Serum beta 2-microglobulin binds to a T-cell differentiation antigen and increases its expression

The human T-cell leukaemia and differentiation antigen HTA 1 is defined by the monoclonal antibody NA1/34 (ref. 1) and also recognized by the monoclonal antibody OKT6. Like class I products of the human major histocompatibility complex, it has a glycosylated heavy (alpha) chain of approximately 45-50,000 molecular weight (MW) in non-covalent association with beta 2-microglobulin (beta 2m) (MW 11,900). A particular feature of HTA 1 is the presence in significant amounts of an additional beta 2m-like subunit, called beta t (refs 3, 4). Top facilitate biochemical studies we have prepared a high HTA 1 expressor variant (NH17) of the human thymoma line MOLT-4. The N-terminal amino acid sequence of the beta t purified from this cell line was shown to be indistinguishable from that of bovine beta 2m. Further, beta t was present when the cells were grown in medium containing fetal calf serum (FCS), but absent from cells grown with human serum (HuS). We show here that addition of human and bovine beta 2m to MOLT-4 and NH17 cells grown in serum-free medium produces a significant elevation of HTA 1 antigen expression, providing evidence for a regulatory or stabilizing function for the exchange of extracellular beta 2m with a cell-surface antigen.     

Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria

Haemoglobin C, which carries a glutamate-to-lysine mutation in the beta-globin chain, protects West African children against Plasmodium falciparum malaria. Mechanisms of protection are not established for the heterozygous (haemoglobin AC) or homozygous (haemoglobin CC) states. Here we report a marked effect of haemoglobin C on the cell-surface properties of P. falciparum-infected erythrocytes involved in pathogenesis. Relative to parasite-infected normal erythrocytes (haemoglobin AA), parasitized AC and CC erythrocytes show reduced adhesion to endothelial monolayers expressing CD36 and intercellular adhesion molecule-1 (ICAM-1). They also show impaired rosetting interactions with non-parasitized erythrocytes, and reduced agglutination in the presence of pooled sera from malaria-immune adults. Abnormal cell-surface display of the main variable cytoadherence ligand, PfEMP-1 (P. falciparum erythrocyte membrane protein-1), correlates with these findings. The abnormalities in PfEMP-1 display are associated with markers of erythrocyte senescence, and are greater in CC than in AC erythrocytes. Haemoglobin C might protect against malaria by reducing PfEMP-1-mediated adherence of parasitized erythrocytes, thereby mitigating the effects of their sequestration in the microvasculature.     

A gamma-chain complex forms a functional receptor on cloned human lymphocytes with natural killer-like activity

We have recently derived from human fetal blood (25 wks) a series of cloned cell lines that were selected for their ability to kill the conventional natural killer (NK) target cell K562. It was found that a fraction of these clones express CD3 proteins but not the monomorphic Ti alpha beta determinant recognized by WT31 antibody. One interleukin-2-dependent CD3+ WT31- clone, termed F6C7, was used for immunization of mice to generate monoclonal antibodies directed at a potentially novel recognition receptor. It was shown that F6C7 cells, which transcribe Ti beta but not Ti alpha genes, surface-express a clonotypic structure, termed NKFi. Immunoprecipitations performed with anti-NKFi monoclonal antibody (mAb) indicated that the corresponding molecule is resolved in SDS-polyacrylamide gel electrophoresis (PAGE) as a single band of relative molecular mass approximately 85,000 (Mr approximately 85K). After reduction, a major band was detected at 44K and a faint band was present at 41K. The present study was designed to characterize this structure. It was found that NKFi represents either two 44K disulphide-linked gamma (TCR) chains, or possibly one gamma chain associated to an additional undetected molecule, and that the 41K material corresponds to a partially glycosylated fraction of the gamma protein. Anti-NKFi mAb both induces a specific autocrine proliferative response and blocks cytotoxic function, demonstrating that gamma chains serve as functional receptor structures on subpopulations of normal human lymphocytes.     

Rudimentary study on humanization of porcine red blood cells:Enzymatic removal of galactose-α1,3-galactose antigen from porcine red blood cell

The serological and biochemical characterization of porcine red blood cells (pRBCs) are similar to human red blood cells,Porcine erythrocytes are considered as an alternative source for human blood transfusion.But there exist galactose-α1,3-galactose antigens (Galα1,3Galβ1,4galNAc-R,abbreviated αGal antigen ) on pRBCs,which can induce anti-αGai antibodies in human serum ,The αGal epitopes are the major antigen responsible for hyperacute rejection in exnotransfusion .In this study ,recombined soybean α-galactosidase (rSα-GalE) was used to remove the αGal antigens from pPRCs for humanization .The results showed that αGal antigen was eleared by rSα-GalE and the structure and function of rSα-GalE treated pRBC were normal.     

A haemoglobin switching activity modulates hereditary persistence of fetal haemoglobin

During human development there is a switch from fetal to adult haemoglobin formation, reflecting the differential expression of fetal (G gamma and A gamma) and adult (beta and delta) globin genes. Mutations that inhibit this switch produce variants of the syndrome of hereditary persistence of fetal haemoglobin (HPFH). Adult heterozygotes for these mutants produce 15-30% fetal haemoglobin (HbF) in their red cells. The general assumption is that the mutations result in a permanent switching on of gamma-globin genes. Here, however, we show that fetal globin expression can be turned off in cultures of HPFH cells by an uncharacterized factor in fetal sheep serum. This is the first demonstration that mutations affecting the developmental expression of globin genes can be modulated by exogenous factors. The findings raise the possibility that the phenotype of HPFH is not simply the direct result of mutations in or around globin genes but the consequence of the mutations on the interaction of globin genes with trans-acting regulatory factors.     

Beta 2-microglobulin from serum associates with MHC class I antigens on the surface of cultured cells

Beta 2-microglobulin (beta 2-m) is a highly conserved polypeptide (12,000 molecular weight; 12K) noncovalently associated with the heavy chain (45-48K) of the major histocompatibility complex (MHC) class I antigens. Its synthesis is required for expression of the HLA-A/B and H-2K/D heavy chains at the cell surface; beta 2-m is also associated with the human cell-surface antigens T6 and M241 isolated from thymocytes. However, on the T leukaemic cell line MOLT-4 some of the T6 antigens contain a different 12K subunit, termed beta t (refs 3, 7, 8). Purified human beta 2-m can exchange partially both with human beta 2-m associated with HLA-antigens, and with mouse beta 2-m associated with murine alloantigens. As MOLT-4 cells were grown in the presence of fetal calf serum (FCS) and as serum is known to contain some free beta 2-m, we examined whether beta t was bovine beta 2-m which had replaced endogenous beta 2-m on the surface of the cell. Here we show both that beta 2-m from FCS or human serum (HuS) used in cell culture can exchange with beta 2-m on the cell surface, and that beta t is in fact bovine beta 2-m.     

Control of haemoglobin switching by a developmental clock?

The pattern of haemoglobin production changes at the embryonic, fetal and postnatal stages of human development, reflecting the expression of different globin genes in both the alpha-like and beta-like gene clusters. Recent studies have identified alterations in the state of DNA methylation and sensitivity to nuclease digestion associated with developmental expression of the globin genes in red blood cell precursors, but the mechanism initiating these changes remains unknown. Despite the screening of large numbers of blood samples from newborn infants, no mutants have been found which affect the timing of these changes (with one possible exception involving a chromosomal translocation), thus necessitating alternative approaches to analysing the cellular basis for the timing of haemoglobin switching. Although many mechanisms are possible, the initiation of the switch from fetal to adult haemoglobin could be regulated essentially either by a developmental clock inherent to haematopoietic stem cells or by an inductive environment, and in an attempt to distinguish between these possibilities, we have transplanted sheep fetal haematopoietic tissue into adult animals. Although previous experiments of this type produced conflicting results, the accumulated results presented here demonstrate that the pattern of haemoglobin production after transplantation is determined largely by the gestational age of the fetal donor cells.     

Qa-1 restricted recognition of foreign antigen by a gamma delta T-cell hybridoma

Distinct T-lymphocyte subsets recognize antigens in conjunction with different classes of major histocompatibility complex (MHC) glycoproteins using the T-cell receptor (TCR), a disulphide-linked heterodimer associated with the CD3 complex on the cell surface. In general, class I and class II MHC products provide a context for the recognition of foreign antigens by CD8+ and CD4+ T cells, respectively. This recognition seems to be largely dependent on alpha beta TCR heterodimers, whereas the function of the second gamma delta TCR, present on a minor subpopulation of cells, is still unknown. In the mouse, the existence of six cell-surface MHC class I products (K, D, L, Qa-1, Qa-2 and Tla) has been firmly established by serological, biochemical and genetic evidence. So far, only the most polymorphic of them, K, D and L ('classical' class I) have been reported as restriction elements for T-cell recognition of foreign antigens. The function of the relatively invariant Qa and Tla molecules remains unknown. We have made a T-helper cell hybridoma clone (DGT3) that recognizes synthetic copolymer poly(Glu50Tyr50) in the context of Qa-1 cell surface product, and has a CD4-CD8- phenotype. Our studies indicate that DGT3 cells express the gamma delta TCR on the cell surface, implicating its role in Qa-1-restricted antigen recognition. This is the first evidence that T cells can recognize foreign antigen in association with self Qa product, confirming that Qa molecules not only topologically, but also functionally, belong to the MHC.     

Differential expression of two distinct T-cell receptors during thymocyte development

The product of the T-cell receptor (TCR) gamma-gene has recently been found to be expressed on a subset of both peripheral cells and thymocytes. As an initial approach to understanding the role of this gamma-chain of TCR (TCR gamma) in T-cell development, we have studied the ontogeny of TCR expression at the protein level in the developing murine thymus. We show here that the first T3-associated TCR to be expressed in the developing thymus is a disulphide-linked heterodimer composed of a gamma-chain of relative molecular mass 35,000 (Mr 35K) and a 45K partner (termed TCR delta). This TCR gamma delta is first detected approximately two days before the appearance of cell-surface TCR alpha beta heterodimers. We report that N-glycosidase digestions reveal that all of the gamma-protein expressed on fetal thymocytes, as in adult CD4-8-(L3T4-, Lyt2-) thymocytes, bear N-linked carbohydrate side chains. The major gamma-gene transcribed in mature, alpha beta-bearing T cells (V gamma 1.2C gamma 2)encodes no N-linked glycosylation site so these results suggest that the fetal gamma delta receptor defines a distinct T-cell lineage whose development in the thymus precedes classical alpha beta-bearing cells.     

Multiple mRNA species with distinct 3' termini are transcribed from the beta 2-microglobulin gene

beta 2-Microglobulin is the small, relatively invariant subunit of a family of cell-surface glycoproteins encoded within the major histocompatibility complex (MHC). Proteins associated with beta 2-microglobulin in the mouse include the classical transplantation antigens (H-2K, D and L), the thymus leukaemia antigen (TL) and certain haematopoietic cell differentiation antigens (Qa-1 and Qa-2). The genes encoding these proteins are members of a large, multigene family. In contrast, beta 2-microglobulin is encoded by a single copy gene on mouse chromosome 2 (refs 5, 6). We have shown that this gene consists of four coding blocks separated by three intervening sequences. We now demonstrate that the single beta 2-microglobulin gene is transcribed into at least two different size classes of mRNA that differ in the lengths of their 3' untranslated regions. We further show that three polyadenylation signals and a poly (A) tail are encoded at the 3' end of the gene.     

Erythrocyte receptors for Mycoplasma pneumoniae are sialylated oligosaccharides of Ii antigen type

Among the pathological effects in man following infection with Mycoplasma pneumoniae is a transient autoimmune disorder characterized by the presence of high-titre erythrocyte autoantibodies (cold agglutinins). These autoantibodies are usually directed against the carbohydrate antigen termed I (ref. 3) which consists of a branched oligosaccharide. The mechanism by which the anti-I antibodies are elicited is unknown. However, sialic acid-containing receptors have been implicated in the adherence of M. pneumoniae to erythrocytes and other cell types, and both I and the related antigen i occur on erythrocytes in sialylated form: i is the predominant antigen on fetal erythrocytes and I is predominant in adults. Anti-I antibodies might arise in M. pneumoniae infection in response to a modification of the 'self' antigen-I as a result of its interaction with this agent. Here we report our study of the specificity of the interaction of M. pneumoniae with human erythrocytes. We found that this interaction is mediated by long chain oligosaccharides of sialic acid joined by alpha 2-3 linkage to the terminal galactose residues of poly-N-acetyllactosamine sequences of Ii antigen type.     

Increased gamma-globin expression in a nondeletion HPFH mediated by an erythroid-specific DNA-binding factor

In man, a shift from gamma- to beta-globin gene expression in erythroblasts underlies a switch from fetal to adult haemoglobin during development. In hereditary persistence of fetal haemoglobin (HPFH), inappropriately high gamma-globin expression in adult life is associated with deletions in the beta-globin cluster or with single-base changes upstream of the gamma-globin genes. To account for enhanced gamma-gene expression in HPFH of the non-deletion type, we tested the nuclear proteins of human erythroleukaemia cells that bind gamma-promoter sequences in vitro by correlating specific mutations in their binding sites with promoter activity. An erythroid-specific factor (GF-1) binds as a single molecule to the -195 to -170 region and contacts two TATCT(AGATA) motifs, but not the conserved octamer (ATGCAAAT) that separates them. We observe that a single change (at -175, T----C) found in HPFH leads to increased promoter activity only in erythroid cells. This effect is mediated by GF-1, the human counterpart of the chicken erythroid factor Eryf 1. The form of HPFH we studied here is an inherited disorder which can be ascribed to the action of a cell-specific DNA-binding factor on a mutant promoter.     

Accumulation of a heat shock-like protein during differentiation of human erythroid cell line K562

The human erythroid cell line K562 provides a model system for studying erythroid differentiation and eukaryotic gene regulation. These cells express glycophorin A, spectrin and i antigen. They accumulate embryonic and fetal haemoglobins on induction of erythroid differentiation with haemin, sodium butyrate or hydroxyurea. In the present study, the protein composition of K562 cells during haemin-mediated induction of erythroid maturation was analysed by two-dimensional gel electrophoresis. Under conditions in which haemin did not effect cell viability and proliferation, a protein of approximately 70,000 molecular weight (MW) accumulated in the differentiated K562 cells. The accumulation appears to be due to an increase in the rate of RNA synthesis for this protein. The protein is related in sequence to a 70,000-MW heat shock protein. An antigenically related protein was also demonstrated in human bone marrow and accumulates at particular stages of human erythroid maturation.     

Importance of IL-2 receptors in intra-thymic generation of cells expressing T-cell receptors

During development, lymphoid stem cells migrate into the thymic rudiment where they proliferate, rearrange their antigen receptor genes and become differentiated into functionally mature T cells. At present, the regulation of these processes is poorly understood, although recent studies have shown that early fetal and adult immature thymocytes express receptors for the T-cell growth factor, interleukin-2 (IL-2). We now present direct evidence that IL-2 receptors have a function in intra-thymic development by demonstrating that proliferation and the generation of cells expressing the T-cell antigen receptor (alpha beta TCR), which is responsible for the recognition of antigens in the context of MHC, are inhibited when antibodies to IL-2 receptors are added to fetal thymus organ cultures. The inhibition is specific in that it does not affect pre-thymic stem cells and can be partially reversed by addition of exogenous recombinant IL-2.     

Human gamma delta+ T cells respond to mycobacterial heat-shock protein

Most T cells recognize antigen through the T-cell antigen receptor (TCR)alpha beta-CD3 complex on the T-cell surface. A small percentage of T cells, however, do not express alpha beta but a second type of TCR complex designated gamma delta (ref. 2). Unlike alpha beta+ lymphocytes, gamma delta+ lymphocytes do not generally express CD4 or CD8 molecules, and the nature of antigen recognition by these cells is unknown. To study antigen recognition by gamma delta+ lymphocytes we raised a gamma delta+ alpha beta- -CD4-CD8- line from an individual immune to PPD (purified protein derivative). This line showed a specific proliferative response to PPD and to a recombinant mycobacterial heat-shock protein (HSP) of relative molecular mass 65,000 (65K). The gamma delta+ line was shown to exhibit a major response to HSP in the presence of autologous antigen-presenting cells (APCs). Minor responses occurred, however, with APCs matched for some HLA class I or II antigens, whereas no response occurred with HLA-mismatched APCs. These findings, therefore, document the requirement of HSP-reactive gamma delta+ lymphocytes for histocompatible APCs.     

Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes

The vast majority of mature T lymphocytes in the peripheral blood and lymphoid organs use the CD3-associated alpha, beta T-cell receptor (TCR) heterodimer for antigen recognition. A second class of TCRs consists of disulphide-linked gamma and delta proteins that are also CD3-associated. A subset of early CD3+ fetal and adult CD4- 8- thymocytes express gamma, delta TCRs before alpha, beta TCRs are detectable. In addition, a minor (1-5%) subpopulation of peripheral T lymphocytes, and some spleen cells from nude mice express gamma, delta TCRs. Notably, dendritic epidermal cells have also been shown to express gamma, delta TCRs. All of these populations lack CD4 and CD8 molecules. We now report that most mature T cells residing in the murine intestinal epithelium express CD3-associated TCRs composed of gamma-chains disulphide-linked to a protein resembling the delta-chain. The striking feature of these intraepithelial lymphocytes (IEL) was that they were exclusively CD4-8+. In addition, approximately half of CD3-bearing IEL lacked detectable Thy-1 on the cell surface, which is unprecedented for murine T cells. In contrast to other CD8+ peripheral T cells, freshly isolated IEL could be induced to display cytolytic activity by engaging the CD3 molecule, indicating that activation had occurred in vivo. Thus, CD8+ IEL are a phenotypically diverse and anatomically restricted population of lymphocytes that use gamma-chain containing heterodimers for antigen recognition.     

Importance of FSH-releasing protein and inhibin in erythrodifferentiation

Inhibin is a hypophysiotropic hormone which selectively suppresses the secretion of pituitary follicle-stimulating hormone. It has been isolated from gonadal fluids and characterized as a protein heterodimer consisting of an alpha subunit and one of two beta subunits (beta A or beta B). FSH-releasing protein (FRP), also named activin, is a dimer consisting of two inhibin beta-chains. A factor from conditioned medium of a leukaemia cell line has been isolated which can induce mouse Friend cells to become benzidine-positive, and which shares a similar N-terminal sequence with porcine FRP. In this report, we find that FRP and inhibin modulate both the induction of haemoglobin accumulation in a human erythroleukaemic cell line, K562, and the proliferation of erythroid progenitor cells in human bone marrow culture. These two proteins could constitute a novel humoral regulatory control of erythropoiesis which would involve two types of related protein dimers with functionally opposite effects.     

A new subunit of the human T-cell antigen receptor complex

The T-cell antigen receptor binds antigen in association with a cell surface molecule encoded by the major histocompatibility complex (MHC). MHC restricted recognition of antigen by this receptor leads to the complex pattern of programmed gene expression that characterizes T-cell activation. The eventual understanding of human T-cell function will require the complete elucidation of the structure of the human T-cell antigen receptor. On human T cells, clonally determined, disulphide-linked alpha and beta chains of the receptor are non-covalently and stoichiometrically associated with three additional polypeptides known as the T3 complex. These receptor subunits are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20K (gamma and delta) and a non-glycosylated 20K protein (epsilon). Our studies of murine T cells show that the mouse T-cell antigen receptor consists of at least seven distinct polypeptide chains. In addition to clonotypic alpha and beta chains, the murine complex consists of glycoproteins of 26K and 21K and endoglycosaminidase F (endo F)-insensitive polypeptides of 25K, 21K and 16K. The latter, which we have termed zeta (zeta), exists as a homodimer within the complex. The 26K component (gp26) has been shown to be the murine analogue of the human delta chain. Other cross species homologies remain to be established, however none of the described human receptor components appear similar to the murine zeta polypeptide. We report here the use of an antiserum raised against the murine zeta subunit to identify a previously unrecognized component of the human T-cell antigen receptor. This human protein is T-cell specific and biochemically similar to the murine zeta polypeptide.     

Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2–fucosyltransferase gene

The polycarbohydrate structure of Galα1- 3Ga1β1-4GluNAc-R （known as αGal epitopes of xenoantigen）, produced by α1-3-galactosyltransferase （α1,3-GT） in the course of animal development, is the major xenoantigen on the cell surface of porcine which causes hyperacute rejection in pig-to-human xenotransplantation. Alpha-1,3-galactosidase （AGL）, a hydrolytic enzyme, can remove the terminal α1,3-galactosyi from the Galα1-3Galβ1-4GluNAc-R structure resulting in cleaning αGai epitopes from the porcine cells. Aipha-1,2-fucosyitransferase （HT） can modify the surface carbohydrate phenotype of porcine cells, bringing about reduction of αGai epitopes expression. In this study, human AGL and HT gene were co-transfected to porcine fetal fibroblast （PFFb） in equimolar concentration to reduce the xenoantigen. Gene and protein of hAGL and HT were both detected to express at high level by RT-PCR and Western blot, respectively. There was an 84% reduction in αGai xenoantigen and an 82% increase in H antigen as assayed by flow cytometry in the AGL and HT gene co-transfected PFFb. The number and morphology of transgenic PFFb chromosome were normal. Findings indicate that Galα1-3Gal epitopes of PFFb could be down regulated by AGL and HT co-transfection without deleterious effects on the chromosomal profile of the transgenic ceil.     

Positive selection of antigen-specific T cells in thymus by restricting MHC molecules

Thymus-derived lymphocytes (T cells) recognize antigen in the context of class I or class II molecules encoded by the major histocompatibility complex (MHC) by virtue of the heterodimeric alpha beta T-cell receptor (TCR). CD4 and CD8 molecules expressed on the surface of T cells bind to nonpolymorphic portions of class II and class I MHC molecules and assist the TCR in binding and possibly in signalling. The analysis of T-cell development in TCR transgenic mice has shown that the CD4/CD8 phenotype of T cells is determined by the interaction of the alpha beta TCR expressed on immature CD4+8+ thymocytes with polymorphic domains of thymic MHC molecules in the absence of nominal antigen. Here we provide direct evidence that positive selection of antigen-specific, class I MHC-restricted CD4-8+ T cells in the thymus requires the specific interaction of the alpha beta TCR with the restricting class I MHC molecule.