Preferential Expression of VH5 and VH6 Immunoglobulin Genes in Early Human B-Cell Ontogeny

Two veto active allospecific cytotoxic T-cell clones (CTL), 4A2 and 4B3, were studied for the development of autoreactivity. The 4A2 CTL became autoreactive within 6 days when cultured without appropriate allospecific stimulator cells, whereas the 4B3 CTL retained its alloreactivity without becoming autoreactive when cultured under these conditions. Simultaneously with the development of autoreactivity, the 4A2 CTL lost its veto activity. Limiting dilution cultures of normal allospecific 4A2 CTL cells and autoreactive 4A2 CTL cells and mixtures of both showed that the former CTL cells were capable of inhibiting clonal growth of the autoreactive CTL cells in a dose-dependent manner. We suggest that this growth inhibition reflects veto activity, i.e. cells capable of growing are inhibited by autorecognition of veto active allospecific 4A2 CTL cells. Taken together, the present results indicate that the existence of intraclonal down-regulation of potentially autoreactive cells is a normal feature of allospecific cytotoxic T-cell clones.     

Modifying Antibody Specificity by Chain Shuffling of VH / VL between Antibodies with Related Specificities

Histo-blood group antigens are important markers of developmental stages and as such also often of tumours. Generation of antibodies towards these carbohydrate structures is still a challenging task as they may lack specificity, affinity or are only of the IgM class. We have examined four own antibodies to Lewis Y/H type 2 for their fine specificities using a large panel of mono- and oligosaccharides. Sequence alignment to other antibodies with similar specificity revealed an overall limited variation, and that our antibodies constitute a novel set. Based on produced and analysed chimeric mouse–human antibodies, extensive chain shuffling experiments were performed in order to analyse influences of the respective H and L chains on the specificity of the antibodies, and to generate modified antibodies with improved properties. One chIgG1 out of the shuffled antibodies revealed improved specificity and markedly enhanced functional affinity to Lewis Y compared to the parental chIgG1 antibodies. Therefore, the combinatorial approach of chain shuffling provides a platform to improve specificity and/or affinity of anti-carbohydrate antibodies.     

The molecular basis and biological significance of VH replacement

Summary:  First observed in mouse pre-B-cell lines and then in knock-in mice carrying self-reactive IgH transgenes, V _H replacement has now been shown to contribute to the primary B-cell repertoire in humans. Through recombination-activating gene (RAG)-mediated recombination between a cryptic recombination signal sequence (RSS) present in almost all V _H genes and the flanking 23 base pair RSS of an upstream V _H gene, V _H replacement renews the entire V _H -coding region, while leaving behind a short stretch of nucleotides as a V _H replacement footprint. In addition to extending the CDR3 region, the V _H replacement footprints preferentially contribute charged amino acids. V _H replacement rearrangement in immature B cells may either eliminate a self-reactive B-cell receptor or contribute to the generation of self-reactive antibodies. V _H replacement may also rescue non-productive or dysfunctional V _H DJ _H rearrangement in pro-B and pre-B cells. Conversely, V _H replacement of a productive immunoglobulin H gene may generate non-productive V _H replacement to disrupt or temporarily reverse the B-cell differentiation process. V _H replacement can thus play a complex role in the generation of the primary B-cell repertoire.     

Genetic diversity of the human immunoglobulin heavy chain VH region

Summary: The human immunoglobulin heavy chain V_H region is one of the most complex regions in the human genome. The high level of diversity of this region has been shown by a number of studies. However, because of the limitations of the conventional experimental methods, it has been difficult to learn the extent of the diversity and the underlying mechanisms. This review describes a number of new genetic approaches developed in the authors' laboratory. By using these approaches, significant progress has been made in assigning different V_H sequences to their respective loci, in learning the diversity of gene segment number and composition among the V_H haplotypes, and in learning V_H gene segment organization in individual haplotypes. Information obtained toward this direction could help in understanding the mechanisms underlying V_H region diversity and the biological impact of the V_H region diversity.     

Distribution of Heavy-Chain Variable-Region (VH) Subgroups on Human Lymphocytes

Lymphocytes from 20 normal blood donors were stained with fluorescein isothiocyanate (FITC)-conjugated anti-F(ab')₂, anti-V_HI, anti-V_HII, and anti-V_HIII subgroup antisera. The means of the percentages of staining were 2.9%, 5.0%, and 5.5% for the V_HI, V_HII, and V_HIII subgroups, respectively. The sum of the percentages of the lymphocytes stained with each of the V_H subgroup-specific antisera corresponded well with the percentages of lymphocytes stained with an anti-F(ab')₂ antiserum. In addition, it was shown by double immunofluorescence staining and in various depletion experiments and tests with thymocytes and lymphocytes from patients with hypogammaglobulinemia that the cells staining with anti-V_H antisera corresponded to the membrane Ig-positive cells—that is, the B-lymphocyte population.     

Expression of the Human Immunoglobulin Heavy Chain VH6 Gene Element by Fetal B Lymphocytes

Fetal B lymphocytes in mice and humans use a limited number of the available V_H gene segments. Mouse fetal B cells primarily utilize 3' V_H elements, suggesting that the localization of these elements determines their rearrangement frequency. The previously reported non-random usage of human V_H genes has been more difficult to explain. In this study the authors analysed the expression of the most proximal 3' human V_H element (V_H6) using a monoclonal antibody (JE-6). V_H6 expression was assessed in various B cell differentiation stages from fetal liver, bone marrow and spleen at 12–20 weeks of gestation. The authors demonstrate that the level of V_H6 expression does not exceed a stochastic usage frequency. This suggests that the localization of V_H6 does not significantly promote its expression during human fetal life, and that other factors must affect the usage of V_H genes during human fetal development.     

VH-Gene Family Dominance in Ageing Mice

The cellular composition and Vn-gene family repertoire were compared in different B-cell compartments from young adult (8–12 weeks) and old (18–24 months) C57BL/6 and BALB/c mice. Ageing mice were found to have a higher frequency of peripheral mature B cells utilizing genes from a single V_H-gene family. While in each individual old C57BL/6 mice cells expressing the V_H J558 gene family consistently were over-represented, a marked individual variation was observed in old BALB/c mice with increased frequency of either the V_h J558, Q52 or J606 families. Aged mice were found also to have a reduced number of bone-marrow pre-B cells and an augmented number of splenic Ig-secreting cells. These results suggest that old mice express less diversified antibody repertoires possibly as a consequence of reduced input from precursors and increased peripheral selection, which may be responsible for the progressive establishment of immunodeficiency.     

A Human Rheumatoid Factor C304 Shares VH and VL Gene Usage with Antibodies Specific for Ubiquitous Human Viral Pathogens

Analysis of the variable region getie sequetices of a human hybridoma rheumatoid factor (RF), derived from a patient with rheumatoid arthritis (RA), revealed the expression of genes from the V_βI and V_H3 families. Specifically, the C304 RF had rearranged the DPL8/Humlv1042 and VH26 germline V_L and V_H genes, respectively. This gene usage has been observed in the rearrangement of human anti-viral antibodies specific for the herpes group of viruses. This overlap between the autoimmune and anti-viral antibody gene repertoires suggests a possible structural relationship between the immune response directed against ubiquitous pathogens and the induction of RF production.     

Idiotype-Anti-Idiotype Interactions of VHIX-Coded Anti-Progesterone and Anti-Arsonate Antibodies

The reactivity and specificity of potyctonal and monoclonal anti-idiotypic antibodies raised against monoclonal anti-progesterone and anti-arsonate antibodies have been studied by solid phase radioimmunoassay (RIA) with immobilized idiotype and by passive haemagglutination with idiotype-coupled red cells. The sensitivity of the two methods was comparable, though some cross-reactions were only detected by RIA. Passive haemagglutination was found to be especially suitable in screening for monoclonal anti-idiotypes in hybridoma supernatants and ascites. and had advantages over RIA in detection of syngeneic anti-idiotypes. Demonstration of binding site-associated idiotopes was possible by haemagglutination inhibition. RIA and haemagglutination were used to investigate the idiotypic relationships between BALB/c antiprogesterone and anti-arsonate monoclonal antibodies which share heavy chains encoded by V_HIX variable region genes.     

A VH-Associated Idiotype in Human Anti-Tetanus Antibodies

An anti-idiotypic (α Id) serum was induced against anti-tetanus toxoid (α TET)-reactivc F(ab')₂ fragments of a single donor. After appropriate adsorption the serum was shown to react only with α TET F(ab')₂ fragments and not with F(ab')₂ fragments deplcted of α TET reactivity or anti-diphtheria toxoid (DIP) or anti-purified protein derivative (α PPD) F(ab')₂ fragments of this donor. Inhibition studies with soluble TET indicated that the anti-idiotypic serum partly reacts with determinants close to or within the antigcnic binding site. When tested on a small panel of α TET F(ab')₂ fragments derived from unrelated donors, one out of four donors tested showed cross-reactivity. When tested on α TET F(ab')₂ fragments derived from related family members, the donor's father and two sisters were found to cross-react, whereas the mother and two other sisters did not. The observed cross-reactivity was independent of the litres of α TET antibodies in the serum. Furthermore, two-dimensional gel electrophoretic studies carried oui on α TET IgG of Id-positive or Id-negative family members indicated that the expression of idiotypic determinants is linked to a particular heavy chain. Immunofluorescent staining with α Id and cytofluoro-graphic analysis of donor T-lymphoblast proliferation in response to TET showed that the T cells did not express Id determinants found on his α TET antibodies.     

VH Subgroup Restriction in Human Erythrocyte Antibodies: Studies of Anti-A, Anti-B, and Anti-Duffy Antibodies

IgG from 13 anti-A, 26 anti-B, and 10 anti-Duffy antisera were used to coat human erythrocytes. With antisera specific for each of the three VH subgroups, VHI, VHII, and VHIII, a clear VH subgroup restriction was shown in these antibody preparations. Of the 26 IgG anti-Bcoated cell preparations, 21 were agglutinated exclusively by the anti-VHIII antiserum, and 5 were agglutinated mainly by the anti-VHIII antiserum but showed also some reactions using anti-VHI or anti-VHII antiserum, or both. Similarly, 11 out of the 13 IgG anti-A antibodies belonged to VHI, and only 2 to VHIII. The 6 Igg anti-Fy(a)antibodies were restricted to subgroups VHI and VHII, and of the 4 IgG anti-Fy(b) antibodies, 3 belonged to subgroup VHIII and one to VHII. Additional experiments indicated that IgM anti-A and IgM anti-B antibodies showed the same type of restriction to VH subgroups as the corresponding IgG antibody preparations.     

Neonatal Pattern of VH Gene Utilization in Nonobese Diabetic Mice Does not Correlate with Development of Insulitis

The nonobese diabetic (NOD) mouse model is a model of human autoimmune insulin dependent diabetes, IDDM. The effector cells of the disease have been shown to be T cells, but also B cells seem to contribute. Adult NOD mice have been shown to display a bias in their utilization of immunoglobulin (Ig) variable heavy (V(H)) genes. In this study the analysis of VH gene utilization in NOD mice protected from insulitis by transgenic insertion of a major histocompatibility complex (MHC) class II E(alpha) gene, point out that the bias in V(H) gene expression is not correlated to disease development. The aberrant V(H) gene utilization pattern in mice with the NOD genetic background is instead suggested to be a consequence of a deregulation of the apoptosis inhibiting gene bcl-2. We also investigated if prolonged in vitro survival of NOD lymphocytes is correlated to disease development. The E(alpha) transgenic NOD mice were shown to display a prolonged in vitro survival of spleen T cells, similar to normal NOD mice. These results indicate that defective death mechanisms of T cells may not be primarily involved in the development of autoimmune disease in these mice. However, in contrast to results from other groups, no difference in in vitro survival could be detected for B cells from mice with NOD genetic background compared to C57BL/6 mice.     

Identical VHD and DJH Junctions in Monoclonal Antibodies Derived in Response to Dextran B512 Could be the Result of Developmental Selection

We describe here the CDR3s of a collection of monoclonal antibodies (MoAb) with specificity for the carbohydrate dextran B512 produced in the mouse strain C57BL/6. In spite of the postulated mechanisms for variability in this region, a high proportion of these monoclonals displayed identical V_HD (24/30) and DJ_H (21/30) junctions and 21 of them were identical in the whole CDR3. These 21 independently generated identical CDR3s could be ordered in eight groups indicating that not a particular CDR3, but instead the mechanism for generating identical junctions was preserved. Two of the CDR3s in this study were found to be identical to the CDR3 of the monoclonal B1-8 produced in C57BL/6 in response to proteins bearing the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). This and other parameters support the notion that the generation of identical junctions could be independent of antigenic selection. We also report here the association between J_H usage and amino acid (aa) residues at the V_HD and DJ_H junctions. Since these MoAb were generated in response to dextran B512, immunoglobulin conformation has to be compatible with antigen binding. Nevertheless, no aa residue of CDR3 could be directly related to antigen binding. We postulate therefore, that the observed selection of CDR3s could be directed to the production of variable regions with protein configuration most suitable with immunoglobulin folding and may occur prior to antigenic selection. Selection for junctional residues in relation to J_H usage and the generation of identical CDR3s are probably different events. Possible genetic mechanisms operating for CDR3 construction and/or selection by cellular ligands are discussed.     

Positive selection focuses the VH12 B-cell repertoire towards a single B1 specificity with survival function

Summary:  B cells of varying antigen specificities are consistently present in the unmanipulated repertoire. These B cells appear to belong to the marginal zone (MZ) and B1 B-cell subsets and provide protection to the blood and lymph, respectively. Some are specific for self-antigens, suggesting that they are selected based on specificity for self but have a protective role against foreign pathogens. One of these specificities is for phosphatidylcholine (PtC). Anti-PtC B cells comprise 5–8% of the B1 repertoire and are protective against bacterial pathogens. In general, they are restricted to the expression of two V_H/Vκ combinations, V_H11/Vκ9 and V_H12/Vκ4/5H. This review focuses on the differentiation of V_H12 anti-PtC B cells. They undergo a series of positive selection events beginning at the pre-B-cell stage that enriches for those with a V_HCDR3 and L chain required for PtC binding and eliminating the majority of V_H12 B cells that lack the ability to bind PtC. Thus, positive selection focuses the V_H12 repertoire toward PtC, ensuring that anti-PtC V_H12 B cells are a significant component of the B1-cell repertoire in all individuals.     

The Primary Antibody Repertoire of κ-Deficient Mice is Characterized by Non-Stochastic Vλ1 + VH Gene Family Pairings and a Higher Degree of Self-Reactivity

We have investigated the primary antibody repertoire of genetically manipulated 129/Sv κ-deficient (JC_κD) mice, in order to understand the contributions of the λ-light chain, in the absence of an otherwise predominant κ-light chain, to the development of humoral immunity. The expression of V_λ1 gene (λ₁ and λ₃ subtypes) and the V_λ1 + V_H (J558, 36–60, V_H11 and S107) gene family associations were studied in 7.43 × 10³ mitogen-activated splenic B-lymphocyte clones of JC_κD origin. Furthermore, the functional significance of the exclusive expression of the λ-light chain, in the peripheral B-cell repertoire of JC_κD mice, was analysed by determining natural autoantibody specificities in the circulating serum immunoglobulin and the frequency of autoreactive B-lymphocyte clones in the peripheral B-lymphocyte repertoire. These experiments revealed that: first, of the three available V_λ genes at the λ locus, the V_λ1 gene is the one that is expressed most frequently (59.9%); second, non-random V_λ1 + V_H (J558, 36–60) gene family pairings occur in κ-deficient mice; and third, a higher degree of self-reactivity is generated as a result of exclusive use of the λ-light chain, as evidenced by higher levels of serum natural autoantibodies as well as a high frequency of autoreactive B-lymphocyte clones in κ-deficient (129/Sv JC_κD) mice. These observations suggest that the high murine κ/λ ratio in mice may, apart from high sequence diversity at the κ-locus, be a result of endogenous selection against the λ-light chain to restrict self-reactivity within the homeostatic threshold.     

Specific detection of blaVIM and blaIMP metallo-β-lactamase genes in a single real-time PCR

This study describes the development of a real-time PCR protocol for rapid detection of the most common bla(VIM) (bla(VIM-1), bla(VIM-2), bla(VIM-3), bla(VIM-4), bla(VIM-5), bla(VIM-6), bla(VIM-10), bla(VIM-11), bla(VIM-12)) and bla(IMP) (bla(IMP-1), bla(IMP-2), bla(IMP-6), bla(IMP-8), bla(IMP-10), bla(IMP-15), bla(IMP-19), bla(IMP-20)) genes in a single reaction. The genes were specifically detected and clearly differentiated into four groups, i.e., (i) bla(VIM-1)-like (bla(VIM-1), bla(VIM-4), bla(VIM-5), bla(VIM-12)); (ii) bla(VIM-2)-like (bla(VIM-2), bla(VIM-3), bla(VIM-6), bla(VIM-10), bla(VIM-11)); (iii) bla(IMP-1)-like (bla(IMP-1), bla(IMP-6), bla(IMP-10)); and (iv) bla(IMP-2)-like (bla(IMP-2), bla(IMP-8), bla(IMP-15), bla(IMP-19), bla(IMP-20)), by melting curve analysis of the real-time PCR products. The protocol was used to screen positive bla(VIM-1), bla(VIM-2) and bla(IMP-1) control strains, 70 Gram-negative isolates resistant to carbapenems, and 30 Gram-negative isolates susceptible to carbapenems (negative controls).     

Nucleotide Sequence Analysis of Rheumatoid Factors and Polyreactive Antibodies derived from Patients with Rheumatoid Arthritis Reveals Diverse Use of VH and VL Gene Segments and Extensive Variability in CDR-3

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogren's syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of V_H and V_L gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain 'restrictions' do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of V_H3 genes beyond that one would expect based on random utilization.     

Selection during development of VH11+ B cells: a model for natural autoantibody-producing CD5+ B cells

Summary:  Natural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from a distinctive subset of B cells, termed B-1, that express CD5. Here, we describe our studies with a representative CD5⁺ B-cell-derived natural autoantibody, the V_H11V_κ9 B-cell receptor (BCR) that binds a determinant on senescent erythrocytes. This specificity represents 5–10% of the CD5⁺ B-cell subset, with a large portion accounted for by two novel BCRs, V_H11V_κ9 and V_H12V_κ4. We have found that the development of B-lineage cells with a V_H11 rearrangement is surprisingly restricted at several crucial bottlenecks: (i) one of the most common V_H11 rearrangements generates a heavy-chain protein that only inefficiently assembles a pre-BCR, key for recombinase-activating gene downregulation/allelic exclusion and pre-B-clonal expansion; (ii) cells containing V_H11-µ chains lacking N-addition are favored for progression to the B-cell stage, eliminating most bone marrow V_H11 rearrangements; and (iii) only a subset of V_κ-light chains combine with V_H11 heavy chain to foster progression to the mature B-cell stage. Together, these constrain V_H11 generation to fetal development and may favor production of B cells with the prototype V_H11V_κ9 BCR.