Molecular Analysis of Immunoglobulin Variable Genes in HIV-Related Non-Hodgkin Lymphoma: Implications for Disease Pathogenesis and Histogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 330-330
Author(s):  
Daniela Capello ◽  
Eva Berra ◽  
Michaela Cerri ◽  
Annunziata Gloghini ◽  
Davide Rossi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Molecular Analysis ◽  
Light Chain ◽  
Mutation Frequency ◽  
Aggressive Lymphoma ◽  
Antigen Binding ◽  
High Affinity ◽  
Aggressive Lymphomas ◽  
Selection For

Abstract Molecular analysis of immunoglobulin variable region (IGV) genes can provide insights into the histogenesis and clonal history of B-cell NHL. We investigated 67 HIV-related NHL (HIV-NHL), including 30 HIV-diffuse large B cell lymphomas (DLBCL), 21 HIV-Burkitt lymphoma (BL), 6 HIV-primary effusion lymphomas (PEL) and 10 HIV-plasmablastic lymphomas (PBL) for usage, mutation frequency and intratumoral heterogeneity of clonal IGV rearrangements as well as mutation profile and CDR3 structure of IGHV, IGKV and IGLV genes. Results were compared to 200 IGV rearrangements from aggressive lymphomas of immunocompetent hosts and to the normal B-cell repertoire. We identified a total of 65 IGHV and 56 IGV light chain rearrangements in HIV-NHL. A functional IGHV rearrangement was found in 60/67 (90%) cases, a functional IGKV chain rearrangement in 17/38 (44.7%) cases and a functional IGLV rearrangement in 21/38 cases (55.3%). Fifty-three of 60 HIV-NHL (88.3%) showed somatic hypermutation in IGHV and/or IGV light chain genes. The average mutation frequency was 9.42% (median 7.50%, range 2.04%–23.3%) for IGHV genes and 5.42% (median 4.20%, range 2.01%–12.5%) for IGV light chain genes. IGV germline rearrangements selectively associated with HIV-PBL (p<0.001). Among mutated cases, average mutation frequencies did not differ among HIV-NHL groups. HIV-NHL showed a significant overrepresentation of the IGHV4 family (28/60; 46.6%) and a significant underrepresentation of IGHV3 family (18/60, 30.0%) compared to aggressive lymphomas of immunocompetent hosts (p<0.05) and to normal B-cells (p<0.05). IGHV4–34 was the IGHV gene most frequently rearranged (17/60; 28.3%) and was overrepresented in HIV-NHL versus aggressive lymphoma of immunocompetent hosts (17%; p<0.03) and normal B-cells (4%; p<0.001). IGHV4-34 expressing cells preferentially associated with lambda chain rearrangements (70%). The IGKV4-1 gene was the IGKV segment most frequently rearranged (6/17; 35.3%) and its usage was biased in HIV-NHL compared to normal B-cells (5.30; p<0.001). The single IGLV gene most frequently encountered was IGLV1-44 (6/17; 35,3%). Distribution of replacement and silent mutations in IGHV sequences showed tendency to conserve FR sequences and maintain antigen binding in 34/52 (65.4%) cases. A higher than expected number of CDR replacement mutations, suggesting selection for high affinity antigen binding, occurred in 17/52 (32.7%) cases. Analysis of intraclonal heterogeneity showed the presence of ongoing mutations in only 1 HIV-BL and 2 HIV-DLBCL. Implications of these data are multifold. First, most HIV-NHL derive from B-cells persistently subjected to GC reaction, suggesting a potential role for antigen stimulation in the pathogenesis of these lymphomas. This hypothesis is supported by the finding of antigen binding preservation in the majority of HIV-NHL and selection for high affinity antigen binding in a fraction of cases. Second, the preferential usage of IGHV4-34 and IGKV4-1 genes in a fraction of HIV-NHL may suggest a role for stimulation of pre-neoplastic B-cells with polyreactive and/or autoreactive antigens. Finally, at variance with NHL of immucompetent hosts, the presence of intraclonal heterogeneity is a rare finding in HIV-NHL, suggesting a derivation from B-cells that have concluded the GC-reaction.

scholarly journals Light chain skewing in autoantibodies and B-cell receptors of the citrullinated antigen-binding B-cell response in rheumatoid arthritis

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0247847
Author(s):  
Linda M. Slot ◽  
Rochelle D. Vergroesen ◽  
Priscilla F. Kerkman ◽  
Ellen Staudinger ◽  
Sanne Reijm ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Cell Response ◽  
World Population ◽  
Antigen Binding ◽  
Cell Repertoire ◽  
Citrullinated Antigen ◽  
B Cell Response

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting 1% of the world population. RA is associated with the presence of autoantibodies, of which anti-citrullinated protein antibodies (ACPA) are most prominent. ACPA are produced by citrullinated antigen-binding B cells that have presumably survived tolerance checkpoints. So far, it is unclear how and when such autoreactive B cells emerge. Light chain (LC) rearrangement and mutation rates can be informative with regard to selection steps during B-cell development. Therefore, we studied LC characteristics of ACPA-expressing B cells and secreted ACPA with the aim to better understand the development of this disease-specific, autoreactive B-cell response. Paired ACPA-IgG and ACPA-depleted IgG were isolated from serum (n = 87) and synovial fluid (SF, n = 21) of patients with established RA. We determined the LC composition for each fraction by ELISA using kappa(Igκ)- and lambda(Igλ) LC-specific antibodies. Cellular LC expression was determined using flow cytometry. In addition, we used a B-cell receptor (BCR)-specific PCR to obtain LC variable region sequences of citrullinated antigen- and tetanus toxoid (TT)-binding B cells. In serum, we observed an increased frequency of lambda LC in ACPA-IgG (1.64:1) compared to control IgG (2.03:1) and to the κ/λ ratio reported for healthy individuals (2:1). A similar trend towards higher frequencies of lambda LCs was observed for ACPA-IgG in SF (1.84:1). Additionally, the percentage of Igλ-expressing B cells was higher for citrullinated antigen-binding B cells (51%) compared to TT-specific (43%) and total CD19+CD20+ B cells (36%). Moreover, an increased Igλ percentage was observed in BCR-sequences derived from ACPA-expressing (49%) compared to TT-specific B cells (34%). Taken together, we report an enhanced frequency of lambda LCs in the secreted ACPA-IgG repertoire and, on the cellular level, in BCR sequences of ACPA-expressing B cells compared to control. This skewing in the autoreactive B-cell repertoire could reflect a process of active selection.

The Pattern of Immunoglobulin Variable Genes Indicates That Most Post-Transplant Lymphoproliferative Disorders Derive from B-Cells That Have Failed the Germinal Center Reaction.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 702-702 ◽  
Author(s):  
Daniela Capello ◽  
Michaela Cerri ◽  
Eva Berra ◽  
Davide Rossi ◽  
Enrica Morra ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Germinal Center ◽  
Mutation Frequency ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Lymphoproliferative Disorders ◽  
Antigen Binding ◽  
Germinal Center Reaction

Abstract Monoclonal posttransplant lymphoproliferative disorders (PTLD) comprise polymorphic PTLD (P-PTLD), diffuse large B cell lymphoma (DLBCL) and Burkitt/Burkitt-like lymphoma (BL/BLL). Recent studies have elucidated the germinal center-origin (GC) of PTLD, yet a detailed analysis of IgVH and IgVL chain genes is lacking. We investigated 54 PTLD, including 16 P-PTLD, 35 DLBCL and 3 BL/BLL for usage, mutation frequency and mutation pattern of clonal IgVH and IgVL rearrangements. A functional IgVH rearrangement was identified in 47/54 (87.0%) cases. Four cases yeilded only an out of frame IgVH rearrangement or a rearrangement rendered nonfunctional by crippling mutations. Three cases showed hybrid Ig VDJ rearrangements: two cases with a V-V fusion rearrangement and one case with a J-J fusion rearrangement, suggesting a failed attempt of heavy chain receptor revision in GC reaction. Despite extensive investigation by multiple PCR strategies, a functional IgVL rearrangement was found in only 25/54 (46.3%) cases. Eleven out of 25 (44.0%) cases harbored IgV kappa rearrangements and 12/25 (48.0%) cases harbored functional IgV lambda rearrangements. Two cases showed the presence of both IgV kappa and IgV lambda functional rearrangements. Among PTLD carrying solely nonfunctional IgVL rearrangements, 7/54 (13.0%) cases showed a crippled rearrangement and 11/54 (20.4%) cases harbored only an out of frame and/or inactivated IgV kappa gene. Inactivation occurred by rearrangement involving the kappa-deleting element (KDE). In 11/54 (20.4%) cases, no IgVL rearrangement was identified. Overall, only 23/54 (42.6%) PTLD displayed both a functional IgVH and a functional IgVL rearrangement. Analysis of somatic hypermutation showed the presence of somatically hypermutated IgVH and/or IgVL genes in 45/54 PTLD (83.3%). Conversely, IgV rearrangements of 9/54 (16.6%) PTLD were in germline configuration, suggesting a derivation from B-cells that have not experienced the GC-reaction. Among mutated cases, the average mutation frequency was 8.83% (median 8.43%, range 2.10%–24.1%) for IgVH genes and 7.37% (median 6.71%, range 2.30%–26.0%) for IgVL genes. Thirty-two cases (71.1%) showed highly mutated (mutation frequency >6%) IgVH and/or IgVL genes, a condition that, in normal B-cell, results in lower affinity for antigen and apoptosis. Analysis of the distribution of replacement and silent mutations in functional IgVH and/or IgVL sequences showed tendency to conserve FR sequences and maintain antigen binding in 20/34 (58.8%) cases. Selection for high affinity antigen binding occurred in 14/34 (41.2%) cases. Our data suggest that most PTLD arise from B-cells that have experienced the GC-reaction and frequently display impaired B-cell receptors (BCR). Since a functional receptor is required for normal B-cell survival during GC transit, PTLD development may implicate rescue from apoptosis and expansion of B-cells that have failed the GC-reaction. Notably, virtually all PTLD with nonfunctional IgVH and/or IgVL rearrangements carried EBV infection, which may promote cell survival.

The expression of Vpre-B/λ5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice

Cell ◽  
1994 ◽  
Vol 77 (1) ◽  
pp. 133-143 ◽  
Author(s):  
Hajime Karasuyama ◽  
Antonius Rolink ◽  
Yoichi Shinkal ◽  
Faith Young ◽  
Frederick W. Alt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Mutant Mice ◽  
Deficient Mutant ◽  
Surrogate Light Chain ◽  
Bone Marrow Precursor ◽  
Precursor B Cells

scholarly journals Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244158
Author(s):  
WeiYu Lin ◽  
Wei-Ching Liang ◽  
Trung Nguy ◽  
Mauricio Maia ◽  
Tulika Tyagi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Sorting ◽  
Assay Development ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fab Fragment ◽  
Variable Regions ◽  
High Affinity ◽  
Clinical Assay ◽  
Cloning Technology

The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic antibodies with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture and cloning technology by immunizing rabbits with therapeutic drug Fab fragment and sorting complementarity determining regions (CDRs) specific B cells using designed framework control as a negative gate to exclude non-CDRs-specific B cells. The supernatants of cultured B cells were subsequently screened for binding to drug-molecule by enzyme-linked immunosorbent assay and the positive hits of B cell lysates were selected for cloning of their immunoglobulin G (IgG) variable regions. The recombinant monoclonal anti-IDs generated with this method have high affinity and specificity with broad epitope coverage and different types. The recombinant anti-IDs were available for assay development to support pharmacokinetic (PK) and immunogenicity studies within 12 weeks from the start of rabbit immunization. Using this novel rapid and efficient in-house approach we have generated a large panel of anti-IDs against a series of 11 therapeutic antibody drugs and successfully applied them to the clinical assay development.

scholarly journals Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development

1997 ◽  
Vol 185 (4) ◽  
pp. 609-620 ◽  
Author(s):  
Andrei Constantinescu ◽  
Mark S. Schlissel
Keyword(s):  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Light Chain ◽  
Immunoglobulin Heavy Chain ◽  
Cell Development ◽  
B Cell Development ◽  
Allelic Exclusion ◽  
Chain Locus ◽  
Heavy Chain Locus

The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an Eμ-myc transgene.

scholarly journals gamma-c gene transfer into SCID X1 patients' B-cell lines restores normal high-affinity interleukin-2 receptor expression and function

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3108-3116 ◽  
Author(s):  
H Hacein-Bey ◽  
M Cavazzana-Calvo ◽  
F Le Deist ◽  
A Dautry-Varsat ◽  
C Hivroz ◽  
...  
Keyword(s):  
B Cells ◽  
Gene Transfer ◽  
B Cell ◽  
Cell Lines ◽  
Killer Cell ◽  
Interleukin 2 ◽  
Receptor Expression ◽  
Chain Gene ◽  
High Affinity ◽  
And Function

SCID X1 is characterized by faulty T-cell and natural killer cell differentiation caused by mutation of the gamma-c chain gene encoding a number of multiple cytokine receptors (interleukin-2 [IL-2], IL-4, IL- 7, IL-9, and IL-15 receptors). To assess the feasibility of inducing long-term expression and function of the gamma-c chain, Epstein-Barr virus (EBV)-transformed B-cell lines from two patients with SCID X1 were transduced with a Moloney-derived retroviral vector containing the gamma-c chain cDNA. The viral LTR was used as the promoter. Immediately after two cycles of coculture with the psi-crip clone producing the MFG(B2)-gamma-c cDNA vector, gamma-c expression, assessed by detection of the mRNA and membrane protein expression, was found in 15% to 20% of cells. The degree of membrane expression was similar to that in control EBV-B cells. Expression increased steadily over 6 months, becoming detectable in 100% of cells, and remained stable thereafter for a total of 9 months, reflecting positive selection of transduced cells. A study of provirus integration sites showed multiple integration. The expressed gamma-c was functional, because it restored high-affinity IL- 2 receptor binding, IL-2 endocytosis, and IL-2-triggered phosphorylation of JAK-3 tyrosine kinase. Similar results were obtained with the two B-cell lines. These results show that efficient gamma-c gene transfer into B-cells lacking functional gamma-c is feasible and results in strong and stable expression of a functional gamma-c chain, apparently conferring a selective growth advantage in culture. Further in vitro studies of gamma-c gene transfer into gamma-c- hematopoietic progenitors are being conducted to assess the feasibility of correcting lymphocyte differentiation defects.

GA101, a Novel Humanized Type II CD20 Antibody with Glycoengineered Fc and Enhanced Cell Death Induction, Exhibits Superior Anti-Tumor Efficacy and Superior Tissue B Cell Depletion In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2348-2348 ◽  
Author(s):  
Pablo Umana ◽  
Moessner Ekkehard ◽  
Bruenker Peter ◽  
Klingner Gabriele ◽  
Puentener Ursula ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Lymph Nodes ◽  
B Cell ◽  
Cell Depletion ◽  
Type Ii ◽  
B Cell Depletion ◽  
Cynomolgus Monkeys ◽  
High Affinity ◽  
Cd20 Antibody

Abstract GA101 is a novel monoclonal antibody of IgG1 type which binds with high affinity and selectivity to the extracellular domain of the human CD20 antigen on B cells. In contrast to rituximab which is a chimeric antibody and recognizes a type I epitope, GA101 is humanized and recognizes a type II epitope which is also localized in the extracellular loop of CD20. The recognition of the type II epitope together with a modification of the elbow hinge region results in enhanced direct non-caspase dependent cell death induction, and concomitant reduction in CDC upon binding to CD20. In addition, using GlycoMab technology, the Fc-region of GA101 was glycoengineered to contain bisected, afucosylated carbohydrates. As a result GA101 has increased affinity for the low and high affinity FcγRIIIa receptor expressed on natural killer cells, macrophages and monocytes. Consequently, GA101 mediated a 5–50 fold enhanced induction of effector cell mediated ADCC. In B-cell depletion assays with whole blood from healthy donors, an assay combining all mechanisms of action, GA101 was significantly more potent and efficacious in depleting B cells than rituximab. In preclinical NHL testing these properties translated into superior anti-tumoral efficacy of GA101 in direct comparison to rituximab against a number of aggressive NHL xenograft models. In cynomolgus monkeys the induction of B cell depletion mediated by GA101 and subsequent B cell recovery were investigated. GA101 induced complete, rapid and long-lasting B cell depletion both in peripheral blood and in lymphoid tissue e.g. spleen and lymph nodes. The efficacy of GA101 (10 and 30 mg/kg) at depleting B cells in different lymphoid tissues of cynomolgus monkeys was compared with that of rituximab (10 mg/kg) following 2 i.v. doses administered on days 0 and 7. Notably, GA101 showed statistically superior depletion of total B cells from lymph nodes compared to Rituximab from day 9 to 35 onwards with B cell numbers decreased by over 95%. These results demonstrated that GA101 was more efficacious at depleting B cells from lymph nodes and spleen of cynomolgus monkeys compared to rituximab. Compared to existing antibodies, GA101 constitutes the first type II CD20 antibody engineered for increased ADCC with significantly enhanced efficacy in a variety of preclinical models. Based on these data it is assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

Characterization of Antibodies Direct against the Idiotypic VK3 Chain of HCV-Related Type-II Mixed Cryoglobulinemia and B-Cell Proliferations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4988-4988
Author(s):  
Valli De Re ◽  
Maria Paola Simula ◽  
Alessandro Pavan ◽  
Dolores Marin ◽  
Laura Caggiari ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
B Cells ◽  
Western Blot ◽  
B Cell ◽  
Rheumatoid Factor ◽  
Light Chain ◽  
B Cell Lymphoma ◽  
Type Ii ◽  
Cell Proliferations

Abstract Autoimmune type-II cryoglobulinemia (MC) is sustained by clonal/oligoclonal B-cell populations such the disease may be considered an “indolent B-cell lymphoma (NHL) “ and may favor overt NHL development. HCV-antigen driven mechanism induces B-cell proliferations. Clonal B-cells demonstrate a restricted used of variable genes to construct the B-cell receptor (BCR) and a homology between BCR functional regions and autoimmune rheumatoid factor (RF) activity. We underline the BCR unique repertoire with frequent rheumatoid factor activity also in other autoimmune disorders associated to NHL development as Sjogren’s syndrome and rheumatoid arthritis. All together these BCRs are characterized by their high degree of idiotypic (Id) cross reactivity. Particularly the K chain V3-20/15 is frequently found in subject with positive HCV antibody. Id is a clonamarker expressedby B cells, thus is an ideal target for immunotherapy. The evidence of few Id presented in the NHL subgroup above reported constitute the rational for the development of antibodies and recombinant proteins that use shared Id among different NHL. Five monoclonal antibodies have been produced in our laboratory toward the VK3-20 region of a subject HCV+ with NHL. Epitopes recognized has been performed using epitope excision approach. A fine determination of the antibodies activities toward specific amino acids, possibly common to different individuals, are in progress. Monoclonal antibodies reactivity has been tested in vitro in ELISA, western blot and cytofluorimetry. Apoptosis and SyK/ERK phosphorylation pathways induced following BCR cross-linking of antiId murine IgG will be performed. All the antibodies were reactive in Elisa against the VK3-Ig light chain. Two of these antibodies showed a reactivity against the light chain of cryoprecipitated immunocomplexes from several patients in western blot, and by cytofluorimter they recognised two lymphoma B-cell lines.

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