B2M Genes Research Articles

Combination of HPLC and solid‐phase binding assay for isolation and purification of MHC class I and associated peptides using a bladder tumour cell line

The major histocompatibility complex (MHC) class I molecules present processed self and non-self peptides to T lymphocytes. Given that the class I peptide complex plays a critical role in cell-mediated immunity, it is important to identify the nature of class I-associated peptides unique to malignant cells as a prelude to the development of vaccines. The aim of this study was to combine immuno-bead purification (using anti-class I antibody W6/32) technique, sequential ultra-filtration and high performance liquid chromatography (HPLC) to isolate class I antigens and associated peptides from an in-house established bladder tumour cell line (Fen) whose missing class I antigens had been restored by β2-microglobulin (β2-m) gene transfaction. The results were as follows: (a) class I antigens could be separated from tumour cell lysate but only from the class I positive Fen cells; (b) treatment of CNBr-W6/32 beads pre-exposed to class I positive Fen lysate and eluted with dissociation agent (mild acid) resulted in the release of more than 20 peptides at an approximate molecular weight of between 700 and 3000 Da based on SDS–PAGE and silver staining analysis; (c) purified and eluted peptides from class I antigens showed distinct peaks when analysed by HPLC. The data presented in this investigation demonstrated the feasibility of isolating class I antigens and associated peptides from a bladder tumour cell line. The extension of these approaches to isolate peptides from tissue tumour biopsies may help the future of vaccine therapy in cancer patients. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: FCS foetal calf serum MHC major histo compatibility complex PAGE polyacrylamide gel electrophoresis RP reverse-phase SDS sodium dodecyl sulphate.

Effect of B2m gene disruption on MHC-determined odortypes.

Major histocompatibility complex (MHC) genes confer individual olfactory identity that can be detected with exquisite accuracy by mice. The fact that MHC genes themselves generate the characteristic odortype, rather than dedicated odor-determining genes, was supported in studies of point mutations in H2K and HLA transgenic mice, which evinced distinct odor profiles in olfactory assays. In this article we provide further evidence for a central role of MHC genes themselves in odortype specification by demonstrating that mice that are unable to express their genomic class I MHC genes because they lack beta2-microglobulin are distinguishable by scent from otherwise identical mice which possess an intact B2m gene. This odortype disparity appears at 9-12 days of gestational age, the period in which the MHC is first detectable in fetal cells of normal mice.

Of metals, mice, and men: what animal models can teach us about body iron loading.

Of metals, mice, and men: what animal models can teach us about body iron loading.

Mutation in the β2m gene is not a frequent event in head and neck squamous cell carcinomas

In cryostat sections of 84 head and neck squamous cell carcinomas (HNSCC) HLA class I and β2m expression was analysed using monomorphic and locus specific monoclonal antibodies. Loss of expression was heterogeneous and none of the tumours tested showed a total loss of HLA class I and/or β2m when analysed with W6/32, which recognises HLA class I determinants and anti-β2m MoAbs. Weak HLA class I and β2m expression was found in 9 tumours (11%) and heterogeneous expression was found in 2 tumours (2%). When analysed with locus-specific antibodies (HCA2 and HC10, anti-HLA-A and anti-HLA-B/C, respectively) 37 tumours (44%) showed a loss, weak or heterogeneous expression of one or both loci. Tumours showing a down-regulated HLA class I expression were analysed for mutations in either allele of the β2m gene by sequencing based mutation analysis (SBMA). Exon 1 and exons 2 and 3 were amplified separately by PCR using M13-tailed intron-specific primers. PCR products were sequenced in two directions. In none of the tumours mutations in the β2m gene were detected. In 59% of the tumours with down-regulated HLA class I expression, lost or down-regulated TAP 1 expression was found when analysed with anti-TAP 1 antibodies. This indicates an important role for TAP in down-regulation of HLA class I expression in HNSCC.

Quantitative analysis of NF1 and OMGP gene transcripts in sporadic gliomas, sporadic meningiomas and neurofibromatosis type 1-associated plexiform neurofibromas.

The close association of neurofibromatosis type 1 (NF1) with gliomas raises the question of whether the NF1 gene may be involved in the pathogenesis of sporadic astrocytic brain tumors. However, no frequent mutations within NF1 have been described in these tumors. Recent data on a limited series of gliomas indicate that NF1 expression may even be increased, thereby questioning the role of NF1 as a tumor suppressor in astrocytomas. In the present study, we examined the expression of NF1 in a series of 96 tumors including astrocytomas, meningiomas and plexiform neurofibromas. NF1 RNA transcription levels were compared to those of the reference genes B2M, ACTB and GAPD. The expression of OMGP, which is interposed in the NF1 gene, served as an additional control. NF1 expression did not significantly diverge among different malignancy stages of astrocytomas. As expected, the plexiform neurofibromas showed only very low NF1 expression. A striking finding was the highly variable expression of those genes selected to serve as references. While B2M and ACTB exhibited comparable levels of expression within different grades of astrocytomas and meningiomas, GAPD showed an inverse pattern in these tumors. In conclusion, NF1 expression is strongly reduced in NF1-associated plexiform neurofibromas but not in astrocytic tumors. The significant differences between B2M, ACTB and GAPD transcript levels brings into question the common practice of defining gene expression as a ratio between the transcripts of interest and those of these reference genes.

Gastric cancers of the microsatellite mutator phenotype display characteristic genetic and clinical features

Background & Aims: Colon cancer of the microsatellite mutator phenotype (MMP) exhibits significant genotype differences from cancer without the MMP. Twenty-nine MMP-positive gastric cancers were analyzed to clarify if these genotype differences were also associated with distinctive clinicopathologic features. Methods: Alterations of p53, β 2-microglobulin ( β2M), hMLH1, and hMSH2 genes were analyzed by using polymerase chain reaction, single-strand conformational polymorphism, sequencing, microallelotyping, hypermethylation assays, and immunostaining. The results were contrasted with mutations in BAX, hMSH3, and hMSH6, genes target for the MMP. Results: Tumors with the MMP had a significantly lower incidence of p53 gene mutations than the other tumors and often contained β2M gene somatic mutations. Many tumors contained concomitant genetic and epigenetic alterations in DNA mismatch repair genes, hMLH1, hMSH2, hMSH3, and hMSH6. Gastric cancer of the MMP was associated with well/moderate differentiation, distal location, and better survival. Conclusions: Analysis of somatic alterations in microsatellite sequences and in cancer genes target for the MMP is useful for the classification of groups of gastric cancers with different prognosis. The results further support the concept that (gastric) cancer of the MMP represents a distinctive oncogenic pathway because the mutated cancer genes are usually different from those found in tumors without the MMP. GASTROENTEROLOGY 1999;116:1348-1357

Residue 3 of beta2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody.

Previous studies of class I MHC molecules have shown that the owl monkey (Aotus) possesses at least two variants of the beta2-microglobulin (beta2m) protein. These two variants have different isoelectric points, and exhibit differential reactivity with the monoclonal antibody W6/32. We report cDNA sequences of the B2m gene, from W6/32-positive and W6/32-negative Aotus cell lines. The two beta2m variants we identified exhibit a single amino acid difference at position three. An arginine residue at position 3 was correlated with W6/32 reactivity, whereas histidine was associated with non-reactivity. W6/32 reactivity was conferred to a W6/32-negative Aotus cell line when it was transfected with the B2m from the W6/32-positive cell line. Residue 3 of beta2m is located at the surface of the class I molecule. It is also close to position 121 of the MHC class I heavy chain, which has previously been shown to influence W6/32 antibody binding. We conclude that W6/32 binds a compact epitope on the class I molecule that includes both residue 3 of beta2m and residue 121 of the heavy chain. We examined the distribution of the two beta2m motifs in a sample Aotus population using an allele-specific polymerase chain reaction assay. The pattern of beta2m segregation we observed matches that which was defined previously by serology. Additionally, we identified laboratory-born hybrid animals who possess both variants of beta2m.

β2-Microglobulin-Dependent T Cells Are Not Necessary for Alloantigen-Induced Th2 Responses After Neonatal Induction of Lymphoid Chimerism in Mice

AbstractWe have analyzed the requirement for β2-microglobulin (β2m)-dependent T cells in the generation of allogeneic Th2 responses in vivo. A neonatal injection of semiallogeneic cells in BALB/c mice induces a state of chimerism that promotes the differentiation of donor-specific CD4+ T cells toward the Th2 phenotype. Polyclonal T-B cell interactions occur in this model between host Th2 and donor B cells, resulting in the production of IgE Abs. IgE production and Th2-priming are critically dependent upon the early production of IL-4. Our data in the present paper demonstrate that: 1) IgE synthesis and the up-regulation of MHC class II and CD23 molecules on B cells are independent of β2m expression in the host, 2) no difference in the induction of CD4 alloreactive Th2 cells could be observed between β2m−/− and their wild-type control littermates when Th2-priming was measured in adult mice, and 3) the Th2 response and IgE production is induced in the complete absence of β2m-dependent T cells both in the host and in the inoculum. Therefore, using a variety of assays, we could not demonstrate diminished responses in mice with a disrupted β2m gene in this model of Th2-mediated allogeneic interaction, indicating that β2m-dependent NK1.1+ and CD8+ T cells are not required for the generation of alloreactive Th2 responses in vivo.

THE HLA CLASS I DEFICIENT BURKITT LYMPHOMA CELL LINE DAUDI FAILS TO EXPRESS GPI-ANCHORED CELL SURFACE PROTEINS

267 The Burkitt lymphoma-derived Daudi B cell line is the best known HLA class I deficient human cell line and has extensively been used as a target in cell mediated cytolysis and other functional assays. During a routine screening of an extensive panel of leukemic cell lines for cell surface expression of the cellular prion protein (PrPc) we noted a complete absence of this protein on the surface of Daudi cells. It was the purpose of this study to characterize in detail the nature of this unexpected defect. We used RT-PCR to demonstrate transcripts of equal size and specificity in human brain tissue, EBV-transformed JY25 and Daudi B cells. Permeabilization with L-α-lysophosphatidyl choline of cell membranes of Daudi, JY5 B cells and CEM C7E2A T cells, respectively, permitted binding of the PrP-specific mAb 3F4 to intracellular PrPc thus demonstrating translation of transcripts into immunoreactive PrPc protein. We speculated that the lack of PrPc expression might be linked to the known β2m gene defect. However, other β2m deficient cells (human melanoma line FO-1, murine T cell line R1E/TL8x.1, splenocytes from β2m gene-deleted mice) displayed unequivocal cell surface expression of PrPc. Transfection of a functional β2m gene into Daudi cells restored HLA class I but not PrPc cell surface expression thus refuting our hypothesis. PrPc belongs to the family of GPI-anchored cell surface proteins. Therefore, we examined the expression of other GPI-linked proteins on Daudi cells. We were unable to demonstrate antibody binding to CD55 and CD59 on Daudi and control B cells with known defects in GPI anchor formation. We finally used somatic hybridization of Daudi cells with murine spleen cells to successfully demonstrate de novo cell surface coexpression of HLA class I and PrPc, CD55 and CD59, respectively. We demonstrate here a hitherto unknown defect of GPI anchor synthesis in Daudi cells. Since Daudi cells are commonly used in cellular assays experimental results must be interpreted in light of this novel finding. Finally, Daudi cells may be useful in future studies of GPI anchor synthesis.

Normal beta-2-microglobulin (β2m) gene in patients with hemochromatosis and no evidence of linkage to chromosome 6

Normal beta-2-microglobulin (β2m) gene in patients with hemochromatosis and no evidence of linkage to chromosome 6

Probing the stability of class I major histocompatibility complex (MHC) molecules on the surface of human cells.

We used binding of a fluorescent adduct of beta2-microglobulin, fluorescein beta2m, to probe the stability of class I HLA molecules on the surface of human cells. The weight of the literature suggests that this ligand binds to heavy chains that have lost beta2m and possibly peptide as well. Hence Fl-beta2m reports on the stability of the class I HLA trimer. A small fraction of HLA molecules, approximately 5%, binds Fl-beta2m on both resting and activated T cells. A larger fraction of all HLA molecules binds Fl-beta2m in FO-1 cells, beta2m-deficient cells, transfected with various B2m genes. HLA molecules of FO-1 cells are more stable when expressed with human beta2m, than when expressed with mouse beta2m. The non-covalent association of HLA heavy chains, beta2m and peptide implies that eventually every molecule of HLA trimer ought to dissociate and bind Fl-beta2m. In fact, the extent of exchange is limited by the lifetime of a given molecule at the cell surface. beta2m exchange decreases as cell concentration increases, suggesting that some density-dependent process acts to enhance degradation or denaturation of beta2m-free HLA heavy chains.

Of mice and Marfan: genetic linkage analyses of the fibrillin genes, Fbn1 and Fbn2, in the mouse genome

The fibrillin genes, FBN1 and FBN2, encode large extracellular matrix glycoproteins involved in the structure and function of microfibrils. Mutations in FBN1 are found in patients with Marfan syndrome, a heritable connective tissue disease that primarily affects the cardiovascular, ocular, and skeletal systems. We extended the studies of these genes by determining their chromosomal position in the mouse genome. Restriction fragment length polymorphisms (RFLPs) between the progenitors of an interspecific backcross involving AEJ/Gn and Mus spretus mice were used to establish the segregation patterns of the murine homologs, Fbn1 and Fbn2, in the backcross progeny. The results position Fbn1 between the B2m and Illa genes on mouse Chromosome (Chr) 2 and establish its candidacy for the Tight skin (Tsk) mutation. The results position Fbn2 between the D18Mit35 and Pdgfrb loci in the central region of mouse Chr 18. Fbn2 maps near three mutations [bouncy (bc), plucked (pk), and shaker with syndactyly (sy)] and may be a candidate for the pk mutation.

Major Histocompatibility Complex Class I-Deficient NOD-B2mnull Mice are Diabetes and Insulitis Resistant

Specific allelic combinations within the class II region of the major histocompatibility complex (MHC) represent a major genetic component for susceptibility to autoimmune insulin-dependent diabetes mellitus (IDDM) in humans. We produced and used a stock of NOD/Lt mice congenic for a functionally inactivated beta 2-microglobulin (B2mnull) locus to assess whether there was an absolute requirement for MHC class I expression and/or CD8+ T-cells in diabetogenesis. These NOD-B2mnull mice do not express cell surface MHC class I molecules or produce detectable levels of CD8+ T-cells and are diabetes and insulitis resistant. Previous results from transgenic mouse models indicated that intracellular accumulation of MHC class I molecules negatively affects pancreatic beta-cell function and can result in the development of nonautoimmune insulin-dependent diabetes mellitus (IDDM). MHC class I molecules have been shown to accumulate intracellularly in the presence of a disrupted B2m locus, but this mutation does not negatively affect plasma insulin levels in either NOD/Lt mice or in those of a mixed 129 and C57BL/6 genetic background. Interestingly, 14% of the male mice in this mixed background did develop hyperinsulinemia (> 1,500 pM) independent of the disrupted B2m locus, suggesting that these mice could conceivably develop insulin-resistant diabetes. However, none of these mice became diabetic at up to 22 months of age. Thus, elimination of cell surface MHC class I expression with a disrupted B2m gene blocks autoimmune diabetes in NOD/Lt mice, without engendering a separate, distinct form of glucose intolerance.

Gene mapping in a murine cell line by immunoselection with cytotoxic T lymphocytes.

Minor histocompatibility (H) loci encode alloantigens that are recognized by cytotoxic T (Tc) lymphocytes. A (C57BL/10 x 129)F1-derived transformed lymphocyte cell line was immunoselected in vitro with cloned Tc cells that were specific for H-3aa, a Chromosome 2-encoded minor H antigen. This cell line is heterozygous at H-3a (former symbol, Cd-1) and other loci. Three groups of antigen-loss variants were identified. One group contained mutations affecting only the antigen-encoding gene. Another group probably arose through a single homologous interchromosomal exchange, resulting in extensive regions of loss of heterozygosity (LOH). The third group of variants contained an interstitial LOH, one of which was shown to be a significant deletion. Several deletion boundaries were identified, one of which ordered the closely linked H-3a and beta 2-microglobulin (B2m) genes. We suggest that Tc immunoselection against minor H antigens is a promising approach for targeting negative selection to specified chromosomal regions and can provide high-resolution genetic map information.

The Tight Skin (Tsk) Mutation in the Mouse, a Model for Human Fibrotic Diseases, Is Tightly Linked to the β2-Microglobulin (B2m) Gene on Chromosome 2

The Tsk mutation in the mouse is characterized by the excessive accumulation of collagen in skin and various internal organs, including the heart and lungs. These connective tissue abnormalities are similar to those present in human systemic sclerosis or scleroderma. The Tsk mutation provides an opportunity to investigate, at the molecular level, the pathogenesis of tissue fibrosis. As a first step to cloning the Tsk gene, we report the localization of the Tsk mutation with respect to known molecular markers on mouse chromosome 2. N2 progeny carrying the Tsk mutation were obtained from an intersubspecific backcross of [(C57BL/6-pa +/+ Tsk × Mus castaneus)F1 × M. castaneus ] mice. Genomic DNA from each N2 mouse was subjected to Southern and PCR analyses to identify restriction fragment length polymorphisms and simple sequence length polymorphisms, respectively. Our results refine the location of Tsk to a 3-cM region, eliminate several genes from consideration as the Tsk mutation, identify molecular probes tightly linked with Tsk, and suggest candidate genes responsible for the Tsk phenotype.

Lack of HLA class I antigen expression by melanoma cells SK-MEL-33 caused by a reading frameshift in beta 2-microglobulin messenger RNA.

The lack of HLA class I antigen expression by the melanoma cell line SK-MEL-33 is caused by a unique lesion in beta 2-microglobulin (beta 2-mu). Sequencing of beta 2-mu mRNA detected a guanosine deletion at position 323 in codon 76 that causes a frameshift with a subsequent introduction of a stop codon at a position 54 base upstream of the normal position of the stop codon in the message. The loss of 18 amino acids and the change of 6 amino acids, including a cysteine at position 80 in the carboxy terminus of beta 2-mu, are likely to cause marked changes in the structure of the polypeptide. The latter may account for the inability of beta 2-mu to associate with HLA class I heavy chains and for its lack of reactivity with the anti-beta 2-mu mAb tested. HLA class I antigen expression on SK-MEL-33 cells was reconstituted after transfection with a wild-type B2m gene, therefore indicating that the abnormality of endogenous B2m gene is the only mechanism underlying lack of HLA class I antigen expression by SK-MEL-33 cells. The guanosine deletion in B2m gene was detected also in the melanoma tissue from which SK-MEL-33 cells had originated. Therefore, the molecular lesion identified in the SK-MEL-33 melanoma cell line is not caused by a mutation acquired during growth in vitro but is likely to reflect a somatic mutation during tumor progression.

The chromatin structure of the mouse beta-2-microglobulin locus

Abstract Within the promoter regions of both major histocompatibility complex (MHC) class I genes and the β 2-microglobulin (β2m) gene, there are a number of common regulatory elements suggesting co-ordinate control. However, there is also evidence to suggest that β2m and class I are differentially regulated, indicating that- these genes may have distinct regulatory elements. We sought to explore this question by analysing DNase I hypersensitive (DH) sites flanking the β2m gene. Five DH sites have been found within the vicinity of the β2m gene. One of these sites (DH1) located within the promoter region, correlates with the transcriptional activity of β2m since it is weak in embryonal (β2m negative) cell lines. The remaining DH sites (2–5) are located downstream of the β2m gene. The most proximal downstream site, (DH2) located 5.5 kb from the last exon, was observed only in embryonal cell lines, indicating possible involvement in the downregulation of β2m. Furthermore, this site is markedly diminished in differentiated F9 cells. Possible roles for the remaining sites are discussed, in particular relationship to a second transcriptional unit identified in the vicinity. In addition, a similar analysis reveals a cluster of DH sites located downstream from the last exon of the human β2m gene.

CD8 + T Lymphocytes Are Not Required for Murine Resistance to Human Filarial Parasites

Mice are resistant to the establishment of infection with the nematode parasite Brugia malayi, an etiologic agent of human lymphatic filariasis. We have recently shown that T and B lymphocyte-deficient C.B.-17 scid/scid mice are permissive for infection with this parasite, whereas coisogenic C.B.-17+/+ mice are resistant. This observation suggests that T and B lymphocytes that comprise the antigen-specific immune system orchestrate murine resistance to B. malayi. In order to define the component of the antigen-specific immune response that is responsible for this resistance, we have tested the susceptibility of beta 2M-/- mice to infection with B. malayi L3 larvae. These mice are homozygous for insertional disruption of their B2m genes, which encode beta 2-microglobulin, the small subunit of the major histocompatibility (MHC) antigens. They do not express beta 2-microglobulin and, as a consequence, fail to express the class I major histocompatibility antigens, and they do not develop the CD8+ class I MHC-restricted cytotoxic T cell subset. We find that these mice are completely resistant to B. malayi, indicating that the CD8+ T lymphocyte subset is not an obligate requirement for murine resistance to human filarial parasites.

Damage in B2m genes and DNA methylation of H-2 genes are involved in loss of expression of class I MHC products on the membrane of LR.4, a cell line derivative of the T-cell lymphoma L5178Y.

We have isolated an H-2 deficient cell line (LR.4) from the T-cell lymphoma L5178Y which grew without restrictions in the peritoneal cavity of different inbred strains of mice. The use of polyclonal anti-H-2 antiserum and complement indicated that LR.4 cells did not express class I determinants on the cell membrane. Southern blots of genomic DNA of LR.4 cells showed that B2m genes were severely damaged and that class I H-2 genes were extensively methylated. Consequently, LR.4 cells failed to transcribe mRNAs for both B2m and class I H-2 genes. On the other hand, specific immunity to LR.4 was demonstrated in C57BL/6J mice since, in subsequent challenges with either LR.4 or EL4.4, LR.4 did not grow, whereas EL4.4 grew and killed the mice. In C57BL/6J mice, rejection of LR.4 was accompanied by the production of cytotoxic antibodies. The immune response induced in C57BL/6J mice was determined by non-H-2 antigenic determinants in LR.4 cells.

Immunologic and genetic characterization of S180, a cell line of murine origin capable of growing in different inbred strains of mice

Some of the immunologic and genetic properties of the cell line S180 have been examined. These cells grew without restrictions in the peritoneal cavity of different inbred strains of mice and invariably killed the animals. With Northern blots it was demonstrated that S180 cells contained class I mRNAs but failed to transcribe B2m genes. However, under experimental conditions, a protective humoral immune response mediated by cytotoxic antibodies and complement against S180 cells was obtained through non-H-2 antigens in C57BL/6J mice.