Biomedical subjects

F K Stevenson

Publications and source records attributed to F K Stevenson.

At least 91 records · Page 5Linked to original sources

PubMed1995

Differential usage of an autoantibody-associated VH gene, VH4-21, by human B-cell tumors.

Selection of immunoglobulin variable region genes for recombination in B cells takes place from among those VH and VL gene segments available in the unrearranged germ line repertoire. In the case of neoplastic B cells, there is apparent deviation in the use of V-genes from that expected on a random basis, both for VH and for VL. Also, the preferred V-genes, and their patterns of mutation, differ among the various categories of B-cell tumor possibly reflecting the distinct origins and clonal histories on the individual tumor cells. This review focuses on a single VH gene, VH4-21, which is a member of the VH4 family, and which appears selectively to encode immunoglobulins with autoantibody activity, particularly anti-red cell antibodies. The pattern of usage of this VH gene by B-cell tumors demonstrates clear asymmetry among different tumor types. Also, the mutations detected in this relatively non-polymorphic gene indicate that antigen, possibly autoantigen, may influence the behavior of the tumor cell.

Agglutinins↗

PubMed1995

Dual recognition of lipid A and DNA by human antibodies encoded by the VH4-21 gene: a possible link between infection and lupus.

The VH4-21 (V4-34) gene segment, a member of the VH4 family, is expressed early in B-cell maturation and is utilized by approximately 6% of normal adult B lymphocytes. This prevalence indicates an importance of VH4-21 in the B-cell repertoire. The gene also encodes certain autoantibodies being mandatory for pathological IgM anti-red cell antibodies directed against the I/i antigen, and also capable of encoding anti-DNA antibodies. Recognition of I/i antigen or DNA appears to be via two distinct sites on VH, with I/i binding mediated by sequences in the framework region, and DNA binding correlating with the presence of positively charged amino acids in complementarity-determining region 3. However, these positively charged residues appear to suppress the ability of the framework region to interact with I/i, rendering a single sequence monospecific for I/i or DNA. The IgM anti-DNA antibodies also recognize bacterial lipid A, whereas the anti-I/i antibodies do not, indicating that CDR3 may be involved in binding the negatively charged lipid A. Structural similarities between the DNA backbone and lipid A provide a possible explanation for this cross-reactivity. This dual recognition of bacterial antigen and autoantigen provides a potential link between infection and autoimmunity.

Adult↗

PubMed1994

Biased utilization of immunoglobulin variable region heavy- and light-chain genes by the malignant CD5- B lymphocytes from patients with Burkitt's lymphoma.

Twenty-six established Burkitt's lymphoma (BL) cell lines from endemic or sporadic groups of patients were examined for the expression of cross-reactive idiotypes (CRI) associated with VHI, VHIII, VHIV, VHVI, VKIIIa and VKIIIb heavy- and light-chain gene products, using a panel of anti-CRI and anti-subgroup monoclonal antibodies (MAbs). Membrane, cytoplasmic and secreted immunoglobulins (Ig) were analysed by immunofluorescence, immunoperoxidase and ELISA respectively. While 35% of the lines expressed either of the VHIV-associated CRI, recognised by the MAbs 9G4 or LCI, none expressed the other VH-associated CRI included in our study. Of the kappa light chain expressing BL lines 54% and 46% belonged to the VKIII subgroup and VKIIIb sub-subgroups respectively. None, however, was found to express the VKIIIa or VKIIIb-associated CRI, recognised by the 6B6.6 and 17-109 MAbs. A significant association has been observed between the expression of the VHIV-associated CRI and the VKIII subgroup within the BL lines derived from the sporadic group of patients as compared with their endemic counterparts. Our results suggest that the expressed repertoire of Ig variable region genes within the malignant B lymphocytes of BL is not random and that a highly selective mechanism(s) may operate on this subset of B lymphocytes, as evidenced by the expression of the VH and VK gene products.

Animals↗

PubMed1994

Idiotypic vaccination against human B-cell lymphoma. Rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines.

Idiotypic determinants on neoplastic B cells could provide tumor antigens for vaccination of patients with B-cell tumors. Because this approach requires an individual vaccine for each patient, simple methods for obtaining idiotypic antigen are desirable. Using polymerase chain reaction (PCR) with family-based V-gene and J-region primers, the variable region genes of heavy and light chains (VH and VL) of Ig have been obtained from biopsy material from 13 patients with B-cell tumors. In each case, analysis of random clones derived from the PCR product showed repeated, clonally-related sequences, whereas normal lymphoid tissue generated no repeated sequences. In 3/3 cases, the repeated sequences were found to be the same as those in a tumor-derived hybridoma. Mutational patterns in the V-genes differed among the tumors, with follicular lymphoma tending to be more highly mutated. The individual VH and VL sequences have been assembled with a flexible linker sequence to encode single-chain Fv (scFv). The scFv sequences can be cloned into bacterial expression vectors to produce protein, or into vectors suitable for direct vaccination using naked DNA. In a model system, expressed scFv protein retained all idiotypic determinants defined by a panel of five anti-idiotypic monoclonal antibodies (MoAbs). Similarly, expressed scFv proteins from two patients were shown to react with anti-idiotypic antibodies. This approach allows production of potential vaccines from surgical biopsies within 2 to 3 weeks.

Amino Acid Sequence↗

PubMed1994

Clonal history of a human follicular lymphoma as revealed in the immunoglobulin variable region genes.

The variable region genes used to encode the immunoglobulin expressed by tumour cells of a patient with follicular lymphoma have been identified and sequenced. Initially, a lymph node biopsy was analysed and revealed usage of VH and V kappa genes which had numerous substitutions as compared with the closest germ line genes. The pattern of mutations in VH was consistent with a role for positive selection by antigen. In addition, there was evidence in both VH and V kappa sequences for clonal heterogeneity. After 5 years, which included treatment with chemotherapy, the patient relapsed with tumour cells present in the blood. Analysis of the V-genes used by the emerging tumour revealed a single homogeneous sequence for both VH and VL, which, in each case, matched closely one of the sequences in the original lymph node biopsy. These results indicate that selection, possibly mediated by antigen, can operate on a cell destined to give rise to lymphoma, and that intraclonal variation can occur after the neoplastic event. However, in this case, late relapse in the blood is dominated by a single clone.

Amino Acid Sequence↗

PubMed1994

Anti-j: human cold agglutinins recognizing linear (i) and branched (I) type 2 chains.

Two IgM lambda cold agglutinins (CAs) reacted with protease- and sialidase-resistant antigens expressed in equal strength on human adult (I), newborn (i), i adult, rabbit (I) and rhesus monkey (i) erythrocytes. The antibodies were inhibited by the linear type 2 sequence lacto-N-neotetraose and the branched type 2 sequence lacto-N-neohexaose. Endo-beta-galactosidase treatment of red cells, which splits type 2 chains from the surface, abolished CA reactivity. The CAs expressed the idiotype recognized by the anti-idiotype 9G4 specific for anti-I and anti-i CAs. The data suggest that the two CAs recognize linear (i) as well as branched (I) type 2 chains. It is proposed to term these CAs anti-j.

Aged↗

PubMed1994

Assessment of the role of clonogenic B lymphocytes in the pathogenesis of multiple myeloma.

The identifiable neoplastic cell in multiple myeloma is the plasma cell, which usually synthesizes and secretes a monoclonal immunoglobulin. However, there exists the possibility that the neoplastic event has occurred in a less mature clonally-related cell, such as a B lymphocyte, prior to Ig class switching. Since the presence of such a clonogenic cell could influence design of therapy, particularly with monoclonal antibodies, we have used the analysis of tumour-related VH genes to approach this question. Cloning and sequencing of PCR products from VH genes of tumour cells obtained from 4/4 patients with myeloma revealed significant mutation of the genes as compared to germ line sequences. In all cases the mutations were scattered throughout the variable region, with a pattern which did not indicate a role for antigen in selection. Importantly for therapy, multiple VH sequences from all patients were completely homogeneous, with no intraclonal variation. These findings indicate that, although IgM-positive clonogenic cells may exist, it is unlikely that they are involved in continuous maintenance of the malignant isotype-switched cell population. One possibility is that the B-cell progenitor population has to undergo further chromosomal changes to generate the malignant cell, and that this occurs at a more mature stage; in this case, antibody therapy should be aimed primarily at the more differentiated cells.

Amino Acid Sequence↗

PubMed1993

Human antilipid A monoclonal antibodies bind to human B cells and the i antigen on cord red blood cells.

We describe two independently derived human mAb, A6(H4C5) and 216, initially selected for their reactivity to the lipid A domain of bacterial LPS, which also react with the following Ag: the i Ag present on cord RBC, a ligand on human B lymphocytes, and to certain autoantigens, defining these mAb as polyreactive. Both mAb have specific affinity for a carbohydrate epitope consisting minimally of a disaccharide with an acyl substitution at the 2-carbon position. Structural examination of the diverse Ag recognized by the two antibodies reveals the presence of this carbohydrate structure required for antibody binding. A6(H4C5) and 216 are IgM in isotype, but differ in their L chain expression. Molecular analysis shows that both the mAb are encoded by a highly conserved VH4 gene, designated VH4-21. This gene encodes a number of autoantibodies, particularly cold agglutinins. Specific recognition of lipid A and of a carbohydrate epitope on B lymphocytes by the two human mAb suggests a dual function for the highly conserved VH4-21 gene in antibacterial response and in B cell development and regulation.

Agglutinins↗

PubMed1993

Molecular characterization of a cross-reactive idiotope on human immunoglobulins utilizing the VH4-21 gene segment.

The anti-idiotypic (anti-Id) antibody (Ab) 9G4 binds a cross-reactive idiotope (CRI) present in a select group of human autoantibodies. This Id has been localized to the portion of immunoglobulin (Ig) heavy (H) chains encoded by the VH4-21 gene segment, a member of the human VH4 family. This gene segment is utilized by essentially all cold agglutinin (CA) Abs with I/i specificity isolated from patients with CA disease stemming from chronic lymphoproliferative disorders. In this study, mutational analysis of a CA has been used to determine the structural basis for 9G4 binding to Abs utilizing the VH4-21 gene segment. Recombinant CA H chain mutants were produced and their 9G4 reactivity determined. Mutants were generated by exchanging VH4-21 sequences in the FR1, CDR1, and CDR2 with corresponding sequences from a closely related gene segment V71-2, a VH4 family member that is associated neither with Abs having CA activity nor with Abs that react with 9G4. The results indicate that the motif AVY at amino acid positions 23-25 in FR1 defines the 9G4 idiotope. Reaction of these recombinant Abs with a polyclonal rabbit anti-CA antiserum absorbed to render it specific for a CA CRI also maps predominantly to FR1. These findings indicate that the solvent-exposed FR1 plays an important role in eliciting an immune response to Igs.

Agglutinins↗

PubMed1993

Autoanti-red cell antibodies synthesized by patients with infectious mononucleosis utilize the VH4-21 gene segment.

A significant proportion of patients with B cell tumors secrete IgM mAb that recognize a carbohydrate autoantigen (li) on the red cell surface. The majority bear an idiotope (Id) that arises from heavy chains encoded by the VH4-21 gene segment, indicating unusual restriction of autoantibody specificity to a single VH gene. Polyclonal anti-li antibodies are also synthesized transiently by normal B cells following certain infections, and we have analyzed the role of the VH4-21 gene in encoding these antibodies. Levels of Id in sera of patients following infection with EBV or Mycoplasma pneumoniae were significantly raised (p < 0.01), and the Id-positive Ig reacted with patients' red cells. Id-positive B cell clones were established from four EBV-infected patients, and 6/6 of the IgM-Id agglutinated red cells. Nucleotide sequence analysis revealed involvement of the VH4-21 gene in all cases, with remarkably little change from the germ-line sequence. However, D segments were heterogeneous, and light chains differed. These results indicate that the same VH gene is used both by polyclonal autoantibodies produced in response to infection, and by B cell tumors. However, the lack of mutations would not appear to give an opportunity for Ag selection to drive affinity maturation of these autoantibodies.

Adolescent↗

PubMed1993

Differential usage of an Ig heavy chain variable region gene by human B-cell tumors.

A monoclonal anti-idiotypic antibody has been raised that recognizes Igs with heavy chains encoded by a member of the VH4 family, the VH4-21 gene segment. The idiotope (Id) is detectable on a high percentage of early B cells in fetal spleen, and is expressed by certain autoantibodies, particularly cold-reactive anti-red blood cell antibodies. Therefore, it was of interest to investigate usage of this VH gene by neoplastic B cells; 81 chronic lymphocytic leukemias (CLLs) involving CD5+ B cells and 62 B-cell lymphomas of varying histologic type have been analyzed. The Id was expressed by only 3 of 81 (3.7%) of the CLLs, indicating a relatively low usage by these tumors. In contrast, the Id was expressed by 9 of 62 (14.5%) of the lymphomas across a range of histologic types, indicating a differential use of the VH4-21 gene among B-cell neoplasms. For three of the Id-positive lymphomas, each of a different histologic class, the nucleotide sequence of the tumor-derived VH gene was determined; the VH4-21 gene was identified, as expected. The sequence from the CLL was identical to the germline sequence, and the marginal zone lymphoma showed only 3 nucleotide changes, 2 of which gave rise to amino acid substitutions. In contrast, the sequence from the follicular lymphoma showed 29 nucleotide changes giving rise to 14 amino acid substitutions, which were scattered among the CDR and FW regions.

Antibodies, Monoclonal↗

PubMed1993

Idiotypic IgM on a B-cell surface requires processing for recognition by anti-idiotypic T cells.

Immunization with purified idiotypic IgM derived from the BCL1 lymphoma generates CD4-positive T cells which proliferate specifically in response to idiotypic antigen expressed at the surface of the BCL1 tumor cells. These T cells have been hybridized and two cloned hybridomas have been used to study the molecular nature of the idiotypic antigen recognized. Endogenous presentation of idiotype by the B cells generated IL-2 from the T-cell hybridomas, as measured by a CTLL response. Presentation was inhibited in a dose-dependent manner by chloroquine or ammonium chloride, both of which affect proteolytic degradation of antigen in the endosomes. Similar results were obtained with inhibitors of metabolism or protein synthesis. However, none of these reagents affected expression of idiotypic IgM at the cell surface under the conditions used, indicating that whole IgM is not interacting directly with the T-cell receptor. Interaction between the presenting B cells and T-cell hybridomas was inhibited by anti-CD4 and, in one case, by a monoclonal antibody directed against a determinant in the I-Ed region of the MHC Class II. Inhibition also occurred with antibodies against IgM, either anti-constant region or anti-idiotype, and the univalent Fab' gamma fragment was an effective inhibitor. These data indicate that the B cell constitutively presents its endogenous idiotypic immunoglobulin to CD4-positive T cells following endocytosis from the surface, proteolytic degradation, and interaction with MHC Class II molecules. This endogenous pathway is perturbed by binding of exogenous anti-immunoglobulin antibody, perhaps mimicking what might occur when surface immunoglobulin encounters antigen.

Animals↗

PubMed1993

Utilization of the VH4-21 gene segment by anti-DNA antibodies from patients with systemic lupus erythematosus.

A monoclonal antibody (9G4) which detects an idiotope (Id) rising from heavy chains encoded by the VH4-21 gene segment has been utilized to investigate the role of this gene in encoding anti-DNA antibodies in SLE. Two hybridomas secreting Id-positive anti-DNA antibodies were established from two patients with SLE, with one (RT-79) an IgM kappa and the other (D-5) IgG kappa. Nucleotide sequence analysis of the heavy chain variable regions revealed involvement of the VH4-21 gene; the IgM antibody used the gene in germ line configuration, whereas the IgG antibody had 18 nucleotide changes. The CDR3 sequences for both the antibodies had a predominance of basic amino acids, with RT-79 having five and D-5 two arginine residues respectively. The VH4-21 gene segment, often in germ line configuration, is also used by IgM autoantibodies against red cell Ii antigens. However, IgM from a panel of six hybridomas secreting antibodies of Ii specificity, had no detectable activity against DNA. Conversely, RT-79 had only a weak ability to agglutinate red cells. Comparisons of Ig variable regions indicate that the amino acids in the CDR3 region of the mu chain influence the ability of IgM encoded by the VH4-21 gene segment to discriminate between a carbohydrate Ii antigen and DNA, and strongly support the suggested role of arginine in interaction with DNA. Sera from patients with SLE were found to have significantly raised levels of Id which was expressed by anti-DNA antibodies against both ss and dsDNA.

Amino Acid Sequence↗

PubMed1993

A genetic approach to idiotypic vaccination.

Treatment of cancer with vaccines is an attractive prospect, but few tumours express suitable target antigens. With B-cell lymphomas, the idiotypic immunoglobulin (Ig) of the malignant B-cell should provide a suitable target but this requires a vaccine to be created for each patient. We propose a strategy for making such vaccines: first to clone the V genes of the idiotypic Ig, and second to inject the patient with the cloned DNA (genetic immunisation) in order to elicit an immune response against the encoded Ig. We have previously shown that the V genes of the idiotypic Ig can be identified from human lymph node biopsies by polymerase chain reaction amplification, cloning, and sequencing. In this report, we show that anti-idiotypic antibodies can be elicited by direct injection of an expression vector that encodes the V genes of murine antibodies (the V genes of B1.8, a murine hybridoma or of BCL1, a murine lymphoma line). This finding suggests a simple approach to the preparation of idiotypic vaccines for patients with B-cell lymphoma, which also circumvents the need for adjuvants.

Animals↗

PubMed1993

The genetic basis of human VH4 gene family-associated cross-reactive idiotype expression in CD5+ and CD5- cord blood B-lymphocyte clones.

In a recent study we have observed a high frequency expression of cross-reactive idiotypes encoded by genes from the relatively small VH4 family of immunoglobulin heavy chain genes in cord blood B-lymphocyte lines. Furthermore, we have demonstrated a selective pattern of expression of two VH4-associated cross-reactive idiotype (CRI) in B-lymphocyte lines established from CD5+ and CD5- cord blood B-lymphocytes. There was a restricted expression of one CRI marker recognized by the 9G4 monoclonal antibody in lines established from CD5+ B-lymphocytes but not in those established from the CD5- population. In the current study we examine the molecular basis for the selective pattern of CRI expression. Nucleotide-sequence analysis of functional immunoglobulin heavy chain (IgH) gene rearrangements in three CD5+ lines expressing the CRI recognised by 9G4 reveal that all use a single gene from the VH4 family, the V4.21 gene. However, all three lines have distinct third complementarity determining regions (CDR3) implying different clonal origins. In contrast, four cord blood cell lines (two established from CD5+ B-lymphocytes) expressing the CRI recognized by MoAb Lc1 have functional IgH gene rearrangements involving two different genes from the VH4 family, the V71-4, and V2-1 genes. Antigen specificity analysis reveals that all three 9G4-reactive lines produce antibodies that react with the I and/or i red blood cell carbohydrate antigens. These data suggest that the distinction in VH4 gene use in CD5+ B-lymphocytes in cord blood results from a selection process in vivo that shapes the repertoire of CD5+ B-lymphocytes. This study extends recent observations that the monoclonal anti-CRI antibodies 9G4 and Lc1 are markers of two distinct subgroups of proteins encoded by two subsets of genes within the VH4 family. Furthermore, it appears that amino acid residues in framework region one and complementarity determining region two are critical for the expression of the cross reactive idiotypes and the serological distinction between the two subgroups of proteins.

Amino Acid Sequence↗

PubMed1992

VH restriction among human cold agglutinins. The VH4-21 gene segment is required to encode anti-I and anti-i specificities.

We previously reported that human autoantibodies with cold agglutinin activity contained a single human VH gene segment (VH4-21) which was also responsible for the cross-idiotypic specificity characteristic of the cold agglutinin response. To confirm and extend this observation we have analyzed at the nucleotide level the H and L chains of six new cold agglutinin molecules derived from different patients. We found that regardless of whether the antibody recognizes the i or the I red cell Ag, restriction at the VH gene segment level is absolute. We also found that even in the absence of somatic mutation the VH4-21 gene segment can encode both anti-i and anti-I specificities. Finally, although the VH4-21 gene segment is essential for cold agglutinin activity, the other genetic elements that contribute to the V region of the antibody molecules can be extremely diverse. The structural information provided in this report sharply restricts the requirement for encoding pathogenic cold agglutinin activity to one of the components of the H chain V region, specifically the VH gene segment. The implications of this apparently absolute requirement for a single VH gene segment, unprecedented in the human autoimmune response, are discussed.

Aged↗

PubMed1992

Update on tumor vaccines.

Vaccination against tumor has always been an attractive idea for the treatment of patients bearing tumor. By harnessing the host's own immune response the attack on tumor cells would act on a continuing basis, with emerging tumor cells stimulating their own destruction. However, the approach has been hampered by our poor understanding of the nature of tumor antigens and of the pathways by which immune cells might operate against tumor growth. Recent developments in molecular biology and immunology are remedying this deficiency and bringing vaccination to the forefront of new approaches to treatment of a range of tumors. Results obtained in B-cell tumors, where the idiotypic immunoglobulin at the cell surface provides a well-defined tumor antigen, are already indicating exciting possibilities as well as delineating problems. There is considerable clinical evidence that patients have some intrinsic ability to control tumor growth and that certain tumors remain dormant for long periods. Attempts to understand and perhaps stimulate the mechanisms involved are being made through the use of biological modifiers and by manipulating potential effector cells in vitro. Ideally this approach, which may include non-specific and specific elements, could be combined with specific vaccination in order to combat the apparent ability of many tumor cells to evade host defences.

Antibodies, Neoplasm↗

PubMed1991

Selective expression of a VHIV subfamily of immunoglobulin genes in human CD5+ B lymphocytes from cord blood.

Human B lymphocytes expressing the CD5 surface antigen (CD5+ B cells) constitute a subset capable of producing polyspecific antibodies recognizing a variety of self antigens. The repertoire of antibodies produced by CD5+ and CD5- B cells is different. However, it is not yet established whether this distribution is reflected in different immunoglobulin variable region gene (IgV) use. Rearrangement of heavy chain IgV (IgVH) genes represents one of the first identifiable stages in the maturation of B cells, and occurs in a developmentally ordered fashion. The repertoire of IgVH gene expression is highly restricted during fetal life but diversifies progressively after birth. A high frequency of VH gene use from the relatively small VHIV gene family has previously been demonstrated in human fetal liver B cells. In the present study, 102 B cell lines established by Epstein-Barr Virus-transformation of separated CD5+ and CD5- cord blood B cells, were examined for the frequency of IgV expression using monoclonal antibodies to cross-reactive idiotypes (CRI). The results demonstrate a relatively high frequency of VHIV gene use (30%) in B cells from cord blood. Furthermore, two mutually exclusive CRI associated with distinct subgroups of the VHIV family are segregated in their association with either subset of B cells. One CRI is exclusively expressed in lines established from CD5+ B cells while the other is associated with lines established from CD5- B cells.

Antigens, CD↗