Search

High-level expression of the transcription factor T-bet characterizes a phenotypically distinct murine B cell population known as “age-associated B cells” (ABCs). T-bet–deficient mice have reduced ABCs and impaired humoral immunity. We describe a patient with inherited T-bet deficiency and largely normal humoral immunity including intact somatic hypermutation, affinity maturation and memory B cell formation in vivo, and B cell differentiation into Ig-producing plasmablasts in vitro. Nevertheless, the patient exhibited skewed class switching to IgG1, IgG4, and IgE, along with reduced IgG2, both in vivo and in vitro. Moreover, T-bet was required for the in vivo and in vitro development of a distinct subset of human B cells characterized by reduced expression of CD21 and the concomitantly high expression of CD19, CD20, CD11c, FCRL5, and T-bet, a phenotype that shares many features with murine ABCs. Mechanistically, human T-bet governed CD21 lo CD11c hi B cell differentiation by controlling the chromatin accessibility of lineage-defining genes in these cells: FAS , IL21R , SEC61B , DUSP4 , DAPP1 , SOX5 , CD79B , and CXCR4 . Thus, human T-bet is largely redundant for long-lived protective humoral immunity but is essential for the development of a distinct subset of human CD11c hi CD21 lo B cells.

Kids armed with anti-coronavirus B cells It remains unclear whether B cell repertoires against coronaviruses and other pathogens differ between adults and children and how important these distinctions are. Yang et al. analyzed blood samples from young children and adults, as well as tissues from deceased organ donors, characterizing the B cell receptor (BCR) repertoires specific to six common pathogens and two viruses that they had not seen before: Ebola virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Children had higher frequencies of B cells with convergent BCR heavy chains against previously encountered pathogens and higher frequencies of class-switched convergent B cell clones against SARS-CoV-2 and related coronaviruses. These findings suggest that encounters with coronaviruses in early life may produce cross-reactive memory B cell populations that contribute to divergent COVID-19 susceptibilities. Science, this issue p. 738, Blood taken from children before the COVID-19 pandemic contains memory B cells that bind SARS-CoV-2., Vaccination and infection promote the formation, tissue distribution, and clonal evolution of B cells, which encode humoral immune memory. We evaluated pediatric and adult blood and deceased adult organ donor tissues to determine convergent antigen-specific antibody genes of similar sequences shared between individuals. B cell memory varied for different pathogens. Polysaccharide antigenspecific clones were not exclusive to the spleen. Adults had higher clone frequencies and greater class switching in lymphoid tissues than blood, while pediatric blood had abundant class-switched convergent clones. Consistent with reported serology, prepandemic children had class-switched convergent clones to severe acute respiratory syndrome coronavirus 2 with weak cross-reactivity to other coronaviruses, while adult blood or tissues showed few such clones. These results highlight the prominence of early childhood B cell clonal expansions and cross-reactivity for future responses to novel pathogens.

Immunity after the flu shot The seasonal flu shot is currently recommended each year because the influenza viral strains in circulation are continuously changing and because the antibody responses produced by the vaccine decline over time. In a human study of healthy volunteers, Davis et al. tracked antibody responses after flu vaccination. They investigated whether the vaccine led to the generation of antibody-secreting plasma cells in the bone marrow, a lymphoid organ that supports the survival of these cells for years. Although vaccination did generate influenza-specific cells, most were short-lived and lost within 1 year. The fact that a small number did persist over 1 year raises prospects that the longevity of flu vaccines can be improved and provides key information for the development of universal vaccines against influenza. Science , this issue p. 237

The immunoglobulin genes of inbred mouse strains that are commonly used in models of antibody-mediated human diseases are poorly characterized. This compromises data analysis. To infer the immunoglobulin genes of BALB/c mice, we used long-read SMRT sequencing to amplify VDJ-C sequences from F1 (BALB/c x C57BL/6) hybrid animals. Strain variations were identified in theIghmandIghg2bgenes, and analysis of VDJ rearrangements led to the inference of 278 germline IGHV alleles. 169 alleles are not present in the C57BL/6 genome reference sequence. To establish a set of expressed BALB/c IGHV germline gene sequences, we computationally retrieved IGHV haplotypes from the IgM dataset. Haplotyping led to the confirmation of 162 BALB/c IGHV gene sequences. A musIGHV398 pseudogene variant also appears to be present in the BALB/cByJ substrain, while a functional musIGHV398 gene is highly expressed in the BALB/cJ substrain. Only four of the BALB/c alleles were also observed in the C57BL/6 haplotype. The full set of inferred BALB/c sequences has been used to establish a BALB/c IGHV reference set, hosted athttps://ogrdb.airr-community.org. We assessed whether assemblies from the Mouse Genome Project (MGP) are suitable for the determination of the genes of the IGH loci. Only 37 (43.5%) of the 85 confirmed IMGT-named BALB/c IGHV and 33 (42.9%) of the 77 confirmed non-IMGT IGHV were found in a search of the MGP BALB/cJ genome assembly. This suggests that current MGP assemblies are unsuitable for the comprehensive documentation of germline IGHVs and more efforts will be needed to establish strain-specific reference sets.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies., Graphical Abstract, Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions.

Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.

Vaccination and infection promote the formation, tissue distribution, and clonal evolution of B cells encoding humoral immune memory. We evaluated convergent antigen-specific antibody genes of similar sequences shared between individuals in pediatric and adult blood, and deceased organ donor tissues. B cell memory varied for different pathogens. Polysaccharide antigen-specific clones were not exclusive to the spleen. Adults’ convergent clones often express mutated IgM or IgD in blood and are class-switched in lymphoid tissues; in contrast, children have abundant class-switched convergent clones in blood. Consistent with serological reports, pre-pandemic children had class-switched convergent clones to SARS-CoV-2, enriched in cross-reactive clones for seasonal coronaviruses, while adults showed few such clones in blood or lymphoid tissues. These results extend age-related and anatomical mapping of human humoral pathogen-specific immunity.One Sentence SummaryChildren have elevated frequencies of pathogen-specific class-switched memory B cells, including SARS-CoV-2-binding clones.

B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV., Graphical Abstract, Highlights • Human IGH repertoire sequencing identifies SARS-CoV-2-specific B cell clones • Convergent virus-specific antibody sequences are shared between COVID-19 patients • SARS-CoV antibodies are detected by sequence in COVID-19 patients, B cells produce antibodies and provide protective immunity to viruses. In longitudinal data from COVID-19 patients, Nielsen et al. analyze the development of antibody gene repertoires responding to SARS-CoV-2. COVID-19 patients share subsets of similar antibody sequences that bind SARS-CoV-2 antigens, with rare antibodies that also recognize SARS-CoV.

A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth., A subset of HIV-infected individuals can develop broadly neutralizing antibodies. Boyd and colleagues studied such HIV-infected individuals and found significant perturbations in their antibody repertoires, including increased frequencies of B cells expressing antibodies with features associated with autoreactivity.

In patients, gain-of-function (GOF) mutations in PIK3CD break tolerance, causing highly penetrant secretion of autoreactive IgM. Mouse models reveal that Pik3cd GOF subverts the response to self-antigen, preventing the induction of anergy and instead stimulating plasmablast and GC formation., Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity., Graphical Abstract

Objective Rheumatoid factors (RFs) are associated with systemic disease in primary Sjogren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking. Methods Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. Results Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig VH ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig VH 4-34 IgM-RF decreased following immunosuppression and remission of MC. Conclusion Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.

The development of obesity-associated insulin resistance is associated with B-lymphocyte accumulation in visceral adipose tissue (VAT) and is prevented by B-cell ablation. To characterize potentially pathogenic B-cell repertoires in this disorder, we performed high-throughput immunoglobulin (Ig) sequencing from multiple tissues of mice fed high-fat diet (HFD) and regular diet (RD). HFD significantly changed the biochemical properties of Ig heavy-chain complementarity-determining region-3 (CDRH3) sequences, selecting for IgA antibodies with shorter and more hydrophobic CDRH3 in multiple tissues. A set of convergent antibodies of highly similar sequences found in the VAT of HFD mice but not RD mice showed significant somatic mutation, suggesting a response shared between mice to a common antigen or antigens. These findings indicate that a simple high-fat dietary intervention has a major impact on mouse B-cell repertoires, particularly in adipose tissues.

Mammalian immunoglobulin (IG) genes are found in complex loci that contain hundreds of highly similar pseudogenes, functional genes and repetitive elements, which has made their investigation particularly challenging. High-throughput sequencing has provided new avenues for the investigation of these loci, and has recently been applied to study the IG genes of important inbred mouse strains, revealing unexpected differences between their IG loci. This demonstrated that the structural differences are of such magnitude that they call into question the merits of the current mouse IG gene nomenclatures. Three nomenclatures for the mouse IG heavy chain locus (Igh) are presently in use, and they are all positional nomenclatures using the C57BL/6 genome reference sequence as their template. The continued use of these nomenclatures requires that genes of other inbred strains be confidently identified as allelic variants of C57BL/6 genes, but this is clearly impossible. The unusual breeding histories of inbred mouse strains mean that, regardless of the genetics of wild mice, no single ancestral origin for the IG loci exists for laboratory mice. Here we present a general discussion of the challenges this presents for any IG nomenclature. Furthermore, we describe principles that could be followed in the formulation of a solution to these challenges. Finally, we propose a non-positional nomenclature that accords with the guidelines of the International Mouse Nomenclature Committee, and outline strategies that can be adopted to meet the nomenclature challenges if three systems are to give way to a new one.

Mammalian immunoglobulin (IG) genes are found in complex loci that contain hundreds of highly similar pseudogenes, functional genes and repetitive elements, which has made their investigation particularly challenging. High-throughput sequencing has provided new avenues for the investigation of these loci, and has recently been applied to study the IG genes of important inbred mouse strains, revealing unexpected differences between their IG loci. This demonstrated that the structural differences are of such magnitude that they call into question the merits of the current mouse IG gene nomenclatures. Three nomenclatures for the mouse IG heavy chain locus (Igh) are presently in use, and they are all positional nomenclatures using the C57BL/6 genome reference sequence as their template. The continued use of these nomenclatures requires that genes of other inbred strains be confidently identified as allelic variants of C57BL/6 genes, but this is clearly impossible. The unusual breeding histories of inbred mouse strains mean that, regardless of the genetics of wild mice, no single ancestral origin for the IG loci exists for laboratory mice. Here we present a general discussion of the challenges this presents for any IG nomenclature. Furthermore, we describe principles that could be followed in the formulation of a solution to these challenges. Finally, we propose a non-positional nomenclature that accords with the guidelines of the International Mouse Nomenclature Committee, and outline strategies that can be adopted to meet the nomenclature challenges if three systems are to give way to a new one.

The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus. We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflects differences in subspecies origin. To investigate this hypothesis, we conducted high-throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ) and diversity (IGHD) genes in four inbred wild-derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ and PWD/PhJ) and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline IGHV sequences were inferred in the wild-derived strains, including 247 not curated in the international ImMunoGeneTics information system. By contrast, 83/84 inferred NOD IGHV genes had previously been observed in C57BL/6 mice. Variability among the strains examined was observed for only a single IGHJ gene, involving a description of a novel allele. By contrast, unexpected variation was found in the IGHD gene loci, with four previously unreported IGHD gene sequences being documented. Very few IGHV sequences of C57BL/6 and BALB/c mice were shared with strains representing major subspecies, suggesting that their IGH loci may be complex mosaics of genes of disparate origins. This suggests a similar level of diversity is likely present in the IGH loci of other classical inbred strains. This must now be documented if we are to properly understand interstrain variation in models of antibody-mediated disease.

Background Autoantibodies appear in the serum years before the development of clinical disease in patients ultimately diagnosed with systemic lupus erythematosus (SLE) or Sjogrens syndrome. The transition from benign to pathogenic autoimmunity is not understood. Anti-IgG rheumatoid factors provide an illuminating example of an autoantibody that persists, on average, more than four years prior to the development of systemic vasculitis and type II cryoglobuinemia. The aim of this study was to identify the pathogenic changes occurring in B cells making rheumatoid factor autoantibody during this transition. Methods Massively parallel sequencing of peripheral blood B cell receptors (BCR) was combined with mass spectrometry peptide sequencing of serum rheumatoid factor autoantibodies to identify circulating B cells expressing IgM rheumatoid factors in 4 patients with SLE or Sjogrens syndrome. In a patient presenting with primary Sjogrens syndrome evolving to cutaneous vasculitis, blood samples before and after transition were subjected to single cell analysis of mRNA, genome methylation, and lymphocyte regulatory genes recurrently mutated in lymphoma and leukemia. Results Circulating rheumatoid factor B cell clones and their matched serum autoantibodies were identified in 4 patients, 3 of whom, produced the stereotypic Wa-type rheumatoid factor with IGHV1–69 and IGKV3–20 variable domains. Longitudinal analysis of a patient before and after vasculitis revealed a single Wa-type rogue clone was responsible for the rheumatoid factor over a period of 6 years. Compared to the patients normal memory B cells, the rogue cells had aberrant gene expression corresponding to CD11c+CD21 low B cells observed in autoimmunity, and hypomethylation of gene regions aberrantly hypomethylated in chronic lymphocytic leukemia (CLL). A loss-of-function somatic mutation in a Kelch-like protein gene, KLHL6 identical to recurring CLL and lymphoma mutations was acquired by half of the rheumatoid factor B cells. KLHL6 mutant cells slowly accumulated antibody variable domain replacement mutations that did not alter binding affinity but caused IgM-IgG immune complexes to precipitate at temperatures below 25C. Conclusions Transition from a benign to pathogenic rheumatoid factor was preceded by a lymphoproliferative disease mutation in the responsible B cells and resulted in accumulation of antibody somatic mutations that diminish immune complex solubility. The ability to detect clones of the rheumatoid factor that are accumulating replacement mutations years prior to the patient developing cryoglobulinemia represents an opportunity to interfere with the conversion from asymptomatic seropositivity to clinical disease. Funding Source(s): Australian National Health and Medical Research Council and New South Wales Department of Health

Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT®, the international ImMunoGeneTics information system® (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT®. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC—focusing, to begin with, on human IGHV genes—with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species.

Background Specific immunotherapy (SIT) is the only treatment with proved long-term curative potential in patients with allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B-cell repertoires are not well understood. Objective We sought to characterize the IgE sequences expressed by allergen-specific B cells and track the fate of these B-cell clones during SIT. Methods We used high-throughput antibody gene sequencing and identification of allergen-specific IgE with combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and the nasal mucosa from aeroallergen-sensitized subjects before and during the first year of subcutaneous SIT. Results Of 52 distinct allergen-specific IgE heavy chains from 8 allergic donors, 37 were also detected by using high-throughput antibody gene sequencing of blood samples, nasal mucosal samples, or both. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. Conclusion In the future, combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies might aid assessment of SIT mechanisms and efficacy.

Human IgH diversity is influenced by biases in the pairing of IGHD and IGHJ genes, but these biases have not been described in detail. We used high-throughput sequencing of VDJ rearrangements to explore DJ pairing biases in 29 individuals. It was possible to infer three contrasting IGHD-IGHJ haplotypes in nine of these individuals, and two of these haplotypes include deletion polymorphisms involving multiple contiguous IGHD genes. Therefore, we were able to explore how the underlying genetic makeup of the H chain locus influences the formation of particular DJ pairs. Analysis of nonproductive rearrangements demonstrates that 3′ IGHD genes tend to pair preferentially with 5′ IGHJ genes, whereas 5′ IGHD genes pair preferentially with 3′ IGHJ genes; the relationship between IGHD gene pairing frequencies and IGHD gene position is a near linear one for each IGHJ gene. However, striking differences are seen in individuals who carry deletion polymorphisms in the D locus. The absence of different blocks of IGHD genes leads to increases in the utilization frequencies of just a handful of genes, and these genes have no clear positional relationships to the deleted genes. This suggests that pairing frequencies may be influenced by additional complex positional relationships that perhaps arise from chromatin structure. In contrast to IGHD gene usage, IGHJ gene usage is unaffected by the IGHD gene–deletion polymorphisms. Such an outcome would be expected if the recombinase complex associates with an IGHJ gene before associating with an IGHD gene partner.

Background The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major pathogenic and therapeutic significance. Objective We sought to characterize peanut allergen–specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy. Methods B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones. Results Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG 4 members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase. Conclusion Most peanut allergen–binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG 4 .

Antigenic exposures at epithelial sites in infancy and early childhood are thought to influence the maturation of humoral immunity and modulate the risk of developing immunoglobulin E (IgE)-mediated allergic disease. How different kinds of environmental exposures influence B cell isotype switching to IgE, IgG, or IgA, and the somatic mutation maturation of these antibody pools, is not fully understood. We sequenced antibody repertoires in longitudinal blood samples in a birth cohort from infancy through the first 3 years of life and found that, whereas IgG and IgA show linear increases in mutational maturation with age, IgM and IgD mutations are more closely tied to pathogen exposure. IgE mutation frequencies are primarily increased in children with impaired skin barrier conditions such as eczema, suggesting that IgE affinity maturation could provide a mechanistic link between epithelial barrier failure and allergy development.

In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS) of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation.

Vaccination has saved many lives and prevented needless suffering from disease, but it is not always effective. Immune responses are a highly "personalized" aspect of an individual's biology, as they are subject to germline genetic influences but are embodied in cell populations that continuously sample the environment. Additionally, immunity is shaped by memory of prior infectious diseases and other antigenic exposures. Here, we review examples of recent technical advances and insights into human vaccine responses that are helping to define the features associated with successful vaccination and that may enable a more predictive vaccinology in the future.

The immune systems of all mammals include populations of B cells producing antibodies with incredibly diverse specificities. Repertoire diversity has been described as the "miracle of immunology," and it was long thought to be the result of essentially stochastic processes. Recently, however, analysis of high throughput gene sequencing data has shown that hard-wired biases in these processes result in antibody repertoires that are broadly predictable. The repertoires of mice and humans are both predictable, but they are strikingly different. In this review, features of the naive antibody repertoires of the two species are contrasted. We show that the mouse repertoire includes a conspicuous population of public clonotypes that are shared by different individuals of an inbred strain. These clonotypes are the result of gene rearrangements that involve little gene processing. By skewing repertoire formation toward such sequences, which probably target commonly encountered pathogens, it may be that the relatively small mouse repertoire is appropriate and effective despite its size. Species like the mouse face challenges that are a direct consequence of their small body sizes and the limitations this places on the antibody arsenal-particularly early in ontogeny. We propose that it is the differences in the naive repertoires of mice and humans, and the differences in the ways these repertoires are used, which ensure that the very different biological needs of the two species are met. The processes that contribute to repertoire formation may appear to be stochastic, but in both species, evolution has left little to chance.

It was with great interest that we read the recently published article by Yu et al. ([1][1]), which proposes a solution to the problem of building complete and accurate databases of germline Ig (IG) and TCR genes and alleles. This highlights one of the most formidable challenges in the

SummaryB cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual’s secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.

Somatic point mutations provide glimpses into B-cell histories, and mutation numbers generally correlate with antibody affinity. We recently proposed a model of human isotype function, based in part on mutation analysis, in which the dominant pathway of isotype switching involves B cells moving sequentially through the four immunoglobulin (Ig) G subclasses. This should result in predictable differences in affinity between isotypes, and this helps explain how different isotypes work together. The model built on analysis of rearranged immunoglobulin heavy chain sequences amplified from Papua New Guinean villagers, which showed highly significant differences in the mean number of V-REGION mutations in sequences, associated with the different IgG subclasses. To determine whether this relationship between mutation levels and isotypes is a more general phenomenon, the present study was conducted in healthy, urban residents of Sydney, Australia. VDJ sequences were generated from eight individuals, using 454 pyrosequencing, from cells expressing all isotypes except IgD and IgE. This resulted in 35 118 unique, productive VDJ sequences for the study. The data confirm that VDJ genes associated with progressively more 3' Ig heavy chain gamma (IGHG) constant region genes show increasing levels of point mutation. Mean V-REGION mutations in IgA1 and IgA2 sequences were similar. Patterns of mutations also differed between isotypes. Despite their association with T-independent responses, IgG2 sequences showed significantly more mutational evidence of antigen selection than other IgG isotypes. Antigen selection was also significantly higher in IgA2 than in IgA1 sequences, raising the possibility of a preferential switch pathway from IGHG2 to IGHA2.

Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by cytomegalovirus (CMV) is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or Epstein-Barr virus (EBV) infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of immunoglobulin heavy chain (IGH) gene rearrangements to study the B cell receptor repertoires over two successive years in 27 individuals ranging in age from 20 to 89 years. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG immunoglobulin genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, while CMV infection correlates with the proportion of highly mutated antibody genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires, and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.

Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody.

We have analysed the transcribed immunoglobulin kappa (IGK) repertoire of peripheral blood B cells from four individuals from two genetically distinct populations, Papua New Guinean and Australian, using high-throughput DNA sequencing. The depth of sequencing data for each individual averaged 5,548 high-quality IGK reads, and permitted genotyping of the inferred IGKV and IGKJ germline gene segments for each individual. All individuals were homozygous at each IGKJ locus and had highly similar inferred IGKV genotypes. Preferential gene usage was seen at both the IGKV and IGKJ loci, but only IGKV segment usage varied significantly between individuals. Despite the differences in IGKV gene utilisation, the rearranged IGK repertoires showed extensive identity at the amino acid level. Public rearrangements (those shared by two or more individuals) made up 60.2% of the total sequenced IGK rearrangements. The total diversity of IGK rearrangements of each individual was estimated to range from just 340 to 549 unique amino acid sequences. Thus, the repertoire of unique expressed IGK rearrangements is dramatically less than previous theoretical estimates of IGK diversity, and the majority of expressed IGK rearrangements are likely to be extensively shared in individual human beings.

Patterns of somatic mutation in IgE genes from allergic individuals have been a focus of study for many years, but IgE sequences have never been reported from parasitized individuals. To study the role of antigen selection in the evolution of the anti-parasite response, we therefore generated 118 IgE sequences from donors living in Papua New Guinea (PNG), an area of endemic parasitism. For comparison, we also generated IgG1, IgG2, IgG3 and IgG4 sequences from these donors, as well as IgG1 sequences from Australian donors. IgE sequences had, on average, 23.0 mutations. PNG IgG sequences had average mutation levels that varied from 17.7 (IgG3) to 27.1 (IgG4). Mean mutation levels correlated significantly with the position of their genes in the constant region gene locus (IgG3 < IgG1 < IgG2 < IgG4). Interestingly, given the heavy, life-long antigen burden experienced by PNG villagers, average mutation levels in IgG sequences were little different to that seen in Australian IgG1 sequences (19.2). Patterns of mutation provide clear evidence of antigen selection in many IgG sequences. The percentage of IgG sequences that showed significant accumulations of replacement mutations in the complementarity determining regions ranged from 22% of IgG3 sequences to 39% of IgG2 sequences. By contrast, only 12% of IgE sequences had such evidence of antigen selection, and this was significantly less than in PNG IgG1, IgG2 and IgG4 subclass sequences (P < 0.01). The anti-parasite IgE response therefore has the reduced evidence of antigen selection that has previously been reported in studies of IgE sequences from allergic individuals.

Complete and accurate knowledge of the genes and allelic variants of the human immunoglobulin gene loci is critical for studies of B cell repertoire development and somatic point mutation, but evidence from studies of VDJ rearrangements suggests that our knowledge of the available immunoglobulin gene repertoire is far from complete. The reported repertoire has changed little over the last 15 years. This is, in part, a consequence of the inefficiencies involved in searching for new members of large, multigenic gene families by cloning and sequencing. The advent of high-throughput sequencing provides a new avenue by which the germline repertoire can be explored. In this report, we describe pyrosequencing studies of the heavy chain IGHV1, IGHV3 and IGHV4 gene subgroups in ten Papua New Guineans. Thousands of 454 reads aligned with complete identity to 51 previously reported functional IGHV genes and allelic variants. A new gene, IGHV3-NL1*01, was identified, which differs from the nearest previously reported gene by 15 nucleotides. Sixteen new IGHV alleles were also identified, 15 of which varied from previously reported functional IGHV genes by between one and four nucleotides, while one sequence appears to be a functional variant of the pseudogene IGHV3-25. BLAST searches suggest that at least six of these new genes are carried within the relatively well-studied populations of North America, Europe or Asia. This study substantially expands the known immunoglobulin gene repertoire and demonstrates that genetic variation of immunoglobulin genes can now be efficiently explored in different human populations using high-throughput pyrosequencing.

Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.

The reliable identification of IGHD genes within human immunoglobulin heavy chains is challenging with up to one third of rearrangements having no identifiable IGHD gene. The short, mutated IGHD genes are generally assumed to be indistinguishable from the N-REGIONS of non-template encoded nucleotides that surround them. In this study we have characterised N-REGIONS, demonstrating the importance of nucleotide composition biases in the addition process, including the formation of homopolymer tracts. We then use a simulation approach to determine the likelihood of misidentification of highly mutated IGHD genes among the JUNCTION nucleotides. These likelihoods provide general rules for the identification of mutated D-REGIONs, and suggest that longer D-REGIONs (>25 nucleotides) with as many as ten mutations can be identified with a low risk of error. Shorter D-REGIONs (>16 nucleotides) with as many as four mutations are also identifiable. The reliability of different alignments is dependent upon the junction length (combined N-REGIONs and D-REGION). Data is presented that can guide the alignment of sequences with junction lengths from 5 to 50 nucleotides, including explicit selection between two D-REGION possibilities. The use of such a statistically-based approach to the alignment of IGHD genes will improve the reliability of the partitioning of immunoglobulin sequences, and this in turn will facilitate the study of the many processes that contribute to the diversity of the immunoglobulin repertoire.

The human immunoglobulin repertoire is vast, producing billions of unique antibodies from a limited number of germline immunoglobulin genes. The immunoglobulin heavy chain variable region (IGHV) is central to antigen binding and is comprised of 48 functional genes. Here we analyzed whether HIV-1 infected individuals who develop broadly neutralizing antibodies show a distinctive germline IGHV profile. Using both 454 and Illumina technologies we sequenced the IGHV repertoire of 28 HIV-infected South African women from the Center for the AIDS Programme of Research in South African (CAPRISA) 002 and 004 cohorts, 13 of whom developed broadly neutralizing antibodies. Of the 259 IGHV alleles identified in this study, approximately half were not found in the International Immunogenetics Database (IMGT). This included 85 entirely novel alleles and 38 alleles that matched rearranged sequences in non-IMGT databases. Analysis of the rearranged H chain V region genes of monoclonal antibodies isolated from 7 of the CAPRISA women and previously isolated broadly neutralizing antibodies from other donors provided evidence that at least 8 novel or non-IMGT alleles contributed to functional antibodies. Importantly, we found that despite a wide range in the number of IGHV alleles in each individual, including alleles used by known broadly neutralizing antibodies, there were no significant differences in germline IGHV repertoires between individuals who do and do not develop broadly neutralizing antibodies. This study reports novel IGHV repertoires and highlights the importance of a fully comprehensive immunoglobulin database for germline gene usage prediction. Furthermore, these data suggest a lack of genetic bias in broadly neutralizing antibody development in HIV-1 infection, with implications for HIV vaccine design.

The diversity of the human antibody repertoire that is generated by V(D)J gene rearrangement is extended by nine constant region genes that give antibodies their complex array of effector functions. The application of high throughput sequencing to the study of V(D)J gene rearrangements has led to significant recent advances in our understanding of the antigen-binding repertoire. In contrast, our understanding of antibody function has changed little, and mystery still surrounds the existence of four distinctive IgG subclasses. Recent observations from murine models and from human studies of VDJ somatic point mutations suggest that the timing of emergence of cells from the germinal centre may vary as a consequence of class switching. This should lead to predictable differences in affinity between isotypes. These differences, and varying abilities of the isotypes to fix complement and bind FcRs, could help coordinate the humoral defences over the time course of a response. We therefore propose a Temporal Model of human IgE and IgG function in which early emergence of IgE sensitises sentinel mast cells while switching to IgG3 recruits FcγR-mediated functions to the early response. IgG1 then emerges as the major effector of antigen clearance, and subsequently IgG2 competes with IgG1 to produce immune complexes that slow the inflammatory drive. Persisting antigen may finally stimulate high affinity IgG4 that outcompetes other isotypes and can terminate IgG1 / FcγR-mediated activation via the inhibitory FcγRIIB. In this way, IgG antibodies of different subclasses, at different concentrations and with sometimes opposing functions deliver cohesive, protective immune function.

Both the B cell receptor (BCR) and the T cell receptor (TCR) repertoires are generated through essentially identical processes of V(D)J recombination, exonuclease trimming of germline genes, and the random addition of non-template encoded nucleotides. The naïve TCR repertoire is constrained by thymic selection, and TCR repertoire studies have therefore focused strongly on the diversity of MHC-binding complementarity determining region (CDR) CDR3. The process of somatic point mutations has given B cell studies a major focus on variable (IGHV, IGLV, and IGKV) genes. This in turn has influenced how both the naïve and memory BCR repertoires have been studied. Diversity (D) genes are also more easily identified in BCR VDJ rearrangements than in TCR VDJ rearrangements, and this has allowed the processes and elements that contribute to the incredible diversity of the immunoglobulin heavy chain CDR3 to be analyzed in detail. This diversity can be contrasted with that of the light chain where a small number of polypeptide sequences dominate the repertoire. Biases in the use of different germline genes, in gene processing, and in the addition of non-template encoded nucleotides appear to be intrinsic to the recombination process, imparting "shape" to the repertoire of rearranged genes as a result of differences spanning many orders of magnitude in the probabilities that different BCRs will be generated. This may function to increase the precursor frequency of naïve B cells with important specificities, and the likely emergence of such B cell lineages upon antigen exposure is discussed with reference to public and private T cell clonotypes.

The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased “haplotypes” has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.

The identification of the genes that make up rearranged immunoglobulin genes is critical to many studies. For example, the enumeration of mutations in immunoglobulin genes is important for the prognosis of chronic lymphocytic leukemia, and this requires the accurate identification of the germline genes from which a particular sequence is derived. The immunoglobulin heavy-chain variable (IGHV) gene repertoire is generally considered to be highly polymorphic. In this report, we describe a bioinformatic analysis of germline and rearranged immunoglobulin gene sequences which casts doubt on the existence of a substantial proportion of reported germline polymorphisms. We report a five-level classification system for IGHV genes, which indicates the likelihood that the genes have been reported accurately. The classification scheme also reflects the likelihood that germline genes could be incorrectly identified in mutated VDJ rearrangements, because of similarities to other alleles. Of the 226 IGHV alleles that have previously been reported, our analysis suggests that 104 of these alleles almost certainly include sequence errors, and should be removed from the available repertoire. The analysis also highlights the presence of common mismatches, with respect to the germline, in many rearranged heavy-chain sequences, suggesting the existence of twelve previously unreported alleles. Sequencing of IGHV genes from six individuals in this study confirmed the existence of three of these alleles, which we designate IGHV3-49*04, IGHV3-49*05 and IGHV4-39*07. We therefore present a revised repertoire of expressed IGHV genes, which should substantially improve the accuracy of immunoglobulin gene analysis.

Antigen selection of B cells within the germinal center reaction generally leads to the accumulation of replacement mutations in the complementarity-determining regions (CDRs) of immunoglobulin genes. Studies of mutations in IgE-associated VDJ gene sequences have cast doubt on the role of antigen selection in the evolution of the human IgE response, and it may be that selection for high affinity antibodies is a feature of some but not all allergic diseases. The severity of IgE-mediated anaphylaxis is such that it could result from higher affinity IgE antibodies. We therefore investigated IGHV mutations in IgE-associated sequences derived from ten individuals with a history of anaphylactic reactions to bee or wasp venom or peanut allergens. IgG sequences, which more certainly experience antigen selection, served as a control dataset. A total of 6025 unique IgE and 5396 unique IgG sequences were generated using high throughput 454 pyrosequencing. The proportion of replacement mutations seen in the CDRs of the IgG dataset was significantly higher than that of the IgE dataset, and the IgE sequences showed little evidence of antigen selection. To exclude the possibility that 454 errors had compromised analysis, rigorous filtering of the datasets led to datasets of 90 core IgE sequences and 411 IgG sequences. These sequences were present as both forward and reverse reads, and so were most unlikely to include sequencing errors. The filtered datasets confirmed that antigen selection plays a greater role in the evolution of IgG sequences than of IgE sequences derived from the study participants.

Immunoglobulin rearrangement involves random and imprecise processes that act to both create and constrain diversity. Two such processes are the loss of nucleotides through the action of unknown exonuclease(s) and the addition of P nucleotides. The study of such processes has been compromised by difficulties in reliably aligning immunoglobulin genes and in the partitioning of nucleotides between segment ends, and between N and P nucleotides. A dataset of 294 human IgM sequences was created and partitioned with the aid of a probabilistic model. Non-random removal of nucleotides is seen between the three IGH gene types with the IGHV gene averaging removals of 1.2 nucleotides compared to 4.7 for the other gene ends (p < 0.001). Individual IGHV, IGHD and IGHJ gene subgroups also display statistical differences in the level of nucleotide loss. For example, within the IGHJ group, IGHJ3 has average removals of 1.3 nucleotides compared to 6.4 nucleotides for IGHJ6 genes (p < 0.002). Analysis of putative P nucleotides within the IgM and pooled datasets revealed only a single putative P nucleotide motif (GTT at the 3' D-REGION end) to occur at a frequency significantly higher then would be expected from random N nucleotide addition. The loss of nucleotides due to the action of exonucleases is not random, but is influenced by the nucleotide composition of the genes. P nucleotides do not make a significant contribution to diversity of immunoglobulin sequences. Although palindromic sequences are present in 10% of immunologlobulin rearrangements, most of the 'palindromic' nucleotides are likely to have been inserted into the junction during the process of N nucleotide addition. P nucleotides can only be stated with confidence to contribute to diversity of less than 1% of sequences. Any attempt to identify P nucleotides in immunoglobulins is therefore likely to introduce errors into the partitioning of such sequences.