however, one peptide that bound to HLA-B27 was 15 amino acids long. All peptides that bound to HLA-B27 in the direct-binding assay also competed with antigenic peptides for binding to HLA-B27 on the surface of intact cells, as determined by a standard cytotoxic T-lymphocyte functional assay. Thus, we conclude that HLA-A2 and HLA-B27 bind distinct but partially overlapping sets of peptides and that, at least in vitro, the assembly of HLA heavy chain-beta 2-microglobulin complexes requires specific peptides.     

Dependence of elevated human leukocyte antigen class I molecule expression on increased heavy chain, light chain (beta 2-microglobulin), transporter associated with antigen processing, tapasin, and peptide

Human leukocyte antigen (HLA) class I molecule expression was investigated by DNA-mediated gene transfer. Cell surface expression was increased up to 75% by transfection of HLA-A2 or HLA-B8 heavy chain genes but not genes encoding light chains (beta(2)-microglobulin (beta(2)m)), transporter associated with antigen processing (TAP), or tapasin. Interferon (IFN) treatment further increased expression of transfected heavy chains, suggesting that IFN inducible molecules support heavy chain expression. IFN induces beta(2)m, TAP, and tapasin mRNAs. Transfected heavy chain expression increased upon cotransfection with genes encoding TAP1 and TAP2 but not individual TAP subunits, beta(2)m, or tapasin. Tetracycline inducible heavy chain gene expression was also increased by IFN treatment or TAP cotransfection, suggesting that IFN-induced TAP supports heavy chain maturation. Expression of a mutant that does not interact strongly with TAP, HLA-A2-T134K, was also increased by IFN. Inhibition of TAP-dependent peptide transport by ICP47 reduced heavy chain expression. Expression of HLA-A2, but not HLA-B8, was restored in ICP47 cells by HLA-A2-binding (IP-30) signal peptides. However, these peptides did not further increase transfected HLA-A2 expression, suggesting that peptide availability does not limit heavy chain expression in the absence of ICP47. These results suggest that cytokine-induced TAP supports maturation of HLA class I molecules through combined chaperone and peptide supply functions.     

Coexpression of the human HLA-A2 or HLA-B7 heavy chain gene and human beta 2-microglobulin gene in L cells

L cells expressing human HLA-A2 or HLA-B7 class I antigen heavy chains are not recognized by human cytotoxic T lymphocytes directed at HLA-A2 or HLA-B7 antigens. To test whether the absence of human beta 2-m was the cause of the lack of recognition by the human cytotoxic T lymphocytes, coexpression of the human beta 2-m gene and the HLA-A2 or HLA-B7 heavy chain in L cells ("double transfectants") was obtained. In addition, L cells expressing HLA-A2 or HLA-B7 antigens in association with human beta 2-m were obtained by an exchange reaction, in which human beta 2-m from serum replaced the endogenous murine beta 2-m. Both types of transfectant cells were used in 51Cr-release assays and cold target inhibition assays for human cytotoxic T cell clones which were directed at HLA-A2 or HLA-B7. Neither human CTL clones nor a mixture of CTL specific for HLA-A2 and HLA-B7 were able to recognize these cells. Several alternative explanations for these observations are discussed.     

Acquisition of HLA class I W6/32 defined antigenic determinant by heavy chains from different species following association with bovine beta 2-microglobulin

Murine, rat, rabbit and guinea pig class I heavy chains, which do not react with W6/32 monoclonal antibody when they are expressed in association with autologous beta 2-microglobulin (beta 2-m), can acquire such a reactivity once they are expressed at the surface of cells cultured in conditions which allow their association with bovine beta 2-m. Sequence comparison of beta 2-ms suggests that glutamine at position 89 might be critical for the induction of the W6/32 defined antigenic determinant. However, in the murine species, certain class I heavy chains, in spite of their association with bovine beta 2-m, do not express this determinant. Using genetically engineered hybrid class I molecules and selected congenic strains of mice this negative property was shown to be related to the presence of a cysteine residue at position 121 which allows covalent association of beta 2-m to class I heavy chains (Bushkin, Y., J-S. Tung, A. Pinter, J. Michaelson, and E. A. Boyse. 1986. Unusual association of beta 2-microglobulin with certain class I heavy chains of the murine major histocompatibility complex. Proc. Natl. Acad. Sci. USA 83:432). Therefore, expression of the W6/32 defined antigenic determinant implicates both the beta 2-m and the second domain of the heavy chain, but its expression (or exposure) is prevented by the covalent fixation on cysteine 121 of the light chain.     

An antigenic determinant of human beta 2-microglobulin masked by the association with HLA heavy chains at the cell surface: analysis using monoclonal antibodies

Four anti-human beta 2-microglobulin monoclonal antibodies were produced against whole lymphoid cells or against urinary beta 2-microglobulin. Their reactivity was fully inhibited by purified soluble beta 2-microglobulin. The B1.1G6, C23.24.2, and B2.62.2 antibodies bound either to free or to HLA-complexed beta 2m. They recognized the same or very close determinants, since they achieved mutual blocking. In contrast, the C21.48A1 antibody did not bind to the cell surface and did not recognize the same determinant on the purified beta 2-microglobulin molecule as the others. It was able to bind and to form a specific complex with membrane beta 2-microglobulin only, once separated from the HLA heavy chain. These data strongly suggest that the C21.48A1 antigenic determinant might be hidden when the beta 2-microglobulin is complexed with the HLA heavy chains at the cell surface.     

The epitope recognized by pan-HLA class I-reactive monoclonal antibody W6/32 and its relationship to unusual stability of the HLA-B27/β2-microglobulin complex

A broadly used pan-HLA class I-reactive monoclonal antibody W6/32 is believed to recognize a conformational epitope dependent on association between heavy chains and beta2-microglobulin (beta2m). However, in the present study we report that W6/32 does recognize at least some free HLA class I heavy chains under the partially denaturating conditions of nonreducing Western blotting, namely nearly all HLA-B allelic products. Furthermore, we confirm and largely extend our previous observation that complexes of beta2m with heavy chains of a few HLA class I allelic forms (most notably HLA-B27) exhibit unusual resistance to dissociation by SDS, which is reminiscent of MHC class II molecules. In addition, our data indicate the existence of covalent (disulfide-linked) heterodimers of certain HLA class I heavy chains (namely Cw1 and Cw4) and beta2m.     

Formation of HLA-B27 homodimers and their relationship to assembly kinetics

The human HLA-B27 class I molecule exhibits a strong association with the inflammatory arthritic disorder ankylosing spondylitis and other related arthropathies. Major histocompatibility complex class I heavy chains normally associate with beta(2)-microglobulin and peptide in the endoplasmic reticulum before transit to the cell surface. However, an unusual characteristic of HLA-B27 is its ability to form heavy chain homodimers through an unpaired cysteine at position 67 in the peptide groove. Homodimers have previously been detected within the ER and at the cell surface, but their mechanism of formation and role in disease remain undefined. Here we demonstrate, in the rat C58 thymoma cell line and in human HeLa cells transfected with HLA-B27, that homodimer formation involves not only cysteine at position 67 but also the conserved structural cysteine at position 164. We also show that homodimer formation can be induced in the non-disease-associated HLA class I allele HLA-A2 by slowing its assembly rate by incubation of cells at 26 degrees C, suggesting that homodimer formation in the endoplasmic reticulum may occur as a result of the slower folding kinetics of HLA-B27. Finally, we report an association between unfolded HLA-B27 molecules and immunoglobulin-binding protein at the cell surface.     

Altered structure of HLA class I heavy chains associated with mouse beta-2 microglobulin

The serological reactivities of HLA-A3, -B7, and -CW3 heavy chains associated with either mouse, bovine, or human beta-2 microglobulin ( ₂m) and expressed on the surface of transfected mouse fibroblasts were analyzed. All reactivities associated with one cluster (defined by monoclonal antibody W6/32) of antigenic determinants expressed by these HLA class I molecules were lost, or profoundly reduced, after each heavy chain associated with mouse ₂-m. Expression by the transfected fibroblasts of the HLA-A3, -B7, and -CW3 heavy chains in association with human ₂m restores these reactivities. Since most of the amino acid differences between mouse and human ₂m probably correspond to externally oriented hydrophilic residues, these results suggest that critical interactions in the three-dimensional structure of HLA class I molecules occur between the light chain and the first two external domains of the class I heavy chains, to which some of the altered reactivities have been mapped.     

Analysis of the HLA-Cw3-specific cytotoxic T lymphocyte response of HLA-B7 X human beta 2m double transgenic mice

The cytolytic responses of either normal (non transgenic), HLA-B7 (single transgenic) or HLA-B7 x human beta 2 microglobulin (double transgenic) DBA/2 mice induced by transfected HLA-Cw3 P815 (H-2d) mouse mastocytoma cells were compared, to evaluate whether the expression of an HLA class I molecule in responder mice would favor the emergence of HLA-specific, H-2-unrestricted CTL. Only 8 of 300 HLA-Cw3-specific CTL clones tested could selectively lyse HLA-Cw3-transfected cells in an H-2-unrestricted manner, all having been isolated after hyperimmunization of double transgenic mice. These clones also lysed HLA-Cw3+ human cells. Unexpectedly, the lysis of the human but not that of the murine HLA-Cw3 cells was inhibited by Ly-2,3-specific mAb. Despite significant expression of HLA-B7 class I molecules on transgenic lymphoid cells, including thymic cells, limiting dilution analysis and comparative study of TCR-alpha and -beta gene rearrangements of the eight isolated clones (which suggested that they all derived from the same CTL precursor) indicated that the frequency of HLA-Cw3-specific H-2 unrestricted cytotoxic T lymphocytes remained low (even in HLA-B7 x human beta 2-microglobulin double transgenic mice). This suggests that coexpression of HLA class I H and L chain in transgenic mice is not the only requirement for significant positive selection of HLA class I-restricted cytotoxic mouse T lymphocytes.     

Translocation of peptides through microsomal membranes is a rapid process and promotes assembly of HLA-B27 heavy chain and beta 2-microglobulin translated in vitro

We have translated major histocompatibility complex (MHC) class I heavy chains and human beta 2-microglobulin in vitro in the presence of microsomal membranes and a peptide from the nucleoprotein of influenza A. This peptide stimulates assembly of HLA-B27 heavy chain and beta 2-microglobulin about fivefold. By modifying this peptide to contain biotin at its amino terminus, we could precipitate HLA-B27 heavy chains with immobilized streptavidin, thereby directly demonstrating class I heavy chain-peptide association under close to physiological conditions. The biotin-modified peptide stimulates assembly to the same extent as the unmodified peptide. Both peptides bind to the same site on the HLA-B27 molecule. Immediately after synthesis of the HLA-B27 heavy chain has been completed, it assembles with beta 2-microglobulin and peptide. These interactions occur in the lumen of the microsomes (endoplasmic reticulum), demonstrating that the peptide must cross the microsomal membrane in order to promote assembly. The transfer of peptide across the microsomal membrane is a rapid process, as peptide binding to heavy chain-beta 2-microglobulin complexes is observed in less than 1 min after addition of peptide. By using microsomes deficient of beta 2-microglobulin (from Daudi cells), we find a strict requirement of beta 2-microglobulin for detection of peptide interaction with the MHC class I heavy chain. Furthermore, we show that heavy chain interaction with beta 2-microglobulin is likely to precede peptide binding. Biotin-modified peptides are likely to become a valuable tool in studying MHC antigen interaction and assembly.     

Construction of bioactive chimeric MHC class I tetramer by expression and purification of human-murine chimeric MHC heavy chain and beta(2)m as a fusion protein in Escherichia coli

Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a beta(2)-microglobulin (beta(2)m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and beta(2)m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 alpha1, HLA-A2 alpha2 and MHC-H2D alpha3) and beta(2)m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18-27 FLPSDFFPSI, HBx52-60 HLSLRGLPV, and HBx92-100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.     

Chemical, physical-chemical, and immunological properties of papain-solubilized human transplatation antigens.

Papain-solubilized HLA-A, -B, and -C antigens have been isolated from cadaveric spleens. The isolated material was homogeneous and comprised subunits with the apparent molecular weights 33,000 and 12,000. Amino acid analyses of a mixture of HLA antigen heavy chains obtained from a great number of spleens with different HLA antigen phenotypes revealed a composition that is very similar to that of individual HLA-A and -B antigens. Likewise, the NH2-terminal 30 residues of the HLA-antigen heavy chain mixture were virtually identical with that recorded for individual specificities. The circular dichroism spectra for the isolated HLA antigens and for free beta2-microglobulin revealed similarities with spectra recorded for immunoglobulin chains and domains. The HLA-antigen heavy chain may contain an appreciable amount of beta structure. Antibodies raised against free beta2-microglobulin react better with beta2-microglobulin in free form than when bound to the HLA-A, -B, and -C antigen heavy chains. This is due to the fact that free beta2-microglobulin can bind a maximum of four Fab fragments simultaneously, whereas the HLA-antigen-associated beta2-microglobulin can bind only two Fab fragments without dissociating from the heavy HLA-antigen subunit.     

Expression of the major histocompatibility antigens HLA-A2 and HLA-B7 by DNA-mediated gene transfer

Genes coding for the heavy chain of the class I antigens HLA-A2 or HLA-B7 of the human major histocompatibility complex have been introduced into mouse LtK- cells by cotransfection with the herpes simplex virus thymidine kinase gene. HAT-resistant colonies were isolated expressing either HLA-A2 or HLA-B7 as monitored by indirect immunofluorescence. Immunoprecipitation analysis of both antigens by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing (IEF) showed that they were identical to the HLA-A2 and HLA-B7 expressed in the human lymphoblastoid cell line JY (homozygous HLA-A2, HLA-B7). However, human cytotoxic T lymphocytes (CTL) generated against JY and CTL clones specific for HLA-A2 or HLA-B7 were unable to recognize the transfectants as targets. These results indicate that the human HLA-A2 (or B7) complexed with the murine beta 2-microglobulin could be an inappropriate target structure for the CTL. However, because the transfectants are not killed by human CTL even in the presence of lectins, it is suggested that other molecules that are not able to overcome the human-mouse species barrier may be involved in the killing mechanism.     

Human MHC class I transgenic mice deficient for H2 class I expression facilitate identification and characterization of new HLA class I-restricted viral T cell epitopes

Although mice transgenic (Tg) for human MHC (HLA) class I alleles could provide an important model for characterizing HLA-restricted viral and tumor Ag CTL epitopes, the extent to which Tg mouse T cells become HLA restricted in the presence of endogenous H2 class I and recognize the same peptides as in HLA allele-matched humans is not clear. We previously described Tg mice carrying the HLA-B27, HLA-B7, or HLA-A2 alleles expressed as fully native (HLA(nat)) (with human beta(2)-microglobulin) and as hybrid human/mouse (HLA(hyb)) molecules on the H2(b) background. To eliminate the influence of H2(b) class I, each HLA Tg strain was bred with a H2-K(b)/H2-D(b)-double knockout (DKO) strain to generate mice in which the only classical class I expression was the human molecule. Expression of each HLA(hyb) molecule and HLA-B27(nat)/human beta(2)-microglobulin led to peripheral CD8(+) T cell levels comparable with that for mice expressing a single H2-K(b) or H2-D(b) gene. Influenza A infection of Tg HLA-B27(hyb)/DKO generated a strong CD8(+) T cell response directed at the same peptide (flu nucleoprotein NP383-391) recognized by CTLs from flu-infected B27(+) humans. As HLA-B7/flu epitopes were not known from human studies, we used flu-infected Tg HLA-B7(hyb)/DKO mice to examine the CTL response to candidate peptides identified based on the B7 binding motif. We have identified flu NP418-426 as a major HLA-B7-restricted flu CTL epitope. In summary, the HLA class I Tg/H2-K/H2-D DKO mouse model described in this study provides a sensitive and specific approach for identifying and characterizing HLA-restricted CTL epitopes for a variety of human disease-associated Ags.     

Studies on biosynthesis, assembly and expression of human major transplantation antigens

Biosynthesis and regulation of expression of transplantation as detected by a monoclonal antibody to HLA-A,B,C antigens (human leucocytic antigen) and a polyclonal antiserum to beta 2-microglobulin have been investigated using radioactive amino acids and sugars to label human lymphoid cells. We found unbalanced synthesis of HLA heavy chains and beta 2-microglobulin, the latter being in excess and secreted to the extracellular medium. In DAUDI cells, which are defective in beta 2-microglobulin, no HLA-A,B,C could be detected intracellularly even in the presence of added beta 2-microglobulin. Treatment of BRI-8 cells with tunicamycin, an antibiotic which inhibits glycosylation of polypeptides, almost had no effect on the levels of beta 2-microglobulin, while it markedly decreased that of HLA heavy chains, both on the cell surface and intracellularly. Glycosylation of the HLA heavy chains appeared to be an essential requirement for the normal expression of HLA-A,B,C antigens. The translation in vitro in a messenger-dependent reticulocyte system with total polysomes obtained from BRI-8 cells showed that beta 2-microglobulin was synthesized as a precursor. This larger polypeptide was converted into mature beta 2-microglobulin when protein synthesis was performed with microsomes instead of polysomes.     

Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones

Human cytolytic T lymphocytes (CTL) clones and HLA-A2- and HLA-B7-transfected human, monkey, and mouse cell lines were used to investigate the basis for species-restricted antigen recognition. Most allospecific CTL clones obtained after stimulation with the human JY cell line (source of HLA-A2 and HLA-B7 genomic clones) recognized HLA antigens expressed in human and monkey cell lines but did not recognize HLA expressed in murine cells. By initially stimulating the responder cells with HLA-transfected mouse cells, two CTL clones were obtained that recognized HLA expressed in murine cells. Functional inhibition of these CTL clones with anti-class I monoclonal antibodies (MAb) indicated that clones reactive with HLA+ murine cells were of higher avidity than clones that did not recognize HLA+ murine target cells. MAb inhibition of accessory molecule interactions demonstrated that the LFA-1 and T8 surface molecules were involved in CTL-target cell interactions in all three species. In contrast, the LFA-2/CD2 molecule, previously shown to participate in a distinct activation pathway, was involved in the cytolysis of transfected human and monkey target cells, but not in the lysis of HLA+ murine cells. Thus transfection of HLA genes into different recipient species cell lines provides us with the ability to additionally delineate the functional requirements for allospecific CTL recognition and lysis.