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J Borst

Publications and source records attributed to J Borst.

At least 55 records · Page 3Linked to original sources
PubMed1995

Assembly and intracellular transport of the human B cell antigen receptor complex.

The B cell antigen receptor (BCR) complex consists of transmembrane (m) Ig, in non-covalent association with a disulphide-linked heterodimer of mb-1 and B29 gene products. The MB-1-B29 heterodimer is required for deposition of the BCR at the plasma membrane, as well as for coupling of the antigen receptor to intracellular signal transduction cascades. We have performed biosynthetic labelling studies using the mature B cell line Ramos to investigate the process of assembly of the BCR components. We conclude that association of the four components, Ig-heavy chain (HC) and -light chain (LC), MB-1 and B29, is required and sufficient to permit exit of the BCR complex out of the endoplasmic reticulum (ER). With the short pulse labelling procedures used, no evidence was found for transient participation of other molecules in complex formation. A 32 kDa glycoprotein was identified, which is serologically related to MB-1, but has a more acidic isoelectric point (pl) and a protein backbone of 21 kDa, as compared with 25 kDa for MB-1. This protein did not appear to participate in BCR complex formation and is most likely degraded prior to reaching the cis-Golgi. The MB-1 component was found to be the rate-limiting step in BCR complex formation, while Ig-HC, -LC and B29 are synthesized in excess. Ig-HC and -LC form disulphide-linked tetrameric complexes within 3 min after biosynthesis, with which B29 and MB-1 components associate independently, followed by disulphide bond formation between these heterodimeric partners. While partial BCR complexes containing B29 and mlg-H2L2 tetramers are rapidly formed and have a half-life of a few hours in the ER, entry of MB-1 into these complexes controls exit out of this compartment.

Antigens, CD↗
PubMed1995

Novel mAbs reveal potent co-stimulatory activity of murine CD27.

Members of the tumor necrosis factor receptor (TNFR) family are emerging as important molecules implicated in the regulation of proliferation, differentiation and survival of T and B lymphocytes. Among these receptors is CD27, the function of which has thus far only been studied in the human system, where it amplifies the T cell proliferative response induced by TCR triggering. We report here the generation of mAbs to murine CD27, by an efficient method involving the use of transfected Armenian hamster fibroblasts. Previous analysis had already indicated that murine CD27 mRNA is uniquely expressed in lymphoid cells. As determined with one of the newly developed antibodies, murine CD27 is expressed on the great majority of both alpha beta and gamma delta T lymphocytes, on a small population of peripheral B cells, and on a very small subset of B220+ cells in the bone marrow. This distribution largely corresponds to that in the human system. However, unlike human CD27, which is primarily expressed in mature, medullary thymocytes, murine CD27 is found on all thymocytes, except a subset of CD4-CD8- precursors. Upon cross-linking, anti-CD27 mAb amplified the proliferative response of purified T lymphocytes to suboptimal stimulation with concanavalin A at least 4-fold. This indicates that such mAbs can mimick ligand binding and demonstrates that CD27 also acts as a potent co-stimulatory molecule in the murine system.

Amino Acid Sequence↗
PubMed1994

B-cell antigen receptor stimulation activates the human Bruton's tyrosine kinase, which is deficient in X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is an inherited human immunodeficiency disease, characterized by an arrest in B-cell development, which results in a dramatic decrease in immunoglobulin production. The gene product defective in XLA has been identified as a cytoplasmic protein tyrosine kinase, named Bruton's tyrosine kinase (Btk). The dramatic XLA phenotype indicates a critical role for Btk in the regulation of B-cell development. However, neither external stimuli leading to Btk activation nor any of its in vivo substrates have thus far been identified, and the mechanism of disease induction remains unexplained. We report here that stimulation of the B-cell antigen receptor (membrane immunoglobulin) on mature B-cells induces tyrosine phosphorylation of Btk in vivo, accompanied by an increase in its kinase activity in vitro. These results place Btk in the B-cell receptor signal transduction pathway, which is known to be essential in driving B-cell differentiation.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

B cell antigen receptor stimulation induces formation of a Shc-Grb2 complex containing multiple tyrosine-phosphorylated proteins.

Activation of growth factor receptor tyrosine kinases, such as the epidermal growth factor and insulin receptors, induces tyrosine phosphorylation of Shc proteins and their association with the SH2 domain-containing adaptor protein Grb2. The Shc-Grb2 complex has been implicated in coupling these receptors to p21ras. The B cell antigen receptor plays a key role in directing B cell proliferation and differentiation. Although the B cell receptor lacks intrinsic tyrosine kinase activity, its mode of action parallels that of receptor tyrosine kinases in many aspects. B cell receptor stimulation activates src-related tyrosine kinases and the tyrosine kinase syk, which leads to phosphorylation of various cytoplasmic proteins and initiates multiple signaling events, including p21ras activation. Therefore, we have investigated whether Shc proteins are targets for the activated B cell receptor. It was found that the 52- and 46-kDa forms of Shc are expressed in mature human B cells and become rapidly phosphorylated on tyrosine upon B cell receptor stimulation. Also, Shc is induced to associate with the Grb2 molecule and an undefined 130-kDa protein. In a specific response to B cell activation, the Shc-Grb2 complex associates with several tyrosine-phosphorylated proteins, including two prominent phosphoproteins with molecular masses of 130 and 110 kDa. These observations strongly suggest that the Shc and Grb2 adaptor proteins are involved in coupling the B cell antigen receptor to one or multiple signal transduction pathways.

3T3 Cells↗
PubMed1994

CD3 components at the surface of pro-T cells can mediate pre-T cell development in vivo.

Developmentally arrested pro-T cells (CD4-8-, IL-2R+, HSA++) of RAG-1-deficient mice appear to express low levels of CD3 molecules in the absence of T cell receptor (TcR) chains at their surface, while developmentally arrested pre-T cells of TcR alpha-deficient mice express low levels of a disulfide-linked TcR beta chain in association with CD3 molecules. Cross-linking of the CD3 modules on pro-T cells of RAG-1-/- mice in vivo, with either of two different CD3 epsilon-specific monoclonal antibodies, induces differentiation of these pro-T cells into pre-T cells (CD4+8+, IL-2R-, HSA+), concomitant with a rapid expansion of the thymic T cell compartment, up to 175-fold within 12 days. The same effects can be produced by introduction of a mutant TcR beta transgene lacking most of the variable domain (delta V-TcR beta) into the RAG-1-/- background. These experiments suggest that cross-linking of the CD3 modules on pro-T cells mimics the signaling function expected of the pre-TcR complex, which is found at the surface of pre-T cells prior to functional TcR alpha gene rearrangement. The variable domain of the TcR beta chain is apparently not essential for inducing these aspects of T cell development.

Animals↗
PubMed1993

Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins.

The human serine proteases granzymes A and B are expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzymes A and B in the cytotoxic response in vivo.

Animals↗
PubMed1993

Cloning and expression of murine CD27: comparison with 4-1BB, another lymphocyte-specific member of the nerve growth factor receptor family.

CD27 is a member of the nerve growth factor receptor family, that includes two types of tumor necrosis factor receptor, CD40 and Fas/Apo-1. Human CD27 has been found only on lymphocytes. In T cells, its expression strongly increases in a transient fashion upon antigenic stimulation, suggesting that CD27 plays a role during T cell activation. To analyze the function of CD27, we have identified the murine CD27 at the cDNA and protein level. Murine CD27 shows an identity of 65% compared with human CD27. The amino-terminal cysteine-rich region, i.e. the putative ligand-binding domain, and the carboxy-terminal part of the cytoplasmic domain are approximately 80% identical in man and mouse. Murine CD27 has 29% identity to 4-1BB, another lymphocyte-specific member of the receptor family defined only at the cDNA level. Murine CD27 and 4-1BB have 39% homology in the cysteine-rich domain and share a conserved region in the cytoplasmic tail. Expression studies identified murine CD27 mRNA in thymus and spleen, but not in non-lymphoid tissues, while 4-1BB mRNA was not detected in any tissue tested. In resting T cells, only murine CD27 mRNA was found, while in activated T cells murine CD27 as well as 4-1BB were present at high levels. Murine CD27 and 4-1BB mRNA are expressed with different kinetics during T cell activation, suggesting that these molecules play different roles in this process. Peptide antisera identified murine CD27 as a 45-kDa protein on thymocytes and activated T cells, while 4-1BB was precipitated as a 35-40-kDa protein from activated T cells.

Amino Acid Sequence↗
PubMed1993

The structure of the mu/pseudo light chain complex on human pre-B cells is consistent with a function in signal transduction.

Prior to immunoglobulin (Ig) light (L) chain rearrangement, pre-B cells can express mu heavy (H) chains at the cell surface in association with pseudo (psi) L chains. This complex may be essential for B cell development. We have investigated the composition of the mu/psi L chain complex of a human pre-B cell line, in view of its potential role in transmembrane signal transduction. The mu/lambda receptor of a mature B cell line was analyzed in comparison. The mu/psi L chain complex is associated with disulfide-linked molecules that are homologous or identical to the mb-1 and B29 proteins, known to be integral components of membrane Ig receptors on mature B cells. Both receptors contain tyrosine (Tyr) kinase activity. In the mu/lambda receptor, the lyn and lck Tyr kinases could clearly be identified. The mb-1 and B29 proteins in both mu/lambda and mu/psi L chain receptors are substrates for in vitro phosphorylation on Tyr, but also on serine (Ser) and threonine (Thr) residues. The undefined mu-associated Ser/Thr kinase also phosphorylates the src-related kinases in the mu/lambda receptor and a 43-kDa mu-associated protein that is present in both complexes. The 43-kDa protein may be an integral part of both receptor types, or a transiently associated molecule instrumental in the signaling process. We conclude that the mu/psi L receptor on human pre-B cells fulfills the presently known criteria to function as a signal transduction unit.

Amino Acid Sequence↗
PubMed1993

Transcription and protein expression of mb-1 and B29 genes in human hematopoietic malignancies and cell lines.

The transmembrane forms of all immunoglobulin (Ig) classes are associated with two glycoproteins, mb-1 and B29, that are crucial for signal transduction following antigen binding to the Ig molecule. We have investigated the transcription and protein expression of mb-1 and B29 genes during B-cell development. Sixty human continuous cell lines (35 B-lineage, 11 T-lineage, 11 myeloid-lineage and three non-hematopoietic) and 75 hematopoietic malignancies (55 B-lineage, 12 T-lineage and eight myeloid-lineage), were tested for RNA expression by Northern blotting experiments with the mb-1 pRA3 cDNA probe, and a newly isolated B29 cDNA probe. Protein expression was analyzed by immunofluorescence microscopy of cytocentrifuge preparations, which were labeled with the anti-mb-1 HM57 monoclonal antibody (mAb) and an anti-B29 polyclonal antiserum, directed against intracellular epitopes of these polypeptides. Except for two early precursor B-cell lines, mb-1 and B29 transcripts and proteins were detected in all B-cell lines and B-cell malignancies, i.e. from immature to more mature B cells, irrespective of their Ig class expression. Transcription of mb-1 genes seems to be down-regulated at the plasma cell stage, because no mb-1 transcripts and mb-1 proteins could be detected in the four plasma cell lines and two plasma cell leukemias tested. B29 transcripts were detectable in these cell samples, but low levels of B29 proteins were only detected in one plasma cell line. The HM57 mAb gave strong labeling on fresh cytocentrifuge preparations of all B-cell samples, and this mb-1 protein expression appeared to be B-cell specific. We therefore conclude that the HM57 mAb is well suited for the detection of the mb-1 molecule as a pan-B-cell marker for the diagnosis of immature and mature B-cell malignancies. The expression pattern of the mb-1 protein is comparable to that of the CD19 and CD22 antigens, but has the advantage of being B-lineage specific. Although B29 protein expression was restricted to B-lineage cells, the anti-B29 antiserum is less suitable for diagnosis of B-cell malignancies, because of the variable and generally weak signals on cytocentrifuge preparations.

Antigens, CD↗
PubMed1992

Genomic organization and chromosomal localization of the human CD27 gene.

CD27 is a lymphocyte-specific member of a recently identified receptor family with at least 10 members that includes the receptors for nerve growth factor and TNF, CD40, and Fas. Several members of this family play a role in cell differentiation, proliferation, and survival. Within the amino terminal ligand binding domain of these receptors, repeat motifs have been identified. These repeats contain many cysteine residues in a conserved pattern, characteristic of this family. We have isolated and characterized the human CD27 gene to gain insight into the evolution of this type of receptor domain. The gene was localized on chromosome 12, band 12p13. Sequence analysis showed no correlation between the intron/exon organization and the subdivision of the protein into distinct domains. Structural information for the cysteine-rich domain is contained within three exons. In addition, the splice sites in the CD27 gene are located in a different position from those in the related nerve growth factor receptor gene. However, a comparison of the splice sites within the regions encoding the respective ligand-binding domains of the CD27 and nerve growth factor receptor genes identifies the archetypal cysteine-rich building blocks, from which the members of this family may have arisen during the course of evolution. From this observation, we propose a new organization of the repeat motifs.

Amino Acid Sequence↗
PubMed1992

T cell receptor-alpha beta lacking the beta-chain V domain can be expressed at the cell surface but prohibits T cell maturation.

A TCR-beta gene lacking V domain sequences (delta V-TCR-beta) was inserted into the germline of mice. Expression of the transgene inhibited endogenous TCR-beta, but not TCR-alpha gene rearrangement and expression. The mutated TCR-beta gene affected alpha beta T cell development: the common thymocyte pool was normal in cell number, with cells expressing CD4 and CD8, but the mature, "CD3bright" population expressing either CD4 or CD8 molecules was reduced by 90%. To help understand these effects on TCR-beta gene rearrangement and T cell development, biosynthesis of the delta V-TCR-beta protein was analyzed in a tumor cell line derived from a transgenic mouse. Despite absence of the V domain, the delta V-TCR-beta chain paired with endogenous TCR-alpha chains and assembled with CD3 gamma, -delta, -epsilon, and -zeta components in the endoplasmatic reticulum, followed by transport through the Golgi complex to the plasma membrane. Therefore, assembly of the complex, and even cell surface expression, may be relevant for allelic exclusion of the TCR-beta gene. In the common thymocyte population, the CD3 components, endogenous TCR-alpha, and the delta V-TCR-beta gene product were expressed at the RNA level, but endogenous TCR-beta was not. The TCR-alpha delta beta/CD3 complex was present at the cell surface at low levels and was functional in terms of anti-CD3-induced Ca2+ mobilization. The observed arrest of alpha beta T cell development at the CD4+8+ thymocyte stage indicates that ligand recognition by the TCR, with contribution of the beta-chain V domain, is not required for transition of CD4-8- thymocytes to the CD4+8+ phenotype, but necessary for entry into the "single positive," CD3bright differentiation stage.

Animals↗
PubMed1992

Comparison of human B cell antigen receptor complexes: membrane-expressed forms of immunoglobulin (Ig)M, IgD, and IgG are associated with structurally related heterodimers.

We have recently reported that on human B lymphocytes, membrane IgM (mIgM) associates with a heterodimer of 47- and 37-kD polypeptides, the 47-kD subunit being encoded by the mb-1 gene. We show here that expression of mb-1, both at the mRNA and the protein level, is not restricted to IgM+ B cells but can also be found in IgM- pre-B cells and mIgM-IgG+ B cells. Membrane forms of IgD and IgG, isolated from freshly isolated human B cells and B cell lines, are expressed together with heterodimeric protein structures biochemically similar to the mIgM-associated polypeptides, and these were shown to comprise the products of the mb-1 and B29 genes, or homologous genes. Finally, all three classes of antigen receptors are linked to protein kinases, capable of phosphorylating the Ig-associated heterodimers. Our findings provide insight in the structural organization of the different antigen receptors on human B cells and have implications for their function.

Antibodies, Monoclonal↗
PubMed1992

The CD27 membrane receptor, a lymphocyte-specific member of the nerve growth factor receptor family, gives rise to a soluble form by protein processing that does not involve receptor endocytosis.

CD27 is a transmembrane glycoprotein found exclusively on human T and B lymphocytes. It belongs to a recently identified receptor family, whose members are involved in cell differentiation and survival. This family includes the nerve growth factor receptor, two different types of tumor necrosis factor, receptors the Fas antigen, and the B cell-specific protein CD40. T cell activation via the antigen receptor strongly enhances CD27 membrane expression, suggesting a role for CD27 during T cell differentiation. A soluble form of CD27 (sCD27) is released into the supernatant of activated T cells, and detected in serum and urine of healthy individuals and patients. We have investigated the mechanism underlying the generation of sCD27. One mRNA encodes both the transmembrane receptor and sCD27, as shown by cDNA transfection. In line with this, only one CD27 precursor protein is found, that is processed to the mature receptor by extensive O-linked glycosylation. All newly synthesized protein is rapidly transported to the plasma membrane; no internal pool of mature protein is detectable. The transmembrane form gives rise to sCD27 after arrival at the cell surface, most likely via a proteolytic event, that does not involve receptor internalization.

Antigens, CD↗
PubMed1992

Both LFA-1-positive and -deficient T cell clones require the CD2/LFA-3 interaction for specific cytolytic activation.

We investigated the capacity of T lymphocytes from a leukocyte adhesion-deficient (LAD) patient to respond to alloantigen. Leukocytes of this patient completely lacked LFA-1 surface expression due to the absence of mRNA coding for the LFA-1 beta chain. Despite the absence of LFA-1, T lymphocytes obtained from this patient, cultured with allogeneic stimulator cells (lymphoblastoid B cells JY), were capable of lysing JY cells. Furthermore, two T cell clones (one CD4+ and one CD8+), generated from this lymphocyte culture, specifically lysed the allogeneic lymphoblastoid JY cells. The cytolytic capacity of LFA-1-negative T lymphocytes and T cell clones was comparable to that of control LFA-1-positive T cells with allospecificity against JY. Detailed analysis of the CD4 positive and LFA-1-negative T cell clone demonstrated that it specifically recognized HLA-DQ. Antibody inhibition studies showed that the CTL/target cell interaction was mediated through the CD2/LFA-3 adhesion pathway. LFA-1 expressed by the target cells did not participate in the CTL/target cell conjugate formation and contributed only minimally to the cytotoxic activity. Moreover, when allogeneic LFA-1-deficient B cells, bearing the appropriate HLA-DQ alloantigen, were used as target cells, significant levels of specific cytotoxicity were measured, further excluding a role for LFA-1 in this interaction. The adhesion molecules, VLA-4, CD44 and L-selectin (LECAM1) were not involved. These results demonstrate that LFA-1-negative T lymphocytes can exert allospecific cytotoxicity and that CTL/target cell contact is mediated through the CD2/LFA-3 route. This observation may explain in part why in LAD patients viral infections, cleared largely by T cells, are less frequently observed than bacterial infections, in which phagocytic cells play a major role.

Antibodies, Monoclonal↗
PubMed1992

Polyclonal expansion of T-cell receptor-gamma delta+ T lymphocytes associated with neutropenia and thrombocytopenia.

A patient with large granular lymphocyte (LGL) expansion (T-gamma lymphocytosis), neutropenia and thrombocytopenia was studied longitudinally. Analysis of peripheral blood mononuclear cells (PBMC) demonstrated an unusual large proportion of CD3+ T-lymphocytes expressing a gamma delta T-cell receptor (TcR-gamma delta). Immunofluorescence (IF) stainings with subset-specific monoclonal antibodies showed a fluctuating expansion of TcR-gamma delta+ T-cells expressing V gamma 9 and V delta 2 variable (V) gene segments. Biochemical characterization of PBMC showed the presence of a disulphide-linked TcR-gamma delta. TcR gene rearrangement studies on sorted TcR-gamma delta+ T-cells showed rearrangements of V gamma 9-J gamma 1.2 and V delta 2-J delta 1 V and joining (J) gene segments, thereby confirming the IF staining results. These data alone did not allow us to determine whether the TcR-gamma delta+ LGL expansion represented a polyclonal or monoclonal proliferation, because the combinatorial repertoire of TcR-gamma delta receptors is limited due to the availability of only a few V and J segments within the TcR-gamma and TcR-delta genes and because of the preferential usage of V gamma 9-J gamma 1.2 and V delta 2-J delta 1 rearrangements by TcR-gamma delta+ T-cells in blood of healthy individuals. We therefore used polymerase chain reaction (PCR)-mediated amplification of the TcR-gamma delta rearrangements, using specific V gamma 9, J gamma 1.2, V delta 2, and J delta 1 oligonucleotides to determine the junctional diversity of the TcR-gamma delta+ T-cell population. Sequence analysis of the PCR products obtained revealed a mixture of different junctional region sequences compatible with a polyclonal expansion. This is in contrast to the few reported TcR-gamma delta+ LGL and the majority of TcR-alpha beta+ LGL expansions, which appeared to consist of monoclonal proliferations.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells.

Recent evidence indicates that the transmembrane form of IgM on murine and human B lymphocytes is physically associated with at least two proteins, forming a disulfide-linked dimer, which may control cell surface expression of IgM and also play a role in signal transduction after Ag binding (by analogy with the TCR-associated CD3 components in T lymphocytes). We have used mAb and polyclonal antibodies against an intracytoplasmic epitope on one of these polypeptides (previously identified in murine B cells as the product of the B cell specific mb-1 gene) to study the distribution of the IgM-associated dimer in human cells. By immunocytochemical staining of normal and neoplastic B cells, we show that the human mb-1 protein appears early in B cell differentiation, probably before expression of cytoplasmic mu-chain, and persists until the plasma cell stage, where it is seen as an intracytoplasmic component. According to immunohistologic analysis of reactive lymphoid tissue and lymphoma samples, mb-1 protein is completely B cell specific. Anti-mb-1 also labels B cell areas in tissues from seven different mammalian species. Finally, the Ig-associated dimer could be isolated from human hairy-cell leukemia cells in high purity and yield by affinity chromatography using anti-mb-1 antibody. Mice immunized with this material have produced a strong polyclonal response, so that it should now be possible to prepare a panel of new mAb reactive with different epitopes on both mb-1 and on its associated polypeptide(s).

Animals↗
PubMed1991

The T cell activation antigen CD27 is a member of the nerve growth factor/tumor necrosis factor receptor gene family.

CD27 is a dimeric membrane glycoprotein found on the surface of most human T lymphocytes. Activation of T cells by engagement of the Ag receptor increases CD27 surface expression, and anti-CD27 antibodies augment Ag receptor-mediated T cell proliferation. In this study a cDNA-encoding CD27 was isolated by expression and immunoselection in COS cells. The predicted polypeptide was found to belong to a recently characterized family of cysteine-rich receptors whose known ligands include nerve growth factor and TNF-alpha and -beta. Structural similarities suggest that CD27 belongs to a lymphocyte-specific subgroup of the family, comprised of the B cell Ag CD40, the rat T cell subset Ag OX40, and the mouse T cell activation Ag 4-1BB. Recent studies suggest some of these molecules may play a role in the survival of activated cells.

Antigens, CD↗