VH replacement in primary immunoglobulin repertoire diversification

The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. V_H replacement represents a RAG-mediated secondary rearrangement in which an upstream V_H element recombines with a rearranged V_HD_HJ_H joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original V_H element and the conventional RSS of the invading V_H gene, leaving behind a footprint of up to five base pairs (bps) of the original V_H gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that V_H replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive V_HD_HJ_H allele. Using this mouse model, we characterized the role of V_H replacement in the diversification of the primary Ig repertoire through the modification of productive V_HD_HJ_H rearrangements. Our results indicate that V_H replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.A hallmark of the adaptive immune system is its ability to generate a large antibody repertoire despite a limited genome through the rearrangement of variable (V), diversity (D), and joining (J) genes during B-cell development in a process called V(D)J recombination (1). Each antibody-producing cell expresses a single pair of heavy and light chains generated by this process during the pro–B-cell and pre–B-cell stages of development, respectively. V(D)J recombination is mediated by the recombination activating proteins RAG1 and RAG2, which bind and cleave recombination recognition sequences (RSSs) flanking V, D, and J genes. Because there are several members of V, D (for the heavy chain), and J segments, the combinatorial nature of V(D)J rearrangement allows for the generation of a vastly diverse antibody repertoire from a relatively modest amount of genetic information present in the germline DNA.Ig gene rearrangement progresses in an ordered stepwise manner during B-cell development, with Ig heavy (IgH) chain assembly before Ig light (IgL) chain assembly. In-frame rearrangement of a V_HD_HJ_H joint leads to expression of an IgH chain that pairs with an invariant surrogate light chain, and, in association with the Igα and Igβ signal-transducing subunits, these proteins form the pre–B-cell receptor (pre-BCR) (2, 3). Signaling through a functional pre-BCR allows the cell to progress from the pro–B-cell stage to the pre–B-cell stage, where IgL rearrangement can occur. This progression is accompanied by a burst of proliferation, which ensures that the functional IgH rearrangement is not lost in the event of unsuccessful IgL recombination and increases diversity by allowing a given IgH chain to pair with multiple IgL chains. Only cells that acquire a functional B-cell receptor (BCR), with an in-frame rearranged IgH chain that successfully pairs with a productively rearranged IgL chain, progress to become mature B cells.The imprecise nature of the joining process in V(D)J recombination contributes to the diversity of the antibody repertoire, but also leads to a significant number of nonfunctional rearrangements, with approximately two-thirds of the joints being out-of-frame. At the IgH locus, V_H replacement can rescue “dead-end” pro-B cells that have acquired nonproductive IgH joints on both alleles by rearranging an upstream V_H gene with a nonfunctional V_HD_HJ_H (4–7).Ordered rearrangement of the IgH and IgL loci is mediated by the RAG1/2 proteins that recognize RSSs flanking V, D, and J segments. The consensus RSS is composed of a heptamer (CACTGTG) and a nonamer (GGTTTTTGT) separated by either a 12-bp or 23-bp spacer (8). V(D)J recombination occurs preferentially between gene segments flanked by RSSs of dissimilar lengths, thus directing the order of recombination; this is known as the 12/23 rule. Because the V_HD_HJ_H recombination process eliminates any remaining D_H genes and their flanking 12-bp RSSs, further editing of this locus requires a recombination event that violates the 12/23 rule. V_H replacement occurs between a 23-bp RSS of an upstream invading V_H gene and a highly conserved 7-bp cryptic RSS (cRSS; TACTGTG) in the body of the recipient V_H gene (6, 7). Because the cRSS is located near the 3′ terminus of the recipient V_H gene, this process leaves behind only 5 bps proximal to the highly variable CDR3 region of the original V_HD_HJ_H joint, a minimal footprint that can be modified by exonuclease “chewback” and N-nucleotide addition (9, 10).V_H replacement was initially observed in mouse B-cell lines in which a small fraction of cells regularly acquired alternate IgH rearrangements, thus “editing” their original specificity (6, 7). Since then, V_H replacement has been studied in transgenic mouse models, human cell lines, and human blood. Recent studies that bioinformatically examined the frequency of 4- to 5-bp V_H footprints at the V_H-D_H vs. D_H-J_H junctions have suggested evidence of V_H replacement in at least 5% of the human Ig repertoire (9). Analysis of V_H footprints in mouse IgH rearrangements revealed a similar frequency of V_H replacement in wild-type (WT) mice, and also demonstrated a significantly higher frequency of V_H replacement footprints in IgH sequences from patients with autoimmune diseases and animals of autoimmune-prone backgrounds (11, 12). However, the random nature of exonuclease and terminal deoxynucleotidyl transferase (TdT) activity at the CDR3 region of IgH joints makes it difficult to assess the contribution of V_H replacement to the repertoire by sequence analysis, owing to loss of the V_H footprint. Furthermore, our earlier work with a mouse model carrying a predefined nonproductive IgH rearrangement in the IgH locus has demonstrated that up to one-third of V_H replacement events are mediated by sequence microhomology of the highly conserved 3′ bases of V_H genes (4). Replacement reactions of this nature lack a detectable footprint and thus escape detection by sequence analysis.Although the nonproductive V_HD_HJ_H mouse model allowed us to study how V_H replacement rescues nonproductive V_HD_HJ_H rearrangements, it did not address the contribution of V_H replacement to the diversification of the primary immune repertoire or its role in editing self-reactive antibody specificities. Thus, we generated a knock-in mouse with a productive V_HD_HJ_H rearrangement knocked into its physiological position within the IgH locus. With this mouse model, we assessed the role of V_H replacement in modifying productive IgH rearrangements in nonautoreactive and autoreactive settings.     

Analysis of VH gene rearrangements from synovial B cells of patients with rheumatoid arthritis reveals infiltration of the synovial membrane by memory B cells

The VH gene (Variable gene segments of the heavy chain locus) repertoire can be investigated by DNA analysis of rearranged immunoglobulin VH genes, which also allows for an indirect estimation of antibody selection by analysis of somatic mutations. Using a polymerase chain reaction (PCR) it is also possible to analyse these genes in small numbers of cells or even single cells. This approach was chosen to investigate germinal centre like lymphocyte follicles in the synovial membranes of two patients with rheumatoid arthritis (RA) in order to analyse the local humoral immune response in RA. Individual B-cell aggregates of synovial membrane of two patients with RA were isolated by micromanipulation from microscopic slides. VH-DH-JH (variable, diversity, and joining segments of the heavy chain locus) rearrangements in all possible VH-JH combinations were amplified from these B cell foci, cloned and subjected to sequence analysis. Sequence analysis revealed that most of the rearranged VH genes were somatically mutated with at least 1% (range 1.3 – 14.9%) somatic mutations and therefore were derived from antigen-selected memory B cells. Intraclonal diversity in one-third of the clones indicated the generation of memory B cells in the synovial membrane and characterized the synovial membrane as lymphatic tissue where secondary immune responses to an as yet unknown antigen take place. Received: 22 April 1997 / Accepted: 25 August 1997     

VH gene analysis of Burkitt's lymphoma in children from north-western Iran

Cases of Burkitt's lymphoma (BL) from north-western Iran were investigated for the usage and somatic mutational pattern of their immunoglobulin variable region genes. Potentially functional V_H genes were amplified from 6/12 of the tumour masses and all of these were derived from the V_H3 family, with 4/6 being derived from the most commonly used V_H3 family member, V_3-23. All of the tumour sequences were mutated from their germline counterparts, to varying degrees, with a mean level of 5.8%, indicating that the cell of origin had encountered the germinal centre. Intraclonal sequence heterogeneity was also evident in 4/6 of the lymphomas, showing that the tumour cells had undergone further somatic mutation following neoplastic transformation. Analysis of the five potentially functional mutated V_H sequences showed a significant clustering of replacement mutations in the complementarity-determining region 2, consistent with a role for antigen in selection of tumour cell sequences. The pattern of extensive somatic mutation, and intraclonal variation, in these mainly EBV+ve tumours, was similar to that previously reported in V_H sequences of EBV+ve endemic BL (eBL) and EBV−ve sporadic BL (sBL), with the mean level of somatic mutation lying between those reported for eBL (7.7%) and sBL (4.0%). However, V_H gene bias and the distribution of mutations in the Iranian cases showed features which differed from those reported for endemic or sporadic BL.     

Next‐generation sequencing of the intrahepatic antibody repertoire delineates a unique B‐cell response in HBV‐associated acute liver failure

Hepatitis B virus (HBV) is a major cause of acute liver failure (ALF) worldwide. While liver damage in classic acute hepatitis B is believed to be T‐cell mediated, the pathogenesis of HBV‐associated ALF remains largely unknown. Access to liver specimens from well‐characterized patients with HBV‐associated ALF provided us with the opportunity to perform next‐generation sequencing (NGS) of the entire VH repertoires of IgM and IgG from the livers of four ALF patients, a control liver donor and a patient with chronic HBV infection. We found that ALF is not associated with expansion of specific B‐cell lineages. However, NGS showed that the intrahepatic VH repertoires from ALF patients were characterized by the abundant presence of antibodies in germline configuration in contrast to their marginal prevalence in controls. Moreover, NGS identified a large number of VH genes in germline configuration with identical VDJ sequences in the IgM and IgG repertoires in all four ALF patients, indicating that isotype switch from IgM to IgG had occurred without somatic hypermutation. The results of this study indicate that the presence of intrahepatic antibodies in unmutated germline configuration is a broad phenomenon in the global antibody repertoire generated from total RNA derived from whole‐liver tissue that is strongly associated with ALF, suggesting a major role of T cell–independent humoral immunity in the pathogenesis of ALF.     

Biased JH usage in plasma cell immunoglobulin gene sequences from colonic mucosa in ulcerative colitis but not in Crohn's disease

BACKGROUND: Ulcerative colitis is an inflammatory disease of the colonic and rectal mucosa. Autoantibodies have been observed in ulcerative colitis which may have a role in the pathogenesis of the disease. Evidence also suggests that there is an hereditary predisposition towards the disease, although no individual genes have been identified. AIMS: This is a pilot study of immunoglobulin heavy chain genes (IgH) in ulcerative colitis to determine whether they have any particular genetic characteristics which may lead to a better understanding of the disease aetiology. SUBJECTS: Colonic or rectal tissue was obtained from five children with ulcerative colitis. Tissue was also obtained from five children with Crohn's disease and five children who did not have inflammatory bowel disease as controls. METHODS: B cells and IgD+ B cells were identified by immunohistochemistry on frozen sections. Areas of lamina propria containing plasma cells, and areas of IgD+ B cells were microdissected. The immunoglobulin genes were PCR amplified, cloned, and sequenced. Sequences were analysed for content of somatic mutations and composition of heavy chain. RESULTS: An increase in the use of JH6 and DXP'1, and a decrease in the use of JH4, gene segments in immunoglobulin genes from lamina propria plasma cells, and from virgin IgD+ B cells, was found in patients with ulcerative colitis. These biases were not present in the control groups. CONCLUSIONS: There is a fundamental difference in the immunoglobulin genes from patients with ulcerative colitis. Whether this is caused by a difference in content of immunoglobulin gene segments in the germline or a difference in the recombination mechanism is not known.     

内毒素耐受小鼠脾脏 CD11clowCD45RBhigh树突状细胞对急性肝功能衰竭锌指蛋白 A20表达的影响

目的：观察内毒素耐受 CD11clow CD45RBhigh DC 对急性肝功能衰竭小鼠锌指蛋白 A20表达的影响,探讨其在急性肝功能衰竭治疗中的作用及可能机制。方法建立内毒素耐受小鼠模型,经免疫磁珠法分选正常小鼠脾脏来源 CD11clow CD45RBhigh DC 和内毒素耐受小鼠脾脏来源 CD11clow CD45RBhigh DC。选取健康雄性 BALB／c 小鼠126只,随机分为正常对照组（A 组6只）、急性肝功能衰竭组（B 组40只）、正常小鼠脾脏 CD11clow CD45RBhigh DC 治疗组（C 组40只）和内毒素耐受小鼠脾脏CD11clow CD45RBhigh DC 治疗组（D 组40只）;B、C、D 三组小鼠同时腹腔注射 D-GalN 600 mg／kg和脂多糖10μg／只,A 组注射等体积0．9％氯化钠溶液;30 min 后 C 组腹腔注射正常小鼠脾脏来源 CD11clow CD45RBhigh DC（1×106／只,0．2 mL／只）,D 组腹腔注射内毒素耐受小鼠脾脏来源 CD11clow CD45RBhigh DC（1×106／只,0．2 mL／只）进行干预,A、B 组则给予相同剂量的0．9％氯化钠溶液。检测各组小鼠各时间点血 ALT、AST 水平;采用 HE 染色观察各组小鼠各时间点肝组织病理变化;反转录（RT）-PCR 和Western 免疫印迹法检测各组小鼠各时间点 TNF-α、核因子-κB、锌指蛋白 A20表达情况。样本均数比较采用单因素方差分析。进行正态性检验和方差齐性分析,方差齐者采用 LSD 检验。结果 B 组在造模2 h 后 ALT、AST 开始逐渐升高,于24 h 达高峰,明显高于 A 组（t 值分别为31．00和11．52,均 P ＜0．05）。C、D 两组于造模2 h 后 ALT、AST 也逐渐升高,于24 h 达高峰,与 B 组比较,差异均有统计学意义（t 值分别为14．60和26．43,均 P ＜0．05）。B 组小鼠 TNF-αmRNA（t ＝427．58）、核因子-κB mRNA （t＝122．42）及蛋白表达量（t 值分别为179．35和165．98）随着时间的推移逐渐升高,至12 h 达高峰,与A 组相比差异有统计学意义（均 P ＜0．05）。B 组小鼠锌指蛋白 A20 mRNA 及蛋白则随着时间的延长逐渐降低,在12 h 达到最低,与 A 组相比差异有统计学意义（t 值分别为90．80和160．43,均 P ＜0．05）;A20 mRNA 及蛋白表达量则随着时间的延长逐渐增加,在12 h 达到最高值,且 D 组增加较 C 组更明显,差异有统计学意义（t 值分别为11．21和24．80,均 P ＜0．05）。结论内毒素耐受小鼠脾脏来源CD11clow CD45RBhigh DC 治疗可减轻肝脏损害。     

Early pregnancy induced expression of prostaglandin E2 receptors EP2 and EP4 in the ovine endometrium and regulated by interferon tau through multiple cell signaling pathways

Prostaglandin E2 (PGE₂) plays pleiotropic roles at fetal-maternal interface during establishment of pregnancy. The objectives of the study were to: (i) determine regulation of PGE2 receptors EP1, EP2, EP3, and EP4 in the endometrium during the estrous cycle and early pregnancy; and (ii) understand endometrial epithelial and stromal cell-specific hormonal regulation of EP2 and EP4 in sheep. Results indicate that: (i) early pregnancy induces expression of EP2 and EP4 but not EP1 and EP3 proteins in the endometrium on days 12-16 compared to that of estrous cycle; (ii) intrauterine infusion of interferon tau (IFNT) increases expression of EP2 and EP4 proteins in endometrium; and (iii) IFNT activates distinct epithelial and stromal cell-specific JAK, EGFR, ERK1/2, AKT, or JNK signaling module to regulate expression of EP2 and EP4 proteins in the ovine endometrium. Our results indicate a role for EP2 and EP4-mediated PGE₂ signaling in endometrial functions and establishment of pregnancy in ruminants.