V-region Genes Research Articles

Structural Basis of a Conventional Recognition Mode of IGHV1-69 Rheumatoid Factors.

Rheumatoid factors (RFs) are autoantibodies that recognize the fragment crystallizable (Fc) region of immunoglobulin G (IgG). Genetically diverse RFs are produced in rheumatoid arthritis patients; however, in hematologic diseases, such as cryoglobulinemia and B cell lymphoma, RFs from a limited combination of heavy chain V-region genes and J-region genes are produced in large quantities and forms immune complexes with IgG. These genetically limited RFs have historically been used for the immunochemical characterization of RFs. Among them, RFs derived from the heavy-chain germline gene IGHV1-69 are the most common. Recently, the crystal structure of an IGHV1-69-derived RF named YES8c was elucidated in complex with human IgG1-Fc. Based on the structure and mutant analyses, a recognition mechanism for the autoantigen (IgG-Fc) common to IGHV1-69-derived RFs was proposed. This review summarizes the immunochemical character of the IGHV1-69-derived RFs, and then focuses on the recognition mechanism of the IGHV1-69-derived RFs, referring the structural features of the IGHV1-69-derived neutralizing antibodies.

Pathogenic Citrulline-Multispecific B Cell Receptor Clades in Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen-specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity. Blood samples were obtained from patients in a University of Minnesota cohort with ACPA-positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α-enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in RA patients. Citrullinated peptide tetramer-bound B cells were subjected to flow cytometric cell sorting and single-cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality. Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V-region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline-dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen-specific mAb with cross-reactive binding profiles also promoted arthritis in mice. Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline-multispecific B cell clades with pathogenic potential.

Human antibody production in transgenic animals.

Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.

Reconstructing a B-cell clonal lineage. I. Statistical inference of unobserved ancestors

One of the key phenomena in the adaptive immune response to infection and immunization is affinity maturation, during which antibody genes are mutated and selected, typically resulting in a substantial increase in binding affinity to the eliciting antigen. Advances in technology on several fronts have made it possible to clone large numbers of heavy-chain light-chain pairs from individual B cells and thereby identify whole sets of clonally related antibodies. These collections could provide the information necessary to reconstruct their own history - the sequence of changes introduced into the lineage during the development of the clone - and to study affinity maturation in detail. But the success of such a program depends entirely on accurately inferring the founding ancestor and the other unobserved intermediates. Given a set of clonally related immunoglobulin V-region genes, the method described here allows one to compute the posterior distribution over their possible ancestors, thereby giving a thorough accounting of the uncertainty inherent in the reconstruction.I demonstrate the application of this method on heavy-chain and light-chain clones, assess the reliability of the inference, and discuss the sources of uncertainty.

Potential of Nasopharynx-associated Lymphoid Tissue for Vaccine Responses in the Airways

Nasopharynx-associated lymphoid tissue (NALT), constituting Waldeyer's ring in humans, is a unique inductive site for B-cell responses and plasma cell generation. This makes the nasal route of vaccine administration interesting for induction of mucosal and systemic antibodies. The unpaired nasopharyngeal tonsil (adenoids) and the paired palatine tonsils are prominent NALT structures, functionally similar to the paired rodent NALT structures located dorsal to the cartilaginous soft palate. Human NALT is more strategically located, however, because its elements are exposed to both airborne and alimentary antigens and have antigen-retaining crypts. It also shows similarities with lymph nodes and participates both in systemic- and secretory-type mucosal immunity. Primary follicles occur at 16 weeks of gestation, which is similar to Peyer's patches but different from rodent NALT whose organogenesis begins at birth. The formation of germinal centers reflecting B-cell activation does not take place until shortly after birth, and terminal differentiation of plasma cell can be seen about 2 weeks postnatally. Germinal centers arise in T cell-dependent B-cell responses and are associated with somatic hypermutation of Ig V-region genes. Downstream switching to various Ig isotypes also takes place, with or without concurrent expression of the J-chain gene. The J chain is a crucial part of dimeric IgA and pentameric IgM, making these Ig polymers able to interact with the epithelial polymeric Ig receptor. This interaction is central in the formation of secretory IgA and secretory IgM. Accumulating evidence suggests a major role for NALT in antibody immunity of the respiratory tract and associated glands.

Clonal relationships between thyroid‐stimulating hormone receptor‐stimulating antibodies illustrate the effect of hypermutation on antibody function

Graves' disease is characterized by production of agonist antibodies to the thyroid-stimulating hormone receptor (TSHR), but knowledge of the genetic and somatic events leading to their aberrant production is limited. We describe the genetic analysis of two monoclonal antibodies (mAbs) with thyroid-stimulating activity (TSAb) obtained from a single mouse with experimental Graves' disease. The mAbs were class switched, but used the same rearrangement of immunoglobulin heavy chain, variable region (IGHV) and immunoglobulin light chain, variable region (IGLV) germline genes, implying a clonal relationship and derivation from a single precursor B-cell clone. The IGHV-region genes of the two mAbs underwent high degrees of somatic hypermutation by sharing numerous mutations before diverging, while the IGLV genes evolved separately. Interestingly, the mutations were present in both the complementarity-determining regions (CDRs) and the framework regions. The cloned IGHV and IGLV genes were confirmed to have TSAb properties in experiments in which they were expressed as recombinant Fabs (rFabs). In other experiments, we swapped the IGLV genes with IGHV genes by constructing chimeric rFabs and showed that the chimeras retained TSAb activities, confirming the close functional relatedness of the V-region genes. Importantly, the IGLV genes in chimeric rFabs had a dominant stimulatory effect at low concentrations, while the IGHV genes had a dominant effect at higher concentrations. Our findings demonstrate that, in experimentally immunized mice, multiple pathogenic antibodies to TSHR can arise from a single clone by a series of somatic mutations in the V-region genes and may give an insight into how such antibodies develop spontaneously in autoimmune Graves' disease.

Generation of High Affinity Monoclonal Antibodies for the Prevention of HIV Infection

The most critical issue for the application of high affinity monoclonal antibodies is their creation. Here, we summarize the cellular and molecular mechanisms by which high affinity antibodies are generated, and then review the attempts of many investigators to create high affinity monoclonal antibodies against various target molecules. High affinity monoclonal antibodies are generated by one or a combination of the following three major methods. (1) The improvement of antibody affinity by introducing mutations in the immunoglobulin V-region genes by in vitro mutagenesis. (2) Screening many clones from a random combinatory repertoire of IgV-region genes using a phage library established in yeast or bacteria. (3) Attempting to introduce many somatic hypermutation of IgV-region genes. We summarize the advantages and applications of each of these methods including recent patents to facilitate informed individual choice. We also extend our review to the current creation of antibodies for HIV research.

Characterization of C-, J- and V-region-genes of the feline T-cell receptor γ

Lymphomas and leukemias are important neoplasias of domestic cats and human beings. In some cases it can be difficult to differentiate these tumors from reactive lymphatic hyperplasia. To overcome this problem, the diagnosis of lymphomas and leukemias in man is often supported by molecular techniques. To be able to establish such a technique in the cat we had to sequence the genes coding for the antigen receptors. As primary target in this study we choose the T-cell receptor γ. Using 5′-and 3′-RACE techniques we were able to clone and sequence four different V-region genes, which can be clustered into two subgroups as well as six variants of the C-region gene. Additionally, we found eight J-region genes which can be classified into three subgroups. One of the V-region genes, six of the J-region genes and all C-region genes had not been described previously. All together we analysed 112 clones containing V- and J-region genes and 31 clones containing C-region genes. Sixty-six of these clones were full length containing the L-region as well as the 5′-UTR of the feline T-cell receptor γ. The sequences of the V-region- and J-region-genes show sufficiently homologous areas that can be used to establish a small number of consensus-primers to be applied in molecular diagnosis of feline lymphomas and leukemias.

Antibody repertoire development in teleosts—a review with emphasis on salmonids and Gadus morhua L

The group of teleosts is highly diverse, comprising more than 23000 extant species. Studies of the teleost antibody repertoire have been conducted in many different species within different orders, though some species and families have been better characterised than others. The Atlantic cod ( Gadus morhua L.) and several species within the Salmoninae (e.g. Salmo salar and Oncorynchus mykiss) are among the best-studied teleosts in terms of the antibody repertoire. The estimated size of the repertoire, the organisation of immunoglobulin (IG) gene segments, the expressed IG repertoire, the IgM serum concentration, and the serum antibody responses reveal some fundamental differences between these species. The serum IgM concentration of G. morhua is some ten times higher than that of S. salar, though G. morhua is characterised as a ‘low’ (or ‘non’) responder in terms of specific antibody production. In contrast, an antibody response is readily induced in S. salar, although the response is strongly regulated by antigen induced suppression. The IGHD gene of G. morhua has a unique structure, while the IGHM and IGHD genes of S. salar have a characteristic genomic organisation in two parallel loci. In addition, salmonids, express a broad repertoire of IGH and IGI V-region gene segments, while a single V gene family dominates the expressed heavy and light chain repertoire of G. morhua. Little is known about the developing antibody repertoire during ontogeny, in different stages of B-cell maturation, or in separate B-cell subsets. Information on the establishment of the preimmune repertoire, and the possible role of environmental antigens is also sparse.

Mechanisms Controlling Termination of V-J Recombination at the TCRγ Locus: Implications for Allelic and Isotypic Exclusion of TCRγ Chains

Analyses of Vgamma-Jgamma rearrangements producing the most commonly expressed TCRgamma chains in over 200 gammadelta TCR(+) thymocytes showed that assembly of TCRgamma V-region genes display properties of allelic exclusion. Moreover, introduction of functionally rearranged TCRgamma and delta transgenes results in a profound inhibition of endogenous TCRgamma rearrangements in progenitor cells. The extent of TCRgamma rearrangements in these cells is best explained by a model in which initiation of TCRgamma rearrangements at both alleles is asymmetric, occurs at different frequencies depending on the V or J segments involved, and is terminated upon production of a functional gammadelta TCR. Approximately 10% of the cells studied contained two functional TCRgamma chains involving different V and Jgamma gene segments, thus defining a certain degree of isotypic inclusion. However, these cells are isotypically excluded at the level of cell surface expression possibly due to pairing restrictions between different TCRgamma and delta chains.

Molecular case report: IgVH analysis in acute humoral and cellular liver allograft rejection suggests a selected accumulation of effector B cells and plasma cells

Acute cellular (CLR) and humoral liver allograft rejection (HLR) are the most important immunological obstacles to successful liver transplantation. In HLR, serum antibodies play the central pathogenetic role. In CLR, CD3+ T lymphocytes drive the destructive immune response. Although CLR and HLR show different clinical symptoms and can be kept apart in most cases, they share histomorphological similarities. In CLR, hepatic B lymphocytes and plasma cells as well as B-cell-activating cytokines have recently been described, indicating that, in addition to T cells, antibody-mediated mechanisms might be involved. To analyze the impact of hepatic B cells in CLR and HLR, the immunoglobulin (Ig) variable (V)-region gene repertoire was determined from tissue of one case of CLR and one case of HLR. Complement deposits and lymphocytic infiltrate were determined using immunohistochemistry. T cells, B lymphocytes and plasma cells could be detected in both cases, whereas C3c and C4d deposits could only be demonstrated in the HLR case. The molecular analysis of 63 V-region genes showed that B cells in both allografts expressed selected V-gene repertoires. All sequences differed from the putative germline sequences by multiple somatic mutations. This suggests a clonal expansion of selected effector B cells in the portal tracts of liver allografts. Locally accumulated B cells and their antibodies might be involved in IgG-mediated complement activation in CLR and HLR.

23 The Repertoire of Igg Expressing B-Cells Develops Much Slower in Preterm Than in Term Infants

Background: Even very immature preterm infants in the first weeks of life develop a repertoire of B cells with IgG-type antigen receptors (1). Yet, the different kinetics of the postnatal evolution of B effector cells in preterm an term neonates are unknown. Hypothesis: The variable (V) region gene diversity of the IgG repertoire and the somatic diversity increases slower in preterm than in term neonates. Study design: Crossover observational study. Methods: Heavy chain V-region genes coding for the antigen binding site of the IgG-type B-cell receptor were amplified, cloned and sequenced. The diversity of the V-region gene repertoire was determined from the percentage of different amplificates in the total number of amplificates. In addition, the number of somatic mutations per V-region gene was calculated. Results: We analyzed peripheral blood samples of 8 preterm infants (GA 24–27 weeks, postnatal age 0–14 weeks) and of 16 term infants (postnatal age 0–16 weeks). In preterm infants V-region gene diversity increased slowly with postnatal age (r=0.82; p<0.000) from 30% at birth to 47% at 4 weeks, and to 82% at 12 weeks. In contrast, in term neonates already at two weeks of age diversity was higher than 80%. Furthermore the frequency of somatic mutations per V-region gene increased faster in preterm (r=0.712; p<0.004) than in term infants (r=0.83; p<0.000). Per postnatal week the accumulation of somatic mutations was three times higher in term than in preterm infants (0.12% per week vs 0.04% per week). Conclusion: In the first weeks of life both preterm and term infants respond to environmental antigens. However, preterm infants are much slower in developing an IgG repertoire. (1) Bauer K et al. Diversification of Ig heavy chain genes in human preterm neonates prematurely exposed to environmental antigens. J Immunol 2002;169:1349–1356

The different process of class switching and somatic hypermutation; a novel analysis by CD27− naive B cells

AbstractThe relationship between class switch recombination (CSR) and somatic hypermutation has been unclear. By using human CD27− naive B cells, we investigated the somatic hypermutation and producibility of immunoglobulins (Igs) that occur after CSR. Although neither adult CD27− nor cord blood B cells, which showed the unmutated Ig V-region genes, produced IgG, IgM, or IgA in response to conventional stimuli, they produced IgG and IgM but not IgA in the presence of Staphylococcus aureus Cowan strain (SAC) + interleukin-2 (IL-2) + IL-10 + anti-CD40 mAb + CD32 transfectants (CD40/CD32T). The naive B cells also produced IgE when combined with IL-4 + CD40/CD32T. In parallel with IgG production, the expression of mature γ1 and γ 2 transcripts was induced from naive B cells by the stimuli. The CD27 expression on human naive B cells was induced remarkably by CD40 signaling or B-cell receptor engagement, but somatic hypermutation could not be induced. The proliferation and differentiation into plasma cells were induced from naive B cells, whereas most of the plasma cells displayed very low levels of mutations in Ig V-region genes. CD27− naive B cells expressed activation-induced cytidine deaminase messenger RNA by the stimuli later than CD27+memory B cells. Our results demonstrate that CSR, but not noticeable somatic hypermutation, can be induced from CD27− naive B cells upon B-cell receptor engagement and CD40 signaling in cooperation with cytokines, suggesting that CSR and somatic hypermutation processes can occur independently, and the antibodies produced in this in vitro system are low-affinity antibodies.

Combinatorial library cloning of human antibodies to Streptococcus pneumoniae capsular polysaccharides: variable region primary structures and evidence for somatic mutation of Fab fragments specific for capsular serotypes 6B, 14, and 23F.

Antibodies specific for capsular polysaccharides play a central role in immunity to encapsulated Streptococcus pneumoniae, but little is known about their genetics or the variable (V) region polymorphisms that affect their protective function. To begin to address these issues, we used combinatorial library cloning to isolate pneumococcal polysaccharide (PPS)-specific Fab fragments from two vaccinated adults. We determined complete V region primary structures and performed antigen binding analyses of seven Fab fragments specific for PPS serotype 6B, 14, or 23F. Fabs were of the immunoglobulin G2 or A isotype. Several V(H)III gene segments (HV 3-7, 3-15, 3-23, and 3-11) were identified. V(L) regions were encoded by several kappa genes (KV 4-1, 3-15, 2-24, and 2D-29) and a lambda gene (LV 1-51). Deviation of the V(H) and V(L) regions from their assigned germ line counterparts indicated that they were somatically mutated. Fabs of the same serotype specificity isolated from a single individual differed in affinity, and these differences could be accounted for either by the extent of mutation among clonal relatives or by usage of different V-region genes. Thus, functionally disparate anti-PPS antibodies can arise within individuals both by activation of independent clones and by intraclonal somatic mutation. For one pair of clonally related Fabs, the more extensively mutated V(H) was associated with lower affinity for PPS 14, a result suggesting that somatic mutation could lead to diminished protective efficacy. These findings indicate that the PPS repertoire in the adult derives from memory B-cell populations that have class switched and undergone extensive hypermutation.

T-Cell-dependent antibody response to the dominant epitope of streptococcal polysaccharide, N-acetyl-glucosamine, is cross-reactive with cardiac myosin.

Autoantibodies against myosin are associated with myocarditis and rheumatic heart disease. In this study, the antigenic cross-reactivity of myosin and N-acetyl-glucosamine (GlcNAc), the dominant epitope of Group A streptococcal polysaccharide, was examined. Six antimyosin monoclonal antibodies (MAbs) derived from mice with cardiac myosin-induced myocarditis were characterized. All MAbs cross-reacted with GlcNAc, mimicking a subset of MAbs derived from rheumatic carditis patients that bind both myosin and streptococcal polysaccharide. Variable (V) region gene usage was diverse, with five of six MAb heavy-chain V regions encoded by distinct members of the J558 family and the sixth encoded by a member of the VGAM3.8 family. Light-chain V-region segments were derived from the Vk1, Vk4/5, Vk10, and Vk21 families. These antimyosin, anti-GlcNac MAbs demonstrated several T-cell-dependent features: they were predominantly immunoglobulin G, were encoded by V-region genes expressed late in development, and displayed somatic mutation. A direct correlation between the extent of somatic mutation and the affinity for myosin was observed. Affinity for GlcNAc also increased with the frequency of mutation, demonstrating that affinity maturation can occur simultaneously for both self antigen and foreign antigen. Based on these observations, we immunized mice with GlcNAc coupled to bovine serum albumin and demonstrated that a T-cell-dependent response to GlcNAc leads to antimyosin reactivity. We speculate that the pathogenic antibody response in rheumatic carditis may reflect the conversion of a T-cell-independent response to GlcNAc to a T-cell-dependent cross-reactive response to GlcNAc and myosin.

Analysis of ‘natural’ and vaccine-induced Haemophilusinfluenzae type B capsular polysaccharide serum antibodies for 3H1, a V3-23-associated idiotope

The variable (V-) region repertoire of antibodies (Abs) to Haemophilus influenzae capsular polysaccharide (Hib PS) has been extensively studied in individuals vaccinated against the microbe, but to a lesser extent in subjects who generated such Abs in response to a ‘natural’ encounter with this microbe or its antigenic mimics. To gain an insight into the repertoire of Hib PS-reactive Abs in vaccinated and non-vaccinated individuals, we used a monoclonal Ab, 3H1, which detects an idiotypic marker associated with an Ab V-region gene, V3–23. We show here that Hib PS-reactive Abs with detectable 3H1 idiotope can be quantified by an indirect inimunoezymatic assay in serum samples of non-vaccinated healthy adults as well as of recently vaccinated healthy infants. The percentage of Abs that was simultaneously Hib PS-reactive and 3H1-positive ranged widely (from 0 to 68%) among individual serum samples from both groups of subjects. No dramatic differences in the expression of 3H1 idiotope on Hib PS-reactive Abs were found between vaccinated and non-vaccinated individuals. Our results are consistent with the hypothesis that the utilization of V-region genes in Hib PS-reactive Abs that individuals generate after a ‘natural’ encounter with Hib PS or its mimics is similar to that in these Abs elicited by Hib PS conjugate vaccines.

Dynamics of One-pass Germinal Center Models: Implications for Affinity Maturation

During an immune response, the affinity of antibodies that react with the antigen that triggered the response increases with time, a phenomenon known as affinity maturation. The molecular basis of affinity maturation has been partially elucidated. It involves the somatic mutation of immunoglobulin V-region genes within antigen-stimulated germinal center B cells and the subsequent selection of high affinity variants. This mutation and selection process is extremely efficient and produces large numbers of high affinity variants. Studies of the architecture of germinal centers suggested that B cells divide in the dark zone of the germinal center, then migrate to the light zone, where they undergo selection based on their interaction with antigen-loaded follicular dendritic cells, after which they exit the germinal center through the mantle zone. Kepler and Perelson questioned this architecturally driven view of the germinal center reaction. They, as well as others, argued that the large number of point mutations observed in germinal center B cell V-region genes, frequently 5 to 10 and sometimes higher, would most likely render cells incapable of binding the antigen, if no selection step was interposed between rounds of mutations. To clarify this issue, we address the question of whether a mechanism in which mutants are generated and then selected in one pass, with no post-selection amplification, can account for the observed efficiency of affinity maturation. We analyse a set of one-pass models of the germinal center reaction, with decaying antigen, and mutation occurring at transcription or at replication. We show that under all the scenarios, the proportion of high affinity cells in the output of a germinal center varies logarithmically with their selection probability. For biologically realistic parameters, the efficiency of this process is in clear disagreement with the experimental data. Furthermore, we discuss a set of, possibly counterintuitive, more general features of one-pass selection models that follow from our analysis. We believe that these results may also provide useful intuitions in other cases where a population is subjected to selection mediated by a selective force that decays over time.

Identification of cation-independent mannose 6-phosphate receptor/insulin-like growth factor type-2 receptor as a novel target of autoantibodies.

Two human monoclonal autoantibodies, B-33 and B-24, were generated from the B cells of a patient with scleroderma. Both monoclonal antibodies (mAbs) were composed of mu and lambda chains, and recognized cytoplasmic vesicular structures by indirect immunofluorescence on Hep-2 cell line slides, although mAb B-24 showed an additional diffuse cytoplasmic staining pattern. By Western blot, mAb B-24 exhibited a polyreactive-like binding pattern, whereas mAb B-33 failed to recognize any electroblotted Hep-2 antigen. The polyreactive versus monospecific behaviour of mAbs B-24 and B-33 was further confirmed by enzyme-linked immunosorbent assay (ELISA) with a variety of foreign and autoantigens. The N-terminal sequence of a protein band isolated by affinity chromatography with mAb B-33 was identical to that of cation-independent mannose 6-phosphate receptor (CI-MPR), also known as the insulin-like growth factor type-2 receptor (IGF-2R). Immunofluorescence experiments on Hep-2 cell line slides demonstrated a striking co-localization between the staining pattern exhibited by these mAbs and the pattern obtained using a goat anti-CI-MPR serum, indicating the recognition by B-24 and B-33 of a structure located predominantly in late endosomes. Sequence analysis of the V-region gene segments of B-33 and B-24 showed both to be identical, except for the existence of a point mutation in B-33 located in the H-complementarity-determining region 3 (H-CDR3) (position 100D), which produces a non-conservative replacement of Gly by Ser. This single replacement appears to be responsible for the dramatic change in reactivity of human mAb B-33. The data shown here provide new evidence of the critical role played by the H-CDR3 region in distinguishing a polyspecific from a monospecific antibody. A population study demonstrated the existence of immunoglobulin G (IgG) reactivity against CI-MPR/IGF-2R in serum specimens from five individuals with different pathological conditions, thus indicating that this molecule is a potential target for the human autoimmune response.

Phenotypic and molecular characterization of human peripheral blood B-cell subsets with special reference to N-region addition and J kappa-usage in V kappa J kappa-joints and kappa/lambda-ratios in naive versus memory B-cell subsets to identify traces of receptor editing processes.

We identified a population of IgM+IgD+ B-cells in the peripheral blood (PB) of humans that express somatically mutated V-region genes like classical class switched or IgM-only memory B-cells and comprise around 15% of PB B-cells in adults. Mutated IgM+IgD+ cells differ from unmutated naive IgM+IgD+ cells in that they express the CD27 cell surface antigen. In addition, a very small subset of IgD-only B-cells was identified in the PB that carried rearranged VH-genes with an extremely high load of somatic mutations (up to 60 mutations per gene). A common characteristic of the four somatically mutated subsets, which altogether comprise 40% of PB B-lymphocytes in adults, is the surface expression of CD27. This antigen may thus represent a general marker for memory B-cells in the human. Somatically mutated and unmutated PB B-cell subsets were analyzed for N-region addition and J kappa-usage in V kappa J kappa-joints, and in addition for the respective kappa/lambda-ratios: N-nucleotides could be identified in a large fraction of V kappa-regions of all B-cell subsets, indicating that N-region insertion already occurs in the pre-germinal center (GC) phase of B-cell development. Both the J kappa-usage in expressed V kappa J kappa-joints and the kappa/lambda-ratio from somatically mutated B-cells do not differ substantially from those of the unmutated cells, so that in terms of these parameters, a contribution of secondary V kappa J kappa-rearrangements in shaping the memory B-cell repertoire is not detectable.

Intestinal IgA plasma cells of the B1 lineage are IL-5 dependent.

Two lineages of B cells, designated B1 and B2 cells, have been identified based upon their origins, anatomical distribution, cell surface markers, antibody repertoire and self-replenishing potential. B1 cells are maintained by self-renewal of cells resident in the peritoneal cavity (PerC) and they utilize a limited repertoire of germline V-region genes, mostly directed against ubiquitous bacterial antigens such as phosphoryl choline (PC). B2 cells are replenished from bone marrow precursors and use a larger repertoire of immunoglobulin V-region genes. Whereas most immunoglobulin A (IgA) plasma cells in the intestine derive from B2 lineage precursors in the Peyer's patch, a subpopulation of Per C-derived B1 cells populate the intestinal lamina propria where they mature into IgA plasma cells. In previous in vivo studies we have shown that whereas IgA+ B2 cells are interleukin (IL)-6 dependent, B1 cells are IL-6 independent. In view of the in vitro evidence that IL-5 is also involved in IgA expression, in the studies reported here we have used IL-5-deficient mice to evaluate the role of IL-5 in vivo in IgA expression in the gut. The results demonstrate that although total IgA cell numbers are only marginally depressed in IL-5-deficient mice, there is a marked selective depletion of IgA+ cells of the B1 lineage in the gut and a corresponding depression in the capacity of these mice to mount an intestinal response to a B1 antigen (PC) but not to a B2 antigen (oralbumin; OVA), reflecting intact B2-derived IgA cell function but a defect in the B1 cell contribution to IgA responses in IL-5 deficient mice. Collectively these data demonstrate differential cytokine regulation of subsets of IgA+ cells in the gut in that IgA+ cells of the B2 lineage are IL-6 dependent but IL-5 independent, but B1-derived IgA+ cells are IL-5 dependent and IL-6 independent.