Amino acid sequences in the alpha 1 domain and not glycosylation are important in HLA-A2/beta 2-microglobulin association and cell surface expression.

The role of the single carbohydrate moiety present on the HLA-A2 molecule was studied by introducing several amino acid substitutions (by site-directed mutagenesis of the HLA-A2 gene) in the consensus glycosylation sequence Asn-X-Ser. Two different amino acid substitutions of the asparagine residue at position 86 (glutamine and aspartic acid) resulted in the synthesis of ca. 39,000-molecular-weight nonglycosylated heavy chains that were detected in the cytoplasm but not on the surface of mouse L-cell transfectants. However, a low level of surface expression was detected following transfection of human (rhabdomyosarcoma) cells or mouse L cells containing human beta 2-microglobulin. The defect in surface expression was not due to the absence of the glycan moiety, since the substitution of a glycine for a serine at amino acid 88 did not have the same drastic effect in the presence of human beta 2-microglobulin. These and other data suggest that the asparagine residue may play a critical role in the conformation of the HLA heavy chain and its interaction with beta 2-microglobulin. Immunofluorescence microscopy following permeabilization of the transfectants demonstrated that the unglycosylated HLA heavy chains are sequestered in an unidentified cellular compartment that is different from the Golgi structure.     

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.     

Somatic cell genetic analysis of HLA-A, B, C and human beta 2-microglobulin expression

We have examined the cell surface expression of the human histocompatibility antigens HLA-A, B, C and beta 2-microglobulin (beta 2m) on a human-mouse somatic cell hybrid line. Using specific antibodies and the fluorescence-activated cell sorter (FACS), we viably fractionated and characterized four separate hybrid subpopulations (HLA+,beta 2m+; HLA+,beta 2m-; HLA-,beta 2m+; HLA-,beta 2m-). Hybrid selection based on surface antigen expression resulted in corresponding genetic selection for and against human chromosomes 6 and 15. Studies of the homogeneous hybrid sublines revealed that the presence of human beta 2m in a hybrid cell dramatically increased the surface expression of human HLA-A, B, C and mouse H-2Kk antigens. The results demonstrate the importance of human chromosome-specific surface markers and the fluorescence-activated cell sorter in somatic cell genetic analysis.     

The association between murine beta 2-microglobulin and HLA class I heavy chains results in serologically detectable conformational changes of both chains

HLA-A3-, HLA-B7-, and HLA-CW3-transfected L cells, maintained in medium supplemented with murine serum so as to ensure that the human heavy chains were associated with murine beta 2-microglobulin, were subjected to a systematic serologic analysis for an evaluation of the structural consequences of such an heterologous association. The hybrid molecules exhibited alterations of their serologic reactivities that suggest the occurrence of structural modifications of both light and heavy chains. Thus, reactivity of HLA-A3-, HLA-B7-, and HLA-Cw3-transfected L cells with a monoclonal antibody (B1.1G6) directed at a human beta 2-microglobulin specific antigenic determinant was observed; this implies structural modifications of murine beta 2-microglobulin after its association with HLA class I heavy chains. Conversely, a profound reduction of the reactivity of the same transfectants with a monoclonal antibody (W6/32) directed at a monomorphic heavy chain related epitope was observed. The W6/32 reactivity was restored after replacement of the murine by the human light chain, indicating that the conformation adopted by the HLA class I heavy chain depends on the origin of the beta 2-microglobulin associated. Therefore it appears that the complex interactions that develop between the extracellular domains (including the one formed by the light chain) markedly influence the overall structure and the antigenic properties of HLA class I molecules.     

The absence of β 2-microglobulin in Daudi cells: Active gene but inactive messenger RNA

The Daudi cell line is characterized by an absence of HLA antigen on its surface. This has been attributed to a lack of beta 2-microglobulin (beta 2m) while the heavy chain of HLA is present intracellularly. Karyotype analysis of Daudi cells has shown a deletion involving one of the beta 2-microglobulin alleles. It was generally believed that the absence of beta 2-microglobulin in that cell line resulted from an absence of expression of the remaining gene. We report here the unexpected finding of a normal amount of beta 2-microglobulin messenger RNA in Daudi cells. This was demonstrated by "Northern blot" hybridization with cDNA plasmid clones as a probe. This mRNA, however, when purified by hybridization-selection with beta 2-microglobulin plasmid DNA, is unable to function as messenger in protein synthesis and is therefore an inactive mRNA. The finding of a translationally inactive beta 2-microglobulin mRNA provides a new explanation for the absence of beta 2-microglobulin and therefore of HLA antigens in Daudi cells.     

Role of beta2-microglobulin in the intracellular processing of HLA antigens

The biosynthesis of HLA-A, -B, and -C antigens was examined in the two lymphoblastoid cell lines DAUDI and RAJI. In RAJI cells the HLA-A, -B, and -C antigen heavy chains become core-glycosylated in the endoplasmic reticulum as evidenced by their sensitivity to endo-H digestion and tunicamycin treatment. Beta2-Microglobulin is present in excess in the endoplasmic reticulum of the RAJI cells and associates with the heavy chain at the time of synthesis of the heavy chain. Pulse-chase experiments demonstrated that the RAJI HLA-A, -B, and -C antigen heavy chains become terminally glycosylated since their changed characteristics included resistance to endo-H digestion, sensitivity to neuraminidase treatment, and incorporation fucose. DAUDI HLA-A, -B, and -C antigen heavy chains are synthesized normally and become core-glycosylated but not terminally glycosylated. Other glycosylated cell surface proteins, like the HLA-DR antigens, display normal glycosylation in DAUDI cells. Therefore it is unlikely that the absence of terminally glycosylated HLA-A, -B, and -C antigen heavy chains is the result of a general defect in the biosynthetic machinery of DAUDI cells. However, DAUDI cells lack the ability to synthesize beta2-microglobulin, the common subunit of all HLA-A, -B, and -C antigens. Therefore, it seems reasonable to conclude that beta2-microglobulin is of importance for intracellular transport of newly synthesized HLA-A, -B, and -C antigens.     

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.     

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.     

Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid.

Biosynthesis of HLA class I antigens has been studied in a variant B-LCLxT-LCL hybrid, 174XCEM.T2. This cell line encodes HLA-A2 and -B5, but expresses only small amounts of A2 antigen and undetectable B5 antigen at the cell surface due to a mutation inactivating a trans-acting regulatory gene encoded within the class II region of the human major histocompatibility complex. Northern blot analysis with HLA-A- and HLA-B-specific probes shows that 174XCEM.T2 synthesizes quantities of A and B locus mRNA comparable with its class I antigen-positive parent cell line. Immune precipitation studies indicate that 174XCEM.T2 synthesizes normal HLA heavy chains and beta 2-microglobulin which fail to form dimers. The heavy chains are N-glycosylated normally, but processing of the glycan to the complex form does not occur. In addition, free heavy chains in this cell line are not phosphorylated. Thus, the majority of class I heavy chains in 174XCEM.T2 do not combine with beta 2-microglobulin, and are not processed or transported to the cell surface. As both subunits are synthesized in normal amounts, we propose that an additional molecule absent from 174XCEM.T2 and encoded by an HLA-linked gene is necessary for efficient assembly of class I antigen subunits.     

Correlation between classification of human urothelial cell lines and HLA-A,B,C expression

Summary Quantitative changes in major histocompatibility class I antigen expression in tumour cells are believed to affect the host immune response against the tumour. In tumourigenic (TGrIII) human urothelial cell lines the apparent loss of polymorphic HLA-A,B epitopes has previously been demonstrated. In the present study, 3 non-tumourigenic (TGrII) and 6 tumourigenic (TGrIII) human urothelial cell lines have been investigated for their quantitative expression of monomorphic HLA-A,B,C and B₂-microglobulin. Evidence is provided that an inverse correlation exists between tumourigenicity and HLA-A,B,C and B₂-microglobulin expression. Furthermore, treatment of the cells with neuraminidase partly restored the expression of monomorphic HLA-A,B,C suggesting that at least some of the observed quantitative differences could be due to masking of the membrane bound HLA antigens by sialic acid-containing glycoconjugates.     

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.     

Major histocompatibility antigens and mononuclear inflammatory infiltrate in benign nevomelanocytic proliferations and malignant melanoma

The presence of major histocompatibility antigens in malignant melanoma and benign nevomelanocytic lesions and the nature of associated mononuclear inflammatory cells were studied in situ by using monoclonal antibodies and an immunoperoxidase technique. HLA-A,B,C (HLA) and beta 2-microglobulin (beta 2m) were found on malignant melanocytes in primary cutaneous and metastatic melanomas. In contrast, HLA antigens were not identified on nevomelanocytes in benign hyperplasia or nevocellular nevi, although in some cases faint staining for beta 2m was present. The staining of nevomelanocytes for HLA and beta 2 was variable in cases of nevomelanocytic dysplasia. The degree of mononuclear cellular response correlated with the expression of HLA (or beta 2m) on nevomelanocytes. Most of the inflammatory cells were identified as T cells. The majority of T cells were of helper/inducer phenotype, whereas a lesser number were phenotypically suppressor/cytotoxic T cells. The findings suggest that expression of HLA may be involved in triggering or eliciting a cellular immune response against dysplastic or malignant nevomelanocytes.     

Biologic and chemical characterization of HLA antigens in human serum.

HLA antigens of both the A and B loci were shown to be associated with the high density lipoprotein fraction of serum prepared by ultracentrifugal flotation. HLA-A9 antigens were purified 100-fold with essentially complete recovery by a simple procedure of high density lipoprotein preparation involving precipitation with polyanions and ultracentrifugal flotation. The purified lipid-associated antigen was immunogenic since it elicited the formation of cytotoxic xenoantibodies in rabbits. Serum HLA-A9 antigens were found by immunoprecipitation and gel electrophoresis to consist of a 45,000 m.w. heavy chain associated with beta2-microglobulin. The size of the HLA-lipid complex (less than 190,000 m.w.) and of the HLA-deoxycholate complex (less than 102,000 m.w.) suggests that HLA antigens are shed into plasma as a complex of a single HLA molecule and a single beta2-microglobulin chain, associated with boundary lipid.     

Monomorphic anti-HLA-A,B,C monoclonal antibodies detecting molecular subunits and combinatorial determinants

Thirteen monoclonal antibodies that react with monomorphic determinants on the HLA-A,B,C-beta 2-microglobulin (beta 2m) molecule were characterized. Analysis of antibody activity included inhibition by papain-solubilized HLA antigens and free beta 2m, antibody binding to mouse-human somatic cell hybrids containing human chromosome 6 or 15, and antibody cross-reactivity with lymphocytes from nonhuman species. Two criteria for monomorphism were established: 1) equal inhibition or absorption of antibody activity by all papain-solubilized HLA antigens or cell lines of different HLA specificities tested; and 2) nonpolymorphic cross-reactivity within another species or subspecies. On the basis of soluble antigen inhibition and binding to somatic cell hybrids, 3 classes of antibodies were detected: anti-beta 2m, anti-heavy chain, and anti-complex (against a combinatorial determinant formed by heavy chain and beta 2m). Antibody cross-reaction patterns in nonhuman species were suggestive that these monomorphic antibodies detect a limited number of determinants, minimally one on each chain and 2 combinatorial determinants. Examination of the known primary sequences for HLA-A2, HLA-B7, H-2Kb, and mouse, rabbit and human beta 2m provides a molecular explanation for this limited mouse anti-HLA monomorphic antibody activity.     

Differential effect of interferon on the expression of tumor-associated antigens and histocompatibility antigens on human melanoma cells: relationship to susceptibility to immune lysis mediated by monoclonal antibodies

Incubation of cultured human melanoma cells with human leukocyte interferon did not change the expression of melanoma-associated antigens (MAA) recognized by monoclonal antibodies and of Ia-like antigens but significantly increased the expression of HLA-A,B antigens and of beta 2-microglobulin (beta 2-mu). The effect is dependent on the dose of interferon and on the incubation time. Interferon-treated melanoma cells showed an increased susceptibility to lysis mediated by monoclonal antibodies to HLA-A,B antigens and to human beta 2-mu; on the other hand, interferon-treated melanoma cells did not change in their susceptibility to murine natural killer (NK) cell lysis and to immune lysis mediated by monoclonal antibodies to MAA and to Ia-like antigens, and they displayed a reduced susceptibility to human NK cell lysis. Therefore, the increased susceptibility of interferon-treated melanoma cells to lysis mediated by anti HLA-A,B and anti beta 2-mu monoclonal antibodies is likely to reflect the increase in cell surface expression of the corresponding antigens.     

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 class I histocompatibility antigens (HLA-A,B,C). A small proportion only is phosphorylated

Human Class I HLA antigens (HLA-A,B,C) were isolated by immune precipitation from cells labelled with 32P, [35S]methionine or 125I (by lactoperoxidase-catalysed cell-surface iodination) and were analysed using both one- and two-dimensional electrophoretic systems. In several B-lymphoblastoid cell lines and in human peripheral blood lymphocytes the electrophoretic mobility of the 32P-labelled HLA-A,B,C heavy chains consistently differed from that of molecules labelled by other means. Thus the 32P-labelled heavy chains appeared to be larger and to possess a more acidic pI than did heavy chains labelled with [35S]methionine or 125I, or identified by Coomassie Blue staining. Phosphatase treatment of immunoprecipitates, under conditions where 32P-labelled antigens were shown to be dephosphorylated, did not affect the mobilities of the [35S]methionine-labelled heavy chains. On glycosidase treatment, the positions of the 32P-labelled heavy chains were affected by neuraminidase but not by endo-beta-N-acetylglucosaminidase H. These results imply that phosphorylated HLA-A,B,C antigens comprise only a small proportion (relative to the total cellular HLA-A,B,C antigens) of the biosynthetically mature molecules. The possible significance of such heterogeneity is discussed.     

Monoclonal antibody analysis of MHC expression in human brain biopsies: tissue ranging from "histologically normal" to that showing different levels of glial tumor involvement

The expression of class I and class II MHC products in human brain was studied. Radioimmunoassay confirmed weak expression of HLA-A,B,C and beta 2-microglobulin (beta 2-m) in brain extract. Quantitative inhibition assay showed brain had 1/70 as much activity as spleen, per microgram of extract protein. Immunoblot assay confirmed that HLA chains and beta 2-m were present in the brain extract. Class II was not detected. Microscopic analysis was performed on eight brain biopsies. The histologic appearance ranged from "apparently normal," to the presence of reactive astrocytes, to the presence of glial tumor. In every case, HLA-A,B,C and beta 2-m activity was concentrated at blood vessel walls. Small and medium-sized vessels were uniformly stained. Cell body staining was not seen in neurons, glia, oligodendrocytes, microglia, reactive astrocytes, or the majority of glial tumor cells. Class II activity was seen in occasional cell bodies in both grey matter and white matter in the microscopic assays. These cells had the morphologic appearance of microglia or reactive astrocytes. Occasional blood vessels also showed class II activity. Unlike the class I activity, the class II blood vessel stain was often discontinuous. More class II+ cell bodies were seen in tumor-associated tissue.