Article

The Genetic Control of HLA‐A and B Antigens in Somatic Cell Hybrids: Requirement for β2 Microglobulin

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

The lymphoblastoid cell line Daudi lacks both HLA—A and B antigens and β2 microglobulin. Somatic cell hybrids derived from a fusion between this line and D98/AH-2 were shown to express four HLA antigens not detectable on either parent cell, A1, A10(Aw26), Bw16(Bw38, Bw17. The initial definition by direct cytotoxicity assay was confirmed by absorption of reactions against target T lymphocytes, thus avoiding problems due to contaminating Ia antibodies, and by blocking the reactions by pretreatment with a chicken anti-human β2 microglobulin serum. That the new specificities were due to the Daudi HLA region was confirmed by the finding that interspecific hybrids between Daudi and A9L, containing a single human chromosome 6, expressed A10 and Bw17. This also defined the haplotypes of Daudi as A10(Aw26), Bw17 and A1, Bw16(Bw38). The re-expression of the Daudi HLA—A and B antigens in two independent sets of hybrids indicates that it does not carry a mutation in the HLA region. It has previously been reported that somatic cell hybrids with Daudi, which contain chromosome 15, do not express human β2 microglobulin. These results suggest that the reason for the lack of HLA—A and B antigens on Daudi is a secondary effect due to the mutation(s) in the β2 microglobulin gene.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... CRISPR/Cas9-mediated B2M knockout (B2M −/− ) in DFT1 cells rendered the cells irreversibly deficient for surface expression of B2M despite IFNG and NLRC5 stimulation (DFT1.B2M −/− + IFNG and DFT1.NLRC5.B2M −/− ). Due to the pivotal role of B2M in stability of MHC-I complex formation and surface presentation (Arce-Gomez et al. 1978;Williams et al. 1989;Vitiello et al. 1990;Kozlowski et al. 1991;Boyd et al. 1992), absence of surface B2M is indicative of a lack of surface MHC-I expression. ...
Article
Full-text available
PurposeDownregulation of MHC class I (MHC-I) is a common immune evasion strategy of many cancers. Similarly, two allogeneic clonal transmissible cancers have killed thousands of wild Tasmanian devils (Sarcophilus harrisii) and also modulate MHC-I expression to evade anti-cancer and allograft responses. IFNG treatment restores MHC-I expression on devil facial tumor (DFT) cells but is insufficient to control tumor growth. Transcriptional co-activator NLRC5 is a master regulator of MHC-I in humans and mice but its role in transmissible cancers remains unknown. In this study, we explored the regulation and role of MHC-I in these unique genetically mis-matched tumors.Methods We used transcriptome and flow cytometric analyses to determine how MHC-I shapes allogeneic and anti-tumor responses. Cell lines that overexpress NLRC5 to drive antigen presentation, and B2M-knockout cell lines incapable of presenting antigen on MHC-I were used to probe the role of MHC-I in rare cases of tumor regressions.ResultsTranscriptomic results suggest that NLRC5 plays a major role in MHC-I regulation in devils. NLRC5 was shown to drive the expression of many components of the antigen presentation pathway but did not upregulate PDL1. Serum from devils with tumor regressions showed strong binding to IFNG-treated and NLRC5 cell lines; antibody binding to IFNG-treated and NRLC5 transgenic tumor cells was diminished or absent following B2M knockout.ConclusionMHC-I could be identified as a target for anti-tumor and allogeneic immunity. Consequently, NLRC5 could be a promising target for immunotherapy and vaccines to protect devils from transmissible cancers and inform development of transplant and cancer therapies for humans.
... Major Histocompatibility Complex (MHC) molecules are very stable (half-life up to 10 h) and have a polymorphic nature. This allows them to bind to a vast array of foreign peptides [28]. Based on the accuracy of immunoinformatics tools, we predicted highly reliable T-cell epitopes to MAGE-A11. ...
Article
Full-text available
Immunotherapy is a breakthrough approach for cancer treatment and prevention. By exploiting the fact that cancer cells have overexpression of tumor antigens responsible for its growth and progression, which can be identified and removed by boosting the immune system. In silico techniques have provided efficient ways for developing preventive measures to ward off cancer. Herein, we have designed a potent cytotoxic T-lymphocyte epitope to elicit a desirable immune response against carcinogenic melanoma-associated antigen-A11. Potent epitope was predicted using reliable algorithms and characterized by advanced computational avenue CABS molecular dynamics simulation, for full flexible binding with HLA-A*0201 and androgen receptor to large-scale rearrangements of the complex system. Results showed the potent immunogenic construct (KIIDLVHLL), from top epitopes using five algorithms. Molecular docking analyses showed the strong binding of epitope with HLAA*0201 and androgen receptor with docking score of -780.6 and -641.06 kcal/mol, respectively. Molecular dynamics simulation analysis revealed strong binding of lead epitope with androgen receptor by involvement of 127 elements through atomic-model study. Full flexibility study showed stable binding of epitope with an average RMSD 2.21 Å and maximum RMSD value of 6.48 Å in optimal cluster density area. The epitope also showed remarkable results with radius of gyration 23.0777 Å, world population coverage of 39.08% by immune epitope database, and TAP affinity IC50 value of 2039.65 nm. Moreover, in silico cloning approach confirmed the expression and translation capacity of the construct within a suitable expression vector. This study paves way for a potential immunogenic construct for prevention of cancer.
... [106][107][108] The mechanism underlying this abnormal tumor phenotype has frequently been the loss of the b2-microglobulin (b2m), which is required for the formation of HLA class I H chain-b2m-peptide complexes and their transport to the cell surface. 109 Two genetic eventsnamely, mutations of one copy of the b2m gene and loss of the other copy [i.e., loss of heterozygosity (LOH)]have been found to underlie b2m loss in malignant cells, although the chronological sequence of the 2 events is not known. 22,102 It has been demonstrated that in melanoma patients undergoing immunotherapy, a lack of response to immunotherapy and generation of progressing metastases appears to be associated with immune selection of MHC-I negative melanoma cell variants. ...
Article
Full-text available
Pharmacologic inhibition of the cytotoxic T lymphocyte antigen 4 (CTLA4) and the programmed death receptor-1 (PD1) has resulted in unprecedented durable responses in metastatic melanoma. However, resistance to immunotherapy remains a major challenge. Effective immune surveillance against melanoma requires four essential steps: activation of the T lymphocytes, homing of the activated T lymphocytes to the melanoma microenvironment, identification and attack of melanoma cells by activated T lymphocytes, and the sensitivity of melanoma cells to apoptosis. At each of these steps, there are multiple factors that may interfere with the immune surveillance machinery, thus allowing melanoma cells to escape immune attack and develop resistance to immunotherapy. We provide a comprehensive review of the complex immune surveillance mechanisms at play in melanoma, and a detailed discussion of how these mechanisms may allow for the development of intrinsic or acquired resistance to immunotherapeutic modalities, and potential avenues for overcoming this resistance.
Article
Background and aims: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. Methods: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers (MSI-H CRCs) using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. Results: We detected 101 truncating mutations in 57 (50%) tumors and loss of 61 alleles in 21 (18%) tumors. Based on the integrated analysis that enabled the immunological subclassification of MSI-H CRCs, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. Conclusions: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.
Article
A monoclonal antibody (AAP1) to human intestinal alkaline phosphatase (ALP) was produced by immunizing a mouse with D98/AH-2 (HeLa) cells, which produce the enzyme ectopically. The antibody, which did not inhibit enzyme activity using p-nitrophenyl phosphate as the substrate, was of the IgG2A class and did not show complement-dependent cytotoxicity. In trace binding assays AAP1 bound only to cells that expressed an intestinal-like form of human ALP, including some human intraspecific (D98/AH-2 × human lymphocyte or fibroblast) hybrids. Immuno-precipitation of immune complexes from cell-free extracts of D98/AH-2 cells, using protein A containing 5. aureus and AAP1 antibody, resulted in precipitation of all the ALP activity. The precipitated material had a subunit molecular weight of 80,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. In non-denaturing conditions, AAP1 antibody prevented the migration of ALP activity into the gel when cell-free extracts were made from human adult or fetal intestine, or D98/AH-2 cells. Similarly, AAP1 could be used to precipitate ALP activity from these extracts but not from extracts of human liver, kidney or placenta.
Article
Full-text available
We have found markedly deficient expression of the class I major histocompatibility antigens HLA-A,B,C and beta 2m on human small-cell lung cancer (SCLC) lines and fresh tumor samples. The deficit of HLA-A,B,C and beta 2-microglobulin (beta 2m) antigen expression was demonstrated with both radiobinding assays and indirect immunofluorescence assays. Immunoprecipitation of metabolically labeled cells with antibodies to class I antigens showed most SCLC lines to have synthesized almost no beta 2m and HLA-A,B,C proteins. Northern blot analysis, using human HLA-A,B, and beta 2m cDNA probes, showed that almost all SCLC lines tested had markedly decreased amounts of HLA and beta 2m mRNA, but both gene products could be induced with interferon treatment of SCLC lines. We conclude that human SCLC, in contrast to other lung cancer types, is characterized by greatly reduced transcription of HLA-A,B,C and beta 2m genes, which suggests the existence of a mechanism for evading the host immune response to the tumor and of an E1a-like product in this type of tumor cell.
Article
Full-text available
The response of class I major histocompatibility complex antigen expression to in vitro administration of interferon and tumor necrosis factor alpha (TNF-alpha) was measured using class I major histocompatibility complex-deficient small cell lung cancer cell lines. Significant induction also was observed using gamma interferon (IFN-gamma) alone, whereas TNF-alpha alone yielded only modest induction. Classic small cell lung cancer cell lines NCI-H146 and NCI-H209 best demonstrated synergistic HLA and beta 2-microglobulin antigen induction with IFN-gamma and TNF-alpha with the following dose schedule: 3-6 days of TNF-alpha (200 units/ml) followed by 48 h of IFN-gamma (100 IU/ml). Induction was quantitated using an 125I-Protein A radioimmunoassay. Synergistic induction of the HLA and beta 2-microglobulin surface antigens on NCI-H146 was also possible with alpha interferon and TNF-alpha but required a higher concentration of the interferon, i.e., 3-6 days of TNF-alpha (200 units/ml) followed by 48 h of alpha interferon (1000 units/ml). Small cell lung cancer cell line NCI-H146 was further studied for expression of major histocompatibility complex messenger RNA using the optimal doses and sequence of addition of IFN-gamma and TNF-alpha as indicated above. A significant induction with IFN-gamma alone and synergistic induction with both IFN-gamma and TNF-alpha was quantitated for both HLA-A2 and beta 2-microglobulin transcripts using Northern blot analysis. Incubation with relatively low subcytotoxic doses of IFN-gamma and TNF-alpha also resulted in a marked synergistic decrease in c-myc message.
Article
Transcription profiling of ovarian follicles. Understanding the mechanisms by which a single follicle is selected for further ovulation is important to control fertility in mammals. However, development of new treatments is limited by our poor understanding of molecular mechanisms regulating follicular selection. Our hypothesis is that genes involved in the control of cell proliferation and apoptosis are differentially regulated during follicular selection. Our objective was to identify these new genes. Bovine follicles were collected and gene expression levels were measured using microarrays. First, follicles were allocated to three groups, according to the time spent from the initiation of follicular wave to surgery (24 H, 36 H, and 48-60 H). Fifty-seven genes are differentially expressed at a false discovery rate of 5%. These genes are involved in the control of lipid metabolism (P-value = 0.0005), cell proliferation (0.007), cell death (0.003), cell morphology (0.003), and immune response (0.003). Follicles were also grouped into four categories, according to the expected time of deviation (early deviation; 8 mm, mid-deviation; 8.5 mm, late deviation; 9 mm, dominant follicles; >or=10 mm). One hundred and twenty eight genes are differentially expressed between these four groups, including genes involved in cell proliferation (0.00002), cell death (0.0006), cell-to-cell signaling (0.003), cell morphology (0.003), lipid metabolism (0.0004), and immune response (0.00007). The expression levels of 10 genes were confirmed using quantitative real time PCR. As expected, we identified new differentially regulated genes involved in the control of cell growth and apoptosis. We also discovered a potential role for immune cells, and in particular macrophages, in follicular selection.
Article
One of the most complex and important features of both the nervous and immune systems is their data storage and retrieval capability. Both systems encounter a common and complex challenge on how to overcome the cumbersome task of data management. Because each neuron makes many synapses with other neurons, they are capable of receiving data from thousands of synaptic connections. The immune system B and T cells have to deal with a similar level of complexity because of their unlimited task of recognizing foreign antigens. As for the complexity of memory storage, it has been proposed that both systems may share a common set of molecular mechanisms. Here, we review the molecular bases of memory storage in neurons and immune cells based on recent studies and findings. The expression of certain molecules and mechanisms shared between the two systems, including cytokine networks, and cell surface receptors, are reviewed. Intracellular signaling similarities and certain mechanisms such as diversity, memory storage, and their related molecular properties are briefly discussed. Moreover, two similar genetic mechanisms used by both systems is discussed, putting forward the idea that DNA recombination may be an underlying mechanism involved in CNS memory storage.
Article
A decreased expression of major histocompatibility complex (MHC) class I antigens is a common feature of many experimental and human tumors and can often be correlated with malignancy grade. In fact, reduction of class I antigens is associated in most tumors with an enhanced ability to elude immune surveillance. Loss of HLA-A,B,C antigens ranges from a decrease in the percentage of A,B,C-positive cells to selective loss of particular antigens and total loss of class I molecule expression. In man, this has been documented in melanomas, carcinomas, lymphomas, neuroblastoma and acute leukemias. The reduction in membrane antigens is generally associated with a parallel fall in immunoprecipitable intracellular proteins and the corresponding mRNAs in the absence of structural changes in the coding genes. The literature concerning the above mentioned topics is reviewed and discussed.
Article
When Thy-1− cell lines derived from different Thy-1+ murine thymic lymphomas are analyzed by complementation analysis, most fall into the “A” complementation class. A possible explanation for this result is that the Class A phenotype is due to a mutation in a gene on the X chromosome. To test this idea, selection for 6-thioguanine resistance was carried out on Thy-1+ hybrid cell lines between complementary Class A and Class C Thy-1− mutant cell lines. In some hybrid clones, there was complete concordance between 6-thioguanine resistance and a change of the phenotype of the hybrid from Thy-1+ to Thy-1−. Detailed study of one of these hybrid clones showed that 6-thioguanine resistance was accompanied by loss of hypoxanthine guanine phosphoribosyltransferase activity and that the Thy-1− phenotype was attributable to loss of the gene complementing the Class A Thy-1− mutation. Other hybrid clones, however, had some thioguanine resistant lines which remained Thy-1+. The degree of concordance was a characteristic of the particular hybrid clone examined and subclones which showed complete concordance could be derived from clones showing incomplete concordance. The variability in the degree of concordance between 6-thioguanine resistance and the Thy-1− phenotype in different hybrid cell lines was also seen among individual hybrid clones isolated from a fusion between a Class A mutant and normal spleen cell blasts. We conclude from these results that the basis of the Class A Thy-1− phenotype is genetic, but given the variability in the degree of linkage observed, we cannot determine whether the gene determining the Class A mutant phenotype is X-linked in the normal situation.
Article
Full-text available
The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8(+) T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M(-/-) ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.Molecular Therapy (2013); doi:10.1038/mt.2013.59.
Article
The expression of HLA class I a chains by clones derived from a human x mouse T-cell line containing human chromosomes 6 and 15 was analysed by immunofluorescence. Evidence is presented showing that the expression of HLA class I a chains is associated with HLA-β2m. This is based on the phenotypic and quantitative fluorescence flow cytometric analysis of a series of cloned hybrids. In contrast, HLA and H-2 class I determinants show no evidence of such correlation. Results of cell-sorting indicate that the loss of HLA class I a chain expression is due to phenotypic modulation, in addition to gene segregation.
Article
CHEV, a new Epstein-Barr virus negative Burkitt's lymphoma derived cell line has been studied. Karyotype analyses demonstrated the t (8; 14) characteristic translocation. Cell surface characterisation of this line showed the presence of μ and x immunoglobulin chains and β2-microglobobulin and the absence of the complement receptor. We are unable to detect HLA-A, B alloantigens on the cell surface which was in contrast with an apparently normal expression of these antigens in the cytoplasm.
Article
Human a and ß interferons increase the amount of class I human histocompatibility messenger RNA HLA-A, B, C and ß2-microglobulin in most human cells studied to date. This report concerns the effect of interferons on the Burkitt lymphoma-derived cell line Daudi, which does not express HLA-A, B, C antigens or ß2-microglobulin on its membrane. HLA-A, B, C messenger RNA present in Daudi cells is increased by both a and ß interferons. Furthermore, we have shown that although it was not possible to detect mature ß2-microglobulin protein in the cytoplasm or on the cell membrane of Daudi cells, a poly A+ messenger RNA is present in Daudi cells, which hybridizes with a cDNA clone specific for human ß2-microglobulin. This abnormal messenger RNA is, however, increased normally by interferon. These effects were also observed with human interferon ß on a variant of Daudi cells characterized by a markedly reduced sensitivity to anti-proliferative and anti-cellular effects of human interferon a.[/p]
Article
In this overview of my research, I have aimed to give the background as to how I came to be involved in my various areas of interest, with an emphasis on the early phases of my career, which largely determined my future directions. I had the enormous good fortune to have worked under two of the most outstanding scientists of the twentieth century, R.A. Fisher and Joshua Lederberg. From mathematics and statistics, I went to population genetics and the early use of computers for modeling and simulation. Molecular biology took me into the laboratory and eventually to somatic cell genetics and human gene mapping. One chance encounter led me into the HLA field and another led me into research on cancer, especially colorectal cancer. On the way, I became a champion of the Human Genome Project and of the need for scientists to help promote the public understanding of science. Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 16 is August 31, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Article
Full-text available
Recent studies have indicated the anti-tumor activity and reduced allogeneic response of universal chimeric antigen receptor-modified T (UCAR T) cells lacking endogenous T cell receptors and beta-2 microglobulin (B2M) generated using gene-editing technologies. However, these cells are vulnerable to lysis by allogeneic natural killer (NK) cells due to their lack of human leukocyte antigen (HLA) class I molecule expression. Here, constitutive expression of mutant B2M-HLA-E (mBE) and B2M-HLA-G (mBG) fusion proteins in anti-CD19 UCAR T (UCAR T-19) cells was conducted to protect against allogeneic NK cell-mediated lysis. The ability of cells expressing mBE or mBG to resist NK cell-mediated lysis was observed in gene-edited Jurkat CAR19 cells. UCAR T-19 cells constitutively expressing the mBE and mBG fusion proteins were manufactured and showed effective and specific anti-tumor activity. Constitutive expression of the mBE and mBG fusion proteins in UCAR T-19 cells prevented allogeneic NK cell-mediated lysis. In addition, these cells were not recognizable by allogeneic T cells. Additional experiments, including those in animal models and clinical trials, are required to evaluate the safety and efficacy of UCAR T-19 cells that constitutively express mBE and mBG. This article is protected by copyright. All rights reserved
Article
HLA class I cell surface expression is crucial for normal immune responses, and variability in HLA expression may influence the course of infections. We have previously shown that classical HLA class I expression on many human cell types is biased with greatly reduced expression of HLA-B compared with HLA-A in the absence of inflammatory signals. In the search for the mechanisms responsible for this discrepancy, we have recently reported that the regulation is mainly posttranslational and that the C-terminal part of the α2 domain and the α3 domain contain the molecular determinants that explain most of the variability of expression between common HLA-A and -B allomorphs. In this study, we present a fine mapping of the structural determinants that allow such variability by exchanging key amino acids located within the C-terminal part of the α2 domain and the α3 domain of HLA-A2 and -B8, including Glu/Asp at position 177, Gln/Glu at position 180, Gly/Arg at position 239, and Pro/Ser at position 280. We found that the HLA-A2 and -B8 expression profiles could be interconverted to a large extent by mutual exchange of Gln/Glu at position 180 or by Gly/Arg at position 239. The presence of Gln(180) and Gly(239), as in HLA-A2, led to higher cell surface expression levels when compared with the presence of Glu(180) and Arg(239), as in HLA-B8. This indicates that the amino acids at positions 180 and 239 determine the level of cell surface expression of common HLA-A and -B allomorphs, probably by affecting HLA processing in the Ag presentation pathway.
Article
Full-text available
Malignant melanoma, a very common type of cancer, is a rapidly growing cancer of the skin with an increase in incidence among the Caucasian population. The disease is seen through all age groups and is very common in the younger age groups. Several studies have examined the risk factors and pathophysiological mechanisms of malignant melanoma, which have enlightened our understanding of the development of the disease, but we have still to fully understand the complex immunological interactions. The examination of the interaction between the human leucocyte antigen (HLA) system and prognostic outcome has shown interesting results, and a correlation between the down- or upregulation of these antigens and prognosis has been seen through many different types of cancer. In malignant melanoma, HLA class Ia has been seen to influence the effects of pharmaceutical drug treatment as well as the overall prognosis, and the HLA class Ib and regulatory T cells have been correlated with tumor progression. Although there is still no standardized immunological treatment worldwide, the interaction between the human leucocyte antigen (HLA) system and tumor progression seems to be a promising focus in the way of optimizing the treatment of malignant melanoma.
Article
Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8(+) T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.
Article
Full-text available
The MILL family, composed of MILL1 and MILL2, is a group of nonclassical MHC class I molecules that occur in some orders of mammals. It has been reported that mouse MILL2 is involved in wound healing; however, the molecular mechanisms remain unknown. Here, we determine the crystal structure of MILL2 at 2.15 Å resolution, revealing an organization similar to classical MHC class I. However, the α1-α2 domains are not tightly fixed on the α3-β2m domains, indicating unusual interdomain flexibility. The groove between the two helices in the α1-α2 domains is too narrow to permit ligand binding. Notably, an unusual basic patch on the α3 domain is involved in the binding to heparan sulfate which is essential for MILL2 interactions with fibroblasts. These findings suggest that MILL2 has a unique structural architecture and physiological role, with binding to heparan sulfate proteoglycans on fibroblasts possibly regulating cellular recruitment in biological events.
Article
Full-text available
: The ligation of programmed cell death 1 (PD-1) with programmed cell death ligand PD-L activates the immune checkpoint leading to T-cell dysfunction, exhaustion, and tolerance, especially in Hodgkin lymphoma (HL) where the PD-L/ Janus kinase (Jak) signaling was frequently found altered. Anti-PD-1 or anti-PD-L1 monoclonal antibodies can reverse this immune checkpoint, releasing the brake on T-cell responses. The characterization of the mechanisms regulating both the expression of PD-1 and PD-L and their function(s) in HL is ongoing. We provide in this review the recent findings focused on this aim with special attention on the major research topics, such as adverse events and resistance to PD-1–PD-L1 inhibitor treatment, together with a part about angiogenesis, extracellular vesicles, and microbiome in HL pathogenesis.
Article
Allogeneic immune rejection is a major barrier for the application of human pluripotent stem cells (hPSCs) in regenerative medicine. A broad spectrum of immune cells, including T cells, natural killer (NK) cells, and antigen-presenting cells, which either cause direct cell killing or constitute an immunogenic environment, are involved in allograft immune rejection. A strategy to protect donor cells from cytotoxicity while decreasing the secretion of inflammatory cytokines of lymphocytes is still lacking. Here, we engineered hPSCs with no surface expression of classical human leukocyte antigen (HLA) class I proteins via beta-2 microglobulin (B2M) knockout or biallelic knockin of HLA-G1 within the frame of endogenous B2M loci. Elimination of the surface expression of HLA class I proteins protected the engineered hPSCs from cytotoxicity mediated by T cells. However, this lack of surface expression also resulted in missing-self response and NK cell activation, which were largely compromised by expression of β2m-HLA-G1 fusion proteins. We also proved that the engineered β2m-HLA-G5 fusion proteins were soluble, secretable, and capable of safeguarding low immunogenic environments by lowering inflammatory cytokines secretion in allografts. Our current study reveals a novel strategy that may offer unique advantages to construct hypoimmunogenic hPSCs via the expression of membrane-bound and secreted β2m-HLA-G fusion proteins. These engineered hPSCs are expected to serve as an unlimited cell source for generating universally compatible "off-the-shelf" cell grafts in the future.
Article
The generation of ex vivo functional megakaryocytes and platelets is an important issue in transfusion medicine as donor-dependence implies in limitations, such as shortage of eligible volunteers. Indeed, platelet transfusion is still a procedure that saves the lives of patients with defective platelet production. Recent technological development has enabled the isolation and expansion of stem cells that can be used as a source for the production of functional platelets for transfusion. In this review, we discuss recent approaches of in vitro or ex vivo production of megakaryocytes and platelets, suggesting that, in the near future, donor-independent sources may become a possibility. The feasibility of using these cells in the clinic may be safer, and in vitro manipulation could generate universally compatible products, solving problems related to platelet refractoriness. However, functionality and survival testing of these products in human beings is scarce, therefore additional studies are needed to consolidate this purpose.
Article
Derivatives of human Beta 2 microglobulin (B2M) have been made by attaching various groups to amino groups in the peptide. In this way 125iodine, dinitrobenzyl, and m-maleimidobenzoyl groups have been introduced. These derivatives of B2M were able to associate with HLA chains and could be displaced again by native B2M. The rate of dissociation of HLA chains and derivatives of B2M is comparable with the intrinsically labelled native dimer. In some cases, particularly with [125I]-B2M, the rate of dissociation appears to be smaller than for native B2M.
Article
Full-text available
The HLA-CW2 antigen of the B lymphoblastoid cell line BRI 8 is structurally homologous to the HLA-A and B antigens as judged by various criteria. Each antigen comprised a glycosylated polypeptide of 43 000 molecular weight that is noncovalently associated with beta2-microglobulin (beta2m). Some small differences in molecular parameters were, however, revealed. Thus, the deoxycholate-solubilized HLA-CW2 antigen sedimented at the same rate as the HLA-A antigens but at a slightly faster rate than the HLA-B antigens. This variation is apparently is apparently due to different amounts of bound deoxycholate. Also, whereas essentially all of the HLA-A and B antigens and about half of the HLA-CW2 antigen were adsorbed strongly by Lens culinaris lectin-Sepharose, the remaining HLA-CW2 antigen was bound much more weakly and did not require sugar for elution. This difference reflects some structural heterogeneity in the carbohydrate moiety of the HLA-CW2 antigen. The results of various studies suggest that the HLA-CW2 antigen is expressed to a lower extent than the HLA-A or B antigens and that essentially all of the beta2m of the BRI 8 plasma membrane is associated with the HLA-A, B and C alloantigenic polypeptides.
Article
Full-text available
The genotype of the patient Henrietta Lacks from whose cervical carcinoma the HeLa cell was derived was deduced from the phenotypes of her husband and children, and from studies of the HeLa cell. Hemizygous expression of glucose-6-phosphate dehydrogenase in HeLa, together with the deduced heterozygosity of Mrs. Lacks, is consistent with clonal origin of her neoplasm.
Article
Full-text available
SUMMARY Chromosome studies were done on 18 somatic hybrid cell lines produced by fusing cells of the mouse A 9 line with cells of the human Daudi lymphoblastoid line derived from a patient with Burkitt's lymphoma. The human chromosomes were identified by their quinacrine fluorescent banding patterns. In one hybrid line the human chromosomes were identified also by the centromeric heterochromatin staining technique. Every human chromosome was identified in one or more of the hybrid lines. Some lines were homogeneous in terms of their human chromosome content, while others were quite heterogeneous. Detailed analysis of the A 9 chromosomes in one hybrid line showed very few changes in comparison with the chromo- some constitution of the average A g cell.
Article
Full-text available
A low molecular weight β2-globulin (β2-microglobulin) has been purified from urine of patients with tubular proteinurias. The isolation procedure includes ultrafiltration, zone electrophoresis, gel chromatography, and ion exchange chromatography. The yield of purified protein was 11 to 13% of a total amount of 2.8 to 89 mg of β2-microglobulin per 24-hour urine volume. The β2-microglobulin occurs in small quantities in normal human urine, plasma, and cerebrospinal fluid. The purified protein appeared homogeneous in Ouchterlony immunodiffusion, immunoelectrophoresis, starch gel electrophoresis, and ultracentrifugation. The molecular weight found from equilibrium ultracentrifugation was 11,600. Amino acid analyses indicated the presence of 100 residues, giving a calculated molecular weight of 11,815. The protein appears to consist of a single polypeptide chain with 2 half-cystine residues involved in a disulfide bond. Isoleucine was identified as the NH2-terminal residue. The β2-microglobulin is devoid of carbohydrate.
Article
Different autoimmune diseases often occur together in the same individual or family. Among the clustering autoimmune diseases, those mainly observed are insulin-dependent (type 1) diabetes mellitus (IDDM), diseases of the thyrogastric group [autoimmune thyroid disease (ATD); Graves’ disease, Hashimoto’s disease], and rheumatoid arthritis (RA). This clinical observation suggests a shared etiology due to genetic predisposition which is supported by the fact that these clustering autoimmune diseases are associated with the same HLA antigens: DR3 and DR4. For example, IDDM and ATD are significantly associated with DR3, while DR4-associated diseases include IEpM and RA.
Article
The assembly of the separately synthesized light (L) and heavy (H) chains into immunoglobulin G (IgG) has been studied in murine plasmacytoma 5563. The L chains are autonomously released into a small intracellular pool of free L chains; the labeling and turnover of this pool have been studied, and the results lend strong support to the proposal that free L chains are intermediates in IgG assembly. The time course of the labeling of L and H chains of completed IgG was measured. The flow of label into L chains of IgG shows that free intracellular L chains constitute a randomly mixing pool of intermediates. There is a slight lag in the appearance of radioactivity in H chains of IgG which suggests the existence of an extremely small time-ordered pool of completed (or nearly completed) H chains. The present results are consistent with the proposal that the balanced synthesis of L and H chains, which occurs in both 5563 plasmacytoma cells and lymph node cells from hyperimmune mice, results from control of IgG assembly by the L-chain pool.
Article
Direct cytotoxicity and absorption analysis were used to detect HLA antigens on man/mouse somatic cell hybrids. Confirmation of the assignment of the Major Histocompatibility Complex to chromosome 6 was obtained. The same series of hybrids were used to study the interaction between HLA and ß2m on the cell surface. Results obtained from a series of clones indicated that there was complete independence of expression of HLA and ß2m. Human ß2m was found to be unnecessary for the normal serological expression of HLA, suggesting that mouse ß2m could possibly replace human ß2m as a subunit of the HLA molecule. Evidence is presented demonstrating that this is in fact the case. Heteroantisera to human ß2m and alloantiserum to HLA-A2 were used, together with complement, to selectively remove the chromosome coding for ß2m and HLA from a hybrid clone. Manipulation of the karyotype in this way further confirmed the independence of HLA and ß2m on somatic cell hybrids.
Article
Somatic cell hybrids between D98/AH-2 and fibroblasts or peripheral blood lymphocytes were analysed for the expression of HL-A antigens. The hybrids express the antigens of the donor lymphocytes or fibroblasts as well as other HL-A specificities from D98/AH-2, a Hela derivative on which no HL-A could be detected by direct cytotoxicity. Segregant hybrid clones could be used to determine HL-A haplotypes and define the linkage relationships between these haplotypes and the PGM3 alleles expressed in the hybrids. An analysis of the HL-A antigens on D98/AH-2 and other Hela derivates was done using absorption and a modified cytotoxicity assay.
Article
Antisera have been prepared, in mice and rabbits, to membrane and sub-membrane fractions of human B lymphocyte derived lymphoid lines. Antisera to a protein subfraction were, after only minimal absorption, specific for human peripheral B lymphocytes, monocytes and B cell derived lymphoid lines. The antigen(s) recognised by these antisera were not the same as the previously described B-cell markers; immunoglobulin, Fc receptor, complement receptor and Ia antigens. The antigen(s) could not be removed from cells by lysostrip with anti- β2 microglobulin.
Article
1. Investigation of human tissue extracts by starch gel electrophoresis has vevealed three groups of isozymes with PGM activity. 2. Previous studies on red cell lysates have established that two of these groups of isozymes, PGM1 and PGM2 are determined by two separate autosomal loci, PGM1 and PGM2 respectively. 3. The new series of isozymes, designated PGM3, are electrophoretically faster than the other PGM components, constitute only a small fraction of the total PGM activity, and exhibit person-to-person differences in electrophoretic patten which are independent of te PGM1 and PGM2 variants. Three commonly occuring phenotypes have been recognized : PGM3 1, PGM3 2-1 and PGM3 2. 4. The genetics of this polymorphism have been studied using plancentae derived from pairs of non-identical twins. The reult suggest the occurence of two common alleles PGM1/3 and PGM2/3 at an atosomal locus, with frequencies of 0.74 and 0.26 respectively in the English population and 0.34 and 0.66 respectively in the Nigerian population. 5. Tests for linkage using the twin data indicate that the PGM1 and PGM3 loci are not closely linked. 6. The possibility of structural homologies between the various PGM isozymes is noted.
Article
POSSIBLE evolutionary homology between the genetic regions controlling histocompatibility antigens (such as HL-A in man and H-2 in the mouse) and immunoglobulins has been proposed1,2. Recent studies involving beta2-microglobulin (beta2m) seem to support this idea. Human beta2m was found originally in the urine of patients with renal tubular dysfunction. It is also present in serum and on the surface of most types of cell3,4. It has a molecular weight of about 12,000 and shows substantial sequence homology with the constant region domains of immunoglobulin heavy chains5,6. Partially purified papain7,8 and detergent9 solubilised HL-A molecules, and H-2 molecules10, consist of two chains one of which is invariant and has been identified as beta2m11-13. Chance association during the isolation procedure has been shown to be unlikely by the cocapping on the cell surface of beta2m with the allogeneic chain of HL-A that carries the usual serologically detected determinants14-16.
Article
Somatic cell hybrids between D98/AH-2 and fibroblasts or peripheral blood lymphocytes were analysed for the expression of HL-A antigens. The hybrids express the antigens of the donor lymphocytes or fibroblasts as well as other HL-A specificities from D98/AH-2, a HeLa derivative on which no HL-A could be detected by direct cytotoxicity. Segregant hybrid clones could be used to determine HL-A haplotypes and define the linkage relationships between these haplotypes and the PGM3 alleles expressed in the hybrids. An analysis of the HL-A antigens on D98/AH-2 and other HeLa derivates was done using absorption and a modified cytotoxicity assay.
Article
This is a detailed review of the work by the authors and other investigators on genetically determined differences in T cell responsiveness in mice. The ectromelia, Sendai and lymphocytic choriomeningitis viruses have been used in the majority of experiments. The in vitro 51Cr release assays used have been shown quite rigorously to be measuring immune T cell function. The effector T cells in these virus diseases of mice are specific both for the infecting virus and for the H2 genes of the mouse strains used. Different clones of virus immune T cells are associated with each allele at H 2K or H 2D. Heterozygotes generate four clones of effector T cells, compared with two in homozygotes. This provides a mechanistic basis for maintaining a high level of genetic variability. The nature of the interaction between virus and H 2 genes is still obscure. Experiments with H 2 mutant mice have localized the host DNA required in the virus systems to the same cistrons that code for antigens recognized in the allograft response. (Tonder - Bergen)
Article
ANALOGIES are often made between transplantation and placental immunology. The role of histocompatibility antigens is of undisputed importance in transplantation immunology but it is not clear whether such antigens are important in pregnancy. Indeed, it is not known whether trophoblasts manifest histocompatibility antigens. If HLA antigens are relevant in pregnancy, it is of central importance to know if trophoblasts have these antigens. This is a difficult problem because human anti-HLA sera often contain other antibodies, and heterologous anti-HLA sera often lack precise HLA specificity1. But HLA has been shown to be invariably associated with beta2-microglobulin2, and here we present data in support of the concept that human trophoblasts contain neither beta2 microglobulin nor HLA. Also, treatment with several enzymes has failed to reveal these antigens. In addition, we have not been able to identify either beta2 microglobulin or HLA on trophoblasts from 6-week or 13-week placentae or on placental tissues maintained in vitro for 5 d.
Article
A modification of the NIH cytotoxicity test for recognizing B cell (D-associated) antigens and antibodies, when sera also contain anti-HLA(ABC) activity is described. The method is based on the observation that anti-beta2-microglobulin reagents are able to block lympholysis only when due to HLA(ABC) antigens.
Article
It is now well established that β2-microglobulin constitutes one of the two HL-A antigen subunits. In this study support was obtained for the previous notion that the human lymphoma Daudi does not produce β2-microglobulin (β2m). Papain-solubilized as well as nonidet P-40-solubilized Daudi HL-A antigens do not contain any β2m or any detectable structural analogue of this protein. The chemical and physico-chemical characteristics of highly purified HL-A antigens derived from Daudi cells are indistinguishable from those of the HL-A antigen-carrying polypeptide chain isolated from the P3HRIK cell line. Like P3HRIK-derived HL-A antigens, the HL-A antigens derived from Daudi cells are composed of two identical, heavy, alloantigenic polypeptide chains with a molecular weight of about 50000 each, which are held together by disulfide bridge(s). The HL-A antigens of P3HRIK cells contain, in contrast to Daudi HL-A antigens, two molecules of β2m. Although no evidence was obtained suggesting any β2m synthesis in Daudi cells it was apparent that these cells express the HL-A alloantigenic polypeptide chain in amounts similar to those of other cell lines which produce β2m The present data suggest [1] that β2m and the alloantigenic HL-A polypeptide chain are under separate genetic regulation [2], that the cell surface integration of the HL-A antigen-carrying polypeptide chain is independent of the presence of β2m and [3] that β2m does not constitute a membrane component absolutely necessary to the integrity of the cell membrane.
Article
Human fibroblasts containing a translocation between the X chromosome and chromosome 15 were fused with the 6-thioguanine-resistant mouse cell line, IR. Resulting hybrids, selected in HAT medium, retained the X/15 chromosome. Hybrids which were counterselected in 6-thioguanine lost this chromosome. The X-linked markers glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and hypoxanthine phosphoribosyl transferase (HPRT), and the non-X-linked markers pyruvate kinase (PKM2) mannose phosphate isomerase (MPI), N-acetyl hexosaminidase A (HEXA) and beta2-microglubulin (beta2-m) all segregated in concordance with the X/15 translocation chromosome. The latter markers have been assigned to chromosome 15. Selection against the X/15 chromosome was done using antihuman beta2-m serum. Electrophoretic and immunochemical analyses of the N-acetyl hexosaminidases A and B in these hybrids were performed.
Article
Excerpt The recognition and characterization of products specific to particular subclasses of lymphoid cells are important components of any study of the origins and nature of lymphocyte diversity. Intercellular communication is probably often mediated by cell-surface structures, either directly or by means of surface receptors for soluble factors, and so the study of cell-surface molecules is particularly relevant in this respect. Work with a variety of species, especially man and mouse, has indicated that the products of the major histocompatibility region play an important role in many immunological and other cell-mediated phenomena. In this paper, we discuss this role in relation to cell-surface antigens on human B lymphocytes (Bodmer 1972a; Kissmeyer-Nielson 1975; Katz and Benacerraf 1976). The products of the HL-A A, B, and, to a lesser extent, C loci have been well defined serologically (J. G. Bodmer 1975) and biochemically (Bridgen et al. 1976; Terhorst et al. 1976; Snary et...
Article
Peripheral lymphoid cells, from 12 cases of acute infectious mononucleosis (IM), were tested in a micro chromium-51 release assay for cytotoxic activity against a variety of cell lines that did or did not carry the Epstein-Barr virus (EBV) genome. Unfractionated lymphocytes from these patients were cytotoxic to both types of cell lines, as were lymphocytes from healthy individuals. If, however, lymphocytes bearing complement receptors were removed, the residual IM lymphocyte fraction was specifically cytotoxic for EBV-genome-carrying cell lines. The residual lymphocyte fraction in normal donors had no such effect. Heterophile-positive IM is caused by EBV, and these results indicate that, during the acute phase of this disease, patients harbor killer cells, probably T cells, which specifically kill EBV-genome-carrying B cells in vitro. No such specificity for EBV-genome-psitive target cells was found in normal lymphocytes stimulated in vitro with autologous EBV-genome-positive lymphoblastoid cells. Such stimulated cells were highly cytotoxic to both genome-positive and negative lines after removal of complement receptor-positive lymphocytes.
Article
This article discusses the detection of Ia antibodies in HLA-typing sera using B-cell lymphoid lines, the grouping of the sera into five preliminary clusters, and their associations with D-locus specificities.
Article
EFFECTOR T cells generated during the course of virus diseases of mice interact only with virus-infected target cells sharing H-2K or H-2D antigenic specificities with the mouse strain in which the lymphocytes are sensitised1-3. The current interpretation4 of this phenomenon is that the cell membrane component(s) recognised by the T-cell receptor(s) has characteristics of both self (H-2) and non-self (virus). Phenotypic expression of H-2 genes in the target cell would thus be essential for T cell-mediated immunity. Here we test this hypothesis using two teratocarcinoma cell lines (F-9 and PCC4 derived from strain 129 mice5,6) infected with lymphocytic choriomeningitis virus (LCMV). We show that the F-9 tumour cells apparently do not express H-2K or H-2D antigenic specificities (Fig. 1, Table 1), and are also resistant to attack by lymphocytes sensitised against LCMV. The PCC4 line, however, has remained multipotential and can give rise to various different cell types, at least some of which show evidence of H-2 gene function (Table 1) and are attacked by the appropriately sensitised lymphocytes.
Article
Hybrids between two human lymphoma lines, Raji and Daudi (8A) and Raji BJAB (83) were examined for genetically determined and/or differentiation-related surface markers. HL-A B ce-l alloantigens, Fc and complement receptors, EBV receptors and beta2 microglobulin showed an autonomous ("co-dominant") expression in the hybrid. This is in contrast to most previous studies on other differentiation markers, involving as a rule crosses between cells of different lineages, where the differentiated pattern usually became "eclipsed" in the hybrid. Staining of activated complement and complement consumption tests showed intermediate or partially suppressed expression in the hybrids. This may be viewed in relation to the fact that these reactions do not merely depend on complement binding to the receptor, but also on subsequent activation and binding of the activated complement. A more complex interaction is also suggested for immunoglobulin production. Surface immunoglobulin showed a suppressive or intermediate pattern in both hybrids, whereas intracellular kappa chain production showed an amplification in the 83 hybrid. The beta2 microglobulin deficiency of the Daudi parent was corrected in the Raji/Daudi hybrid. Two new HL-A specificities,A10 and BW17, appeared on this hybrid which were not present on the parental lines. This suggests that the HL-A deficiency of the Daudi cell is due to its lack of beta2 microglobulin.
Histocompatibility (transplantation) antigens were initially described as being responsible for the rejection of tumor and tissue grafts between nonsyngeneic individuals (Gorer, 1938; Snell, 1948). Genetic analyses subsequently established that the major transplantation antigens are the products of genes located at two separate, but closely linked, loci. More recent studies have indicated that these loci delineate a chromosomal region that controls a complex series of cell-mediated reactions. This region is referred to as the major histocompatibility complex.: In mice, it is denoted as H-2;: in humans, as HLA.: The H-2: complex has been more extensively studied than the analogous human region, and many of the functions of the HLA: complex are based on analogies with the mouse data. The exact number of genes present in each complex is not known, although most workers are agreed that the number is large. Thus, for example, Klein (1975) has suggested that the H-2: complex contains sufficient DNA to code for up to 2000 polypeptide chains, each of about 200 amino acids. The relationships among the genes have not been established, but their functions appear to be related and to be primarily concerned with the control of expression of cell-surface antigenic determinants, immune-response differences, certain complement functions, and probably other functions related to cell-cell recognition.
Article
The levels of HLA-A and -B antigens expressed by placenta have been assessed relative to parental and other A and B antigen types that were not shared by the foetus. A purified preparation of placenta plasma membrane was used to estimate the antigen activities. The results indicate that maternally and paternally inherited A and B antigen activities and beta2-microglobulin are expressed to similar extents but at much lower levels than in spleen lymphocytes (less than 5%). The possibility that the amounts detected were caused by contamination with blood or maternal tissue was ruled out. The low levels of A and B antigens may account for the lack of a cellular immune response to the other polymorphic cell surface antigens of the trophoblast. No evidence was obtained for the expression of a significant level of Ia antigens.
Article
Rabbit sera used as a complement source contain cytotoxic antibodies which reacted with cultured human cells. This cytotoxic activity can be inhibited by human IgM. The capacity of IgM mu and L chains, and IgM glycopeptides to inhibit these heterospecific antibodies was determined. The inhibitory activity of several monosaccharides, bacterial polysaccharides and blood group substances was also determined. At least some of the heterospecific antibodies in normal rabbit sera are directed against carbohydrate determinants. Mannose, fucose, and bacterial polysaccharides containing mannose as the immunodominant sugar are particularly effective in inhibiting cytotoxic antibodies.
Article
Human HLA-A, B, C and Ia antigens were labelled by lactoperoxidase-catalysed iodination of the inner surface of lymphocyte plasma membrane and thus are transmembrane proteins. In contrast, membrane-bound human IgM and mouse IgM, IgD and Thy-1 antigen were not labelled on the inner membrane surface.
Article
H-2Kk and H-2Dd molecules were specificially purified from a radioiodinated H-2a preparation obtained by papain digestion of spleen cell membranes of A/J strain mice. The molecules were isolated by binding to H-2 alloantisera of the corresponding private specificity followed by precipitation with rabbit anti-mouse IgG antiserum. The specifically precipitated radioiodinated H-2Kk and H-2Dd molecules were dissociated by acid treatment into large and small components of about 37,000 and 11,000 respectively. These were separated by gel filtration at acid pH or by gel isoelectric focusing in the presence of 6 M urea. Each component separated by gel filtration of the acid-dissociated H-2 molecules showed a high degree of size homogeneity as determined by sodium dodecyl sulphate-acrylamide gel electrophoresis. Upon gel isoelectric focusing, however, the small components showed two peaks of radioactivity closely located together at pH 7-8, both of which had a restricted pH range, while the large components gave one peak of a relatively wide pH range of pH 5-6. The H-2Kk and H-2Dd molecules gave essentially the same pattern in terms of the numbers and the positions of the radioactivity bands. Under the iodination conditions used the large components of H-2Kk molecules contained more radioactivity than the small components, while the reverse was true in case of H-2Dd molecules. Such a difference was also found with H-2Kk and H-2Dd molecules isolated by use of alloantisera of the respective public specificity. The assay of binding of the isolated components with H-2 alloantisera of defined specificity revealed that the large components retain most of the allospecificities of the parental H-2 molecules. No H-2 allospecificities were found on the small components. The small components showed extensive binding with rabbit antiserum against mouse beta2-microglobulin. The same antiserum did not show any binding with the large components. On the other hand, both of the components did bind with rabbit antiserum against papain-solubilized H-2 molecules.
Article
A gene (or gene cluster) controlling 1 (or more) cell surface species antigens has been assigned to chromosome 11. Using hybrids made with cells from a carrier of a balanced reciprocal translocation, 45,XX,t(11;17)(p15;q21), the authors have localized this gene or genes to the short arm of chromosome 11.
The affinity of rabbit thymocytes and a proportion of lymphocytes for homologous and autologous erythrocytes has been investigated. Thymocytes were found to rosette strongly with untreated, washed erythrocytes, although reactions on peripheral lymphocytes from blood and lymph nodes were weaker. Enzyme treatment of the erythrocytes was used in attempts to improve reactions with peripheral lymphocytes. While neuraminidase-treated cells showed no increased reactivity, erythrocytes treated with papain were more firmly bound and reacted with greater numbers of blood and lymph node cells. Evidence was obtained to show that peripheral lymphocytes which have an affinity for papain-treated erythrocytes belong to the thymus-derived T cell population.
Article
THE Ir genetic region of the mouse which lies between the H-2K and H-2D loci that control the original serologically detectable H-2 specificities, has also been shown to contain genes controlling a new set of serological specificities called Ia for immune associated. Antisera raised by cross immunisation between H-2 recombinant strains have been shown to recognise the products of several genes within the Ir region (reviewed in ref. 1). Unlike the H-2K and H-2D antigens the Ia antigens have a tissue distribution that often does not include T lymphocytes.
Article
A number of human intraspecific hybrids were produced by fusing the 8-azaguanine-resistant cell line D98/AH-2 with PHA-stimulated lymphocytes from a normal human male, followed by selection in HAT medium. The parent cells differed in zymogram patterns for 4 enzyme systems. Hypoxanthine-guanine phophoribosyltransferase was missing in D98/AH-2 and was determined in the hybrids by the normal gene derived from the lymphocyte donor's X chromosome. The HL-A antigens of the lymphocyte donor as well as the W28 specificity from HeLa were easily recognized by a cytotoxicity assay on the hybrid cells, while D98/AH-2 itself was not killed in the normal way by any HL-4 typing sera. The initial hybrid karyotype in all lines was relatively stable, but slow loss of chromosomes occurred following extended growth in culture. The importance of the culture conditions for the rate of chromosome loss was demonstrated. The behavior of several chromosomes was followed in the hybrids and their derivatives. There was relatively nonspecific loss of small numbers of chromosomes, showing that loss of chromosomes from both the D98/AH-2 and the normal lymphocyte parent can occur. Cell lines resistant to 6-thioguanine were selected from the sensitive hybrids. Most had lost the lymphocyte donor's X chromosome, thereby losing the only active allele for HGPRT present in the initial hybrids. However, one line, DMR41, apparently retained the X chromosome and may have a mutated allele for HGPRT. Two lines that are the products of spontaneous segregation are also described. DM4CS and DM17A.
Article
KNOWLEDGE of the detailed pattern of fluorescence of the normal human karyotype, showing more than 200 bands per haploid chromosome set1, has enabled us to recognize, both in biopsies and in cell cultures from several Burkitt lymphomas, an extra band in one homologue of D group chromosome pair No. 14. The deviation was seen in all analysable cells of five out of six tumour biopsies and of seven out of nine tumour cell lines examined, representing twelve different tumours from nine male and three female patients. In three tumours both biopsies and cultures were examined, and it was found that all gave results consistent in the two types of samples. Thus, two of them had the marker band in both the biopsy and culture, the third revealed the marker band absent in both cases. The remaining tumours were investigated only in biopsies or only in culture. They were positive in eight cases, negative in one. Of the twelve tumours examined altogether, ten were positive and two negative.
Article
Normal lymphoid cells obtained from hyperimmunized BALB/c mice and cells from most but not all IgG-producing mouse myeloma tumors and cell lines synthesized more light (L) than heavy (H) chains. While most myeloma tumors secreted their excess L chains, two tumors, MOPC 21 and MOPC 173, degraded the excess L chains and did not secrete them. A third group of tumors produced the same number of H and L chains. In the case of the MPC-11 tumor, excess L chains were synthesized by cloned tumors and cell lines.
Article
The capacity of normal and malignant human cells to synthesize β2 microglobulin was studied in vitro. Quantitative determinations of β2 microglobulin by a radioimmunoassay were performed on culture media harvested from 7 freshly explanted lymphoma cells and 37 cell lines of hematopoietic, mesenchymal, or epithelial origin. β2 Microglobulin was detected in all but one lymphoma cell culture. Lymphoma cells, freshly explanted, as well as those of continuously growing lines seem to have a low capacity to synthesize β2 microglobulin while permanent lymphoblastoid lines secrete the protein at approximately the same rate as mesenchymal cells. The highest production rate of β2 microglobulin was observed in some epithelial carcinoma cell lines. No correlation was found between the capacity to synthesize immunoglobulin and β2 microglobulin.
Article
Burkitt's lymphoma biopsy specimens from a 16-year-old African were found to differ from more than forty previously tested biopsy specimens from patients with this disease. Direct immunofluorescence detected a surface accumulation of IgM on living lymphoma cells, with a light-chain specificity. Anti-IgM or anti-light-chain sera killed the cells in the presence of complement even in high dilutions. Surface IgM specificity was maintained in three derived cell lines, grown in suspension for more than 4 months in vitro.
Article
In man-Chinese hamster somatic cell hybrids the segregation of the loci for 27 human enzyme markers and the species-specific surface antigens, including the HL-A histocompatibility antigens, was studied. The results show a synteny of the human loci for phosphoglucomutase 3, cytoplasmic malic enzyme, tetrameric indophenol oxidase, and HL-A. Furthermore, evidence is presented that the loci for the human species-specific antigens are distributed over several chromosomes.
Article
THERE is still no adequate explanation for the fact that the placenta, which is foetal in origin and therefore allogeneic in the mother, is not rejected during pregnancy. A question of fundamental importance is whether human trophoblast cells possess HL-A or ABO antigens. Evidence so far is conflicting1-4. We present evidence which suggests that blood group A antigens may be present on human trophoblast cells, but at low surface density.
Article
When two clonal lines of mouse fibroblasts, each containing a drug-resistant marker, are grown together for 4 days, hybrid cells can be detected by selective conditions. These hybrid cells are presumed to be the result of mating. By the same method evidence can be obtained which suggests that mating may be followed by segregation.
Article
Rabbit antiserum to human beta(2)-microglobulin followed by goat antiserum to rabbit immunoglobulin induces aggregation of beta(2)-microglobulin at the lymphocyte surface, as shown by immunofluorescence and by acquisition of resistance to lysis by complement. This treatment also affects all HL-A antigens of the cell, which cap together with beta(2)-microglobulin, so that the cell becomes resistant to lysis upon addition of antibodies to HL-A in the presence of complement. These findings suggest that a physical linkage exists between these two classes of polypeptides at the surface of the living cell and is in agreement with recent biochemical data obtained by others with cell membrane preparations.
Bw17 were detected, both by direct cytotoxicity assay and absorption analysis. The two subclones which had lost the Daudi chromosome 6 (3.7 and 3.9) no longer expressed these specificities
  • Bent0 Arce-Gomez Al
BENT0 ARCE-GOMEZ ET AL, Bw17 were detected, both by direct cytotoxicity assay and absorption analysis. The two subclones which had lost the Daudi chromosome 6 (3.7 and 3.9) no longer expressed these specificities. This result not only confirms the