IL-7 Enables Antibody Responses to Bacterial Polysaccharides by Promoting B Cell Receptor Diversity.

Polysaccharide vaccines such as the Vi polysaccharide (ViPS) of Salmonella enterica serovar Typhi induce efficient Ab responses in adults but not in young children. The reasons for this difference are not understood. IL-7 dependency in B cell development increases progressively with age. IL-7Rα-mediated signals are required for the expression of many VH gene segments that are distal to DH-JH in the IgH locus and for the complete diversification of the BCR repertoire. Therefore, we hypothesized that B cells generated in the absence of IL-7 do not recognize a wide range of Ags because of a restricted BCR repertoire. Compared with adult wildtype mice, young wildtype mice and IL-7-deficient adult mice generated a significantly reduced Ab response to ViPS. Additionally, ViPS-binding B cells in adult wildtype mice predominantly used distal VH gene segments. Transgenic expression of either IL-7 or a BCR encoded by a distal VH gene segment permitted young mice to respond efficiently to bacterial polysaccharides. These results indicate that restricted VH gene usage early in life results in a paucity of Ag-specific B cell precursors, thus limiting antipolysaccharide responses.

Cynomolgus macaque (Macaca fascicularis) immunoglobulin heavy chain locus description.

Cynomolgus macaques (Macaca fascicularis) have become an important animal model for biomedical research. In particular, it is the animal model of choice for the development of vaccine candidates associated with emerging dangerous pathogens. Despite their increasing importance as animal models, the cynomolgus macaque genome is not fully characterized, hindering molecular studies for this model. More importantly, the lack of knowledge about the immunoglobulin (IG) locus organization directly impacts the analysis of the humoral response in cynomolgus macaques. Recent advances in next generation sequencing (NGS) technologies to analyze IG repertoires open the opportunity to deeply characterize the humoral immune response. However, the IG locus organization for the animal is required to completely dissect IG repertoires. Here, we describe the localization and organization of the rearranging IG heavy (IGH) genes on chromosome 7 of the cynomolgus macaque draft genome. Our annotation comprises 108 functional genes which include 63 variable (IGHV), 38 diversity (IGHD), and 7 joining (IGHJ) genes. For validation, we provide RNA transcript data for most of the IGHV genes and all of the annotated IGHJ genes, as well as proteomic data to validate IGH constant genes. The description and annotation of the rearranging IGH genes for the cynomolgus macaques will significantly facilitate scientific research. This is particularly relevant to dissect the immune response during vaccination or infection with dangerous pathogens such as Ebola, Marburg and other emerging pathogens where non-human primate models play a significant role for countermeasure development.

The Eµ enhancer region influences H chain expression and B cell fate without impacting IgVH repertoire and immune response in vivo.

The IgH intronic enhancer region Eµ is a combination of both a 220-bp core enhancer element and two 310-350-bp flanking scaffold/matrix attachment regions named MARsEµ. In the mouse, deletion of the core-enhancer Eµ element mainly affects VDJ recombination with minor effects on class switch recombination. We carried out endogenous deletion of the full-length Eµ region (core plus MARsEµ) in the mouse genome to study VH gene repertoire and IgH expression in developing B-lineage cells. Despite a severe defect in VDJ recombination with partial blockade at the pro-B cell stage, Eµ deletion (core or full length) did not affect VH gene usage. Deletion of this regulatory region induced both a decrease of pre-B cell and newly formed B cell compartments and a strong orientation toward the marginal zone B cell subset. Because Igµ H chain expression was decreased in Eµ-deficient pre-B cells, we propose that modification of B cell homeostasis in deficient animals was caused by "weak" pre-B cell and BCR expression. Besides imbalances in B cell compartments, Ag-specific Ab responses were not impaired in animals carrying the Eµ deletion. In addition to its role in VDJ recombination, our study points out that the full-length Eµ region does not influence VH segment usage but ensures efficient Igµ-chain expression required for strong signaling through pre-B cells and newly formed BCRs and thus participates in B cell inflow and fate.

The V(H) repertoire and clonal diversification of B cells in inflammatory myopathies.

The contribution of antigen-driven B-cell adaptive immune responses within the inflamed muscle of inflammatory myopathies (IMs) is largely unknown. In this study, we investigated the immunoglobulin V(H) gene repertoire, somatic hypermutation, clonal diversification, and selection of infiltrating B cells in muscle biopsies from IM patients (dermatomyositis and polymyositis), to determine whether B cells and/or plasma cells contribute to the associated pathologies of these diseases. The data reveal that Ig V(H) gene repertoires of muscle-infiltrating B cells deviate from the normal V(H) gene repertoire in individual patients, and differ between different types of IMs. Analysis of somatic mutations revealed clonal diversification of muscle-infiltrating B cells and evidence for a chronic B-cell response within the inflamed muscle. We conclude that muscle-infiltrating B cells undergo selection, somatic hypermutation and clonal diversification in situ during antigen-driven immune responses in patients with IMs, providing insight into the contribution of B cells to the pathological mechanisms of these disorders.

AID induces intraclonal diversity and genomic damage in CD86(+) chronic lymphocytic leukemia cells.

The activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off-target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B-cell malignancies. Although it has been shown that AID is expressed in B-cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B-cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sµ), showing ongoing AID activity in vivo during disease progression. This AID-mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ-H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL.

Germline deletion of Igh 3' regulatory region elements hs 5, 6, 7 (hs5-7) affects B cell-specific regulation, rearrangement, and insulation of the Igh locus.

Regulatory elements located within an ∼28-kb region 3' of the Igh gene cluster (3' regulatory region) are required for class switch recombination and for high levels of IgH expression in plasma cells. We previously defined novel DNase I hypersensitive sites (hs) 5, 6, 7 immediately downstream of this region. The hs 5-7 region (hs5-7) contains a high density of binding sites for CCCTC-binding factor (CTCF), a zinc finger protein associated with mammalian insulator activity, and is an anchor for interactions with CTCF sites flanking the D(H) region. To test the function of hs5-7, we generated mice with an 8-kb deletion encompassing all three hs elements. B cells from hs5-7 knockout (KO) (hs5-7KO) mice showed a modest increase in expression of the nearest downstream gene. In addition, Igh alleles in hs5-7KO mice were in a less contracted configuration compared with wild-type Igh alleles and showed a 2-fold increase in the usage of proximal V(H)7183 gene families. Hs5-7KO mice were essentially indistinguishable from wild-type mice in B cell development, allelic regulation, class switch recombination, and chromosomal looping. We conclude that hs5-7, a high-density CTCF-binding region at the 3' end of the Igh locus, impacts usage of V(H) regions as far as 500 kb away.

Translocations activating IRF4 identify a subtype of germinal center-derived B-cell lymphoma affecting predominantly children and young adults.

The prognosis of germinal center-derived B-cell (GCB) lymphomas, including follicular lymphoma and diffuse large-B-cell lymphoma (DLBCL), strongly depends on age. Children have a more favorable outcome than adults. It is not known whether this is because of differences in host characteristics, treatment protocols, or tumor biology, including the presence of chromosomal alterations. By screening for novel IGH translocation partners in pediatric and adult lymphomas, we identified chromosomal translocations juxtaposing the IRF4 oncogene next to one of the immunoglobulin (IG) loci as a novel recurrent aberration in mature B-cell lymphoma. FISH revealed 20 of 427 lymphomas to carry an IG/IRF4-fusion. Those were predominantly GCB-type DLBCL or follicular lymphoma grade 3, shared strong expression of IRF4/MUM1 and BCL6, and lacked PRDM1/BLIMP1 expression and t(14;18)/BCL2 breaks. BCL6 aberrations were common. The gene expression profile of IG/IRF4-positive lymphomas differed from other subtypes of DLBCL. A classifier for IG/IRF4 positivity containing 27 genes allowed accurate prediction. IG/IRF4 positivity was associated with young age and a favorable outcome. Our results suggest IRF4 translocations to be primary alterations in a molecularly defined subset of GCB-derived lymphomas. The probability for this subtype of lymphoma significantly decreases with age, suggesting that diversity in tumor biology might contribute to the age-dependent differences in prognosis of lymphoma.

Central nervous system lymphoma newly developed 12 years after remission of an ocular adnexal lymphoma.

Recurrence of non-Hodgkin's lymphoma more than 5 years after the initial diagnosis is rare. When late relapse occurs, it is difficult to determine whether it is a true recurrence or a new lesion. We experienced a case of an 81-year-old woman who developed central nervous system (CNS) lymphoma 12 years after remission of ocular adnexal lymphoma. Both showed the histology of diffuse large B-cell lymphoma. To elucidate whether the CNS lymphoma was clonally related to the first lymphoma, rearrangement of the immunoglobulin heavy chain genes of each lymphoma was studied using a polymerase chain reaction-based method. The results revealed that the sizes of the amplified products of the rearranged regions from the two lymphomas were different. This suggested different clonal origins of the lymphomas. It is clinically important to determine the origin of a second neoplasm because patients with a clonally related second lymphoma are usually treated with more intensive regimens, while those with a clonally unrelated lymphoma receive standard first-line therapy. The present case shows that, in the case of recurrent non-Hodgkin's lymphoma, not only histological confirmation but also genetic assessment is important to clarify the origin of the second lymphoma.

Granulomatosis with polyangiitis involves sustained mucosal inflammation that is rich in B-cell survival factors and autoantigen.

OBJECTIVE: Granulomatosis with polyangiitis (GPA) is a rare chronic autoimmune disease that may be triggered by upper airway infection. ANCAs specific for PR3 that is expressed by activated neutrophils and macrophages are associated with GPA. Our aim was to investigate regional immune mechanisms that might induce or support the autoimmune response in GPA. METHODS: Biopsy samples from 77 patients including 8 with GPA were studied by immunohistochemistry. B-cell homing subsets in blood samples from 16 patients with GPA and 11 healthy controls were studied by FACS. The distribution of B-cell clones was searched in paired biopsies and blood samples from one patient by analysing immunoglobulin heavy chain gene (IGH) junctional sequences. RESULTS: Activated B cells were located alongside PR3-expressing cells and B-cell survival factors BAFF and APRIL in mucosa from patients with GPA. We detected APRIL production by the granulomas and giant cells. B cells were proliferating in all cases and persistent for 5 years in biopsies obtained from one patient. However, there was no evidence of B-cell clones from the mucosal biopsies circulating in peripheral blood in GPA or any numerical or proportional change in B-cell subsets expressing markers of regional homing in blood in GPA. CONCLUSIONS: Our study illustrates chronically activated B cells alongside autoantigens and B-cell survival factors in the mucosa in GPA.

Multiple myeloma shows no intra-disease clustering of immunoglobulin heavy chain genes.

BACKGROUND: Characterization of the immunoglobulin gene repertoire has improved our understanding of the immunopathogenesis of lymphoid tumors. Early B-lymphocyte precursors of multiple myeloma are known to exist and might be susceptible to antigenic drive. DESIGN AND METHODS: To verify this hypothesis, we collected a database of 345 fully readable multiple myeloma immunoglobulin sequences. We characterized the immunoglobulin repertoire, analyzed the somatic hypermutation load, and investigated for stereotyped receptor clusters. RESULTS: Compared to the normal immunoglobulin repertoire, multiple myeloma displayed only modest differences involving only a few genes, showing that the myeloma immunoglobulin repertoire is the least skewed among mature B-cell tumors. Median somatic hypermutation load was 7.8%; median length of complementarity determining-region 3 was 15.5 amino acids. Clustering analysis showed the absence of myeloma specific clusters and no similarity with published chronic lymphocytic leukemia or lymphoma subsets. CONCLUSIONS: Analysis of multiple myeloma immunoglobulin repertoire does not support a pathogenetic role for antigen selection in this tumor.

In vivo redundant function of the 3' IgH regulatory element HS3b in the mouse.

In the mouse, the regulatory region located at the 3' end of the IgH locus includes four transcriptional enhancers: HS3a, HS1-2, HS3b, and HS4; the first three lie in a quasi-palindromic structure. Although the upstream elements HS3a and HS1-2 proved dispensable for Ig expression and class switch recombination (CSR), the joint deletion of HS3b and HS4 led to a consistent decrease in IgH expression in resting B cells and to a major CSR defect. Within this pair of distal enhancers, it was questionable whether HS3b and HS4 could be considered individually as elements critical for IgH expression and/or CSR. Studies in HS4-deficient mice recently revealed the role of HS4 as restricted to Igmicro-chain expression from the pre-B to the mature B cell stage and left HS3b as the last candidate for CSR regulation. Our present study finally invalidates the hypothesis that CSR could mostly rely on HS3b itself. B cells from HS3b-deficient animals undergo normal proliferation, germline transcription, and CSR upon in vitro stimulation with LPS; in vivo Ag-specific responses are not affected. In conclusion, our study highlights a major effect of the global ambiance of the IgH locus; enhancers demonstrated as being strongly synergistic in transgenes turn out to be redundant in their endogenous context.

Extensive intraclonal diversification in a subgroup of chronic lymphocytic leukemia patients with stereotyped IGHV4-34 receptors: implications for ongoing interactions with antigen.

Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen recognition through the clonotypic B-cell receptors (BCRs). However, it is still unclear whether antigen involvement is restricted to the malignant transformation phase or whether the putative antigen(s) may continuously trigger the CLL clone and affect not only the progenitor cell but also the leukemic cells themselves. To address this issue, we conducted a large-scale subcloning study of rearranged immunoglobulin heavy variable (IGHV) genes of diverse mutational status from 71 CLL cases (total, 1496 subcloned sequences), belonging to both the common IgM/IgD variant and the rare IgG-positive variant. Although most cases showed no or low levels of intraclonal diversification (ID), we report intense ID in the IGHV genes of selected cases, especially a subgroup of 13 IgG-switched cases expressing stereotyped, mutated IGHV4-34 rearrangements (subset 4). We demonstrate that the ID evident in subset 4 cases cannot be attributed to IGHV4-34 usage, IGHV gene-mutated status, class-switch recombination, or BCR stereotypy in general; rather, it represents a unique phenomenon strongly correlated with the distinctive BCR of subset 4. In such cases, the observed ID patterns may imply a stereotyped response to an active, ongoing interaction with antigen(s).

Restricting activation-induced cytidine deaminase tumorigenic activity in B lymphocytes.

DNA breaks play an essential role in germinal centre B cells as intermediates to immunoglobulin class switching, a recombination process initiated by activation-induced cytidine deaminase (AID). Immunoglobulin gene hypermutation is likewise catalysed by AID but is believed to occur via single-strand DNA breaks. When improperly repaired, AID-mediated lesions can promote chromosomal translocations (CTs) that juxtapose the immunoglobulin loci to heterologous genomic sites, including oncogenes. Two of the most studied translocations are the t(8;14) and T(12;15), which deregulate cMyc in human Burkitt's lymphomas and mouse plasmacytomas, respectively. While a complete understanding of the aetiology of such translocations is lacking, recent studies using diverse mouse models have shed light on two important issues: (1) the extent to which non-specific or AID-mediated DNA lesions promote CTs, and (2) the safeguard mechanisms that B cells employ to prevent AID tumorigenic activity. Here we review these advances and discuss the usage of pristane-induced mouse plasmacytomas as a tool to investigate the origin of Igh-cMyc translocations and B-cell tumorigenesis.

Relative seroprevalence of human herpes viruses in patients with chronic lymphocytic leukaemia.

BACKGROUND: Herpes virus infections may have a significant role in chronic lymphocytic leukaemia (CLL) due to their ability to modulate the host's immune system. MATERIALS AND METHODS: We examined the seroprevalence of four herpes viruses [Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), human herpes virus (HHV)-6 and -7] in a cohort of European CLL patients (cohort 1, n = 100) in relation to the immunoglobulin variable heavy (IGHV) chain gene use and compared serological results with those obtained from age- and gender-matched healthy adults (n = 100). RESULTS: CMV-seroprevalence was significantly higher in CLL cohort 1 (79%) than in the control cohort (57%, P = 0.001); the seroprevalence of EBV (89% vs. 94%), HHV-6 (73% vs. 60%), or HHV-7 (35% vs. 35%) was not. In CLL cohort 1, use of IGHV3-30 was more prevalent among CMV-seropositive and of IGHV3-21 among HHV-7-seronegative cases. To investigate the generalizability of these findings, we investigated the herpes virus seroprevalence in a second cohort of age-matched CLL patients from a different geographical area (USA, n = 100, cohort 2). In cohort 2, CMV-seroprevalence was comparable with that of the control cohort (53%). Seroprevalence of EBV, HHV-6 and HHV-7 were 85%, 88% and 73% respectively. In CLL cohort 2, use of IGHV3-30 or IGHV3-21 was not associated with any of the herpes viruses investigated. CONCLUSIONS: CMV-seropositivity is associated with CLL in selected patient cohorts. However, the considerable variation in herpes virus-specific seropositivity between geographically distinct CLL cohorts indicates that seropositivity for any of the four human herpes viruses investigated is not generally associated with CLL.

B cell depletion therapy in systemic lupus erythematosus: relationships among serum B lymphocyte stimulator levels, autoantibody profile and clinical response.

OBJECTIVE: To assess the relationships between serum B lymphocyte stimulator (BLyS) levels, autoantibody profile and clinical response in patients with systemic lupus erythematosus (SLE) following rituximab-based B cell depletion therapy (BCDT). METHODS: A total of 25 patients with active refractory SLE were followed for >or=1 year following BCDT. Disease activity was assessed using the British Isles Lupus Assessment Group (BILAG) system, and serum levels of BLyS and autoantibodies to dsDNA and extractable nuclear antigens (ENA) measured by ELISA. Serum immunoglobulins and anti-dsDNA antibodies were assessed for expression of the 9G4 idiotope (indicating VH4-34 germline gene origin). RESULTS: Following BCDT, all patients depleted in the peripheral blood and improved clinically for >or=3 months. Pre-BCDT BLyS levels were quantifiable (median 1.9 ng/ml) in 18/25 patients and rose in most patients at 3 months post-BCDT (median 4.15 ng/ml). Nine patients, all with quantifiable pre-BCDT serum BLyS, experienced a disease flare within 1 year. This group of patients was more likely to harbour anti-Ro/SSA antibodies (odds ratio 1.76; p = 0.06) with higher serum levels (p = 0.0027; Mann-Whitney U test). Serum levels of anti-ribonucleoprotein (RNP)/Sm were also higher in this group (p<0.05). Expression of VH4-34 by serum immunoglobulins and anti-dsDNA antibodies had no predictive value for the length of clinical response. CONCLUSIONS: Patients with SLE with an expanded autoantibody profile and raised BLyS levels at baseline had shorter clinical responses to BCDT. This may reflect a greater propensity to, and degree of, epitope spreading in such patients and suggests that treatment regimens beyond BCDT may be necessary to induce long-lasting clinical remissions in these individuals.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

Non-coding Class Switch Recombination-Related Transcription in Human Normal and Pathological Immune Responses.

Antibody class switch recombination (CSR) to IgG, IgA, or IgE is a hallmark of adaptive immunity, allowing antibody function diversification beyond IgM. CSR involves a deletion of the IgM/IgD constant region genes placing a new acceptor Constant gene, downstream of the VDJH exon. CSR depends on non-coding (CSRnc) transcription of donor Iµ and acceptor IH exons, located 5' upstream of each CH coding gene. Although, our knowledge of the role of CSRnc transcription has advanced greatly, its extension and importance in healthy and diseased humans is scarce. We analyzed CSRnc transcription in 70,603 publicly available RNA-seq samples, including GTEx, TCGA, and the Sequence Read Archive using recount2, an online resource consisting of normalized RNA-seq gene and exon counts, as well as, coverage BigWig files that can be programmatically accessed through R. CSRnc transcription was validated with a qRT-PCR assay for Iµ, Iγ3, and Iγ1 in humans in response to vaccination. We mapped IH transcription for the human IGH locus, including the less understood IGHD gene. CSRnc transcription was restricted to B cells and is widely distributed in normal adult tissues, but predominant in blood, spleen, MALT-containing tissues, visceral adipose tissue and some so-called "immune privileged" tissues. However, significant Iγ4 expression was found even in non-lymphoid fetal tissues. CSRnc expression in cancer tissues mimicked the expression of their normal counterparts, with notable pattern changes in some common cancer subsets. CSRnc transcription in tumors appears to result from tumor infiltration by B cells, since CSRnc transcription was not detected in corresponding tumor-derived immortal cell lines. Additionally, significantly increased Iδ transcription in ileal mucosa in Crohn's disease with ulceration was found. In conclusion, CSRnc transcription occurs in multiple anatomical locations beyond classical secondary lymphoid organs, representing a potentially useful marker of effector B cell responses in normal and pathological immune responses. The pattern of IH exon expression may reveal clues of the local immune response (i.e., cytokine milieu) in health and disease. This is a great example of how the public recount2 data can be used to further our understanding of transcription, including regions outside the known transcriptome.

Rearrangement of only one human IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses in transgenic mice.

In recent years, mice carrying human IG transgenes are being generated for the production of human monoclonal antibodies as an alternative approach to the conventional use of mouse or chimeric-humanized antibodies. Theoretically, the size of the repertoire of human antibodies that these mice could produce would be critically dependent on the number of human V genes introduced in the transgene. This could be the case for BABkappa and BABkappa,lambda transgenic mice, which carry several genes from the human IGK (BABkappa), and IGK and IGL (BABkappa,lambda) loci, but only five human IGHV genes and the entire IGHD-IGHJ cluster linked to two human IGHC (IGHM-IGHD) genes. We analyzed the expressed human IG genes in 30 IgM-secreting hybridomas generated from transgenic mice immunized either with soluble proteins (human IgM coupled to KLH) or with cells (human PBMC, tumour cell lines or rat cells transfected with human CD69). The results show that all hybridoma cells analyzed rearranged exclusively the IGHV1-2 gene, in contrast with naive spleen B cells that used three out of the five IGHV genes present in the transgene. The configuration of the rearranged CDR3 region revealed a much higher heterogeneity in the heavy chains. A variety of IGHJ and IGHD genes were used in hybridomas, and somatic mutations were also seen in some hybrids. Regarding the rearranged light chains genes, it was a much higher variety in the use of V and J genes in both, kappa and lambda chains, than in the heavy chain, and also in the level of mutation. The results indicate that only one IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses. Thus, efforts aimed at the generation of new transgenic mice should focus more on the integrity of the D/J region and on the DNA regions regulating somatic hypermutation, rather than on the number of V genes present in the transgene.