beta2-Microglobulins: isolation, properties, and distribution.

beta2-Microglobulin is structurally related to immunoglobulin domains and is identical to the light chain of histocompatibility (HL-A) antigens. Similar to free light chains of immunoglobulins, beta2-microglobulin is most easily isolated from urine. We have previously purified human beta2-microglobulin from urine of patients with renal tubular resorption defects. Corresponding proteins have now been obtained from urine of rabbits and guinea pigs treated with sodium chromate. Sequence studies have established that the rabbit protein is rabbit beta2-microglobulin. The guinea pig protein closely resembles the human and rabbit beta2-microglobulins in amino acid composition, charge, molecular size, and also in the presence of an apparently analogous disulfide loop. These findings indicate that this protein is the guinea pig homologue of beta2-microglobulin. Physical-chemical studies suggest that human beta2-microglobulin and isolated immunoglobulin domains are similar not only in amino acid sequence but also in three-dimensional structure. Both types of molecules are compact and globular in shape and apparently contain beta-pleated sheet conformation. beta2-Microglobulin is present in free form in various body fluids and as a subunit of histocompatibility antigens on cell surfaces. Current estimates suggest that the number of beta2-microglobulin molecules on cell surfaces is higher than the number of histocompatibility (HL-A) antigens. Accordingly, beta2-microglobulin is possibly a subunit of additional cellular antigens or receptors.     

Structure of wild-type and mutant mouse beta 2-microglobulin genes

beta 2-Microglobulin is encoded by a single gene per haploid genome in the mouse. This gene was cloned and sequenced and was shown to consist of four coding regions separated by three intervening sequences. Most of the protein (amino acids 3-95) is encoded in a single uninterrupted unit flanked by intervening sequences. In this respect, the genetic organization is similar to that of the evolutionarily related H-2 and immunoglobulin domains. A mutant cell line selected for loss of cell-surface expression of the thymus leukemic antigen and failing to express surface beta 2-microglobulin and H-2 was found to contain no detectable cytoplasmic beta 2-microglobulin. The genetic defect in this mutant was analyzed by Southern blots, which indicated that there are defects in both copies of the beta 2-microglobulin gene present in this cell. The concomitant loss of three cell-surface antigens was thereby linked to genetic defects in the beta 2-microglobulin locus.     

A mutational hot-spot within an intron of the mouse beta 2-microglobulin gene.

beta 2-Microglobulin is the smaller, relatively non-polymorphic chain of class I major histocompatibility complex proteins. We have previously described a mutant mouse cell line which had been selected for loss of the class I thymus leukemia (TL) antigen and had concomitantly lost surface expression of H-2k antigens. Expression of class I antigens on the cell surface was restored by fusion to an antigenically distinct mouse lymphoma line, and the defect in the mutant was shown to be the loss of a functional beta 2-microglobulin gene. We now describe three additional mutants with the same phenotype, all selected for loss of TL but after different types of mutagenesis. All of these mutants have genomic rearrangements resulting in the absence of a functional beta 2-microglobulin gene. These data provide strong evidence for the requirement of beta 2-microglobulin for cell surface expression of the heavy chain of class I major histocompatibility complex proteins. We further show that the defects in at least one beta 2-microglobulin gene in each mutant cell line map to the same small DNA segment within the first intron. The breakpoints of these mutations define a hypermutable site within the mouse beta 2-microglobulin gene.     

Purification of papain-solubilized histocompatibility antigens from a cultured human lymphoblastoid line, RPMI 4265.

HL-A antigens having specificities HL-A2, HL-A7, HL-A12 have been solubilized by papain treatment of membrane preparations from the cultured human lymphoblastoid cell line RPMI 4265 and purified about 80-fold by chromatography on carboxymethylcellulose, Sephadex G-150, and diethylaminoethylcellulose columns. Separation of HL-A2 from a mixture of HL-A7 and HL-A12 was achieved on the final DEAE-cellulose column. The yield was about 1 mg of protein of each antigen preparation per 100 g of packed, frozen cells. On sodium dodecyl sulfate gel electrophoresis both preparations showed two polypeptide bands. The smaller subunit of 12,000 daltons is common to all HL-A preparations and has been shown to be identical with beta2-microglobulin. The larger subunit is a glycopeptide and in the HL-A7, 12 preparation was resolved into a duplex of 34,000 and 37,000 daltons. The HL-A2 preparation showed a single band at 34,000 daltons. On isoelectric focusing under nondenaturing conditions, the preparation showed multiple bands, all of which contained both subunits and retained antigenic activity. On isoelectric focusing in the presence of 6 M urea a single band for beta2-microglobulin and multiple bands for the larger subunit were seen. This charge heterogeneity of the larger subunit has been shown to be due to variable amounts of sialic acid. When HL-A antigen preparations were subjected to Sephadex G-100 chromatography in the presence of 3 M KCl, no separation of the two subunits was observed.     

Cell-free synthesis and membrane insertion of mouse H-2Dd histocompatibility antigen and beta 2-microglobulin.

Messenger RNA from SL2 lymphoma cells was translated in a cell-free system in the presence of microsomal membranes. Mouse H-2Dd histocompatibility antigen was correctly assembled in the microsomal membranes, and transmembrane insertion of the nascent chain was accompanied by glycosylation and cleavage of the signal sequence H-2Kd antigens, synthesized in vivo, comprised a transmembrane glycoprotein and an unglycosylated protein in the cytoplasm. The glycosylated forms of the H-2Dd and H-2Kd antigens were modified during intracellular transport from the endoplasmic reticulum to the cell surface. beta 2-Microglobulin was also synthesized in vitro, and transfer of this protein into microsomal vesicles was accompanied by cleavage of its signal sequence. In the endoplasmic reticulum, beta-microglobulin can bind to newly synthesized H-2d glycoproteins. The mRNAs coding for beta 2-microglobulin and H-2Dd antigen could be separated on aqueous sucrose gradients.     

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.     

Effects of monoclonal antibodies against lymphocyte surface antigens on interleukin 2 excretion by Epstein-Barr virus-specific human T-cell hybridomas

Monoclonal antibodies were used as probes to study the role of cell surface antigens in the response of Epstein-Barr virus (EBV)-specific human T-T hybridomas to autologous EBV-infected B lymphoblasts. Somatic cell hybrids were generated by fusing EBV-primed peripheral blood T lymphocytes with a mutant clone of the JM human T-lymphoblastoid-cell line. When exposed to autologous EBV-infected B lymphoblasts, the resulting hybrid clones released Interleukin 2 into the culture medium. Incubation of the EBV-infected B cells with two monoclonal antibodies against human Ia-like molecules blocked their ability to trigger the hybridomas. Under the same conditions, monoclonal antibodies against beta 2-microglobulin, and a 45,000 MW surface antigen common to EBV-infected B lymphoblasts, did not alter the capacity of the B cells to stimulate the hybridomas. None of four monoclonal antibodies against surface antigens on the T-cell hybridomas impaired their responsiveness to EBV-infected B lymphoblasts. These results suggest the possibility that naturally occurring or exogenously administered antibodies against Ia molecules might interfere with T-cell regulation of EBV-induced B-cell activation.     

Effect of anti-beta2-microglobulin on antigen and allogeneic lymphocyte-induced proliferation of human lymphocytes.

The effect of anti-beta2-microglobulin (beta2m) on the mixed lymphocyte reaction (MLR), and an antigen-induced proliferative response was studied. Anti-beta2m IgG and Fab' fragments completely inhibited the MLR. Preincubation of stimulator or responder cells with anti-beta2m suggested that the major effect of anti-beta2m may be on the responder cell population. A clear-cut effect on responder cells was demonstrated by showing that anti-beta2m completely inhibited a MLR in which the stimulator population was a beta2m negative lymphoblastoid cell line. Anti-beta2m also inhibited PPD-induced proliferation of sensitized lymphocytes. The kinetics of this inhibition indicated that anti-beta2m added within the first 18 hr of stimulation was effective in inhibiting the proliferative response. These data are discussed in light of the hypothesis that beta2m may be a subunit of an antigen receptor on T cells.     

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.     

Structure and significance of beta2-microglobulin.

beta2-Microglobulin shares many structural features with the homology regions of the immunoglobulins. Particularly significant is the fact that its amino acid sequence is homologous to the sequences of the constant regions of both classes of light chains (kappa and lambda) and to the constant homology regions of at least three classes (gamma, mu and epsilon) of heavy chains, especially the carboxyl-terminal regions Cgamma3 Cmu4 and Cepsilon4. Molecules similar to human beta2-microglobulin have been found in other vertebrate species. The properties of beta2-microglobulin suggest that the gene for this protein may have evolved from a precursor gene that by duplication gave rise to immunoglobulin light and heavy chains. Furthermore, the observation that beta2-microglobulin is synthesized by and appears on the surfaces of a variety of cell types, including nonlymphoid cells, suggests that the concepts derived from analysis of the immune system may be applicable to other areas of cell biology. In particular, the close association of this immunoglobulin-like molecule with the histocompatibility antigens has a number of implications for the origin, structure, and function of these as well as other cell surface glycoproteins.     

The activation of guinea pig T lymphocytes by anti-beta 2-microglobulin serum.

In order to study further the role of beta 2-m in the regulation of the immune response, we have examined the effects of a goat anti-guinea pig beta 2-m serum on a number of T lymphocyte functions in vitro. Anti-beta 2-m serum produced a marked inhibition of the response of peritoneal exudate T cells to antigen and mitogen stimulation. Surprisingly, a marked activation of lymph node T lymphocyte proliferation was observed in the absence of antigen or mitogen stimulation. This stimulatory effect of anti-beta 2-m serum was shown to be specific for beta 2-m and required the presence of macrophages. The T cell proliferative response induced by anti-beta 2-m could not be blocked by antisera to the antigens of the guinea pig MHC. These studies suggest that beta2-m may play some critical role in the immune response at the level of T cell activation.     

beta2-Microglobulin: a reevaluation.

Rabbit antiserum to human beta2-microglobulin was used to inhibit the proliferative response of lymphocytes in a variety of in vitro assays and to block the effector phase of the cell mediated lympholysis reaction. The antiserum was able to inhibit both of these reactions; it is not clear whether the cytotoxic reaction that we are studying in a xenogeneic human-rabbit system is based on phytohemagglutinin dependent cytotoxicity or on specific recognition of target cells by receptors on the effector cells, or most likely on a combination of both cytotoxic mechanisms. Whereas the possibility that beta2-microglobulin may be associated with receptors on the thymus-derived lymphocyte surface is considered, it is also pointed out that the effect of the antiserum may be based on other mechanisms of perturbing the membrane so as to inhibit these responses.     

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.     

Class I major histocompatibility proteins are an essential component of the simian virus 40 receptor.

The class I molecules encoded by the major histocompatibility complex (MHC) present endogenously synthesized antigenic peptide fragments to cytotoxic T lymphocytes. We show here that these proteins are an essential component of the cell surface receptor for simian virus 40 (SV40). First, SV40 binding to cells can be blocked by two monoclonal antibodies against class I human lymphocyte antigen (HLA) proteins but not by monoclonal antibodies specific for other cell surface proteins. Second, SV40 does not bind to cells of two different human lymphoblastoid cell lines which do not express surface class I MHC proteins because of genetic defects in the beta 2-microglobulin gene in one line and in the HLA complex in the other. Transfection of these cell lines with cloned genes for beta 2-microglobulin and HLA-B8, respectively, restored expression of their surface class I MHC proteins and resulted in concomitant SV40 binding. Finally, SV40 binds to purified HLA proteins in vitro and selectively binds to class I MHC proteins in a cell surface extract.     

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.     

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.     

Autologous B lymphoblastoid cell lines and long-term cultured T cells as stimulators in the mixed lymphocyte reaction: analysis of the role of HLA class II antigens as stimulatory molecules

Human activated T cells, long-term cultured in the presence of interleukin 2 (IL 2), were compared with autologous Epstein Barr virus-transformed B lymphoblastoid cell lines for expression of human leukocyte (HLA)-HLA-DR and -DQ antigens and for ability to induce proliferative responses in autologous and allogeneic lymphocytes. Immunofluorescence analysis performed with a panel of monoclonal antibodies (mAb) specific for HLA-DR or -DQ antigens did not reveal any significant difference in the expression of HLA-DR antigens but revealed reduced expression of HLA-DQ antigens on two out of four T cell lines tested. No obvious difference could be detected in the two-dimensional gel electrophoretic profile of HLA-DR and -DQ beta-chains synthesized by the autologous pairs of B and T cell lines. In contrast with previous reports, the IL 2-dependent cell lines consistently induced alloproliferative responses in standard 6-day mixed lymphocyte cultures; however, these responses were severalfold lower than those elicited by the autologous B lymphoid lines. Both anti-HLA-DR and anti-HLA-DQ mAb blocked the proliferative responses induced by the B cell lines but did not affect those generated by the T cell lines, suggesting that the latter cells induce T lymphocyte activation via a mechanism independent of HLA-DR or -DQ antigen expression on their surface. Addition of IL 2 to the mixed cultures with B cell lines as stimulators did not affect the outcome of the proliferative responses but partially or completely reversed the blocking activity of the mAb. In contrast, IL 2 significantly enhanced the alloproliferation induced by the T lymphoblastoid cell lines, and the anti-HLA class II mAb partially antagonized this effect. Taken together, these data suggest that unlike the HLA-DR and -DQ gene products on B cells, those on IL 2-dependent long-term cultured T cells do not play a direct or primary stimulatory role in the mixed lymphocyte reaction; the reduced levels of alloproliferation induced by the T cell lines are, at least in part, due to a defective production of endogenous IL 2 by the responder lymphocytes rather than to a defective expression of IL 2 receptors by the alloproliferative T cell subset; and the anti-HLA class II mAb in these cultures act only at the responder cell level, since they can efficiently block the enhancement of T cell proliferation triggered by exogenous IL 2, but not the proliferative responses induced by T cell lines in standard conditions.     

Differential effect of anti-beta 2-microglobulin on IL 2 production and IL 2 receptor expression in the primary mixed lymphocyte culture reaction

The mechanism of inhibition of the proliferative response in primary mixed lymphocyte culture (1 degree MLC) by antibodies to beta 2-microglobulin (beta 2m) was investigated. It is demonstrated that anti-beta 2m antibodies inhibit the production of interleukin 2 (IL 2). In contrast, the expression of IL 2 receptor is not affected by anti-beta 2m. The addition of purified exogenous IL 2 to the antibody-treated 1 degree MLC can completely restore the proliferative response, indicating that anti-beta 2m does not interfere with IL 2 binding to its receptor. Similarly, anti-beta 2m does not interfere with the capacity of IL 2-dependent T cell lines or T cell clones to respond to exogenous IL 2. The inhibition of cell proliferation and IL 2 production by anti-beta 2m is maximal when the antibody is added at the beginning of 1 degree MLC culture, and no effect of anti-beta 2m is seen when added after 3 days of culture. Anti-beta 2m has no effect on mitogen-induced cell proliferation and IL 2 production. Anti-beta 2m acts on the responder cell population, as demonstrated in experiments in which responder cells or stimulator cells are treated separately with the antibody. The expression of HLA-class II antigens (i.e., HLA-DR and DQ (DC) on the T cells activated on 1 degree MLC is not affected by anti-beta 2m. These studies indicate that the HLA-beta 2m class I antigen complex plays a role in T lymphocyte activation via release of IL 2, and suggest the existence of different mechanisms for activation of IL 2 producers and IL 2 responders in 1 degree MLC.