Pairing of variable heavy and variable kappa chains in individual naive and memory B cells

A functional Ig consists of two heterodimers each of which is composed of a heavy and a light chain. Although there is increasing knowledge about the events that govern the rearrangement of the genes encoding each individual chain, only very limited information is available about the mechanisms governing the pairing of variable heavy (V(H)) and variable light (V(L)) chains. Using a single cell PCR, we were able to obtain V(H) and Vkappa chains from 144 individual human CD19+/IgM+ B cells. Pairing of specific V(H) or Vkappa families was not observed, nor was the length or the amino acid composition of the CDR3s of V(H) and Vkappa chains in individual B cells similar. Comparison of V(H) and Vkappa genes in B cells in which one or both contained evidence of somatic hypermutation with those with no mutations revealed a significant decrease in the mean length of the V(H) CDR3. Moreover, there was a significant correlation between the frequencies of mutations in V(H) and Vkappa gene pairs in individual B cells. These results indicate that Ag-mediated selection as opposed to V(H)DJ(H) recombination or subsequent Ig chain pairing tended to approximate the CDR3 lengths and the frequency of mutations of V(H) and Vkappa in individual B cells.     

Clonal expansions of B lymphocytes in old mice

The effect of age on the diversity of the murine Ig heavy chain repertoire has been studied in unimmunized C57BL/6 mice. We examined the heterogeneity of complementarity-determining region 3 (CDR3) sizes of Ig mRNA of the IgM and IgG isotypes using two VH families, VHJ558 and VHQ52, which together account for approximately 65% of the Ab repertoire. The broad and bell-shaped profiles representing the diversity of the VHJ558 family in the spleen of 2- to 6-mo-old C57BL/6 mice becomes significantly less diverse after 12 mo of age and by 18 mo of age, single CDR3 sizes that dominate the profiles can be observed in the spleens of > 85% of the mice. Readable sequences have been obtained from 40 dominant mRNA CDR3 size species indicating that they represent clonal populations of B lineage. There are no significant homologies among these sequences. Clones of B lymphocytes that express a dominant CDR3 mRNA species can also be found in the bone marrow, the mesenteric lymph nodes, and the thymus of C57BL/6 mice > 18 mo of age. Some clones of B cells can be detected in only one lymphoid compartment; others are found in two or more compartments. The splenic B cell clones in C57BL/6 mice > 18 mo of age are stable for at least 2 mo. The CDR3 mRNA species that dominate the splenic repertoire of Ig mRNA-expressing cells in vivo do not dominate the repertoire of splenic B cells activated in vitro by bacterial LPS, suggesting that they represent a modest population of B cells expressing high levels of Ig mRNA.     

Production of env-deficient rous sarcoma virus (RSV) early after infection

In this paper we document a case of peripheral T cell lymphoma (PTCL) that exhibited variable T cell histology at presentation and follow-up. Southern blot analysis for T cell receptor (TCR) and immunoglobulin (Ig) receptor gene rearrangements failed to reveal clonal T or B cell populations. TCR gamma (TCRG) and beta (TCRB) chain gene polymerase chain reaction (PCR) amplification of DNA isolated from biopsies was also consistent with polyclonal T cell populations, however Ig PCR revealed clonal Ig rearrangements in follow-up biopsies but not in the presentation biopsy. There was no histological evidence for a neoplastic B cell population in these biopsies although occasional EB virus positive blasts were present. The significance of a cryptic B cell clone is unknown but suggests a relationship with the proliferating polyclonal T cells in this case of PTCL. These data reflect the complexity of PTCL with implications for treatment and patient management.     

Detection of clonal platelet antibodies in immunologically-mediated thrombocytopenias: association with circulating clonal/oligoclonal B cells

Aware that T and B cells in autoimmune thrombocytopenia are abnormal, including the existence of clonal B cell populations, we sought to characterize this clonal phenomenon in various immunological thrombocytopenias using platelet antibody light chain analysis, flow cytometry, Southern blot analysis, and PCR. Using a monoclonal antibody-antigen capture ELISA, we analysed sera from 21 of 26 patients with autoimmune, alloimmune, or drug-induced immunological thrombocytopenia for the light chain phenotypes of their platelet antibodies. Alloantibodies and drug-dependent antibodies from four and 14 patients, respectively, were found that expressed a predominant type of light chain, suggesting that these platelet-reactive antibodies were monoclonal or oligoclonal in nature. 14 of the 26 patients were available for light chain B cell phenotyping studies. Of these 14 patients, thrombocytopenia was due to autoimmunity in two, drug-induced immunity in four, and alloimmunity in eight. We detected clonal populations of B cells in all 14 patients by flow cytometry. Although six of these latter patients possessed platelet antibodies with clonal characteristics, light chain phenotypes of antibodies in five patients were opposite to those of their B cells. Eight of these patients were further examined for immunoglobulin gene rearrangement using Southern and/or polymerase chain reaction analysis. In all eight patients we detected clonal or oligoclonal B cell populations. Only two of these patients had malignancies (chronic lymphocytic leukaemia) that would be expected to have detectable clonal B cells, and thus the mechanism for clonal expansion in the other six patients did not appear to be related to an obvious neoplastic process. Prior to these studies, detection of clonal B cells in thrombocytopenic patients without known malignancies was limited to individuals with autoimmune thrombocytopenia, prompting the speculation that this particular autoimmune disorder arises from B cell dysregulation, rather than from expansion of specific autoantibody producing B cell clones. In contrast, the current studies provide evidence that clonal B cells are common to patients with any form of immunologically-mediated thrombocytopenia. Moreover, the majority of the platelet antibodies (86%) present in these disorders exhibited monoclonal characteristics in that there was an apparent restriction in light chain usage.     

Analysis of tumor specific immunoglobulin gene rearrangement in peripheral blood B-cells of multiple myeloma patients by a PCR-SSCP method

OBJECTIVE: To analyze the clonal relationship between lymphocytes in peripheral blood (PB) and myeloma cell in bone marrow (BM) for proving the existence of circulating tumor cells in multiple myeloma (MM) patients. METHODS: Eighteen patients with MM who have no cytomorphologic plasma cells and CyIg+ cells in PB demonstrated by anti-kappa and anti delta MoAbs using ABC method were involved in the present study, including 3 cases in phases I-II and 15 cases in phase III. The complementary determining region 3 (CDR3) of immunoglobulin heavy chain (IgH) gene was amplified by polymerase chain reaction (PCR). We further analysed the single strand conformation of the PCR products by single strand conformation polymorphism (SSCP) analysis to detect the mononuclear cells in PB and BM of the patients simultaneously. RESULTS: The same PCR products of IgH-CDR3 gene with BM samples were found in PB of 11 MM patients. The same PCR products and single strand conformation in both PB and BM were found in 9 cases. CONCLUSIONS: This study has proved the presence of identical clonal malignant cells in PB and BM of MM patients. B cells are involved in the pathogenesis of MM.     

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There is still much controversy about the precursor cell type in multiple myeloma (MM). Some authors claim that it is a pre-B cell, others state that it is a memory B cell or plasmablast. We have recently shown that the VDJ region of the MM immunoglobulin heavy chain gene is somatically hypermutated and antigen selected, without intraclonal variation or evolution in time. By using a patient-specific PCR approach we have now obtained evidence that the premyeloma cell can be situated in the pre-switched B-cell compartment and that heavy chain switching can occur without further somatic mutation. Based on the MM immunoglobulin sequences derived from the bone marrow, patient-specific CDR2 and CDR3 oligonucleotides were designed. B lymphocytes were separated from plasma cells based on the expression of CD19 and HLA class II or surface bound IgM using immunomagnetic beads. The expressed Ig sequences were amplified by RT-PCR using patient specific CDR2 primers and isotype specific primers (C mu, C gamma, and C alpha). Myeloma-specific Ig sequences were detected by a myeloma-specific CDR3 probe and sequenced. In one out of five cases we found in the peripheral blood clonally related IgM and IgA sequences with the same somatic mutations as the MM-IgG sequence. In another case of an IgG MM we found in the bone marrow clonally related IgA sequences with the same somatic mutations. These findings, together with the fact that myeloma-Ig genes contain somatic mutations without intraclonal variation, suggest that the clonogenic cell in multiple myeloma can originate from a pre-switched but somatically mutated B cell.     

Nodular lymphocyte predominance Hodgkin's disease: a monoclonal or polyclonal B-cell disorder?

Nodular lymphocyte predominance Hodgkin's disease (NLPHD) is characterized by the presence of atypical putatively neoplastic cells (L & H cells) with a B-cell phenotype. A proportion of patients with NLPHD develop a simultaneous or subsequent large cell B lymphoma (LCL) that is thought to evolve directly from the L & H cells of NLPHD. However, the clonal nature of L & H cells remains controversial, and the relationship between NLPHD and complicating LCL has not been fully established. In an attempt to determine the clonality of L & H cells and to clarify the link between NLPHD and complicating LCL, we used polymerase chain reaction (PCR) to analyze 33 cases of NLPHD, including 15 cases with simultaneous or subsequent LCL, for clonal immunoglobulin (lg) heavy chain variable region (VH) gene rearrangements. PCR amplifications with consensus primers covering framework 2 or framework 3 to joining region were performed on paraffin-embedded tissue sections and, in 12 cases, on microdissection-enriched L & H cells. No clonal Ig rearrangements were detected. In eight of the 15 LCL, monoclonal IgVH regions were amplified, four of which were cloned and sequenced. Clone specific primers were designed based on the unique N region sequences. These allowed detection of LCL clones at a sensitivity up to 1,000 times greater than the consensus primers, as determined by dilution assays. However, no LCL clones were detected in the preceding NLPHD, including microdissection-enriched L & H cells. Our results suggest that populations of L & H cells do not carry monoclonal Ig rearrangements and provide no evidence for a clonal link between NLPHD and complicating LCL.     

B cell deletion, anergy, and receptor editing in "knock in" mice targeted with a germline-encoded or somatically mutated anti-DNA heavy chain

To study the relative contributions of clonal deletion, clonal anergy, and receptor editing to tolerance induction in autoreactive B cells and their dependence on B cell receptor affinity, we have constructed "knock in" mice in which germline encoded or somatically mutated, rearranged anti-DNA heavy (H) chains were targeted to the H chain locus of the mouse. The targeted H chains were expressed on the vast majority of bone marrow (BM) and splenic B cells and were capable of Ig class switching and the acquisition of somatic mutations. A quantitative analysis of B cell populations in the BM as well as of Jkappa utilization and DNA binding of hybridoma Abs suggested that immature B cell deletion and light (L) chain editing were the major mechanisms affecting tolerance. Unexpectedly, these mechanisms were less effective in targeted mice expressing the somatically mutated, anti-DNA H chain than in mice expressing the germline-encoded H chain, possibly due to the greater abundance of high affinity, anti-DNA immature B cells in the BM. Consequently, autoreactive B cells that showed features of clonal anergy could be recovered in the periphery of these mice. Our results suggest that clonal deletion and receptor editing are interrelated mechanisms that act in concert to eliminate autoreactive B cells from the immune system. Clonal anergy may serve as a back-up mechanism for central tolerance, or it may represent an intermediate step in clonal deletion.     

Stabilization and activation of p53 are regulated independently by different phosphorylation events

Rheumatoid factors (RF) recognize conformational determinants located within the Fc portion of IgG. By analyzing a panel of monoclonal rheumatoid arthritis (RA)-derived RFs, we previously demonstrated that the somatically generated light chain complementarity-determining region 3 (CDR3) contributes to RF specificity. We have now generated a panel of heavy chain mutants of the B'20 Ab, a high affinity RA-derived IgM RF. B'20 also binds avidly to protein A and weakly to ssDNA and tetanus toxoid. B9601, a RF negative Ab that is highly homologous to B'20 but does not bind any of the Ags tested, and RC1, a low affinity polyreactive RF, were used to generate heavy chain mutants with framework (FR) and CDR switches. The mutated heavy chains were cotransfected into a myeloma cell line with the germline counterpart of the B'20 light chain, and the expressed Ig tested for antigenic specificity. We show that both RF specificity and polyreactivity of B'20 is dependent on its unique heavy chain CDR3 region. Replacement with a B9601 CDR3 shortened to the same length as the B'20 CDR3, and with only 5 amino acid differences, did not restore Fc binding. Conversely, absence of protein A binding of B9601 is due to the presence of a serine residue at position 82a in the B9601 heavy chain FR3 region. Together, our data suggest that Ig gene recombination events can generate B cells with autoantibody specificities in the preimmune repertoire. Abnormal release, activation, expansion, or mutation of such cells might all contribute to the generation of a high titer RF response in patients with RA.     

Detection of clonal Hodgkin and Reed-Sternberg cells with identical somatically mutated and rearranged VH genes in different biopsies in relapsed Hodgkin's disease

Hodgkin's disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg (H-RS) cells are rare and surrounded by abundant reactive cells. Single-cell analyses showed that H-RS cells regularly bear clonal Ig gene rearrangements. However, there is little information on the clinical evolution of HD in a given patient. In this study, we used the single-cell polymerase chain reaction (PCR) to identify H-RS cells with clonal Ig gene rearrangements in biopsy specimens of patients with relapsed HD. The obtained clonal variable region heavy-chain (VH) gene rearrangements were used to construct tumor-clone-specific oligonucleotides spanning the complementarity determining region (CDR) III and somatically mutated areas in the rearranged VH gene. A number of biopsies were obtained during a period of 3 years from two HD patients. H-RS cells with identical VH rearrangements were detected in two separate infiltrated lymph nodes from one patient with nodular sclerosis HD. In a second patient with mixed cellularity HD subtype, clonal VH rearrangements with identical sequences were detected in infiltrated spleen and two lymph node biopsies. Despite the high sensitivity of the PCR method used (one clonal cell in 10(5) mononuclear cells), residual H-RS cells were not found in peripheral blood, leukapheresis material, purified CD34(+) stem cells or bone marrow. The results show that different specimens from relapsed patients suffering from classical HD carry the same clonotypic IgH rearrangements with identical somatic mutations, demonstrating the persistence and the dissemination of a clonal tumor cell population. Thus, PCR assays with CDRIII-specific probes derived from clonal H-RS cells are of clinical importance in monitoring the dissemination of HD and tumor progression and could be useful for analysis of minimal residual disease after autologous stem cell transplantation.     

Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional mu heavy chains

Ig gene rearrangements could generate V(H)-D-J(H) joining sequences that interfere with the correct folding of a mu-chain, and thus, its capability to pair with IgL chains. Surrogate light (SL) chain might be the ideal molecule to test the capacity of a mu-chain to pair with a L chain early in development, in that only pre-B cells that assemble a membrane mu-SL complex would be permitted to expand and further differentiate. We have previously identified two SL chain nonpairing V(H)81X-mu-chains with distinct V(H)-D-J(H) joining regions. Here, we show that one of these V(H)81X-mu-chains does not rescue B cell development in J(H) knock-out mice, because flow cytometric analysis of bone marrow cells from V(H)81X-mu transgenic J(H) knock-out mice revealed normal numbers of pro-B cells, but essentially no pre-B and surface IgM+ B cells. Immunoprecipitation analysis of transfected pre-B and hybridoma lines revealed that the same mu-chain fails to pair not only with SL chain but also with four distinct kappa L chains. These findings demonstrate that early pre-B cells are selected for maturation on the basis of the structure of a mu-chain, in particular its V(H)-D-J(H) joining or CDR3 sequence, and that one mechanism for this selection is the capacity of a mu-chain to assemble with SL chain. Therefore, we propose a new function of SL chain in early B cell development: SL chain is part of a quality control mechanism that tests a mu-chain for its ability to pair with conventional L chains.     

Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (VH) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.     

Development of pathogenic anti-DNA antibodies in patients with systemic lupus erythematosus

The anti-DNA response is a hallmark of systemic lupus erythematosus (SLE). The precise mechanisms leading to anti-DNA antibody (Ab) production remain to be studied. Nonetheless, it is becoming clear that anti-DNA Abs cause inflammatory lesions not only via deposition of circulating immune complexes (IC) consisting of anti-DNA Ab and antigens (Ags), but also via in situ IC formation by cationic anti-DNA Abs. It is intriguing that cationic anti-DNA Abs are encoded by a unique germline Vkappa gene, A30, which encodes an extraordinary cationic light chain, whereas somatic mutations did not induce a cationic shift of electrical charge in human lupus nephritis, suggesting that the usage of a specific germline gene may confer the cationic charge (or pathogenicity) on anti-DNA Abs and that somatic mutations induce the affinity maturation of Abs. Whether cationic anti-DNA Abs will develop depends at least partly on the presence or absence of the germline A30 gene, since patients who lack this gene in the germline Vkappa repertoire did not develop severe lupus nephritis. Receptor editing, a mechanism for changing the affinity of the B cell Ag receptor [surface immunoglobulin (Ig) receptor] to avoid self-reactivity actually seems defective in patients with SLE because normal B cells edited the A30 gene, whereas SLE B cells express A30 mRNA. Thus, along with the importance of somatic mutations, polymorphisms of Ig Vkappa locus, and genetic predisposition, the failure of receptor editing may contribute to the development of pathogenic anti-DNA responses in humans.     

Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage

The mutational pattern of IgVH and IgVL genes from synovial tissue B cell hybridomas (n = 8) of patients (n = 4) with rheumatoid arthritis (RA) was analysed, which had been produced by the electrofusion technique without prior in vitro stimulation. The molecular data were correlated with immunohistopathological data and parameters of local disease activity. The IgVH genes of the B cell hybridomas belonged to the VH3 family (DP42; DP47, n = 2; DP53), the VH1 family (DP75), the VH4 family (DP71) and the VH5 family (DP73); 7/7 IgVH genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 4/7 IgVH genes and the mean R/S ratio of all IgVH genes was 9.3 (CDR) and 1.0 (FR), suggesting an antigen-dependent selection. The IgVL/lambda genes belonged to the Vlambda1 family (DPL2, DPL5, DPL8nf), the Vlambda2 family (DPL11, n = 2) and to the Vlambda6 family (IGLV6S1); 6/6 IgVL genes showed somatic mutations, the R/S ratio (CDR) was > 3 in 3/6 IgVL genes and the mean R/S ratio of all IgVL was 3.0 (CDR) and 2.3 (FR), suggesting an antigen-dependent selection. The synovial tissue exhibited germinal centres in the follicles (3/4), with the unique distribution of Ki-M4+ follicular dendritic cells and Ki-67+ proliferating cells and a dominance of IgA+ plasma cells (3/3). All patients were positive for RF in serum and exhibited severe local symptoms (swelling 4/4; warmth 4/4; effusion 2/4), whereas the hybridomas were negative for RF. Since B cell hybridomas showed hypermutation and affinity selection for IgVH and IgVL/lambda genes and the patients exhibited severe local symptoms with germinal centres in synovial tissue, this study indicates that an antigen-driven process is behind the B cell expansion in the synovial tissue of clinically affected joints. These mutated B hybridomas were negative for RF, thus suggesting that antigens different from RF are also involved in the local B cell expansion and in the chronic synovitis of RA.     

Abnormal selection of antibody repertoires in retrovirus-induced murine AIDS

The mature B cell repertoire in the course of murine AIDS (MAIDS) was investigated. The polymerase chain reaction (PCR) was used to amplify a large diversity of rearranged Ig H chain genes in normal or infected mice, 2 and 8 wk after virus inoculation. Libraries were constructed from the polymerase chain reaction products. By sequencing V-D-J clones in these libraries and analyzing the respective complementary determining region 3 (CDR3), we have shown at 8 wk the emergence of a population of B cells with significantly less N diversity, some sequences lacking any N addition, a typical feature of fetal repertoires known for degeneracy, and autoreactivities. This decreased N diversity was not present 2 wk after inoculation and could not be related to a defect in terminal deoxytransferase expression because the steady-state levels of terminal deoxytransferase mRNA were found normal in MAIDS bone marrow 8 wk after inoculation. FACS analyses revealed a decreased number of bone marrow B cells (B220+, sIgM+) in MAIDS already present at 2 wk, suggesting an alteration in the pathway of B cell differentiation and resulting in a decrease of peripheral B cells renewal. A relative enrichment of spleen cells in long lived B cells as a consequence of this blockade may participate in the abnormal antibody repertoire selection occurring in MAIDS. These data suggest in the MAIDS pathogeny the relationship between an abnormal repertoire selection and the pathologic process.