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T Logtenberg

Publications and source records attributed to T Logtenberg.

At least 73 records · Page 4Linked to original sources
PubMed1991

Somatic mutations in the variable regions of a human IgG anti-double-stranded DNA autoantibody suggest a role for antigen in the induction of systemic lupus erythematosus.

The processes that govern the generation of pathogenic anti-DNA autoantibodies in human systemic lupus erythematosus (SLE) are largely unknown. Autoantibodies may arise as a consequence of polyclonal B cell activation and/or antigen-driven B cell activation and selection. The role of these processes in humoral autoimmunity may be studied by molecular genetic analysis of immunoglobulin (Ig) variable (V) regions of antibodies that are characteristic of SLE. We have analyzed the gene elements that encode a high affinity, IgG anti-double-stranded DNA autoantibody secreted by a monoclonal Epstein-Barr virus (EBV)-transformed cell line derived from a patient with active SLE. In addition, we have identified, cloned, and sequenced the germline counterparts of the VH and VL genes expressed in this autoantibody. The comparison of both sets of gene elements shows that the autoantibody VH and VL regions harbor numerous somatic mutations characteristic of an antigen-driven immune response. The light chain expressed in this autoantibody is a somatically mutated variant of the kv325 germline gene that is frequently associated with paraproteins having autoantibody activity and with Ig molecules produced by malignant B cells that express the CD5 antigen. Furthermore, the utilized DH segment has been repeatedly found in multireactive, low affinity IgM anti-DNA autoantibodies from SLE patients and healthy individuals. These results suggest that pathogenic IgG anti-DNA autoantibodies in human SLE may arise through antigen-driven selection of somatic mutations in the gene elements that frequently encode multireactive IgM autoantibodies.

Amino Acid Sequence↗
PubMed1991

Conserved nucleotide sequences at the 5' end of T cell receptor variable genes facilitate polymerase chain reaction amplification.

Alignment of all available nucleotide sequences of mouse and rat alpha/beta T cell receptor (TcR) variable (V) regions revealed the presence of relatively conserved sequences at the 5' end of the V gene segments. Based on these conserved sequences, degenerate primers were developed for use in the polymerase chain reaction (PCR). The degenerate primers developed on the basis of the conserved sequences at the 5' end of rat and mouse V gene segments are expected to enable the amplification of all mouse and rat TcR alpha/beta chain V regions. To test their applicability, the primers were used for the amplification of the V region of the TcR alpha/beta expressed by rat T cell lines. After amplification, the TcR V regions expressed were cloned and sequenced. The Z1a T cell line was shown to use the same TcR V gene segments (V alpha 2 and V beta 8.2), as most other experimental allergic encephalomyelitis associated T cell lines, but had different D and J segments. In spite of these differences at the nucleotide level, a remarkable conservation of the amino acid sequence at the V beta D beta J beta junction was found. Alignment of a large number of human V alpha and V beta gene segments revealed the presence of similarly conserved sequences. Degenerate primers based on these conserved sequences enabled the amplification of TcR V regions of human T cell lines.

Amino Acid Sequence↗
PubMed1991

Antibody specificity and immunoglobulin VH gene utilization of human monoclonal CD5+ B cell lines.

Human B lymphocytes that bear the CD5 antigen are relatively abundant in early ontogeny and comprise a small fraction of the B cell population in adults. The CD5 B cell subset has attracted much attention because of its possible involvement in autoimmune disease and certain B cell malignancies. To begin to understand the role of CD5 B cells in disease processes, we have generated a panel of ten human monoclonal B cell lines selected for expression of the CD5 antigen. These cell lines were obtained by Epstein-Barr virus transformation of B lymphocytes isolated from the spleen, liver and bone marrow of a 19-week-old fetus, from cord blood and from peripheral blood of healthy volunteers. In addition, one cell line was isolated from the spleen of a patient with chronic lymphocytic leukemia. Here, we describe the antibody and immunoglobulin VH gene repertoire of this panel of CD5 B cell lines. The results of these experiments show that (a) some but not all CD5 B cell lines secrete polyreactive antibodies that bind to a variety of self- and xenoantigens and (b) members of the small VH4, VH5 and VH6 gene families are overrepresented in this panel of cell lines. Nucleotide sequence analysis revealed the expression of VH gene elements that have been previously reported in the preimmune B cell repertoire, in CD5 B cell tumors and in polyreactive antibodies.

Antibody Specificity↗
PubMed1991

Generation of the (auto)-antibody repertoire: a case of narcissism?

The immune system has the tremendous task of recognizing and eliminating a practically unlimited number of foreign and often harmful substances. To reach that goal, it has evolved as an apparatus generating a vast array of different antigen receptors expressed by T and B lymphocytes. Through these receptors, the cells can recognize, and respond to, foreign structures; nevertheless, the lymphocytes do not normally react to the body's own constituents. The processes that safeguard a sufficiently broad diversity and yet prevent potentially pathogenic autoimmune reactivity are only now beginning to be elucidated. We describe some of the mechanisms that are believed to play a major role in the generation of the B lymphocyte and antibody repertoire, the induction of tolerance against autologous components and the production of pathogenic autoantibodies, once tolerance is broken.

Animals↗
PubMed1990

Molecular analysis of a germ line-encoded idiotypic marker of pathogenic human lupus autoantibodies.

Id-16/6 is an idiotypic marker found in both IgM and IgG antibodies, as well as in the tissue lesions of patients with SLE. The prototypic Id-16/6+ mAb is 18/2, whose VH3-derived H chain is encoded by an unmutated germ-line gene. We found that the H chains of VH3-derived Id-16/6+ antibodies contain the major determinants of Id-16/6. Moreover, B cell clones from which those antibodies were harvested produce RNA that hybridized under conditions of high stringency to oligonucleotide probes corresponding to the CDR of the VH segment of 18/2. Western blots of Id-16/6+ mAbs with anti-Id confirmed the association of the Id with H chains. Id-16/6 can identify a subgroup of VH3-derived antibodies we have termed the 18/2 CDR family. However, Id-16/6 can also be expressed in some antibodies unrelated to the 18/2 CDR family. No characteristic Ag-binding specificity was found among the members of the 18/2 CDR family. The principal phenotypic feature shared by all known members of the family is Id-16/6.

Amino Acid Sequence↗
PubMed1990

Individual VH genes detected with oligonucleotide probes from the complementarity-determining regions.

The germ-line and expressed Ig repertoire was examined with three oligonucleotide probes from the CDR regions of VH18/2, a VH gene from the largest human VH gene family, VHIII. Each oligonucleotide probe detected small numbers of germ-line bands (1-5) under conditions in which single base differences can be detected; more than half of these bands were polymorphic. The combined results from pairs of oligonucleotides from CDR1 and CDR2 identified a single band on Southern blots, as did a probe from the 5' end of CDR2. This band contains the 18/2 germ-line gene. The nucleotide sequence of expressed VH genes that hybridized to both CDR probes or to the 5' CDR2 probe were greater than or equal to 97% homologous to 18/2 in both the framework and CDR regions. This group of closely related VH genes, the 18/2 CDR family, appears to be overexpressed. The role of polymorphisms and differential expression of individual V genes in multigenic autoimmune diseases, as well as the organization and expression of individual V genes, can be examined with pairs of oligonucleotides from CDR1 and the 3' end of CDR2, or with probes from the 5' end of CDR2.

Amino Acid Sequence↗
PubMed1990

CD5 and immunoglobulin V gene expression in B-cell lymphomas and chronic lymphocytic leukemia.

We studied the expression of CD5 and immunoglobulin variable gene families in a panel of monoclonal Epstein-Barr virus (EBV) transformed lines, chronic lymphocytic leukemias (CLLs) and CD5+ and CD5- B-cell lymphomas. The CD5 gene expression was in all cases identical to that of T-cell malignancies. The utilization of the various VH and VK gene families was roughly proportional to the estimated gene family size in EBV lines obtained from adult healthy subjects. In contrast we found a statistically significant biased usage of VH6 in CLL and VH5 in CD5+ lymphomas as compared with EBV lines, and of VKIII in both CLL and CD5+ lymphomas as compared with EBV lines. Some differences in the variable gene usage were also noted when comparing CD5+ and CD5- lymphomas. These findings are analyzed in the context of possible mechanisms involved in the malignant transformation of CD5+ B cells.

Antigens, CD↗
PubMed1989

Autoantibodies encoded by the most Jh-proximal human immunoglobulin heavy chain variable region gene.

Little is known about the utilization of human Ig heavy chain variable gene segments (VH segments) in different B-lineage cell populations or in antibodies of particular specificity and function. We now demonstrate that human antibodies with Ig VH regions encoded by the most JH-proximal human VH segment (VH6) have specificities resembling those of autoantibodies present in sera of patients with systemic lupus erythematosus (e.g., anti-DNA and anticardiolipin). These specificities appear to be encoded by the germline VH6 gene because the activity was found in multiple independent VH6 antibodies in which the light chain varied with respect to isotype and V kappa subgroup. Features of CDR3 length and somatic mutation patterns in several VH6 antibodies suggested that they were selected by the immune system.

Amino Acid Sequence↗
PubMed1989

Frequency of VH-gene utilization in human EBV-transformed B-cell lines: the most JH-proximal VH segment encodes autoantibodies.

We have studied VH-gene utilization in a collection of 187 IgM-secreting EBV-transformed cell lines and have begun to correlate VH-gene family expression with binding properties of the secreted immunoglobulins. The results of these studies demonstrate that (1) frequency of VH-gene utilization in fetal and adult tissue-derived cell lines correlates with the complexity of the family and (2) the single-membered most JH-proximal VH-6 family encodes autoantibodies reminiscent of autoantibodies found in the sera of patients with systemic lupus erythematosus. Nucleotide sequence analysis of VH-6-expressing clones revealed that each clone utilizes a short DH segment, resulting in a CDR3 region of conserved length. Our data suggest that EBV does not selectively transform human B cells on the basis of VH-gene family expression and that the VH-6 family encodes polyspecific autoantibodies that may serve an important regulatory function in the immune system.

Amino Acid Sequence↗
PubMed1989

Immunoglobulin VH gene expression in human B cell lines and tumors: biased VH gene expression in chronic lymphocytic leukemia.

We have studied frequencies of VH gene utilization in a panel of monoclonal Epstein-Barr virus (EBV)-transformed B cell lines derived from human adult and fetal tissues as well as in monoclonal B cells obtained from fresh chronic lymphocytic leukemia (CLL) samples. The results show that IgM-secreting EBV cell lines from both fetal and adult tissues utilize VH genes from particular families roughly in proportion to estimated family size, suggesting that the repertoire of sigM-positive B cells in both fetal and adult organs is 'normalized' with respect to the V(H) gene family. In contrast, we find a highly biased pattern of VH gene expression in CLLs. The significance of these findings is discussed in the context of mechanisms that could be involved in normal B cell repertoire development and in the process of malignant transformation of precursors of CLL.

Alleles↗
PubMed1988

Enumeration of IFN-gamma-producing human lymphocytes by spot-ELISA. A method to detect lymphokine-producing lymphocytes at the single-cell level.

We present a method to detect and enumerate individual interferon (IFN)-producing human lymphocytes. The assay is based on the ELISA-plaque assay developed by Sedgwick and Holt (J. Exp. Med. (1983) 157, 2178; J. Immunol. Methods (1986) 87, 37). Mitogen-stimulated T cells are seeded in anti-IFN-gamma-coated wells. After a 16 h incubation period, the cells are removed. Subsequently a rabbit anti-IFN-gamma-antiserum followed by goat anti-rabbit antiserum conjugated to alkaline phosphatase are used to detect the IFN-gamma spots. Application of the spot-ELISA in combination with the conventional ELISA reveals the amount of IFN-gamma produced per cell. The spot-ELISA is a highly sensitive, easy to perform and rapid assay. Provided specific antisera are available, this method is suitable to detect production of other lymphokines at the single-cell level. To our knowledge, this is the first report of a single, well-defined T cell product measurement by the spot-ELISA.

Adult↗
PubMed1988

Autoreactive B cells in normal humans. Autoantibody production upon lymphocyte stimulation with autoantigen-xenoantigen conjugates.

Mononuclear cells (MNC) from the blood of healthy individuals cannot be stimulated in vitro with the soluble autoantigen thyroglobulin (Tg). However, when Tg or pepsin fragments of Tg were coupled with a carrier protein, tetanus toxoid (TT), MNC from four healthy TT vaccinated individuals responded to the carrier-autoantigen conjugates by generating anti-Tg antibody forming cells (AFC), as shown in a spot enzyme-linked immunosorbent assay. Generation of anti-TT and anti-Tg AFC after stimulation with the conjugates required the donors to be boostered with TT. The autoantibodies were exclusively of the IgM class, in contrast to the carrier-specific anti-TT antibodies, which were predominantly of the IgG isotype. Activation of normal B cells to anti-Tg production was dependent on the presence of T cells in the cultures and required physical linkage of carrier and autoantigen: no anti-Tg AFC could be detected when MNC were stimulated with uncoupled combinations of Tg and TT. The autoreactive and the carrier-reactive B cells exhibited almost identical conjugate dose-response profiles, which suggest that they responded in a similar way to regulatory signals. These findings indicate that normal blood B cells are competent to respond to the autoantigen Tg in conjunction with signals originating from xeno-antigen-stimulated T cells.

Antibodies, Bacterial↗
PubMed1988

Analysis of T-cell-derived factors with the use of B-prolymphocytic leukemia cells.

Mononuclear cells of five patients with B-prolymphocytic leukemia were tested for their functional capacities in vitro after stimulation. Leukemic B cells from these patients have a mature phenotypic marker profile and can be obtained in high numbers from peripheral blood. B cells of all five patients proliferated in response to phorbol myristate acetate. In one patient, this proliferation was strongly enhanced by the addition of T-cell-conditioned medium. Conditioned medium by itself or interleukin 2 in the presence or absence of the phorbol ester was not active. This enabled us to develop an assay for a B-cell growth factor different from interleukin 2. Next to their proliferative capacities, we found that malignant B cells of three of the five patients secreted large amounts of IgM when stimulated with pokeweed mitogen or interleukin 2 in the presence of allogeneic T cells. In the absence of these allogeneic cells, neither pokeweed mitogen nor interleukin 2 had any effect. However, T-cell-conditioned medium as well as the supernatants of two T-cell hybridomas induced strong IgM production in the absence of T cells. Thus, neoplastic B cells of some patients with B-prolymphocytic leukemia can be stimulated to proliferation and differentiation in vitro and can be used as an assay and model system to study the effect of T cells and/or T-cell factors in human B-cell activation.

Aged↗
PubMed1987

Analysis of the antigen- and mitogen-induced differentiation of B lymphocytes from asymptomatic human immunodeficiency virus-seropositive male homosexuals. Discrepancy between T cell-dependent and T cell-independent activation.

Five asymptomatic human immunodeficiency virus (HIV)-seropositive male homosexuals were immunized with the recall antigens tetanus toxoid (TT) and the three types of poliovirus present in diphtheria, tetanus, and polio vaccine. Four weeks after immunization, the in vivo response to booster immunization, the in vitro pokeweed mitogen (PWM)-induced IgG secretion, and the in vitro T cell-dependent and T cell-independent antigen-induced antibody response were assayed. Increase in serum antibody titer to TT and poliovirus was low and normal, respectively. In all five subjects studied, a high rate of spontaneous IgG production, including antibodies directed toward HIV was observed. Addition of PWM to the cultures induced suppression of the spontaneous IgG secretion. Only one donor showed a slightly increased IgG production after stimulation with PWM. Peripheral blood mononuclear cells of four of the five HIV-seropositive individuals did not produce TT, or poliovirus-specific antibodies when stimulated with the respective T cell-dependent antigens. However, stimulation of these peripheral blood mononuclear cells with TT coupled to agarose beads, which was shown to be T cell-independent, resulted in the generation of IgG anti-TT antibody-forming cells.

Adult↗
PubMed1987

Analysis of the human tonsil B cell repertoire by somatic hybridization: occurrence of both "monospecific" and "multispecific" (auto)antibody-secreting cells.

The purpose of our study was to investigate the (auto)antibody repertoire of human tonsil B cells. To this end, unseparated tonsil B cells and "large" and "small" tonsil B cells, obtained after fractionation on a discontinuous Percoll gradient, were fused with the (human X mouse) heterohybridoma K6H6/B5. The IgM antibodies secreted by individual clones were analyzed in spot-enzyme-linked immunosorbent assay for reactivity against a panel of 5 autoantigens and 1 xenoantigen. The results show that both "mono-" and "multispecific" (auto)antibody-secreting clones can be obtained by somatic hybridization of unseparated, "small" and "large" tonsil B cells. The patterns of IgM reactivities in these three collections of hybridomas did not differ significantly. Inhibition experiments revealed that "multispecific" antibodies can bind with high affinity to more than one (auto)antigen. The results of these experiments are discussed in relation to the state of activation of tonsil B lymphocytes.

Antibody Specificity↗
PubMed1986

Production of anti-thyroglobulin antibody by blood lymphocytes from patients with autoimmune thyroiditis, induced by the insolubilized autoantigen.

Mononuclear cells (MNC) from the blood of patients with autoimmune thyroiditis cannot be stimulated in vitro with the autoantigen thyroglobulin (Tg). However, when Tg was coupled with a solid matrix, MNC from 13 of 19 patients tested responded to this insolubilized Tg (i-Tg) by generating cells which produced IgG anti-Tg antibodies, as demonstrated in a "spot ELISA". This response was specific not only in the assay phase but also in the induction phase, because 1) cultures with insolubilized ovalbumin, bovine Tg, or porcine Tg were negative, and 2) i-Tg did not induce polyclonal IgG production. Generation of anti-Tg-producing cells was completely abrogated when free Tg was added to i-Tg cultures. Stimulation with i-Tg after separation of T and non-T MNC revealed that: 1) the response was independent of T cells; 2) in three of the six patients whose MNC did not respond, removal of T cells did result in anti-Tg production; 3) re-addition of the T cells--but not of irradiated T cells--abrogated the response in two of these three patients; and 4) preincubation of monocytes with i-Tg resulted in suboptimal anti-Tg production. These findings indicate that B cells from patients with autoimmune thyroiditis can be specifically stimulated with Tg coupled with a solid matrix, resulting in generation of anti-Tg-producing cells. The essence of this mode of presenting the antigen would appear to be the cross-linkage of surface immunoglobulin. The response was T cell-independent; in some patients, however, the presence of a radiosensitive suppressor T cell population can be demonstrated.

Antibody Specificity↗