The immunoglobulin heavy chain super enhancer controls class switch recombination in developing B cells.

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only.

Two distinct subpopulations of marginal zone B cells exhibit differential antibody-producing capacities and radioresistance.

Marginal zone (MZ) B cells, which are splenic innate-like B cells that rapidly secrete antibodies (Abs) against blood-borne pathogens, are composed of heterogeneous subpopulations. Here, we showed that MZ B cells can be divided into two distinct subpopulations according to their CD80 expression levels. CD80high MZ B cells exhibited greater Ab-producing, proliferative, and IL-10-secreting capacities than did CD80low MZ B cells. Notably, CD80high MZ B cells survived 2-Gy whole-body irradiation, whereas CD80low MZ B cells were depleted by irradiation and then repleted with one month after irradiation. Depletion of CD80low MZ B cells led to accelerated development of type II collagen (CII)-induced arthritis upon immunization with bovine CII. CD80high MZ B cells exhibited higher expression of genes involved in proliferation, plasma cell differentiation, and the antioxidant response. CD80high MZ B cells expressed more autoreactive B cell receptors (BCRs) that recognized double-stranded DNA or CII, expressed more immunoglobulin heavy chain sequences with shorter complementarity-determining region 3 sequences, and included more clonotypes with no N-nucleotides or with B-1a BCR sequences than CD80low MZ B cells. Adoptive transfer experiments showed that CD21+CD23+ transitional 2 MZ precursors preferentially generated CD80low MZ B cells and that a proportion of CD80low MZ B cells were converted into CD80high MZ B cells; in contrast, CD80high MZ B cells stably remained CD80high MZ B cells. In summary, MZ B cells can be divided into two subpopulations according to their CD80 expression levels, Ab-producing capacity, radioresistance, and autoreactivity, and these findings may suggest a hierarchical composition of MZ B cells with differential stability and BCR specificity.

Antibody production relies on the tRNA inosine wobble modification to meet biased codon demand.

Antibodies are produced at high rates to provide immunoprotection, which puts pressure on the B cell translational machinery. Here, we identified a pattern of codon usage conserved across antibody genes. One feature thereof is the hyperutilization of codons that lack genome-encoded Watson-Crick transfer RNAs (tRNAs), instead relying on the posttranscriptional tRNA modification inosine (I34), which expands the decoding capacity of specific tRNAs through wobbling. Antibody-secreting cells had increased I34 levels and were more reliant on I34 for protein production than naïve B cells. Furthermore, antibody I34-dependent codon usage may influence B cell passage through regulatory checkpoints. Our work elucidates the interface between the tRNA pool and protein production in the immune system and has implications for the design and selection of antibodies for vaccines and therapeutics.

IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sµ) and the 3' regulatory region (3'RR) (Sµ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sµ-3'RRrec counts per sample: Sµ-3'RRrecHigh cases (mostly unmutated CLL) and Sµ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sµ-3'RRrec appeared to be ongoing in Sµ-3'RRrecHigh CLL cells and comparison of Sµ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sµ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sµ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sµ-3'RRrec in Sµ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sµ-3'RRrec, even in the absence of AID for the latter.

BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

Minimal residual disease detection by next-generation sequencing of different immunoglobulin gene rearrangements in pediatric B-ALL.

While the prognostic role of immunoglobulin heavy chain locus (IGH) rearrangement in minimal residual disease (MRD) in pediatric B-acute lymphoblastic leukemia (B-ALL) has been reported, the contribution of light chain loci (IGK/IGL) remains elusive. This study is to evaluate the prognosis of IGH and IGK/IGL rearrangement-based MRD detected by next-generation sequencing in B-ALL at the end of induction (EOI) and end of consolidation (EOC). IGK/IGL rearrangements identify 5.5% of patients without trackable IGH clones. Concordance rates for IGH and IGK/IGL are 79.9% (cutoff 0.01%) at EOI and 81.0% (cutoff 0.0001%) at EOC, respectively. Patients with NGS-MRD < 0.01% at EOI or <0.0001% at EOC present excellent outcome, with 3-year event-free survival rates higher than 95%. IGH-MRD is prognostic at EOI/EOC, while IGK-MRD at EOI/EOC and IGL-MRD at EOI are not. At EOI, NGS identifies 26.2% of higher risk patients whose MRD < 0.01% by flow cytometry. However, analyzing IGK/IGL along with IGH fails to identify additional higher risk patients both at EOI and at EOC. In conclusion, IGH is crucial for MRD monitoring while IGK and IGL have relatively limited value.

Shared bias in H chain V-J pairing in naive and memory B cells.

Introduction: H chain rearrangement in B cells is a two-step process where first DH binds JH, and only then VH is joined to the complex. As such, there is no direct rearrangement between VH and JH. Results: Nevertheless, we here show that the VHJH combinations frequency in humans deviates from the one expected based on each gene usage frequency. This bias is observed mainly in functional rearrangements, and much less in out-of-frame rearrangements. The bias cannot be explained by preferred binding for DH genes or a preferred reading frame. Preferred VH JH combinations are shared between donors. Discussion: These results suggest a common structural mechanism for these biases. Through development, thepreferred VH JH combinations evolve during peripheral selection to become stronger, but less shared. We propose that peripheral Heavy chain VH JH usage is initially shaped by a structural selection before the naive B cellstate, followed by pathogen-induced selection for host specific VH-JH pairs.

scBCR-seq revealed a special and novel IG H&L V(D)J allelic inclusion rearrangement and the high proportion dual BCR expressing B cells.

Since the initial report of V (D) J "allelic exclusion/inclusion" (allelic exclusion rearrangement or allelic inclusion rearrangement) and the concept of the "dual B cell receptor (BCR)" in 1961, despite ongoing discoveries, the precise proportion and source mechanism of dual BCR under physiological conditions have been puzzling immuologists. This study takes advantage of the single cell B cell receptor sequencing (scBCR-seq) technology, which can perfectly match the heavy and light chains of BCR at the level of a single B cell, and obtain the full length mRNA sequence of the complementary determining region 3 (CDR3). Through analyzing the pairing of functional IGH (immunoglobulin heavy chain) and IGL (immunoglobulin light chain) in single B cell from both human and mouse bone marrow and peripheral blood, it was observed that dual BCR B cells exhibit stable and high levels of expression. Among them, the human bone marrow and peripheral blood contain about 10% dual (or multiple) BCR B cells, while in mouse peripheral blood and bone marrow memory B cells, this proportion reaches around 20%. At the same time, we innovatively found that in each research sample of humans and mice, there are three (or more) functional rearrangements (mRNA level) of a single chain in a single B cell. By analyzing the position, direction and other compositional characteristics of the V(D)J gene family, we found that at least two (or more) of them are derived from over two (or more) specific allelic inclusion rearrangements of a single chromosome (mRNA molecular level evidence), our findings also highlighted the necessity of classified single cell sequencing data based on single, dual (or multiple) and cannot be assembled into BCR when analyzing the B cell repertoire. The results of this article provides new methods and modeling references for evaluating the proportion and source mechanisms of dual BCR B cells, as well as potential significance of allelic inclusion (exclusion escape) of V(D)J rearrangement.

Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering.

Next-generation DNA sequencing (NGS) technologies have made it possible to interrogate antibody repertoires to unprecedented depths, typically via sequencing of cDNAs encoding immunoglobulin variable domains. In the absence of heavy-light chain pairing, the variable domains of heavy chain-only antibodies (HCAbs), referred to as single-domain antibodies (sdAbs), are uniquely amenable to NGS analyses. In this chapter, we provide simple and rapid protocols for producing and sequencing multiplexed immunoglobulin variable domain (VHH, VH, or VL) amplicons derived from a variety of sources using the Illumina MiSeq platform. Generation of such amplicon libraries is relatively inexpensive, requiring no specialized equipment and only a limited set of PCR primers. We also present several applications of NGS to sdAb discovery and engineering, including: (1) evaluation of phage-displayed sdAb library sequence diversity and monitoring of panning experiments; (2) identification of sdAbs of predetermined epitope specificity following competitive elution of phage-displayed sdAb libraries; (3) direct selection of B cells expressing antigen-specific, membrane-bound HCAb using antigen-coupled magnetic beads and identification of antigen-specific sdAbs, and (4) affinity maturation of lead sdAbs using tandem phage display selection and NGS. These methods can easily be adapted to other types of proteins and libraries and expand the utility of in vitro display technology.

Unique repetitive nucleic acid structures mirror switch regions in the human IgH locus.

Immunoglobulin (Ig) genes carry the unique ability to be reshaped in peripheral B lymphocytes after these cells encounter a specific antigen. B cells can then further improve their affinity, acquire new functions as memory cells and eventually end up as antibody-secreting cells. Ig class switching is an important change that occurs in this context, thanks to local DNA lesions initiated by the enzyme activation-induced deaminase (AID). Several cis-acting elements of the Ig heavy (IgH) chain locus make it accessible to the AID-mediated lesions that promote class switch recombination (CSR). DNA repeats, with a non-template strand rich in G-quadruplexes (G4)-DNA, are prominent cis-targets of AID and define the so-called "switch" (S) regions specifically targeted for CSR. By analyzing the structure of the human IgH locus, we uncover that abundant DNA repeats, some with a putative G4-rich template strand, are additionally present in downstream portions of the IgH coding genes. These like-S (LS) regions stand as 3' mirror-images of S regions and also show analogies to some previously reported repeats associated with the IgH locus 3' super-enhancer. A regulatory role of LS repeats is strongly suggested by their specific localization close to exons encoding the membrane form of Ig molecules, and by their conservation during mammalian evolution.

Leukemic-phase mucosa-associated lymphoid tissue lymphoma.

A 78-year-old man presenting with leukocytosis was admitted to our hospital. The patient was asymptomatic and showed no lymphadenopathy. Peripheral blood flow cytometry revealed a leukemic-phase B-cell lymphoma with medium-to-large abnormal cells with reticulum. Positron emission tomography/computed tomography revealed abnormal uptake in the right orbit, bone marrow, and spleen. We performed immunological staining and fluorescence in situ hybridization on tissues extracted from the right orbit and bone marrow, which led to the diagnosis of mucosa-associated lymphoid tissue (MALT) lymphoma. Polymerase chain reaction analysis of immunoglobulin heavy chain rearrangements in the right orbital mass and bone marrow suggested that they were identical clones. Based on these collective findings, the diagnosis of leukemic-phase MALT lymphoma was confirmed, with sites of involvement including the bone marrow, peripheral blood, right orbit, and spleen. This is a highly rare case of leukemic MALT lymphoma.

Characterizing the dynamics of BCR repertoire from repeated influenza vaccination.

Yearly epidemics of seasonal influenza cause an enormous disease burden around the globe. An understanding of the rules behind the immune response with repeated vaccination still presents a significant challenge, which would be helpful for optimizing the vaccination strategy. In this study, 34 healthy volunteers with 16 vaccinated were recruited, and the dynamics of the BCR repertoire for consecutive vaccinations in two seasons were tracked. In terms of diversity, length, network, V and J gene segments usage, somatic hypermutation (SHM) rate and isotype, it was found that the overall changes were stronger in the acute phase of the first vaccination than the second vaccination. However, the V gene segments of IGHV4-39, IGHV3-9, IGHV3-7 and IGHV1-69 were amplified in the acute phase of the first vaccination, with IGHV3-7 dominant. On the other hand, for the second vaccination, the changes were dominated by IGHV1-69, with potential for coding broad neutralizing antibody. Additional analysis indicates that the application of V gene segment for IGHV3-7 in the acute phase of the first vaccination was due to the elevated usage of isotypes IgM and IgG3. While for IGHV1-69 in the second vaccination, it was contributed by isotypes IgG1 and IgG2. Finally, 41 public BCR clusters were identified in the vaccine group, with both IGHV3-7 and IGHV1-69 were involved and representative complementarity determining region 3 (CDR3) motifs were characterized. This study provides insights into the immune response dynamics following repeated influenza vaccination in humans and can inform universal vaccine design and vaccine strategies in the future.

Clonal composition and differentiation stage of human CD30+ B cells in reactive lymph nodes.

Introduction: Normal CD30+ B cells represent a distinct B-cell differentiation stage with features of strong activation. We lack an in depth understanding of these cells, because they are not present in peripheral blood and are typically very rare in reactive lymphoid organs. CD30+ B cells have been discussed as a potential precursor population for the malignant CD30+ Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma. As CD30+ B cells can be more numerous in some cases of reactive lymphadenitis, we aimed to characterize these CD30+ B cells in terms of their differentiation stage and clonal composition, also as a means to clarify whether such CD30+ B-cell populations may represent potential precursor lesions of Hodgkin lymphoma. Methods: We microdissected single CD30+ B cells from tissue sections of eight reactive lymph nodes with substantial numbers of such cells and sequenced their rearranged immunoglobulin (Ig) heavy chain V region (IGHV) genes. Results: The CD30+ B cells were polyclonal B cells in all instances, and they not only encompass post-germinal center (GC) B cells with mutated IGHV genes, but also include a substantial fraction of pre-germinal center B cells with unmutated IGHV genes. In five of the lymph nodes, mostly small clonal expansions were detected among the CD30+ B cells. Most of the expanded clones carried somatically mutated IGHV genes and about half of the mutated clones showed intraclonal diversity. Discussion: We conclude that in human reactive lymph nodes with relatively many CD30+ B cells, these cells are a heterogenous population of polyclonal B cells encompassing activated pre-GC B cells as well as GC and post-GC B cells, with some clonal expansions. Because of their polyclonality and frequent pre-GC differentiation stage, there is no indication that such cell-rich CD30+ B-cell populations represent precursor lesions of Hodgkin lymphoma.

IGHV allele similarity clustering improves genotype inference from adaptive immune receptor repertoire sequencing data.

In adaptive immune receptor repertoire analysis, determining the germline variable (V) allele associated with each T- and B-cell receptor sequence is a crucial step. This process is highly impacted by allele annotations. Aligning sequences, assigning them to specific germline alleles, and inferring individual genotypes are challenging when the repertoire is highly mutated, or sequence reads do not cover the whole V region. Here, we propose an alternative naming scheme for the V alleles, as well as a novel method to infer individual genotypes. We demonstrate the strengths of the two by comparing their outcomes to other genotype inference methods. We validate the genotype approach with independent genomic long-read data. The naming scheme is compatible with current annotation tools and pipelines. Analysis results can be converted from the proposed naming scheme to the nomenclature determined by the International Union of Immunological Societies (IUIS). Both the naming scheme and the genotype procedure are implemented in a freely available R package (PIgLET https://bitbucket.org/yaarilab/piglet). To allow researchers to further explore the approach on real data and to adapt it for their uses, we also created an interactive website (https://yaarilab.github.io/IGHV_reference_book).

A Novel Strategy for Screening Tumor-Specific Variable Domain of Heavy-Chain Antibodies.

The properties of the variable domain of heavy-chain (VHH) antibodies are particularly relevant in cancer therapy. To isolate tumor cell-specific VHH antibodies, VHH phage libraries were constructed from multiple tumor cells. After enriching the libraries against particular tumor cell lines, a next-generation sequencer was used to screen the pooled phages of each library for potential antibody candidates. Based on high amplification folds, 50 sequences from each library were used to construct phylogenetic trees. Several clusters with identical CDR3 were observed. Groups X, Y, and Z were assigned as common sequences among the different trees. These identical groups over the trees were considered to be cross-reactive antibodies. To obtain monoclonal antibodies, we assembled 200 sequences (top 50 sequences from each library) and rebuilt a combined molecular phylogenetic tree. Groups were categorized as A-G. For each group, we constructed a phagemid and determined its binding specificity with tumor cells. The phage-binding results were consistent with the phylogenetic tree-generated groups, which indicated particular tumor-specific clusters; identical groups showed cross-reactivity. The strategy used in the current study is effective for screening and isolating monoclonal antibodies. Specific antibodies can be identified, even when the target markers of cancer cells are unknown.

Genetic variation in the immunoglobulin heavy chain locus shapes the human antibody repertoire.

Variation in the antibody response has been linked to differential outcomes in disease, and suboptimal vaccine and therapeutic responsiveness, the determinants of which have not been fully elucidated. Countering models that presume antibodies are generated largely by stochastic processes, we demonstrate that polymorphisms within the immunoglobulin heavy chain locus (IGH) impact the naive and antigen-experienced antibody repertoire, indicating that genetics predisposes individuals to mount qualitatively and quantitatively different antibody responses. We pair recently developed long-read genomic sequencing methods with antibody repertoire profiling to comprehensively resolve IGH genetic variation, including novel structural variants, single nucleotide variants, and genes and alleles. We show that IGH germline variants determine the presence and frequency of antibody genes in the expressed repertoire, including those enriched in functional elements linked to V(D)J recombination, and overlapping disease-associated variants. These results illuminate the power of leveraging IGH genetics to better understand the regulation, function, and dynamics of the antibody response in disease.

Attempts to evaluate locus suicide recombination and its potential role in B cell negative selection in the mouse.

Introduction: In mature B cells, activation-induced deaminase reshapes Ig genes through somatic hypermutation and class switch recombination of the Ig heavy chain (IgH) locus under control of its 3' cis-regulatory region (3'RR). The 3'RR is itself transcribed and can undergo "locus suicide recombination" (LSR), then deleting the constant gene cluster and terminating IgH expression. The relative contribution of LSR to B cell negative selection remains to be determined. Methods: Here, we set up a knock-in mouse reporter model for LSR events with the aim to get clearer insights into the circumstances triggering LSR. In order to explore the consequences of LSR defects, we reciprocally explored the presence of autoantibodies in various mutant mouse lines in which LSR was perturbed by the lack of Sµ or of the 3'RR. Results: Evaluation of LSR events in a dedicated reporter mouse model showed their occurrence in various conditions of B cell activation, notably in antigen-experienced B cells Studies of mice with LSR defects evidenced increased amounts of self-reactive antibodies. Discussion: While the activation pathways associated with LSR are diverse, in vivo as well as in vitro, this study suggests that LSR may contribute to the elimination of self-reactive B cells.