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.     

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 Secreted Immunoglobulin Heavy Chain Genes and Immunoglobulin-Secreting Cells in Human Lymphocytes

The numbers of immunoglobulin-secreting cells in peripheral blood mononuclear cells and the expression of mRNA for secreted type of immunoglobulin heavy chains were investigated in healthy children, compared with the percentages of surface immunoglobulin-bearing cells and the expression of mRNA for membrane-bound type of immunoglobulin heavy chains, respectively. Although a difference between expression of μs mRNA and μm mRNA was unclear, μmRNA was well transcribed. The expression of γs mRNA or αs mRNA was markedly higher than that of γm mRNA or αm mRNA. However, although the detection methods could be of different sensitivities, the percentage of IgM-, IgG-, or IgA-secreting cells was markedly low, compared with the percentage of surface IgM-, IgG-, or IgA-bearing cells, respectively. Therefore, additional regulation of the pattern of the immunoglobulin gene expression may be exerted at the translational and post-translational stages.     

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.     

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.     

Stimulation of Human Fetal Lymphocytes by Lipopolysaccharide B in Culture

Cultures of Isopaque-Ficoll-isolated lymphocytes from three human sources were compared with respect to the effect of mitogens. The cell sources were maternal blood immediately after delivery, cord blood, and blood obtained by heart puncture of 10–20-week aborted fetuses. Lipopolysaccharide B (LPS) induced incorporation of tritiated thymidine, blastic transformation, and mitotic activity in cord and fetal, but not maternal, cells. The stimulation reached a maximum on days 4–8 of culture. It was stronger than the spontaneous transformation often displayed by fetal cells. If fetal cells spontaneously occurring in the blood of pregnant women were to react in 3 similar way, it should be possible to selectively stimulate the fetal cells with LPS. Such transformed fetal cells could then be isolated from cultures of maternal blood samples and used for antenatal diagnosis of fetal disease.     

Diversification of rabbit VH genes by gene-conversion-like and hypermutation mechanisms

Summary: Where, when and how does Vu diversification occur in the rabbit? Early diversification by gene-conversion and somatic hypermutation in rabbit appendix and chicken bursa of Fabricius are similar processes; the chicken bursa and the rabbit appendix have homologous functions. However, diversification in bursa starts during embryonic development whereas it starts in rabbit appendix about 2 weeks after birth in the presence of antigens and superantigens that ma) contribute to positive and negative selection, affect B-cell expansion and mold the repertoire. The biochemical steps leading to diversification by gene conversion are unknown. However elevated levels of RAD51 mRNA in both chicken bursa and young rabbit appendix suggest that repair of double stand breaks may be involved. The base changes found in expressed rabbit V_H sequences derived from rearrangement of known germline V_H genes followed by one or more gene conversions occur with frequencies similar to those found in analyses of somatic hypermutation. The Ser codons in CDR1 and CDR2 of rabbit V_Hl I genes are all AGY rather than TCN, suggesting that they may represent intrinsic hotspots for hypermutation comparable to those described in human and mouse V_Hr. Somatic hypermutation may further refine antibody affinities in rabbit germinal centers.     

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.     

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.     

Human Heavy Chain Variable Region Gene Diversity,Organization, and Expression

Elucidation of the cellular and molecular mechanisms which determine the expressed antibody repertoire remains a major challenge in immunology. Knowledge of V gene diversity, organization, and expression is important to an understanding of the formation of the antibody repertoire in normal as well as diseased states. In the last few years, great advances have been made in our understanding of the human heavy chain variable region (V_H) gene locus. In this review we present the current knowledge of V_H gene diversity, organization, and utilization in normal individuals followed by a discussion of the possible relevance of these findings to autoimmunity.     

Expression of Three Immunoglobulin Isotypes by Individual B Cells During Development: Implications for Heavy Chain Switching*

ABSTRACT: Previous studies in mice and humans have shown that the first IgA⁺ and IgG⁺ B cells appearing during ontogeny also bear IgM on the surface. The acquisition of IgD seems to occur at a later stage in differentiation. In this study we have combined autoradiography with two-color immunofluorescence to directly detect human B cells expressing three surface isotypes. We report that the vast majority of IgA⁺ cells in the neonate also bear IgM and IgD; the same holds true for IgG⁺ cells. This phenotype is peculiar of newborns, while in the adult the majority of IgA⁺ and IgG⁺ cells are single. We discuss the genetic implications of such a finding for heavy chain switching.     

Inheritance of a Large Deletion within the Human Immunoglobulin Heavy Chain Constant Region Gene Complex and Immunological Implications

A deletion of the immunoglobulin heavy chain (IgH) pseudo-gamma, gamma-2, gamma-4, epsilon, and alpha-2 constant region gene segments was found to segregate unchanged in three generations of a family. The IgG1 locus on the IgH allele carrying the deletion was expressed to the same extent as its normal counterpart. One individual who was heterozygous for the deletion had an IgG2 deficiency, whereas the four other heterozygous individuals had serum levels of IgG2 and IgG4 within the normal ranges. IgA2 levels were low or below the normal range in all heterozygous individuals. The data indicate that the expression of some Ig isotypes can be decreased by hemizygous deletions, possibly due to a lower probability for switching.     

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.     

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.     

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.     

新生儿免疫球蛋白重链可变区基因多样性分析

目的　构建不同胎龄新生儿IgH基因指纹图谱 ,探讨新生儿成熟度对IgH基因谱型多样性的影响 .方法提取 2 7～ 42周新生儿RNA、进行RT -PCR反应、6%变性聚丙烯酰胺凝胶电泳和银染显色 ,获得不同胎龄新生儿IgH基因指纹图谱 ,用Tiger凝胶图像分析仪进行曲线转化和分析 .结果　各家族指纹图谱转化后的曲线下面积积分值 ,在 2 8～ 3 2周、3 3～ 3 6周和 3 7～ 42周新生儿之间无差异 ;但胎龄 2 7周新生儿曲线下面积小于胎龄 2 8～ 42周新生儿 .结论　 2 8周可能是人类IgH基因谱型发育由不完善到趋于完善的一个转折点 ,2 8周到足月的新生儿IgH基因谱型发育趋于稳定     

Corticosteroid-Induced Modulation of Immunoglobulin Secretion by Human B Lymphocytes: Potentiation of Background Mitogenic Signals

The modulation of immunoglobulin (Ig) secretion of human peripheral blood mononuclear cells by in vitro hydrocortisone (HC) was evaluated. A marked enhancement of Ig secretion was observed in unstimulated cultures in the presence of HC as compared to cultures without HC. The augmented response was not due to a direct induction of Ig secretion by HC, but resulted from an enhancement or unveiling of the background mitogenic signal provided by the supplemental serum used in culture. HC-induced augmentation of Ig secretion was only seen in unstimulated cultures performed in fetal calf serum (FCS) which is known to possess mitogenic properties. In contrast, HC had no significant effect on Ig secretion of cultures performed in human serum, which provides little or no background mitogenic signal. On the other hand, no enhancement of Ig Secretion by HC was seen in cultures maximally stimulated with pokeweed mitogen regardless of whether FCS or human A serum was used. The mechanisms of this modulation of Ig secretion are unclear at present and may include a synergy between corticasteroids and background mitogenic signals (such as FCS) indirectly triggering B cells and/or the dampening of a negative immunoregulatory effect of an accessory cell.