Somatic generation of antigen-receptor diversity: a reprise.

Thirty years ago, in his inaugural article entitled 'The somatic generation of immune recognition', Niels Jerne put forward the hypothesis that the primary antigen (Ag)-receptor repertoire must be restricted towards self-Ags before Ag-mediated selection. The subsequent discovery that Ag receptors are encoded by random rearrangements between discontinuous gene segments was, apparently, at odds with this hypothesis. However, recent findings have begun to reconcile these two concepts. The recombination process is, in fact, relatively precise, exhibiting marked preferences for some gene segments over others, even among members of the same gene family. The result is an intricately patterned primary repertoire that accommodates both sets of predictions, ensuring a balance between the efficiency of selection (requiring limited diversity) and the complexity of the repertoire (requiring maximum diversity).     

Duplicated copies of the bovine JH locus contribute to the Ig repertoire

We report the cloning and analysis of a bovine JH locus comprising a DQ52 segment, six JH segments and sequence to a 5' H chain intronic enhancer. The contig was mapped to BTA 11 and evidence was found for rearrangement of the sixth JH segment at a low but detectable frequency. In contrast, the fourth segment present at a second copy of the bovine JH locus mapping to BTA 21 was found to rearrange at high-frequency, forming FR4 in the majority of bovine Ig H chains. The data thus show that bovine H chains can be generated from segments at two distinct genomic locations. Further investigation should establish if rearrangement takes place at each locus or if the participating segments are brought together from different chromosomal locations by less conventional processes (for example by gene conversion or trans-chromosomal rearrangement).     

Targeted disruption of the VH 81X gene: Influence on the B cell repertoire

We have generated a mutant mouse in which the most D-proximal V_H gene (V_H81X) has been disrupted by introducing a neomycin-resistance gene into the V_H81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the V_H81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of V_H gene rearrangements in the V_H7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of V_H7183 genes is concerned.     

Primary structure of a variable region of the V kappa I subgroup (ISE) in light chain deposition disease.

Although structural abnormalities of monoclonal immunoglobulin light chains (LC) are suspected to play a determinant role in non-amyloid light chain deposition disease (LCDD), this condition is as yet poorly documented at the molecular level, since only three sequences have been reported to date. In a case of myeloma-associated LCDD, the patient's urine contained an unglycosylated kappa Bence Jones protein made up of dimers and monomers with an apparent molecular mass of 25,000 which was assigned to the V kappa I subgroup by N-terminal amino acid sequencing. The complete variable region sequence of the monoclonal kappa chain produced by the malignant plasma cells was amplified by polymerase chain reaction (PCR) using small amounts of material obtained by bone marrow aspiration. The sequence of three independently amplified cDNA clones derived from a normal-sized kappa messenger RNA was identical to that of the urinary kappa chain. The kappa mRNA had an overall normal structure made up of a V kappa I sequence rearranged to J kappa I. Several unusual features of the variable region (the first complete V kappa I sequence reported in LCDD) included three substitutions that introduced hydrophobic residues at spatially close positions. The strategy associating N-terminal sequence determination and cDNA cloning by PCR could help in accumulating new sequence data and improving our understanding of LCDD pathogenesis.     

Polymerase chain reaction screening of immunoglobulin heavy chain and T cell receptor γ gene rearrangements: A practical approach to molecular DNA analysis of non-Hodgkin's lymphoma in a surgical pathology laboratory

Characterization of the clonality of non-Hodgkin's lymphoma (NHL) by the rearranged segments of immunoglobulin heavy chain (Ig(H)) or T cell receptor (TCR) genes is not only useful in the confirmation of the diagnosis but also for the future assessment of how a secondary lymphoma, such as a recurrence or another primary lymphoma, occurs. As a practical approach to obtaining and registering this information in a surgical pathology laboratory, FR3 and FR1 regions of Ig(H) gene and TCRgamma gene were concurrently amplified by polymerase chain reaction (PCR) using each pair of consensus primers and the same PCR protocol. Examined samples consisted of 134 primary NHL (phenotypically, 108 B cell and 26 T cell NHL), 19 reactive lymphadenopathies, as well as five secondary lymphomas whose primary lesions were included in this study. Among the primary NHL, the combined PCR analysis disclosed the clonality in 103 of 134 NHL (77%), by FR3 PCR in 77 B cell and two T cell NHL, by FR1 PCR in 59 B cell and one T cell NHL, and by TCRgamma PCR in 11 B cell and 17 of 26 T cell NHL, but in none of the reactive lymphadenopathies. Among the secondary lymphomas, the same pattern of PCR analysis was obtained in two cases (the durations between first and second lymphomas; 6 and 10 months), which suggested recurrence. In contrast, different results were obtained in three cases (17-37 months), which indicated another primary or emergence of the subclones. The results of Southern blot analysis were concordant with the PCR results of the first and the secondary lymphomas. Although the combined PCR analysis cannot replace Southern blot hybridization because of its lower detection rate, it can select those cases suitable for further Southern blot analysis thus reducing the number of unnecessary examinations by nearly 75%. This approach may also be useful in the comparative evaluation of primary and secondary lymphomas.     

Occult bone marrow involvement in patients with diffuse large B-cell lymphoma: results of a pilot study

AIMS: It is known that advanced stage disease in diffuse large B-cell lymphoma (DLBCL) confers a poor prognosis, and staging investigations are routinely performed at diagnosis, including a bone marrow (BM) biopsy. However, examination of the BM is usually limited to routine light microscopy, with the role of ancillary investigations remaining unestablished. The aim of this pilot study was to estimate the incidence of occult marrow involvement using flow cytometry, immunohistochemistry and immunoglobulin heavy chain (IgH) gene rearrangements, and to determine the impact on survival. METHODS: Clinical and pathological data were obtained on 36 patients diagnosed with DLBCL. Immunohistochemistry using CD3, CD45RO, CD20 and CD79a, and polymerase chain reaction (PCR) to look for IgH gene rearrangements were performed on formalin fixed BM trephines. RESULTS: Nine patients had morphologically apparent BM involvement. Occult marrow involvement was found in seven of 36 (19.4%) patients using the additional diagnostic modalities. When these cases were included with morphologically apparent cases in a proposed new definition of marrow involvement, the median survival of patients with BM involvement was statistically worse (p=0.02) than those without involvement. CONCLUSIONS: The results indicate that use of additional tests on BM at diagnosis can upstage disease for a proportion of patients, which appears to correlate adversely with survival.     

Insights into the regulation of immunoglobulin light chain gene rearrangements via analysis of the kappa light chain locus in lambda myeloma

Accumulating evidence indicates that B cells may undergo sequential rearrangements at the light chain loci, despite already expressing light chain receptors. This phenomenon may occur in the bone marrow and, perhaps, in germinal centers. As immunoglobulin (Ig)kappa light chains usually rearrange before Iglambda light chains, we analysed, by polymerase chain reaction, the Igkappa locus of bone marrow mononuclear cells from 29 patients with Iglambda myeloma to identify earlier recombinations in marrow plasma cells. The results demonstrated that Igkappa alleles were inactivated via the kappa-deleting element, presumably prior to V(kappa)-J(kappa) rearrangement, in many cases. Eighteen alleles (16 myeloma clones, 55%) showed V(kappa)-J(kappa) rearrangements, with increased utilization of 5' distant V(kappa) and 3' distant Jkappa gene segments (Jkappa4, 56%), an indication of multiple sequential rearrangements. In-frame, potentially functional V(kappa)-J(kappa) rearrangements were found in approximately one-third of available rearrangements (as expected by chance), each one in different myeloma clones: three were germline encoded, while one had several nucleotide substitutions, suggesting inactivation after the onset of somatic hypermutation. Three of four potentially functional V(kappa)-J(kappa)rearrangements involved V(kappa)4-1, a segment considered to be associated with autoimmunity. These findings provide insights into the regulation of light chain rearrangements and support the view that B cells may occasionally undergo sequential light chain rearrangements after the onset of somatic hypermutation.     

TET3 dioxygenase modulates gene conversion at the avian immunoglobulin variable region via demethylation of non-CpG sites in pseudogene templates

Diversification of the avian primary immunoglobulin (Ig) repertoire is achieved in developing B cells by somatic hypermutation (SHM) and gene conversion (GCV). GCV is a type of homologous recombination that unidirectionally transfers segments of Ig pseudogenes to Ig variable domains. It is regulated by epigenetic mechanisms like histone modifications, but the role of DNA methylation remains unclear. Here, we demonstrate that the chicken B-cell line DT40 lacking TET3, a member of the TET (Ten-eleven translocation) family dioxygenases that facilitate DNA demethylation, exhibited a marked reduction in GCV activity in Ig variable regions. This was accompanied by a drop in the bulk levels of 5-hydroxymethylcytosine, an oxidized derivative of 5-methylcytosine, whereas TET1-deficient or TET2-deficient DT40 strains did not exhibit such effects. Deletion of TET3 caused little effects on the expression of proteins required for SHM and GCV, but induced hypermethylation in some Ig pseudogene templates. Notably, the enhanced methylation occurred preferably on non-CpG cytosines. Disruption of both TET1 and TET3 significantly inhibited the expression of activation-induced cytidine deaminase (AID), an essential player in Ig diversification. These results uncover unique roles of TET proteins in avian Ig diversification, highlighting the potential importance of TET3 in maintaining hypomethylation In Ig pseudogenes.     

Characterization of a new V gene replacement in the absence of activation‐induced cytidine deaminase and its contribution to human B‐cell receptor diversity

In B cells, B‐cell receptor (BCR) immunoglobulin revision is a common route for modifying unwanted antibody specificities via a mechanism called VH replacement. This in vivo process, mostly affecting heavy‐chain rearrangement, involves the replacement of all or part of a previously rearranged IGHV gene with another germline IGHV gene located upstream. Two different mechanisms of IGHV replacement have been reported: type 1, involving the recombination activating genes complex and requiring a framework region 3 internal recombination signal; and type 2, involving an unidentified mechanism different from that of type 1. In the case of light‐chain loci, BCR immunoglobulin editing ensures that a second V‐J rearrangement occurs. This helps to maintain tolerance, by generating a novel BCR with a new antigenic specificity. We report that human B cells can, surprisingly, undergo type 2 replacement associated with κ light‐chain rearrangements. The de novo IGKV‐IGKJ products result from the partial replacement of a previously rearranged IGKV gene by a new germline IGKV gene, in‐frame and without deletion or addition of nucleotides. There are wrcy/rgyw motifs at the ‘IGKV donor–IGKV recipient chimera junction’ as described for type 2 IGHV replacement, but activation‐induced cytidine deaminase (AID) expression was not detected. This unusual mechanism of homologous recombination seems to be a variant of gene conversion‐like recombination, which does not require AID. The recombination phenomenon described here provides new insight into immunoglobulin locus recombination and BCR immunoglobulin repertoire diversity.     

Identification and localization of two genes on the chicken Z chromosome: implication of evolutionary conservation of the Z chromosome among avian species

A cDNA clone containing an insert of about 3.4 kb, pCIREBP, was isolated from the chicken liver cDNA library and identified as a clone for the chicken homologue of iron-responsive element-binding protein (IREBP). The deduced amino acid sequence showed 88% identity with that of the mouse IREBP and 17 out of the 20 active site residues of the pig heart mitochondrial aconitase were conserved. Another cDNA clone, pZOV3, containing an insert of about 4.5 kb was isolated from the chicken ovary cDNA library. This cDNA contained an open reading frame for 327 amino acid residues, whose sequence had partial similarity to two immunoglobulin superfamily proteins; mouse GP-70 and chicken HT7. Fluorescencein situ hybridization using corresponding genomic clones revealed that both genes are localized on the Z chromosome; the ZOV3 gene at the middle of the short arm and the IREBP gene at the boundary of heterochromatin on the long arm. Southern blot hybridization to male and female genomic DNA preparations from six species representing five avian genera suggested that these two genes are Z-linked in all the species tested.     

The interaction of flavivirus M protein with light chain Tctex-1 of human dynein plays a role in late stages of virus replication

The role of the membrane protein (prM/M) in flavivirus life cycle remains unclear. Here, we identified a cellular interactor to the 40-residue-long ectodomain of prM/M (ectoM) using a yeast two-hybrid screen against a human cDNA library and GST pull-down assays. We showed that dynein light chain Tctex-1 interacts with the ectoM of dengue 1-4, West Nile, and Japanese encephalitis flaviviruses. No interaction was found with yellow fever and tick-borne flaviviruses. This interaction is highly specific since a single amino-acid change in the ectoM abrogates the interaction with Tctex-1. To understand the role of this interaction, silencing of Tctex-1 using siRNA was performed prior to infection. A significant decrease in progeny production was observed for dengue and West Nile viruses. Silencing Tctex-1 inhibited the production of recombinant dengue subviral particles (RSPs). Thus Tctex-1 may play a role in late stages of viral replication through its interaction with the membrane protein.     

A strategy for demonstrating the clonal origin of small numbers of T lymphocytes in histopathological specimens

A strategy is described for detecting large T-cell clones among small numbers of lymphoid cells in fresh or formalin-fixed tissue samples using the polymerase chain reaction (PCR) to amplify and identify rearrangements of the V and J genes of the T-cell gamma receptor. Following hybridization of primers with gene-specific sequences at judiciously selected locations on each of the eight potentially active V and five potentially active J genes, the PCR can theoretically amplify DNA segments which span the join between rearranged V and J genes and are of approximately 384 different sizes, each segment size reflecting different gamma gene clonal rearrangements. Large monoclonal populations of T lymphocytes, indicated by excessive amounts of particular PCR segment sizes, can be further characterized by direct nucleotide sequencing of the hypervariable N regions of these segments, and the presence of such clones can be confirmed directly in tissue sections by in situ hybridization with N region-specific oligonucleotide probes.     

Tumour cell binding by a human monoclonal IgM antibody from the spleen of a non-tumour-associated patient is due to somatic mutations in the VH gene

Recently we described the occurrence of B cells producing polyspecific natural IgM with anti-tumour specificity in the spleen of non-tumour-bearing individuals as well as in fetal organisms. Immunoprecipitation and 2-D electrophoresis showed the binding of such antibodies to a 55-kD (pI 60) membrane surface glycoprotein. In vitro cultivation of human cancer cell lines in the presence of the purified IgM antibodies resulted in growth inhibition and complement-mediated cell lysis. Furthermore, the antibodies were shown to be able to induce MHC class I molecule expression on tumour cells. Because of this, a role for naturally occurring antibodies with anti-tumour specificity in preventing neoplasias had been suggested. We have constructed and expressed in Escherichia coli single-chain fragments (scFv: V_H-linker-V_L) derived from a polyspecific human monoclonal IgM autoantibody produced by a human × mouse heterohybridoma which was obtained from the spleen of an autoimmune patient. The mutated complementarity determining region (CDR) gene segments were replaced by the equivalent germ-line sequences and the CDR3 region was swapped for that from another polyspecific human natural antibody with no binding to tumours. Using these four scFv constructs for binding analyses and in vitro cultivation experiments we found: (i) scFv containing the mutated VH region of the original antibody were able to bind to tumour cells, to induce MHC class I molecule expression, and to inhibit tumour growth in a way similar to what had been described for the complete antibody; (ii) replacement of the mutated by the germ-line V_H gene independently of the CDR3 to which it had been recombined, resulted in failure to bind to tumour cells. Nevertheless, other antigens (ssDNA, tetanus toxin) were still recognized, although with lower affinity. We discuss the significance of the replacement mutations in the V_H gene CDRs. selected probably by B cell contact to an (auto)antigen, for generating a tumour binding capacity, not encoded by the germ-line gene.     

Expression of kappa chains of the Vκ21 group in Mus musculus and related species

The expression of the V_κ21 group in the genus Mus and in other rodents was studied using antisera that divide the six major V_κ21 subgroups into two serologically distinct sets, Serogroup I and Serogroup II. In NZB normal serum Serogroup I constitutes 7.3% of total κ-Ig and Serogroup II constitutes 1.3%. These determinants are expressed in the sera of other inbred strains (BALB/c, C57BL/6J, A/He, C58) and in mice (M. spretus, and subspecies of M. musculus) obtained recently from native environments, at levels that are comparable to NZB. NZB and BALB/c myeloma proteins were tested for their association with V_κ21 serogroup determinants. In the NZB collection 5.7% (of 436 examples) are in Serogroup I and 3.1% (of 477 examples) are in Serogroup II. In the BALB/c collection 3.9% (of 232 examples) are in Serogroup I and 2.5% (of 119 examples) are in Serogroup II. These values correspond closely with the percentage of total serum κ-Ig with V_κ21 determinants. Thus, NZB and BALB/c myeloma proteins appear to reflect accurately the distribution and level of normal V_κ21 Ig. The V_κ21 serogroups determinants are genus-specific because thus far they are found only in the mouse and not in rats and guinea pigs. However, within the genus Mus there are certain species which appear to lack V_κ21 entirely (inclucling M. cookii and M. pahari) and other species (inclucling M. dunni, M. caroli, M. cervicolor and M. shortridgei) that express only the Serogroup I V_κ21 antigen. Based on this distribution of V_κ21 determinants it appears that the genes cocling for Serogroup II arose from the genes encocling Serogroup I. In the normal sera of M. caroli, M. cervicolor, M. shortridgei, M. cookii and M. pahari C_κ was not detectable using a C_κ-specific antiserum.     

Polymerase chain reaction (PCR) detection of B cell clonality in Sjögren's syndrome patients: a diagnostic tool of clonal expansion

Sjögren's syndrome (SS) is an autoimmune disease characterized by clonal B cell attack of the exocrine glands and dysregulated expression of B cell‐activating factor (BAFF). Based upon the current data of increased rates of lymphoid malignancy, as non‐Hodgkin's lymphoma (NHL) is associated with SS, we propose the detection of clonal rearrangements of immunoglobulin heavy chain (IgH) gene in those patients as a predictor of malignant clonal expansion. To test our proposal, we examined the IgH clonal rearrangements in SS patients (60) and healthy control subjects (42) having chronic non‐specific sialadenitis, to determine the presence of clonal B cells in minor labial salivary glands (MSG) of SS patients. Clonal B cell expansion was assessed by two polymerase chain reaction (PCR) assays: (i) semi‐nested PCR, against sequences encoding framework regions FR3, FR2 and FR1c of the variable chain IgH gene in B cells present in the MSG infiltrate; and (ii) the PCR–enzyme‐linked immunosorbent assay (ELISA) technique, against the major and minor breakpoint regions of the Bcl‐2 oncogene coupled with a variable segment of the IgH to assess the Bcl‐2/JH translocation. When FR3, FR2 and FR1c primers were employed, we detected B cell monoclonality in 87% of the SS patients and 19% of the control subjects. The association between inflammation severity of the MSG pattern and the presence of B cell clonality was found to be statistically significant (P < 0·01). We concluded that the presence of B cell clonality in MSG can be used as a index of an altered microenvironment favouring the development of lymphoma in SS patients.     

Polymerase chain reaction in the diagnosis of T-cell lymphoma in paraffin-embedded bone marrow biopsies: a comparative study

AIMS: In routine histological analysis of bone marrow biopsies, the distinction between reactive T-cell infiltrates and T-cell lymphoma can be difficult, even with the use of extensive immunohistochemistry. The aim of this study was to evaluate the diagnostic contribution of TCR-gamma gene rearrangement analysed by PCR. METHODS and RESULTS: The samples studied consisted of 46 paraffin-embedded bone marrow biopsies (diagnosis, staging and follow-up) from 26 patients with T-cell lymphoma. The bone marrow biopsies were categorized into three groups according to the morphological and immunohistochemical results. Group 1, positive for T-cell lymphoma (24 bone marrow biopsies), group 2, suspicion of T-cell lymphoma (15 bone marrow biopsies) and group 3, negative for T-cell lymphoma (seven bone marrow biopsies). DNA could be amplified in 45/46 bone marrow biopsies (98%). Clonal rearrangement was detected in 30/45 bone marrow biopsies tested (67%) including 15/24 bone marrow biopsies (62.5%) of group 1, 11/14 (78.5%) of group 2 and 4/7 (57%) of group 3. In total, PCR analysis supported a diagnosis of T-cell lymphoma in 15/45 bone marrow biopsies (33%), in which histological and/or immunohistochemical examination provided inconclusive evidence of malignancy. CONCLUSIONS: TCR-gamma PCR is a complementary tool for the assessment of T-cell lymphoma in bone marrow biopsies. Optimal evaluation of bone marrow biopsies requires an integrative approach of all available results from morphology, immunohistochemistry, molecular biology and clinical data.     

Slow subcutaneous human intravenous immunoglobulin in the treatment of antibody immunodeficiency: Use of an old method with a new product

J Allergy Clin Immunol 1998;101:848-9.     

Additional diversity within the B70 serotype: identification of B*1580 in a Caucasoid blood donor

Anew B70 variant, B*1580, has been identified in a Swiss Caucasoid blood donor. Sequencing of exons 2 and 3 revealed that the HLA-B*1580 differs from its closest matching allele B*1518 by two substitutions in exon 3, leading to two amino acid changes, threonine to isoleucine and leucine to isoleucine at codons 94 and 95, respectively. The complete human leukocyte antigen type of the donor is: A*2402, A*2601; B*5101, B*1580; Cw*0704, Cw*1402/05; DRB1*0801, DQB1*0402. The B*1580 is a new member of the B70 cluster, characterized by the SEE motif at positions 24, 45, and 46 in the alpha1-domain. Substitutions at codons 94 and 95, also found in some B62 and B75 alleles, do not appear to interfere with the B70 serological reactivity. Based on sequence similarity and linkage with Cw*0704, the rare alleles B*1509, B*1529, B*1564, and B*1580 are possibly derived from the B*1518 haplotype.