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H Kubagawa

Publications and source records attributed to H Kubagawa.

At least 19 recordsLinked to original sources
PubMed1992

Heterogeneity of immunoglobulin-associated molecules on human B cells identified by monoclonal antibodies.

Two covalently linked transmembrane molecules, encoded in mice by the mb-1 and B29 genes, have been defined as integral components of the antibody receptor units expressed on B cells. We have produced monoclonal antibodies against an exposed extracellular epitope on the putative human equivalent of the mouse B29 product. These antibodies, CB3-1 and -2, were used to show that cytoplasmic expression of this molecule begins in human pro-B cells (terminal deoxynucleotidyltransferase-positive, mu chain-negative), whereas surface expression coincides strictly with surface immunoglobulin expression of all isotypes. Immunochemical analysis of the human immunoglobulin-associated molecules revealed greater molecular heterogeneity than has been noted for the murine analogues. This molecular heterogeneity of immunoglobulin-associated molecules varied as a function of differentiation stage and the immunoglobulin isotypes expressed by B-lineage cells. Our data support the hypothesis that biochemical heterogeneity of the surface immunoglobulin-associated molecules may contribute to the variability in biological effects of antigen receptor crosslinkage on B cells of different maturational stages. Because the CB3 antibodies are capable of down-modulating the antigen receptors on all B cells, they may prove therapeutically useful as universal B-cell suppressants.

Antibodies, Monoclonal↗
PubMed1992

Molecular heterogeneity of Fc alpha receptors detected by receptor-specific monoclonal antibodies.

Fc alpha receptors (Fc alpha R) were isolated from a human monocytic cell line and used to raise four mAb with receptor specificity. The antibodies were used to identify the types of white blood cells that express Fc alpha R and the molecular heterogeneity of the receptor molecules. Nonpolymorphic epitopes, outside of the Fc alpha-binding site, were recognized only on blood cells of granulocyte and monocyte/macrophage lineages. The molecules identified, both by the antibodies and by the IgA ligand, were glycoproteins ranging in relative molecular mass from 55 to 75 kDa. However, one antibody detected a subpopulation of Fc alpha R molecules characterized by relatively restricted size heterogeneity. A complex glycosylation pattern was revealed by the resolution of discrete 32- and 36-kDa molecular species after removal of N-linked oligosaccharides and by evidence for O-linked carbohydrate moieties on at least a portion of the Fc alpha R molecules. In biosynthetic studies, all four anti-Fc alpha R antibodies and the IgA ligand bound a single 32-kDa core protein present in tunicamycin-treated cells, and the exceptional antibody again recognized molecules with relatively restricted glycosylation in the nontreated cells. These antibodies and native IgA ligands thus provide complementary reagents for definition of the complex structure and function of Fc alpha R in systemic IgA antibody responses.

Antibodies, Monoclonal↗
PubMed1992

Molecular cloning and expression pattern of a human gene homologous to the murine mb-1 gene.

The mouse mb-1 gene was originally identified based on its restricted expression in B lineage cells. Predicted structural homology with the gamma chain of the CD3 complex on T cells led to the suggestion that the MB-1 protein might associate with surface Ig(sIg) on B cells and be involved in signal transduction. Other studies identified at least two proteins that are noncovalently associated with sIgM, one of which has recently been shown to be the product of the mb-1 gene. To identify genes specifically expressed in normal human B cells we constructed a B minus T lymphocyte subtraction library and isolated a cDNA clone highly homologous to murine mb-1 (m-mb-1). A full-length cDNA was subsequently isolated and found to encode a membrane glycoprotein of 226 amino acids. It included a leader sequence (32 amino acids), an extracytoplasmic domain (111 amino acids) containing six potential N-glycosylation sites and three cysteine residues for potential inter- or intrachain disulfide linkages, a transmembrane domain (22 amino acids), and an intracytoplasmic domain (61 amino acids). The amino acid sequence homology between human and mouse mb-1 was especially striking (approximately 92%) in the intracytoplasmic, transmembrane, and membrane-proximal extracellular domains but was less marked (approximately 42%) in the remaining extracytoplasmic portion. Interestingly, part of the 3'-untranslated region was also highly conserved between species, suggesting an important role for this region in the regulation of mb-1 expression. The human mb-1 (h-mb-1) cDNA hybridized with a mRNA species of approximately 1.2 kb on Northern blots. Similar to m-mb-1, the h-mb-1 transcripts could be detected in pre-B cell lines and fetal bone marrow, in normal, mitogen activated- and transformed B cells but not in myeloma plasma cells. h-mb-1 was not expressed in peripheral T cells nor by cells of other hemopoietic lineages or in brain, heart, muscle, lung, and kidney. Surprisingly, however, low levels of h-mb-1 transcripts were detectable in two early T lineage cell lines and in the fetal thymus. This suggests that mb-1 may have other functions in addition to its role in signal transduction in B lineage cells.

Amino Acid Sequence↗
PubMed1991

VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias.

We show here that analysis of VpreB gene transcription can be a specific way to identify acute leukemias of cells at very early stages of B-cell development. Northern blot analysis of RNAs from 63 leukemia samples showed that VpreB RNA was present in malignancies of precursor B cells, the expression being a feature of both common acute lymphoblastic leukemia (ALL) (CD10+) and null ALL (CD10-). It was absent from malignancies of mature B cells (surface Ig positive), from acute leukemias of the T-cell lineage and granulocyte-macrophage lineages, and from normal tonsil B and T lymphocytes. Chronic myeloid leukemia blast crises of the B-precursor-cell type expressed the VpreB gene while myeloid blast crises did not. VpreB RNA was also expressed in the neoplastic cells of one of three patients with acute undifferentiated leukemias. These data show that VpreB RNA expression is a marker of the malignant forms of precursor B cells, and that it appears at least as early as cytoplasmic CD22 and CD19 in tumors of the B-cell lineage.

Blotting, Northern↗
PubMed1991

Normal pre-B cells express a receptor complex of mu heavy chains and surrogate light-chain proteins.

Precursors of B cells, which constitute a subpopulation of the lymphocytes in bone marrow, can be identified by their surface expression of nonimmunoglobulin markers and the absence of immunoglobulin kappa and lambda light chains. Most pre-B cells synthesize mu heavy chains but, without light-chain partners, these undergo rapid cytoplasmic degradation. In the present study, we demonstrate that late stage pre-B cells, like their neoplastic counterparts, express low levels of a surface receptor composed of mu chains paired with a surrogate light-chain complex formed by Vpre-B and lambda 5-like proteins. The data define a previously suspected but unrecognized stage in normal pre-B-cell differentiation. Expression of a clonally diverse receptor renders this population of immature B-lineage cells potentially vulnerable to clonal selection by antigens and idiotypic interactions.

Antibody Formation↗
PubMed1990

Biochemical nature of an Fc mu receptor on human B-lineage cells.

An IgM-binding protein of approximately 60 kD has been identified on activated B cells, but not on resting and activated T cells, monocytes, or granulocytes. Here, we characterize this IgM-binding protein as a receptor for the Fc portion (CH3 and/or CH4 domains) of IgM molecules (Fc microR). The Fc microR can be expressed as a cell surface activation antigen throughout the pre-B and B cell stages in differentiation. Receptor expression is not directly linked with IgM production, as both mu- pre-B cells and isotype-switched B cells may express the Fc microR. The receptor molecules produced by both pre-B and B cells are identical in size and are characterized as an acidic sialoglycoprotein with O-linked, but no N-linked, oligosaccharide. The Fc microR is anchored to the surface of B-lineage cells via a glycosyl phosphatidylinositol linkage. The Fc microR is thus the third member of a family of Fc receptors expressed on B-lineage cells, and its preferential expression on activated B cells suggests a potential role in the response to antigens.

B-Lymphocytes↗
PubMed1990

Cellular distribution, regulation, and biochemical nature of an Fc alpha receptor in humans.

In these studies, we characterize an Fc receptor (FcR) for IgA that is present on human granulocytes, monocyte/macrophages, and their corresponding cell lines. Receptor expression appears to be constitutive but can be selectively upregulated on monocyte cell lines by stimulation with a phorbol ester and polymeric IgA. Both the induction requirements and ligand specificity of the IgA receptor differ from the IgG receptors, Fc gamma R I, II, and III, that are also expressed on monocytes and granulocytes. IgA binding to the cell surface receptor is mediated via the Fc alpha region. The Fc alpha R is a heterogenously charged, approximately 60-kD molecule with an isoelectric point of 4.5-5.6 that binds monomeric or polymeric IgA1 and IgA2 molecules. This transmembrane glycoprotein appears to be composed of 32- and 36-kD protein cores with multiple N-linked carbohydrate moieties. We conclude that this Fc alpha R represents a novel member of the FcR family that may have a distinctive role in host defense.

Antigens, CD↗
PubMed1990

A mouse monoclonal antibody reactive preferentially with human IgM lambda.

In analyzing mouse monoclonal antibodies (mAb) against a human IgM kappa paraprotein, we found an unusual mAb (LP4; gamma 2b kappa isotype) that reacted in an enzyme linked immunosorbent assay with all 5 IgM lambda but not with 8 IgM kappa or other myelomas. Neither isolated mu heavy nor lambda light chains were reactive with LP4 mAb. By immunofluorescence, LP4 mAb identified approximately 30% of IgM+ B cells and approximately 40% of mitogen-stimulated, IgM+ plasma cells from 4-7 normal blood samples. All LP4+ cells were IgM+. Biosynthetic analysis of the plasma cells revealed that LP4 mAb recognized most IgM lambda and a very minor proportion of IgM kappa molecules. This mAb provides a useful marker for the analysis of pre-B and B cell differentiation.

Animals↗
PubMed1989

Immunofluorescence analysis of IgA binding by human mononuclear cells in blood and lymphoid tissue.

The nature of IgA-binding cells and their tissue distribution was examined by an indirect immunofluorescence assay with the use of IgA1 and IgA2 paraproteins and fluorochrome- or biotin-labeled F(ab')2 fragments of idiotype-specific antibodies. The frequency of IgA-binding mononuclear cells was approximately 13% in blood and spleen samples but less than 1% in tonsil samples. IgA binding could be visualized by flow immunocytometry on monocyte/macrophages, but not on T and B cells. IgA polymers were bound better than IgA dimers and monomers. Nonhomologous IgA myelomas of both IgA1 and IgA2 subclasses inhibited the IgA-binding to monocytes, whereas aggregated normal serum IgG, IgM paraproteins, and an IgG myeloma did not. IgA binding was relatively insensitive to changes in temperature or cation concentration. IgA-binding monocytes were found in IgA-deficient patients at the same frequency as in normal individuals. The results indicate that monocytes constitutively express class-specific binding sites for both IgA1 and IgA2 molecules.

Agammaglobulinemia↗
PubMed1989

Light-chain gene expression before heavy-chain gene rearrangement in pre-B cells transformed by Epstein-Barr virus.

Epstein-Barr virus transformation of B-cell-depleted bone marrow cells from human fetuses allowed us to identify novel cell types characterized by the expression of immunoglobulin kappa or lambda light chain without heavy chains. Four kappa-only clones with normal karyotype were obtained and examined for their immunoglobulin gene configurations and expression. All four clones had kappa-chain gene rearrangements at either one or both alleles, but the heavy-chain gene loci in these clones either were in germ-line context or had undergone only D-JH rearrangements (D and JH represent diversity and joining gene segments). All clones contained kappa mRNA of normal size at levels consistent with the protein level, except for one clone that no longer produced kappa protein. No mu mRNA or immunoglobulin heavy-chain molecules were detected in any of the kappa+ clones. The results suggest that the mu heavy-chain protein is not an obligatory prerequisite for light-chain gene rearrangements.

Antibodies, Monoclonal↗
PubMed1988

Ig isotypes produced by EBV-transformed B cells as a function of age and tissue distribution.

EBV can transform human B cells giving rise to lymphoblastoid cell lines that produce and secrete Ig. Herein B cells from various tissues of newborns and adults were transformed by EBV and their Ig products were analyzed with isotype-specific mAb. Although IgG- and IgA-bearing B cells were present in the newborn, EBV transformed IgM-producing cells almost exclusively in both newborn blood and breast milk. IgM-secreting cells were derived from IgM+ B cells and IgM- pre-B cells present in neonatal blood, but only from IgM+ cells in adult blood. Whereas in adults most EBV-transformed cells produced IgM, producers of IgG and of IgA were present in frequencies that varied according to the tissue source. Precursors of IgG-producing cells were relatively abundant in blood, spleen, and tonsil, and relatively infrequent in bone marrow and appendix. EBV-inducible IgA producers were relatively concentrated in the appendix and to a lesser extent in tonsils and blood. Differences in the subclass composition of EBV-transformed populations of IgG- and IgA-producers were also observed for the various adult lymphoid tissues. IgG1-producing cells predominated in most tissues, and precursors of IgG2 were largely confined to the circulation. Whereas IgA1-producing cells were predominant in all tissues, a marked enrichment in IgA2-producers was observed in the appendix. These results indicate a remarkable heterogeneity in the isotype distribution pattern of EBV-transformable B cells that is determined both by developmental age and tissue localization. We propose that EBV selectively transforms primed B cells, the isotype commitment of which varies according to tissue origin and age.

Adult↗
PubMed1988

Precursor B cells transformed by Epstein-Barr virus undergo sterile plasma-cell differentiation: J-chain expression without immunoglobulin.

Human bone marrow cells were depleted of B lymphocytes to enrich for precursor B cells that could be transformed with Epstein-Barr virus. Transformed immunoglobulin-negative precursors either maintained their immunoglobulin genes in the germ-line configuration or had undergone DJ or abortive VDJ rearrangements (V, D, and J represent variable, diversity, and joining gene segments). All cell lines and their derivative clones, even those with no detectable immunoglobulin gene rearrangements, generated subpopulations of cells that produced high levels of joining (J) chain when analyzed by immunoprecipitation after biosynthetic labeling and by blot hybridization of cytoplasmic RNA. Morphologic and immunofluorescence analyses revealed that J-chain production was confined to clonal progeny that had exited the cell cycle to undergo plasma-cell differentiation. Analysis of cell surface antigens revealed expression of several B-cell maturational markers, including complement receptor type 2 (CR2) and plasma cell antigen 1 (PCA-1). Epstein-Barr virus can thus transform B-cell progenitors, allowing them to proliferate and undergo terminal B-cell differentiation coupled with J-chain expression. These events appear to occur independently of the immunoglobulin gene status of the transformed cells.

Agammaglobulinemia↗
PubMed1988

Bone marrow origin of a B-cell lymphoma.

To search for precursors of the neoplastic B cells in a patient with a nodular lymphoma, we produced a monoclonal antibody to a variable region idiotope on the lymphoma IgM heavy chain. Clonal ancestors of the lymphoma cells were identified by this marker among bone marrow pre-B cells (5% to 26%). A second antiidiotype (anti-Id) antibody specific for the complete lymphoma IgM kappa recognized 10% of B cells in bone marrow and blood and greater than 95% of B cells in lymphomatous lymph nodes, including one obtained after tumor conversion to a diffuse large cell lymphoma. Immunoglobulin gene analysis surprisingly revealed expansion of multiple clones of early B lineage cells in bone marrow, including members of the neoplastic clone. The data suggest that this lymphoma arose through a progression of transformational events beginning in bone marrow: first, creation of an oligoclonal pre-neoplastic pool of pre-B cells, subsequent conversion of a single subclone into low grade neoplastic B cells that homed to the lymph node follicles, and later progression to a more invasive form of the B-cell lymphoma.

Adult↗
PubMed1987

Isotype switching in human B lymphocyte malignancies occurs by DNA deletion: evidence for nonspecific switch recombination.

The mechanism and specificity of isotype switching operative in human B lymphocytes was investigated by a determination of immunophenotype and immunoglobulin heavy and light chain gene status in a panel of human Ig-, IgM, IgG, and IgA B cell malignancies. Regardless of specific tumor type or switched immunophenotype, isotype switching was accompanied by the rearrangement of the expressed CH gene downstream of VDJH, with concomitant deletion of upstream CH genes in all cases. On the allelically excluded chromosome, 25% of the IgG or IgA tumors have retained C mu, and 75% have deleted C mu. The 5' recombination breakpoints for both productive and excluded alleles lie within or near S mu, 3' of the enhancer. No correlation between the extent of allelically excluded CH deletions and the isotype produced by the tumor was observed. Excluded chromosome deletion endpoints were found 5', equal to, or 3' of productive chromosome deletion endpoints. Furthermore, we have identified at least one IgM+ tumor that has undergone abortive CH gene deletions and have observed several unanticipated switch region deletions and potential translocations. The data suggest that isotype switching in human B cells occurs by a nonsubclass- and nonclass-specific switch recombinase.

B-Lymphocytes↗
PubMed1987

Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells.

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.

Antibodies, Anti-Idiotypic↗
PubMed1987

IgM binding protein expressed by activated B cells.

We have identified an IgM binding protein, a single chain polypeptide of Mr 60,000, that is expressed on the surface of B lymphocytes within 18 hr following their activation with phorbol myristate acetate. The IgM binding protein was also detected on fresh leukemic B cells from individuals with chronic lymphocytic leukemia, and the level of its expression was increased after phorbol myristate acetate activation. Resting and phorbol myristate acetate-activated T cells, monocytes, and polymorphonuclear leucocytes did not express detectable amounts of the IgM binding protein. The 60-kDa protein on activated human B cells could bind secreted IgM molecules of both mouse and human origin, as well as endogenous membrane-bound IgM molecules following their cross-linkage with anti-mu antibodies. The binding of soluble IgM molecules to the surface of activated B cells was also demonstrated by indirect immunofluorescence analysis.

B-Lymphocytes↗
PubMed1987

Monoclonal anti-Id antibodies react with varying proportions of human B lineage cells.

Monoclonal antibodies to idiotypic determinants are being used with increasing frequency for analysis and treatment of B cell malignancies. In the present study we have compared the idiotypic specificities of a panel of 39 mouse monoclonal anti-idiotype (anti-Id) antibodies developed against 16 monoclonal human immunoglobulins (Ig). The Id cross-reactivities of these antibodies with Ig products of normal and abnormal B cells were examined by immunofluorescence and immunochemical methods. The reactivity patterns of these anti-Id antibodies with a normal population of plasma cells were highly variable in the immunofluorescence assay. Six were reactive with 2 to 10% of normal plasma cells, 30 with 0.1 to 2% of plasma cells, and three with less than 0.1% of plasma cells from blood, bone marrow, spleen, or tonsils. These reactivity patterns were relatively consistent among samples from 23 Caucasian, black, and Oriental adults. Although the reactivities of most anti-Id antibodies in the panel were not restricted to a particular Ig isotype, several were preferentially reactive with a particular heavy or light chain isotype: one IgM-, two IgA-, two kappa-, and three lambda-restricted antibodies. The immunofluorescence data was confirmed by biosynthetic analysis of Id+ molecules produced by a normal plasma cell population. When the reactivity of this panel of anti-Id antibodies with nonhomologous B cell neoplasms was examined, seven of 30 myelomas or leukemia-derived products and one of nine B cell leukemias or lymphomas without paraproteins were found to be cross-reactive with one or two of the anti-Id antibodies. Although clearly significant, the cross-reactivity between the Id of these paraproteins appeared to be of lower affinity than the reactivity of the homologous Id with their respective anti-Id antibodies. The results reveal a remarkable diversity in the specificities of monoclonal antibodies classified by conventional criteria as anti-Id antibodies, and indicate the potential usefulness of a panel of antibodies for analyzing clonal diversity in normal and abnormal B cell development.

Adult↗