Ig Repertoire Research Articles

The Clonotypic BCR IG of CLL Patients Contain Predicted T-Cell Class I Epitopes with Shared Structural Properties

Targeted therapies have revolutionized the treatment of chronic lymphocytic leukemia (CLL) with remarkable overall response rates. Against that, however, CLL remains incurable, indicating a need for novel strategies towards disease control and eventual eradication, including reinvigoration of anti-tumor immune responses. T cells in CLL display an oligoclonal profile and appear selected by restricted antigens, with recent evidence suggesting that the selecting epitopes may lie within the clonotypic B-cell receptor immunoglobulins (BcR IG). Should this prove to be the case, such neoepitopes could be exploited as idiotypic targets for cellular therapy or for peptide vaccine design, aiming to augment response to current treatments.Here we performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IGs of CLL patients, with an intended bias towards major stereotyped CLL subsets. We selected 27 patients to represent the full spectrum of CLL: (i) with mutated IGHV genes (M-CLL, n=5), (ii) with unmutated IGHV genes (U-CLL, n=5), (iii) assigned to major stereotyped subsets (subset #1, n=7; subset #2, n=5; subset #4, n=5). RT-PCR was performed for the heavy (H) and the light (K/L) IG chains using subgroup-specific Leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant domain (IGHG, IGHM, IGKC, IGLC), in order to produce the full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. PCR products were subjected to direct double-strand Sanger sequencing with a quality-optimized protocol. The amino acid sequences were subsequently parsed in peptides and subjected to NetMHCpan. The rank score was calculated, considering the 4-digit HLA-A and -B typing for each individual patient. High- and medium-binding peptides (rank score <2%) were selected. Exact matches to germline and/or proteome databases were excluded.Overall, 1,007 predicted neoepitopes were identified. All patients had predicted CD8 + T-cell epitopes within the clonotypic BcR IG, either in the heavy chain (26/27 pts, n=632 epitopes) or the light chain (26/27 pts, n=375 epitopes). The majority of the peptides resulted from somatic hypermutations (SHMs) across the IGHV gene outside the complementarity-determining region 3 (CDR3; n=538, 53.4%). With the exception of few peptides located within the FR4 region (n=11, 0.1%), the remaining (n=458, 45.5%) involved (at least part of) the CDR3, which is particularly relevant given its small length (9-27 aa) within the full sequence (331-660 aa). There was no statistically significant difference in the rank score of peptides involving the CDR3 vs. all others. Peptide clustering assigned most of the predicted neoepitopes (970/1007, 96.3%) in 54 clusters of similar length and amino acid composition. Also, it revealed similar or identical predicted neoepitopes among different patients (30 clusters of two, 10 clusters of three, 8 clusters of four, 4 clusters of five, 1 cluster of six and 1 cluster of eight). Importantly, these clusters involved: (i) shared CDR3 patterns in patients assigned to the same stereotyped subset, but also (ii) subset-specific recurrent SHMs across the rearranged IGHV gene, e.g. G-to-E SHM at position 28 in the VH CDR1 of subset #4, a recurrent SHM in this subset. Also of note, the two most highly populated clusters involved peptides within the VL CDR3, and were biased towards specific subsets; the cluster of eight patients contained 4 patients assigned to subset #1 and the cluster of six patients included 4 patients assigned to subset #2.In conclusion, in silico prediction identified a significant number of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. The majority of these neoepitopes can be assigned to clusters based on amino acid similarity and are shared among different patients. Many of them culminate from subset-specific (‘stereotyped‘) CDR3 patterns or recurrent SHMs, suggesting that the targeted SHM which shapes the CLL BcR IG repertoire may produce immunogenic CD8 + T-cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group. DisclosuresAnagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials . Chatzidimitriou: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Access to ultra-long IgG CDRH3 bovine antibody sequences using short read sequencing technology

The advances in high-throughput DNA sequencing and recombinant antibody technologies has presented new methods for characterizing antibody repertoires and significantly increased our understanding on the functional role of antibodies in immunity and their use in diagnostics, vaccine antigen design and as biological therapeutics. A subset of Bos taurus antibodies possesses unique ultra-long third complementary-determining region of the heavy chain (CDRH3) and are of special interest because they are thought to have unique functional abilities of broadly neutralizing properties – a functional role that has not been fully explored in vaccine development. Next generation sequencing technologies that are widely used to profile immunoglobulin (Ig) repertoires are based on short-read methods such as the Illumina technology. Although this technology has worked well in sequencing Ig V-D-J regions of most jawed vertebrates, it has faced serious technical challenges with sequencing regions in bovine Ig bearing ultra-long CDRH3 sequences, which are longer than 120 bp. To overcome this limitation, we have developed a sequencing strategy based on nested PCR products that allows sequence assembly of full-length bovine Ig heavy-chain (IgH) V-D-J regions. We have used this strategy to sequence IgH V-D-J regions of two Bos indicus breeds, Ankole and Boran. We confirm the presence of ultra-long CDRH3 sequences in IgG transcripts in both African cattle breeds, and provide preliminary evidence for differences and preferences in germline VH, DH and JH allele gene usage as well as differences in the length of the VH region in the two bovine breeds. Our method provides tools that should allow more robust analyses of ultra-long CDRH3 sequences aiding antibody and epitope discovery in different cattle breeds and their role in mediating immunity.

Immunoglobulin germline gene variation and its impact on human disease.

Immunoglobulins (Ig) play an important role in the immune system both when expressed as antigen receptors on the cell surface of B cells and as antibodies secreted into extracellular fluids. The advent of high-throughput sequencing methods has enabled the investigation of human Ig repertoires at unprecedented depth. This has led to the discovery of many previously unreported germline Ig alleles. Moreover, it is becoming clear that convergent and stereotypic antibody responses are common where different individuals recognise defined antigenic epitopes with the use of the same Ig V genes. Thus, germline V gene variation is increasingly being linked to the differential capacity of generating an effective immune response, which might lead to varying disease susceptibility. Here, we review recent evidence of how germline variation in Ig genes impacts the Ig repertoire and its subsequent effects on the adaptive immune response in vaccination, infection, and autoimmunity.

CLL-Specific B Cell Receptor Immunoglobulin Stereotypes Are Very Infrequent in Low Count Monoclonal B Cell Lymphocytosis: Implications for Disease Ontogeny

In chronic lymphocytic leukemia (CLL), the existence of subsets of patients with stereotyped B cell receptor immunoglobulin (BcR IG), collectively accounting for ~30% of all CLL, strongly implicates antigen selection in disease ontogeny. That said, it is still inconclusive if and how frequently “CLL-specific” BcR IG gene rearrangements occur in the repertoire of unaffected individuals. This is especially relevant when also considering monoclonal B-cell lymphocytosis (MBL), detected in 3-12% of the general population and characterized by the presence of CLL-like clonal B cells in lower numbers compared to CLL with no evidence of disease. MBL is distinguished into high and low count (HC-MBL and LC-MBL, respectively) with the former considered as a pre-leukemic condition, while the latter having an unclear, if any, link to CLL. Our previous immunogenetic studies have revealed similarities between HC-MBL and CLL, thus setting them apart from LC-MBL that displayed distinct IG gene usage and low incidence of stereotypy. However, these studies were Sanger-based, thus inherently limited regarding sensitivity and accurate quantification.Here we sought to overcome these limitations and obtain a comprehensive view into the composition of the IG repertoire in LC-MBL by NGS immunoprofiling, searching particularly for major CLL IG stereotypes. The study group comprised 23 individuals with CLL-like (CD5+) LC-MBL identified by a standardized flow cytometry protocol. PBMCs were the starting material in 11 cases, whereas sorted MBL cells were studied in the remaining 12 cases; in 9 cases, the normal B cell compartment was also flow sorted. Included in the analysis were also 6 individuals without LC-MBL in whom we sorted different B cell subpopulations, including naïve B cells (n=6) and memory B cells (n=5). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified with leader primers and sequencing libraries were prepared with the TruSeq DNA LT Kit and run on the MiSeq Sequencer using the MiSeq Reagent Kit v3. NGS reads were subjected to an in-house bioinformatics pipeline for clonotype characterization and the identification of CLL-specific BcR IG stereotypes.Overall, 16,857,788 productive filtered-in sequence reads were obtained corresponding to an average of 366,474 reads/sample (range: 67,175-662,994). We identified very few reads (2,470/16,857,788; 0.15%) corresponding to 17/19 major CLL BcR IG stereotypes. Their incidence in LC-MBL did not follow the CLL trend: for instance, the stereotypic IGHV3-21 gene rearrangement defining subset #2, the most frequent in CLL, accounted for only 27/2,470 reads (1.1%) corresponding to major subset stereotypes. Overall, stereotypes were identified in 26/47 samples (55%) from 13/23 LC-MBL and 6/6 non LC-MBL cases. In LC-MBL cases bearing stereotypes the breakdown according to sample type was as follows: (i) PBMCs, n=3 (ii) normal B cell compartment, n=7; (iii) sorted MBL cells, n=5: the stereotypes detected in this particular sample type were present at very low frequencies and concerned rearrangements with unmutated IGHV genes. In individuals without LC-MBL, stereotypes were detected in all 6 samples from naïve B cells versus only 2/5 samples from memory B cells. In general, we did not identify significant associations between specific B cell subpopulations and particular stereotypes, except for the IGHV4-34 rearrangement defining CLL subset #4, an indolent CLL variant with features of BcR-anergized clones, which was found only within naïve B cells.In conclusion, we demonstrate that LC-MBL is immunogenetically distinct from CLL. Major CLL BCR IG stereotypes are very scarce in individuals with or without LC-MBL. This could be taken to imply either that these stereotypes are indeed scarce in the blood of individuals unaffected by CLL or, alternatively, that they might reside within other B cell compartments not assessed in this study. DisclosuresAgathangelidis:Gilead Sciences: Research Funding. Stamatopoulos:Janssen Pharmaceuticals: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Novartis SA: Research Funding. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Research Funding.

Human Cord Blood B Cells Differ from the Adult Counterpart by Conserved Ig Repertoires and Accelerated Response Dynamics.

Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.

Single-cell transcriptome analysis of the B-cell repertoire reveals the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica)

B-cells are key to humoral immunity, are found in multiple lymphoid organs, and have the unique ability to mediate the production of antigen-specific antibodies in the presence of pathogens. The marsupial immunoglobulin (Ig) heavy (H) chain locus encodes four constant region isotypes, IgA, IgG, IgM and IgE, but no IgD, and there are two light (L) chain isotypes, lambda (Igλ) and kappa (Igκ). To gain an understanding of the marsupial humoral immune system, B-cell transcriptomes generated by single-cell RNA sequencing from gray short-tailed opossum (Monodelphis domestica) splenocytes, and peripheral blood mononuclear cells were analysed. The cells used were from a single unimmunized animal and the majority of B-cells were transcribing IgM heavy chains. The ratio of Ig light chain use was roughly 2:1, Igλ:Igκ in this individual. This was not predicted due to Igκ being the more complex of the two L chain loci. The variable (V) gene segment pairs used in individual B-cells confirm greater diversity provided by the L chain V. This study is the first to report on using single cell analysis to investigate Ig repertoires in a marsupial and confirms a number of prior hypothesis, as well as revealing some surprises.

Plasma cell immunoglobulin heavy chain repertoire dynamics before and after tetanus booster vaccination.

Antibody sequence repertoire analysis of plasma cells (PC) isolated before and 1 week after a vaccine provides time-specific snapshots of the antibody response. Comparison of the immunoglobulin (Ig) sequences pre- and post-vaccination allows analysis of maturation over time and identification of antigen specific Ig. Here we compare the Ig heavy chain (Ig-H) repertoire of circulating PCs isolated from 109 peripheral blood mononuclear cells (PBMC) collected by apheresis 1 week after a tetanus toxoid vaccine booster with the Ig-H repertoire of PCs collected 2 and 11 weeks prior to the booster. A total of 21,060 unique Ig nucleotide sequences encoding 14,307 unique heavy chain complementarity determining region 3 (CDR-H3) amino acid sequences, also called clonotypes, were identified. Only 466 clonotypes (3.3%) were present at all 3 time points. In contrast, 90% of the 30 highest frequency CDR-H3 regions at +1w were also identified at another time point and 50% were present at all time points, suggesting the rapid expansion of a memory B cell population. The tetanus toxoid specificity of the CDR-H3 region with the 7th highest frequency at +1w was confirmed using immunoprecipitation and mass spectroscopy, and two public tetanus toxoid-specific CDR-H3 regions were also overrepresented at +1w. In summary, we have used the tetanus vaccine model system to demonstrate that bulk PC Ig repertoire analysis can identify PC populations that expand and mature following antigen exposure. The application of this approach before and after clinical infections should advance our understanding of clinical protection and facilitate vaccine design.

Correlations in Somatic Hypermutation Between Sites in IGHV Genes Can Be Explained by Interactions Between AID and/or Polη Hotspots.

The somatic hypermutation (SHM) of Immunoglobulin (Ig) genes is a key process during antibody affinity maturation in B cells. The mutagenic enzyme activation induced deaminase (AID) is required for SHM and has a preference for WRC hotspots in DNA. Error-prone repair mechanisms acting downstream of AID introduce further mutations, including DNA polymerase eta (Polη), part of the non-canonical mismatch repair pathway (ncMMR), which preferentially generates mutations at WA hotspots. Previously proposed mechanistic models lead to a variety of predictions concerning interactions between hotspots, for example, how mutations in one hotspot will affect another hotspot. Using a large, high-quality, Ig repertoire sequencing dataset, we evaluated pairwise correlations between mutations site-by-site using an unbiased measure similar to mutual information which we termed “mutational association” (MA). Interactions are dominated by relatively strong correlations between nearby sites (short-range MAs), which can be almost entirely explained by interactions between overlapping hotspots for AID and/or Polη. We also found relatively weak dependencies between almost all sites throughout each gene (longer-range MAs), although these arise mostly as a statistical consequence of high pairwise mutation frequencies. The dominant short-range interactions are also highest within the most highly mutating IGHV sub-regions, such as the complementarity determining regions (CDRs), where there is a high hotspot density. Our results suggest that the hotspot preferences for AID and Polη have themselves evolved to allow for greater interactions between AID and/or Polη induced mutations.

TET3 dioxygenase modulates gene conversion at the avian immunoglobulin variable region via demethylation of non-CpG sites in pseudogene templates.

Diversification of the avian primary immunoglobulin (Ig) repertoire is achieved in developing B cells by somatic hypermutation (SHM) and gene conversion (GCV). GCV is a type of homologous recombination that unidirectionally transfers segments of Ig pseudogenes to Ig variable domains. It is regulated by epigenetic mechanisms like histone modifications, but the role of DNA methylation remains unclear. Here, we demonstrate that the chicken B‐cell line DT40 lacking TET3, a member of the TET (Ten‐eleven translocation) family dioxygenases that facilitate DNA demethylation, exhibited a marked reduction in GCV activity in Ig variable regions. This was accompanied by a drop in the bulk levels of 5‐hydroxymethylcytosine, an oxidized derivative of 5‐methylcytosine, whereas TET1‐deficient or TET2‐deficient DT40 strains did not exhibit such effects. Deletion of TET3 caused little effects on the expression of proteins required for SHM and GCV, but induced hypermethylation in some Ig pseudogene templates. Notably, the enhanced methylation occurred preferably on non‐CpG cytosines. Disruption of both TET1 and TET3 significantly inhibited the expression of activation‐induced cytidine deaminase (AID), an essential player in Ig diversification. These results uncover unique roles of TET proteins in avian Ig diversification, highlighting the potential importance of TET3 in maintaining hypomethylation In Ig pseudogenes.

Profiling immunoglobulin repertoires across multiple human tissues using RNA sequencing

Profiling immunoglobulin (Ig) receptor repertoires with specialized assays can be cost-ineffective and time-consuming. Here we report ImReP, a computational method for rapid and accurate profiling of the Ig repertoire, including the complementary-determining region 3 (CDR3), using regular RNA sequencing data such as those from 8,555 samples across 53 tissues types from 544 individuals in the Genotype-Tissue Expression (GTEx v6) project. Using ImReP and GTEx v6 data, we generate a collection of 3.6 million Ig sequences, termed the atlas of immunoglobulin repertoires (TAIR), across a broad range of tissue types that often do not have reported Ig repertoires information. Moreover, the flow of Ig clonotypes and inter-tissue repertoire similarities across immune-related tissues are also evaluated. In summary, TAIR is one of the largest collections of CDR3 sequences and tissue types, and should serve as an important resource for studying immunological diseases.

Systematic Profiling of Full-Length Ig and TCR Repertoire Diversity in Rhesus Macaque through Long Read Transcriptome Sequencing.

The diversity of Ig and TCR repertoires is a focal point of immunological studies. Rhesus macaques (Macaca mulatta) are key for modeling human immune responses, placing critical importance on the accurate annotation and quantification of their Ig and TCR repertoires. However, because of incomplete reference resources, the coverage and accuracy of the traditional targeted amplification strategies for profiling rhesus Ig and TCR repertoires are largely unknown. In this study, using long read sequencing, we sequenced four Indian-origin rhesus macaque tissues and obtained high-quality, full-length sequences for over 6000 unique Ig and TCR transcripts, without the need for sequence assembly. We constructed, to our knowledge, the first complete reference set for the constant regions of all known isotypes and chain types of rhesus Ig and TCR repertoires. We show that sequence diversity exists across the entire variable regions of rhesus Ig and TCR transcripts. Consequently, existing strategies using targeted amplification of rearranged variable regions comprised of V(D)J gene segments miss a significant fraction (27-53% and 42-49%) of rhesus Ig/TCR diversity. To overcome these limitations, we designed new rhesus-specific assays that remove the need for primers conventionally targeting variable regions and allow single cell level Ig and TCR repertoire analysis. Our improved approach will enable future studies to fully capture rhesus Ig and TCR repertoire diversity and is applicable for improving annotations in any model organism.

Immunoglobulin variable domain high-throughput sequencing reveals specific novel mutational patterns in POEMS syndrome

AbstractPolyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare multisystem disease resulting from an underlying plasma cell (PC) dyscrasia. The pathophysiology of the disease remains unclear, but the role of the monoclonal immunoglobulin (Ig) light chain (LC) is strongly suspected because of the highly restrictive usage of 2 λ variable (V) domains (IGLV1-40 and IGLV1-44) and the general improvement of clinical manifestations after PC clone-targeted treatment. However, the diagnostic value of Ig LC sequencing, especially in the case of incomplete forms of the disease, remains to be determined. Using a sensitive high-throughput Ig repertoire sequencing on RNA (rapid amplification of cDNA ends-based repertoire sequencing [RACE-RepSeq]), we detected a λ LC monoclonal expansion in the bone marrow (BM) of 83% of patients with POEMS syndrome, including some in whom BM tests routinely performed to diagnose plasma cell dyscrasia failed to detect λ+ monoclonal PCs. Twenty-four (83%) of the 29 LC clonal sequences found were derived from the IGLV1-40 and IGLV1-44 germline genes, as well as 2 from the closely related IGLV1-36 gene, and all were associated with an IGLJ3*02 junction (J) gene, confirming the high restriction of VJ region usage in POEMS syndrome. RACE-RepSeq VJ full-length sequencing additionally revealed original mutational patterns, the strong specificity of which might crucially help establish or eliminate the diagnosis of POEMS syndrome in uncertain cases. Thus, RACE-RepSeq appears as a sensitive, rapid, and specific tool to detect low-abundance PC clones in BM and assign them to POEMS syndrome, with all the consequences for therapeutic options.

Characteristics of Plasmablast Repertoire in Chronically HIV-Infected Individuals for Immunoglobulin H and L Chain Profiled by Single-Cell Analysis.

Characterization of the diversified immunoglobulin (Ig) repertoire may provide insight into pathways that shape an efficient antibody (Ab) repertoire for immune response against human immunodeficiency virus (HIV) infection. This study aimed to profile characteristics of the plasmablast repertoire during chronic HIV infection. Ig variable regions of plasmablasts from both chronically HIV-infected donors (HIVDs) previously treated with antiretroviral therapy (ART) and healthy donors (HDs) were amplified by single-cell PCR to establish the basis for further repertoire analysis. We compared the plasmablast repertoires expressed in multiple chronically HIVDs after ART treatment cessation and HDs. We also examined the non-productive repertoire to identify the indication of the immediate products of the rearrangement machinery without an impact of selection during HIV infection. We found multiple differences between the productive repertoires of HIVD and HD subjects, including biased usages of VH3-49, VH1-2, VH3-33, VH3-74, and VH5-51 in VH and D1-7, D1-14, D1-20, and D5-5/18 in D segments in the HIVD group, as well as shorter and preferential glycine usages in CDRH3 regions. Gene selections were also detected in light chains. Notably, differences between productive rearrangements of HIVDs and HDs outnumbered those between productive and non-productive rearrangements within HIVDs. HIV infection may exert a dominant impact on the development of the plasmablast repertoire. The impact of selection is of limited significance in shaping the plasmablast repertoire. Overall, the data indicate that the environment in which the plasmablasts live can affect the distribution of the VH and VL genes in the repertoire and the amino acid compositions of the expressed Abs.

Gammaherpesvirus-infected germinal center cells express a distinct immunoglobulin repertoire.

The gammaherpesviruses (γHVs), human Kaposi sarcoma-associated herpesvirus (KSHV), EBV, and murine γHV68 are prevalent infections associated with lymphocyte pathologies. After primary infection, EBV and γHV68 undergo latent expansion in germinal center (GC) B cells and persists in memory cells. The GC reaction evolves and selects antigen-specific B cells for memory development but whether γHV passively transients or manipulates this process in vivo is unknown. Using the γHV68 infection model, we analyzed the Ig repertoire of infected and uninfected GC cells from individual mice. We found that infected cells displayed the hallmarks of affinity maturation, hypermutation, and isotype switching but underwent clonal expansion. Strikingly, infected cells displayed distinct repertoire, not found in uninfected cells, with recurrent utilization of certain Ig heavy V segments including Ighv10-1 In a manner observed with KSHV, γHV68 infected cells also displayed lambda light chain bias. Thus, γHV68 subverts GC selection to expand in a specific B cell subset during the process that develops long-lived immunologic memory.

The Yin and Yang of RNA surveillance in B lymphocytes and antibody-secreting plasma cells

The random V(D)J recombination process ensures the diversity of the primary immunoglobulin (Ig) repertoire. In two thirds of cases, imprecise recombination between variable (V), diversity (D), and joining (J) segments induces a frameshift in the open reading frame that leads to the appearance of premature termination codons (PTCs). Thus, many B lineage cells harbour biallelic V(D)J-rearrangements of Ig heavy or light chain genes, with a productively-recombined allele encoding the functional Ig chain and a nonproductive allele potentially encoding truncated Ig polypeptides. Since the pattern of Ig gene expression is mostly biallelic, transcription initiated from nonproductive Ig alleles generates considerable amounts of primary transcripts with out-of-frame V(D)J junctions. How RNA surveillance pathways cooperate to control the noise from nonproductive Ig genes will be discussed in this review, focusing on the benefits of nonsense-mediated mRNA decay (NMD) activation during B-cell development and detrimental effects of nonsense-associated altered splicing (NAS) in terminally differentiated plasma cells.

Transcription factor binding at Ig enhancers is linked to somatic hypermutation targeting.

Secondary diversification of the Ig repertoire occurs through somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR)-three processes that are initiated by activation-induced cytidine deaminase (AID). AID targets Ig genes at orders of magnitude higher than the rest of the genome, but the basis for this specificity is poorly understood. We have previously demonstrated that enhancers and enhancer-like sequences from Ig genes are capable of stimulating SHM of neighboring genes in a capacity distinct from their roles in increasing transcription. Here, we use an in vitro proteomics approach to identify E-box, MEF2, Ets, and Ikaros transcription factor family members as potential binders of these enhancers. ChIP assays in the hypermutating Ramos B cell line confirmed that many of these factors bound the endogenous Igλ enhancer and/or the IgH intronic enhancer (Eμ) in vivo. Further investigation using SHM reporter assays identified binding sites for E2A and MEF2B in Eμ and demonstrated an association between loss of factor binding and decreases in the SHM stimulating activity of Eμ mutants. Our results provide novel insights into trans-acting factors that dictate SHM targeting and link their activity to specific DNA binding sites within Ig enhancers.

RNA-Seq Based Immunoglobulin Repertoire Analysis of Normal Plasma Cells Generated in an in Vitro B Cell Differentiation System

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC &gt; 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Phenotypic and Ig Repertoire Analyses Indicate a Common Origin of IgD-CD27- Double Negative B Cells in Healthy Individuals and Multiple Sclerosis Patients.

IgD-CD27- double negative (DN) B cells with proinflammatory characteristics are abnormally elevated in a proportion of multiple sclerosis (MS) patients. In this study, the origin and selection characteristics of DN B cells were studied in MS patients and healthy controls (HC). Expression of developmental markers on peripheral blood DN, IgD-CD27+ class-switched memory (CSM) and IgD+CD27- naive B cells of HC (n = 48) and MS patients (n = 96) was determined by flow cytometry. High-throughput adaptive immune receptor repertoire sequencing was performed on peripheral blood DN and CSM B cells of HC and MS patients (n = 3 each). DN B cells from HC and MS patients showed similar phenotypic and Ig repertoire characteristics. Phenotypic analysis indicated a mature state of DN B cells by low CD5, CD10, and CD38 expression. However, the frequency of CD95+ and IgA+ cells was lower in DN versus CSM B cells. DN B cells are Ag experienced, as shown by somatic hypermutation of their Ig genes in adaptive immune receptor repertoire sequencing, although they showed a lower mutation load than CSM B cells. Shared clones were found between DN and CSM B cells, although >95% of the clones were unique to each population, and differences in V(D)J usage and CDR3 physicochemical properties were found. Thus, DN B cells arise in HC and MS patients via a common developmental pathway that is probably linked to immune aging. However, DN and CSM B cells develop through unique differentiation pathways, with most DN B cells representing an earlier maturation state.

Nasal Vaccination Drives Modifications of Nasal and Systemic Antibody Repertoires in Rainbow Trout.

Bony fish represent the most basal vertebrate branch with a dedicated mucosal immune system, which comprises immunologically heterogeneous microenvironments armed with innate and adaptive components. In rainbow trout (Oncorhynchus mykiss), a nasopharynx-associated lymphoid tissue (NALT) was recently described as a diffuse network of myeloid and lymphoid cells located in the olfactory organ of fish. Several studies have demonstrated high levels of protection conferred by nasal vaccines against viral and bacterial pathogens; however, the mechanisms underlying the observed protection are not well understood. We applied 5'RACE and a deep sequencing-based approach to investigate the clonal structure of the systemic and mucosal rainbow trout B cell repertoire. The analysis of Ig repertoire in control trout suggests different structures of IgM and IgT spleen and NALT repertoires, with restricted repertoire diversity in NALT. Nasal and injection vaccination with a bacterial vaccine revealed unique dynamics of IgM and IgT repertoires at systemic and mucosal sites and the remarkable ability of nasal vaccines to induce spleen Ig responses. Our findings provide an important immunological basis for the effectiveness of nasal vaccination in fish and other vertebrate animals and will help the design of future nasal vaccination strategies.

B cell receptor and Toll-like receptor signaling coordinate to control distinct B-1 responses to both self and the microbiota.

B-1a cells play an important role in mediating tissue homeostasis and protecting against infections. They are the main producers of 'natural' IgM, spontaneously secreted serum antibodies predominately reactive to self antigens, like phosphatidylcholine (PtC), or antigens expressed by the intestinal microbiota. The mechanisms that regulate the B-1a immunoglobulin (Ig) repertoire and their antibody secretion remain poorly understood. Here, we use a novel reporter mouse to demonstrate that production of self- and microbiota-reactive antibodies is linked to BCR signaling in B-1a cells. Moreover, we show that Toll-like receptors (TLRs) are critical for shaping the Ig repertoire of B-1a cells as well as regulating their antibody production. Strikingly, we find that both the colonization of a microbiota as well as microbial-sensing TLRs are required for anti-microbiota B-1a responses, whereas nucleic-acid sensing TLRs are required for anti-PtC responses, demonstrating that linked activation of BCR and TLRs controls steady state B-1a responses to both self and microbiota-derived antigens.