Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.
- MeSH
- B-lymfocyty * imunologie MeSH
- CD antigeny imunologie MeSH
- genomika MeSH
- geny pro imunoglobuliny * MeSH
- imunoglobulinový receptor leukocytů B1 imunologie MeSH
- inzerční mutageneze MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- Plasmodium falciparum MeSH
- protilátky protozoální genetika MeSH
- receptory antigenů T-buněk genetika MeSH
- receptory imunologické imunologie MeSH
- rozmanitost protilátek * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.
- MeSH
- antigeny CD20 imunologie MeSH
- chromatografie kapalinová MeSH
- imunoglobulin G chemie MeSH
- kinetika MeSH
- lehké řetězce imunoglobulinů genetika metabolismus MeSH
- lidé MeSH
- monoklonální protilátky chemie genetika MeSH
- nádorové buněčné linie MeSH
- peptidová knihovna MeSH
- psi MeSH
- rekombinantní fúzní proteiny MeSH
- sekvence aminokyselin MeSH
- tandemová hmotnostní spektrometrie MeSH
- těžké řetězce imunoglobulinů genetika metabolismus MeSH
- vazebná místa protilátek MeSH
- vodík/deuteriová výměna a hmotnostní spektrometrie * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- antigeny nádorové genetika MeSH
- chromozomální delece * MeSH
- chronická lymfatická leukemie diagnóza genetika MeSH
- genetické lokusy * MeSH
- imunoglobuliny - lambda-řetězce genetika MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- lidské chromozomy, pár 22 * MeSH
- nádorové biomarkery MeSH
- represorové proteiny genetika MeSH
- srovnávací genomová hybridizace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
Purpose: Unmutated (UM) immunoglobulin heavy chain variable region (IgHV) status or IgHV3-21 gene usage is associated with poor prognosis in chronic lymphocytic leukemia (CLL) patients. Interestingly, IgHV3-21 is often co-expressed with light chain IgLV3-21, which is potentially able to trigger cell-autonomous BCR-mediated signaling. However, this light chain has never been characterized independently of the heavy chain IgHV3-21.Experimental Design: We performed total RNA sequencing in 32 patients and investigated IgLV3-21 prognostic impact in terms of treatment-free survival (TFS) and overall survival (OS) in 3 other independent cohorts for a total of 813 patients. IgLV3-21 presence was tested by real-time PCR and confirmed by Sanger sequencing.Results: Using total RNA sequencing to characterize 32 patients with high-risk CLL, we found a high frequency (28%) of IgLV3-21 rearrangements. Gene set enrichment analysis revealed that these patients express higher levels of genes responsible for ribosome biogenesis and translation initiation (P < 0.0001) as well as MYC target genes (P = 0.0003). Patients with IgLV3-21 rearrangements displayed a significantly shorter TFS and OS (P < 0.05), particularly those with IgHV mutation. In each of the three independent validation cohorts, we showed that IgLV3-21 rearrangements-similar to UM IgHV status-conferred poor prognosis compared with mutated IgHV (P < 0.0001). Importantly, we confirmed by multivariate analysis that this was independent of IgHV mutational status or subset #2 stereotyped receptor (P < 0.0001).Conclusions: We have demonstrated for the first time that a light chain can affect CLL prognosis and that IgLV3-21 light chain usage defines a new subgroup of CLL patients with poor prognosis. Clin Cancer Res; 24(20); 5048-57. ©2018 AACR.
- MeSH
- chromozomální aberace MeSH
- chronická lymfatická leukemie diagnóza genetika mortalita MeSH
- genová ontologie MeSH
- klinické zkoušky jako téma MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- mutace MeSH
- nádorové biomarkery MeSH
- peptidy genetika MeSH
- prognóza MeSH
- regulace genové exprese u leukemie MeSH
- sekvenční analýza DNA MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- variabilní oblast imunoglobulinu genetika MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
The current mammalian paradigm states that 1) rearrangements in the IgH locus precede those in IgL loci, 2) IgLλ genes rearrange only when IgLκ genes are consumed, and 3) the surrogate L chain is necessary for selection of productive IgH gene rearrangements. We show in swine that IgL rearrangements precede IgH gene rearrangements, resulting in the expression of naked IgL on a surface of precursor B cells. Findings also suggest that there is no dependency on the surrogate L chain, and thus the authentic IgL proteins may be used for selection of the IgH repertoire. Although rearrangement starts with IgLκ genes, it is rapidly replaced by IgLλ rearrangement. Fast replacement is characterized by occurrence of IgLλ(lo)IgLκ(lo) dual-expressing precursors in which IgLκ expression is a remnant of a previous translation. Most IgLκ(+) B cells are then generated later, indicating that there are two waves of IgLκ synthesis in different developmental stages with IgLλ gene rearrangements in between. In the absence of stromal cells, the stepwise order of rearrangements is blocked so that IgLλ gene rearrangements predominate in early B cell development. To our knowledge, this is the first evidence that some mammals can use an inverted order of Ig loci rearrangement. Moreover, a situation in which the generation of BCR-bearing IgLκ is delayed until after IgLλ becomes the dominant isotype may help explain the extreme deviations in the IgLκ/IgLλ ratios among mammals.
- MeSH
- B-lymfocyty imunologie fyziologie MeSH
- geny pro imunoglobuliny MeSH
- imunoglobuliny - kappa-řetězce genetika MeSH
- lehké řetězce imunoglobulinů genetika imunologie MeSH
- prasata MeSH
- přestavba genů pro lehké řetězce B-lymfocytů * MeSH
- přestavba genů pro těžké řetězce B-lymfocytů MeSH
- receptory antigenů B-buněk genetika imunologie MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.
- MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- mutace MeSH
- myši MeSH
- řízení kvality MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA metody MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.
- MeSH
- antigeny CD20 * chemie genetika imunologie metabolismus MeSH
- buněčné linie MeSH
- epitopy imunologie MeSH
- exprese genu MeSH
- hybridomy imunologie metabolismus MeSH
- jednořetězcové protilátky imunologie farmakologie MeSH
- klonování DNA MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- peptidová knihovna MeSH
- peptidy chemie metabolismus MeSH
- psi MeSH
- rekombinantní fúzní proteiny farmakologie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- specificita protilátek imunologie MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- tvorba protilátek imunologie MeSH
- vazba proteinů imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Malignant plasma cells in multiple myeloma (MM) are frequently characterized by complex karyotypes and chromosome instability. These cytogenetic changes are considered important prognostic indicators in MM patients. We have studied samples from 68 patients with newly diagnosed MM who were treated with high-dose chemotherapy and autologous stem cell transplantation. G-banding revealed abnormal karyotypes in 14 of 55 patients (25%) who had informative conventional cytogenetics. The combination of cytoplasmic immunoglobulin light chain labeling and interphase fluorescent in situ hybridization (cIg-FISH) revealed the presence of genetic aberrations in 53 of 68 patients (78%). Chromosome 13 abnormalities were found in 33 patients (50%) and IgH rearrangements in 36 patients (56.25%). In IgH positive patients we performed subsequent examinations of IgH affecting translocations t(4;14) and t(11;14) and we found translocation t(11;14) in 8 patients (12.5%) and t(4;14) in 10 patients (15.5%). The occurrences of others chromosomal abnormalities with known prognostic impact in MM were as follows: del(17)(p13) was present in 5 patients (9.8%) and gain 1q21 in 14 patients (36%). Analysis of survival of patients with different cytogenetic abnormalities revealed shorter overall survival (OS) in patients with IgH rearrangements (p=0.020) and trend to shorter OS in patients with gain 1q21 (p=0.064), respectively. Remarkably, patients with two or more aberrations had significantly shorter overall survival (p=0.001), time to progression (p=0.036) and progression free survival (p=0.008). Our results show a high incidence of chromosomal abnormalities in MM patients and confirm the prognostic impact of selected chromosomal aberrations as well as cumulative effect of multiple cytogenetic changes occurring simultaneously.
- MeSH
- autologní transplantace MeSH
- chromozomální aberace MeSH
- cytogenetické vyšetření MeSH
- dospělí MeSH
- hybridizace in situ fluorescenční MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- lidské chromozomy, pár 11 genetika MeSH
- lidské chromozomy, pár 14 genetika MeSH
- lidské chromozomy, pár 4 genetika MeSH
- míra přežití MeSH
- mnohočetný myelom genetika patologie terapie MeSH
- prognóza MeSH
- senioři MeSH
- staging nádorů MeSH
- translokace genetická genetika MeSH
- transplantace kmenových buněk MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
