Light Chain Repertoire Research Articles

Generation of Chicken Antibody Libraries and Selection of Antigen Binders.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.

Exploring the sheep (Ovis aries) immunoglobulin repertoire by next generation sequencing.

Next-generation sequencing (NGS) has revolutionized the way we determine the antibody repertoires encoded by B cells in the blood or lymphoid organs and transformed our understanding of adaptive immune responses in many species. Sheep (Ovis aries) have been widely used as a host for therapeutic antibody production since the early 1980s, however, little is known about their immune repertoires or immunological processes affecting the antibody generation. The objective of this study was to employ NGS for a comprehensive analysis of immunoglobulin heavy and light chain repertoires in four healthy sheep. We obtained >90% complete antibody sequences and nearly 130,000, 48,000 and 218,000 unique CDR3 reads for the heavy chain (IGH), kappa chain (IGK), and lambda chain (IGL) loci, respectively. Consistent with other species, we observed biased usage of germline variable (V), diversity (D) and joining (J) genes in the heavy and kappa loci, but not in the lambda loci. Moreover, the enormous diversity of CDR3 sequences was observed through sequence clustering and convergent recombination. These data will build a foundation for future studies investigating immune repertoires in health and disease as well as contribute to further refinement of ovine-derived therapeutic antibody drugs.

Bone Marrow-Free Sequencing of M Protein Genes in Monoclonal Gammopathies

Introduction In patients with monoclonal gammopathies, M proteins are patient-unique, can cause potentially fatal organ damage and can be used to track the B cell/plasma cell tumor after therapy. The presence of circulating tumor cells and the possibility to analyze the antibody repertoire through NGS and proteomics approaches may provide a window of opportunity to identify patients' specific M protein genes in the peripheral blood, without invasive bone marrow investigations. Methods Mononuclear cells from peripheral blood were subjected to single-molecule real-time sequencing of the M protein (SMaRT M-Seq)1 to obtain the circulating repertoire of the affected light chain isotype in a cohort of patients with MGUS (n=3), MM (n=8) and AL amyloidosis (n=36) at diagnosis. Tryptic digestion peptides from urinary proteins were subjected to mass spectrometry analysis to identify or validate the clonal light chain sequence from the obtained circulating repertoire2. SMaRT M-Seq on matched bone marrow samples to identify the bona fide clonal light chain sequence was performed for confirmatory purposes. Results Reads with 100% identity to the bona fide clonal light chain sequences were identified in the peripheral blood of 44 patients (94%) and were the dominant clonal sequence in 35 cases (75%). In all cases where the most abundant, discrete clonal sequence represented at least 15% of all reads detected within the peripheral blood (n=26, 55%), such sequence invariably coincided with the bona fide clonal light chain sequence as defined by bone marrow sequencing studies (Figure 1A). In a subset of cases (n=21), we also analyzed the urinary proteome and mapped tryptic digestion peptides against each patient's peripheral blood antibody repertoire. In all cases, peptide mapping correctly identified the bona fide clonal light chain sequence as the light chain sequence with the highest relative abundance, sequence coverage, and number of unique peptides among all light chain sequences identified in the patient's peripheral blood (Figure 1B). The percentage of peripheral blood sequences corresponding to the bona fide clonal light chain sequence significantly, but modestly correlated with bone marrow plasma cell infiltration (R2 0.30). Conclusion Sequencing of the circulating light chain repertoire through SMaRT M-Seq, eventually coupled with mass spectrometry-based analysis of the urinary proteome, enables the identification of the full-length clonal light chain sequence in most patients with a monoclonal gammopathy. The possibility of reliably determining disease-related immunoglobulin gene sequences even from the peripheral blood from large cohorts of patients has the potential to uncover molecular mechanisms of M protein-related clinical manifestations which have remained largely unexplored so far2. It could also facilitate approaches to personalized medicine, including the implementation of sequence-based predictive models for the identification of potentially pathogenic M proteins3,4, and the sensitive detection of patients' specific M proteins at diagnosis and MRD after anti-clonal therapy5.

Specificity & Precision of Minimal Residual Disease Monitoring in DLBCL Using Ig-HTS

Background: Dynamic minimal residual disease (MRD) monitoring in the diffuse large B-cell lymphoma (DLBCL) setting has been shown to predict treatment failure at early timepoints and relapse at later timepoints. Precise and reliable approaches are critically important for accurate detection of residual disease. We sought to evaluate the precision of heavy-chain (IGH) and light-chain (IGK/L) MRD measurement within a standard clinical workflow. Methods: We developed a database of clinical clonoSEQ (Adaptive Biotechnologies) and clonoSIGHT reports (Sequenta, Inc.) identifying the dominant malignant IGH or IGK/L clonotypes from the tumor biopsy samples of 285 patients collected as a part of routine clinical care for MRD monitoring. Included patients had been diagnosed with DLBCL at either Stanford University or the National Cancer Institute, 130 of whom had samples sequenced with the clonoSEQ assay and 155 with the clonoSIGHT assay. A control database was generated from all publicly available immunoSEQ (Adaptive Biotechnologies) B-cell repertoires with IGH or IGK/L sequencing, spanning both malignant and healthy patient contexts. The patients within the clinical DLBCL and control databases were distinct without any overlap. We assessed whether each malignant IGH or IGK/L clonotype from the clinical DLBCL database shared overlap with clonotypes present in the control IGH or IGK/L repertoires. A match was considered positive only if the sequence context of the malignant clonotype was an exact match with the sequence of a control clonotype; exact substrings were allowed given the differences in potential primer sites between assays. Results: The clinical database included 341 dominant IGK/L sequences from 187 patients and 271 dominant IGH sequences from 195 patients, where dominance was determined by the clinical assay. We generated a control IGK/L database of 618,775 sequences from 71 subjects and a control IGH database of 65,017,932 sequences from 1698 subjects. IGH demonstrated high precision (99.3%) with only two clinically dominant sequences demonstrating overlap with the control repertoire. Despite the smaller control database, IGK/L demonstrated significantly reduced precision of 92% (Fisher's exact p-value < 0.001) with 29 sequences demonstrating overlap with the control repertoire, all of which were limited to the subset of patients who had been sequenced by clonoSEQ. At the patient level, 19 of the 105 patients with clonoSEQ IGK/L sequencing had clinically trackable (Fig. 1A), putatively malignant sequences that were shared with the control database (precision 84.7%). The frequency of these clinically dominant sequences in the control repertoires ranged from 8.27x10-8 to 0.575 with a mean frequency of 0.000598. Most non-unique clonotypes were shared across multiple control subjects (range 1-37, mean 13, Fig. 1B). Conclusions: The high rate of overlap seen between putatively malignant, dominant IGK/L clonotypes and a publicly available control repertoire suggests substantially reduced validity of approaches relying on IGK/L tracking for minimal residual disease monitoring. The overlapping light chain clonotypes were not only present at relatively high frequencies in both the clinical samples and control repertoire, but were also seen across multiple control subjects, suggesting MRD assays that track only IGK/L may be unable to reliably differentiate malignant clonotypes from background light chain repertoires. Caution should be used when MRD is detected using IGK/IGL given reduced specificity and precision of these sequences. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Stereotyped B-cell responses are linked to IgG constant region polymorphisms in multiple sclerosis.

Clonally related B cells infiltrate the brain, meninges, and cerebrospinal fluid of MS patients, but the mechanisms driving the B-cell response and shaping the immunoglobulin repertoires remain unclear. Here, we used single-cell full-length RNA-seq and BCR reconstruction to simultaneously assess the phenotypes, isotypes, constant region polymorphisms, and the paired heavy- and light-chain repertoires in intrathecal B cells. We detected extensive clonal connections between the memory B cell and antibody-secreting cell (ASC) compartments and observed clonally related cells of different isotypes including IgM/IgG1, IgG1/IgA1, IgG1/IgG2, and IgM/IgA1. There was a strong dominance of the G1m1 allotype constant region polymorphisms in ASCs, but not in memory B cells. Tightly linked to the G1m1 allotype, we found a preferential pairing of the immunoglobulin heavy-chain variable (IGHV)4 gene family with the κ variable (IGKV)1 gene family. The IGHV4-39 gene was most used and showed the highest frequency of pairing with IGKV1-5 and IGKV1(D)-33. These results link IgG constant region polymorphisms to stereotyped B-cell responses in MS and indicate that the intrathecal B-cell response in these patients could be directed against structurally similar epitopes.

The CRL4VPRBP(DCAF1) E3 ubiquitin ligase directs constitutive RAG1 degradation in a non-lymphoid cell line.

The development of B and T lymphocytes critically depends on RAG1/2 endonuclease activity to mediate antigen receptor gene assembly by V(D)J recombination. Although control of RAG1/2 activity through cell cycle- and ubiquitin-dependent degradation of RAG2 has been studied in detail, relatively little is known about mechanisms regulating RAG1 stability. We recently demonstrated that VprBP/DCAF1, a substrate adaptor for the CRL4 E3 ubiquitin ligase complex, is required to maintain physiological levels of RAG1 protein in murine B cells by facilitating RAG1 turnover. Loss of VprBP/DCAF1 in vivo results in elevated RAG1 expression, excessive V(D)J recombination, and immunoglobulin light chain repertoire skewing. Here we show that RAG1 is constitutively degraded when ectopically expressed in a human fibroblast cell line. Consistent with our findings in murine B cells, RAG1 turnover under these conditions is sensitive to loss of VprBP, as well as CRL4 or proteasome inhibition. Further evidence indicates that RAG1 degradation is ubiquitin-dependent and that RAG1 association with the CRL4VPRBP/DCAF1 complex is independent of CUL4 activation status. Taken together, these findings suggest V(D)J recombination co-opts an evolutionarily conserved and constitutively active mechanism to ensure rapid RAG1 turnover to restrain excessive RAG activity.

Lower Somatic Mutation Levels in the λ Light-Chain Repertoires with Chronic HBV Infection

To investigate the characteristics of the immunoglobulin light-chain repertoires with chronic HBV infection, the high-throughput sequencing and IMGT/HighV-QUEST were adapted to analyze the κ (IgK) and λ (IgL) light-chain repertoires from the inactive HBV carriers (IHB) and the healthy adults (HH). The comparative analysis revealed high similarity between the κ light-chain repertoires of the HBV carriers and the healthy adults. Nevertheless, the proportion of IGLV genes with ≥90% identity as the germline genes was higher in the IgL light-chain repertoire of the IHB library compared with that of HH library (74.6% vs. 69.1%). Besides, the frequency of amino acid mutations in the CDR1 regions was significantly lower in the IgL light-chain repertoire of the IHB library than that of the HH library (65.52% vs. 56.0%). These results suggested the lower somatic mutation level in the IgL repertoire of IHB library, which might indicate the biased selection of IGLV genes in the IgL repertoire with chronic HBV infection. These findings might lead to a better understanding of the characteristics of the light-chain repertoires of HBV chronically infected individuals.

Isolation and light chain shuffling of a Plasmodium falciparum AMA1-specific human monoclonal antibody with growth inhibitory activity

BackgroundPlasmodium falciparum, the parasite causing malaria, affects populations in many endemic countries threatening mainly individuals with low malaria immunity, especially children. Despite the approval of the first malaria vaccine Mosquirix™ and very promising data using cryopreserved P. falciparum sporozoites (PfSPZ), further research is needed to elucidate the mechanisms of humoral immunity for the development of next-generation vaccines and alternative malaria therapies including antibody therapy. A high prevalence of antibodies against AMA1 in immune individuals has made this antigen one of the major blood-stage vaccine candidates.Material and methodsUsing antibody phage display, an AMA1-specific growth inhibitory human monoclonal antibody from a malaria-immune Fab library using a set of three AMA1 diversity covering variants (DiCo 1–3), which represents a wide range of AMA1 antigen sequences, was selected. The functionality of the selected clone was tested in vitro using a growth inhibition assay with P. falciparum strain 3D7. To potentially improve affinity and functional activity of the isolated antibody, a phage display mediated light chain shuffling was employed. The parental light chain was replaced with a light chain repertoire derived from the same population of human V genes, these selected antibodies were tested in binding tests and in functionality assays.ResultsThe selected parental antibody achieved a 50% effective concentration (EC50) of 1.25 mg/mL. The subsequent light chain shuffling led to the generation of four derivatives of the parental clone with higher expression levels, similar or increased affinity and improved EC50 against 3D7 of 0.29 mg/mL. Pairwise epitope mapping gave evidence for binding to AMA1 domain II without competing with RON2.ConclusionWe have thus shown that a compact immune human phage display library is sufficient for the isolation of potent inhibitory monoclonal antibodies and that minor sequence mutations dramatically increase expression levels in Nicotiana benthamiana. Interestingly, the antibody blocks parasite inhibition independently of binding to RON2, thus having a yet undescribed mode of action.

Characterization of the Immunoglobulin Heavy- and Light-Chain Repertoires in a Single Reaction

Background B cell repertoire analysis by next-generation sequencing (NGS) has shown particular utility in the field of hematological oncology research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, and the elimination of specifically designed primers often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR assays require separate PCR reactions to survey each immunoglobulin chain (IGH, IGK, IGL), often leading to a longer time-to-answer for samples in which no marker is initially detected. We have developed an assay for receptor analysis based on Ion AmpliSeq technology to circumvent these issues, allowing the effective use of up to thousands of primers in a single reaction. The highly multiplexed, pan-clonality NGS assay provides for efficient detection of IGH, IGK, and IGL chain rearrangements in a single reaction. Methods We developed a single primer panel targeting the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IGK, IGL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IGK loci rearrangements involving Kappa deletion and C intron elements. To evaluate performance, we conducted clonality assessment and limit-of-detection testing used gDNA from a total of 45 research samples representing common B cell malignancies. We included samples derived from peripheral blood, bone marrow, and FFPE-preserved tissues at input levels ranging from 100ng to 2µg. Finally, we further characterized the samples via a separate AmpliSeq-based multiplex PCR assay targeting rearranged TCRB and TCRG chains. Sequencing and clonality analysis was performed using the Ion GeneStudio S5 System and Ion Reporter 5.16. Results Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, and DLBCL) show a &amp;gt;90% overall positive detection rate. Assessment of linearity-of-response and limit-of-detection was carried out using cell lines diluted in PBL to between 10-3 and 10-6 by mass. The multi-receptor assay performs as expected, with linear response to the cell line frequency across the range tested, including the ability to detect clones of interest at 10-6. Conclusions These results demonstrate the robustness of our newly developed Ion AmpliSeq-assay for B cell receptor heavy and light chains. We expect this assay to simplify the workflow for clonality assessment and rare clone detection in B cell malignancy research. For research use only. Disclosures No relevant conflicts of interest to declare.

The analysis of organization and diversity mechanism in goat immunoglobulin light chain gene loci.

The genomic organization of goat immunoglobulin light chains (Igλ and Igκ) loci were annotated based on the goat genome database. The goat Igλ chain located on chromosome 17 contains at least 35 Vλ gene fragments (seven potential functional genes, one ORF and 27 pseudogenes), two Jλ-Cλ clusters arranged in a Vλ(35)-Jλ2-Cλ1-Jλ1-Cλ2 pattern, with another Cλ3 on scaffold. The Igκ locus included 11 Vκ (five potential functional genes, two ORFs and four pseudogene fragments), three Jκ genes and a single Cκ gene ordered in Vκ(35)-Jκ(3)-Cκ pattern on chromosome 11. By analyzing the clonies of Igλ and Igκ, we further found Vλ2 (26.23 %) &Vλ3 (73.11 %), Vκ2 (52.07 %) &Vκ4 (46.15 %) were predominately used in the expression of λ and κ chains respectively. λ chain showed more abundance in connective diversity than κ chain. Besides, somatic hypermutation with higher frequency in both immunoglobulin light chains was the major mechanism for the goat repertoire diversity. These results demonstrated goat immunoglobulin light chain variable region genome loci and repertoire diversity.

Development of a Phage Display Panning Strategy Utilizing Crude Antigens: Isolation of MERS-CoV Nucleoprotein human antibodies

Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.

V2-Directed Vaccine-like Antibodies from HIV-1 Infection Identify an Additional K169-Binding Light Chain Motif with Broad ADCC Activity

SUMMARYAntibodies that bind residue K169 in the V2 region of the HIV-1 envelope correlated with reduced risk of infection in the RV144 vaccine trial but were restricted to two ED-motif-encoding light chain genes. Here, we identify an HIV-infected donor with high-titer V2 peptide-binding antibodies and isolate two antibody lineages (CAP228-16H/19F and CAP228–3D) that mediate potent antibody-dependent cell-mediated cytotoxicity (ADCC). Both lineages use the IGHV5–51 heavy chain germline gene, similar to the RV144 antibody CH58, but one lineage (CAP228-16H/19F) uses a light chain without the ED motif. A cocrystal structure of CAP228-16H bound to a V2 peptide identified a IGLV3–21 gene-encoded DDxD motif that is used to bind K169, with a mechanism that allows CAP228-16H to recognize more globally relevant V2 immunotypes. Overall, these data further our understanding of the development of cross-reactive, V2-binding, antiviral antibodies and effectively expand the human light chain repertoire able to respond to RV144-like immunogens.

Immunoglobulin Light Chain Gene Rearrangements, Receptor Editing and the Development of a Self-Tolerant Antibody Repertoire.

Discussion of the antibody repertoire usually emphasizes diversity, but a conspicuous feature of the light chain repertoire is its lack of diversity. The diversity of reported allelic variants of germline light chain genes is also limited, even in well-studied species. In this review, the implications of this lack of diversity are considered. We explore germline and rearranged light chain genes in a variety of species, with a particular focus on human and mouse genes. The importance of the number, organization and orientation of the genes for the control of repertoire development is discussed, and we consider how primary rearrangements and receptor editing together shape the expressed light chain repertoire. The resulting repertoire is dominated by just a handful of IGKV and IGLV genes. It has been hypothesized that an important function of the light chain is to guard against self-reactivity, and the role of secondary rearrangements in this process could explain the genomic organization of the light chain genes. It could also explain why the light chain repertoire is so limited. Heavy and light chain genes may have co-evolved to ensure that suitable light chain partners are usually available for each heavy chain that forms early in B cell development. We suggest that the co-evolved loci of the house mouse often became separated during the inbreeding of laboratory mice, resulting in new pairings of loci that are derived from different sub-species of the house mouse. A resulting vulnerability to self-reactivity could explain at least some mouse models of autoimmune disease.

Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes

Existing methods for paired antibody heavy- and light-chain repertoire sequencing rely on specialized equipment and are limited by their commercial availability and high costs. Here, we report a novel simple and cost-effective emulsion-based single-cell paired antibody repertoire sequencing method that employs only basic laboratory equipment. We performed a proof-of-concept using mixed mouse hybridoma cells and we also showed that our method can be used for discovery of novel antigen-specific monoclonal antibodies by sequencing human CD19+ B cell IgM and IgG repertoires isolated from peripheral whole blood before and seven days after Td (Tetanus toxoid/Diphtheria toxoid) booster immunization. We anticipate broad applicability of our method for providing insights into adaptive immune responses associated with various diseases, vaccinations, and cancer immunotherapies.

Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

Aortic B cells: Identifying antibodies specific for atherosclerosis in mice

Abstract Atherosclerosis is characterized by the development of arterial plaques containing lipid deposits and immune cells, such as B cells. Once recruited to the site of inflammation, B cells become activated and form germinal centers within the adventitia of the artery adjacent to the plaque. To determine if these B cells produce antibodies specific for atherosclerosis, abdominal aortas were collected, and germinal center B cells were isolated by flow cytometry. We characterized their heavy and light chain repertoires by next generation sequencing to compare non-diseased (C57BL/6) and diseased (ApoE−/−) tissues. Furthermore, the aortic repertoires were compared to the germinal center repertoires from spleens taken from the same mice to address whether local (aortic) and systemic (spleen) B cell responses were similar. Preliminary results suggest that all the splenic repertoires were similar, independent of diet or disease. However, aortic repertoires from ApoE−/− mice indicated that some VH and Vk genes were overexpressed, suggesting selection for particular antigens. We therefore performed single cell analysis and identified over 100 VH and Vk gene pairs, with different frequencies of expression and somatic hypermutation. Antibody gene pairs will then be cloned and expressed in culture using HEK293A cells. The purified recombinant antibodies will be used to identify antigens specific to atherosclerosis by protein array. These results may be useful in creating potential vaccines for its prevention, as well as treatment via antibody immunotherapy.

Transitional B Cells in Early Human B Cell Development - Time to Revisit the Paradigm?

The B cell repertoire is generated in the adult bone marrow by an ordered series of gene rearrangement processes that result in massive diversity of immunoglobulin (Ig) genes and consequently an equally large number of potential specificities for antigen. As the process is essentially random, the cells exhibiting excess reactivity with self-antigens are generated and need to be removed from the repertoire before the cells are fully mature. Some of the cells are deleted, and some will undergo receptor editing to see if changing the light chain can rescue an autoreactive antibody. As a consequence, the binding properties of the B cell receptor are changed as development progresses through pre-B ≫ immature ≫ transitional ≫ naïve phenotypes. Using long-read, high-throughput, sequencing we have produced a unique set of sequences from these four cell types in human bone marrow and matched peripheral blood, and our results describe the effects of tolerance selection on the B cell repertoire at the Ig gene level. Most strong effects of selection are seen within the heavy chain repertoire and can be seen both in gene usage and in CDRH3 characteristics. Age-related changes are small, and only the size of the CDRH3 shows constant and significant change in these data. The paucity of significant changes in either kappa or lambda light chain repertoires implies that either the heavy chain has more influence over autoreactivity than light chain and/or that switching between kappa and lambda light chains, as opposed to switching within the light chain loci, may effect a more successful autoreactive rescue by receptor editing. Our results show that the transitional cell population contains cells other than those that are part of the pre-B ≫ immature ≫ transitional ≫ naïve development pathway, since the population often shows a repertoire that is outside the trajectory of gene loss/gain between pre-B and naïve stages.

Repertoire comparison of the B-cell receptor-encoding loci in humans and rhesus macaques by next-generation sequencing.

Rhesus macaques (RMs) are a widely used model system for the study of vaccines, infectious diseases and microbial pathogenesis. Their value as a model lies in their close evolutionary relationship to humans, which, in theory, allows them to serve as a close approximation of the human immune system. However, despite their prominence as a human surrogate model system, many aspects of the RM immune system remain ill characterized. In particular, B cell-mediated immunity in macaques has not been sufficiently characterized, and the B-cell receptor-encoding loci have not been thoroughly annotated. To address these gaps, we analyzed the circulating heavy- and light-chain repertoires in humans and RMs by next-generation sequencing. By comparing V gene segment usage, J-segment usage and CDR3 lengths between the two species, we identified several important similarities and differences. These differences were especially notable in the IgM+ B-cell repertoire. However, the class-switched, antigen-educated B-cell populations converged on a set of similar characteristics, implying similarities in how each species responds to antigen. Our study provides the first comprehensive overview of the circulating repertoires of the heavy- and light-chain sequences in RMs, and provides insight into how they may perform as a model system for B cell-mediated immunity in humans.

The Effect of BAFF Inhibition on Autoreactive B-Cell Selection in Murine Systemic Lupus Erythematosus.

The goal of this study was to determine how B-cell-activating factor of the TNF family (BAFF) availability influences selection of the autoreactive B-cell repertoire in NZB/W and NZW/BXSB lupus-prone mice bearing the site-directed heavy-chain transgene 3H9 that encodes for anti-dsDNA and anti-cardiolipin (CL) autoantibodies. We used a bone marrow chimera system in which autoreactive 3H9 transgenic B cells were allowed to mature in competition with wild-type cells and could be identified by green fluorescent protein. The light-chain repertoire associated with the 3H9 heavy chain in naive and antigen-activated B-cell subsets was assessed using single-cell polymerase chain reaction. We found that deletion of autoreactive transgenic B cells occurred in the bone marrow of both strains regardless of BAFF availability, and there were only modest and physiologically non-relevant effects on the naive B-cell repertoire. BAFF inhibition had different effects on selection of the germinal center repertoire in the two strains. In the NZW/BXSB strain, BAFF inhibition phenocopied the loss of one TLR7 allele in that it influenced the selection of 3H9-encoded autoreactive B cells in the germinal center but did not prevent somatic mutation. In the NZB/W strain, BAFF inhibition did not alter the selection of 3H9-encoded B cells in the germinal center, but it influenced selection of a subset of germinal center cells into the plasma cell compartment. Our data underscore the complexity of regulation of the autoreactive B-cell repertoire by BAFF and may help to explain the heterogeneity of responses observed after BAFF inhibition in humans.

Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.

In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.