Novel Analysis of Clonal Diversification in Blood B Cell and Bone Marrow Plasma Cell Clones in Immunoglobulin Light Chain Amyloidosis

Immunoglobulin light chain amyloidosis (AL) is characterized by a limited clonal expansion of plasma cells and amyloid formation. Here, we report restriction in the diversity of VL gene usage with a dominance of clonally related B cells in the peripheral blood (PB) isotype-specific repertoire of AL patients. A rigorous quantification of lineage trees reveals presence of intraclonal variations in the PB clones compared to the bone marrow (BM) clones, which suggests a common precursor that is still subject to somatic mutation. When compared to normal BM and PB B cells, AL clones showed significant but incomplete impairment of antigenic selection, which could not be detected by conventional R and S mutation analysis. Therefore, graphical analysis of B cell lineage trees and mathematical quantification of tree properties provide novel insights into the process of B cell clonal evolution in AL.     

A Regulatory Circuit Controlling the Dynamics of NFκB cRel Transitions B Cells from Proliferation to Plasma Cell Differentiation

1. Download high-res image (204KB)
2. Download full-size image

     

Immunoglobulin heavy chain variable region gene utilization by B cell hybridomas derived from rheumatoid synovial tissue.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects synovial joints. Activated B lymphocytes and plasma cells are present in the synovial tissue and are thought to contribute to the immunopathology of the rheumatoid joint. To investigate rheumatoid synovial B lymphocytes, we have generated B cell hybridomas from synovial tissue of an RA patient. Here we describe the immunoglobulin VH gene repertoire of eight IgM- and 10 IgG-secreting synovial-derived hybridomas. The VH4 gene family is highly represented (38.5%) in this panel of hybridomas compared with the frequency of VH4 gene expression in circulating B lymphocytes reported previously (19-22%) and with the VH4 gene frequency we observed in a panel of hybridomas derived in the same manner from the spleen and tonsil of normal individuals (19%). The increased frequency of VH4 gene expression was not due to the expansion of a single B cell clone in vivo as none of these hybridomas was clonally related. Two synovial-derived hybridomas secreted autoantibodies; one (VH3+) secreted an IgM-rheumatoid factor (RF) and the other (VH4+) secreted IgM with polyreactive binding to cytoskeletal proteins and cardiolipin. The antibodies secreted by the remaining synovial-derived hybridomas were not reactive with the autoantigens tested. The VH gene usage in a proportion (5/17) of synovial-derived hybridomas that expressed CD5 antigen provided preliminary evidence that CD5+ B cells in RA synovium have a similar increase of VH4 gene expression reported for CD5+ B cells from normal individuals and patients with chronic lymphocytic leukaemia.     

MicroRNAs和细胞分化

MicroRNAs(miRNAs)是一类大约22个核苷酸大小的非编码RNA,它们通过剪切靶基因的转录产物或者抑制转录产物的翻译从而起到转录后调控靶基因表达的作用。干细胞是指一类能够自我更新的,尚未分化的,但具有形成各种不同功能的终末分化细胞能力的细胞。在干细胞的分化过程中,大量基因参与其中,这些基因的表达水平会影响细胞分化的整个过程。MicroRNAs参与各种干细胞的分化过程,进而影响个体的发育。     

Inhibition of Polyclonally Induced Immunoglobulin Secretion by Aurothiomalate

The influence of sodium aurothiomalate on the secretion of immunoglobulins by normal human lymphocytes in vitro was investigated by means of a reverse hemolytic plaque forming cell (PFC assay, Aurothiomalate inhibited the FFC response induced by pokeweed mitogen (PWM) and by Epstein-Barr virus (EBV) in a dose dependent manner. The inhibition was irreversible, as pre-incubation for 2 h with the drug followed by extensive washing and further culture in gold salt–free medium still caused an inhibition of the PFC response to PWM and to EBV. Cell proliferation was not significantly affected, suggesting that the inhibition of PFC formation was not due to cytotoxicity. Pre–incubation of monocytes/macrophages (Mø's), T lymphocytes and B lymphocytes with the gold compound prior to culture with PWM showed that M0's and B cells were highly sensitive, whereas T lymphocytes where resistant to the drug. The findings indicate that aurothio-malate inhibits the polyclonally induced PFC response by interfering with accessory Me function and by affecting the B lymphocyte itself.     

龟板在体外对SAC诱导肾虚症病人B淋巴细胞增殖的影响

用SAC诱导的B细胞转化试验观察了龟板煎液在体外对肾阳虚,肾阴虚和正常人B细胞转化功能的影响。结果表明肾阳虚病人的SI(t)和Δcpm(t)明显低于肾阴虚病人(p<0.05),而肾阴虚病人与正常人无明显差异(p>0.05)。此外,还比较了龟板煎液体外对PHA诱导淋巴细胞转化的影响,表明龟板煎液对正常人体外PHA诱导淋转有抑制作用。作者认为,龟板煎液体外对肾阴虚病人B细胞有一定的滋养作用,而对肾阳虚病人则有抑制作用。     

Differential activation requirements of isotype-switched B cells

In the present studies, we compared the activation requirements of sIgM⁺/sIgD⁺ B cells with those of isotype-switched sIgM⁻/sIgA⁺ B cells. We found that whereas sIgM⁺ B cells respond to T cell-independent (TI) and T cell-dependent (TD) Ag with no significant bias toward one stimulus, sIgA⁺ B cells were deficient in their ability to respond to antigen receptor cross-linking but responded remarkably well to TD stimuli. Thus, dextran-conjugated anti-IgA antibody (anti-IgA-dextran), anti-kappa-dextran, or various immobilized anti-IgA antibodies (Ab) induced only low-level IgA B cell proliferation and no IgA secretion in the presence of various lymphokines; in marked contrast, sIgA⁺ B cells responded to cognate and noncognate T cell stimulation as well as to stimulation by CD40 ligand-bearing fibroblasts by secreting large amounts of IgA (up to 240 000 ng/ml per 10⁵ cells). This pattern of sIgA⁺ B cell responsiveness was noted with both germinal center peanut agglutinin^hi (PNA^hi) and non-germinal center PNA^lo B cells. In confirmation of these results, whole Peyer's patch or lamina propria cell populations containing less than 15% sIgA⁺ B cells stimulated with a noncognate T cell stimulus or T cell membranes secreted mainly IgA (68%–94% of the total Ig secreted) and relatively little IgM. The strict T cell dependence of IgA B cell activation and differentiation provides important insights into immune responses of mucosal tissues and must be considered in the development of vaccines, particularly those designed to stimulate mucosal tissues containing large numbers of isotype-switched B cells.     

乙型肝炎免疫球蛋白阻断乙型肝炎病毒母婴传播的临床研究

目的探讨乙型肝炎（乙肝）表面抗原（HBsAg）阳性孕妇及其新生儿采用乙肝免疫球蛋白（HBIG）阻断乙型肝炎病毒（HBV）母婴垂直传播的效果。方法将136例HBsAg（＋）的孕妇分为观察组（72例）和对照组（64例）,观察组孕妇于孕28、32与36周分别注射乙型肝炎免疫球蛋白（HBIG）,双阳性注射400IU,单阳性注射200IU;对照组只作随访及常规产检。两组的新生儿在出生6h内、第1、6个月时分别注射乙肝疫苗（HBvac）10μg、5μg、5μg;观察组新生儿在出生6h内臀部肌内注射HBIG 100IU。分别检测两组新生儿及6月龄婴儿血清中HBsAg、乙型肝炎表面抗体（HBsAb）及HBV DNA。结果观察组新生儿HBsAg和HBV DNA阳性率较对照组低,差异有统计学意义（P〈0．05和P〈0．01）。观察组6月龄婴儿HBsAb阳性率较对照组高,而HBV DNA阳性率较对照组低,差异也均有统计学意义（P〈0．01和P〈O．05）。结论HBsAg（＋）的孕妇应用HBIG可有效阻断HBV母婴传播,而新生儿出生时应用HBIG和HBvac联合免疫,可明显提高6月龄婴儿HBsAb阳性率。     

Primary Immune Effects of Eukaryotic Expression Plasmids Encoding Two Hyperactive Mutants of Human Soluble B Lymphocyte Stimulator

B lymphocyte stimulator (BLyS), a ligand belonging to the tumor necrosis factor (TNF) family, plays a critical role in regulating survival and activation of peripheral B cell populations during humeral immune responses. Among the TNF family members, BLyS is unique in that it contains an unpaired Cys residue (Cys146) at the corresponding position where some other members have about 37.5% (6/16) Ala or 37.5% (6/16) Val. Here, with eukaryotic expression vector pcDNA3.1(−), we mutated Cys146 to Ala or Val and constructed two mutant eukaryotic expression plasmids of the human soluble BLyS, pcDNA3.1BY-A and pcDNA3.1BY-V. Following repetitive subcutaneous injection of these expression plasmids in BALb/C mice, the wild-type and mutant BLyS proteins were detectable in the blood of treated animals over several weeks. In addition, the expression of these proteins induced specific IgG but not IgM responses. The implications of the results are discussed.     

乙型肝炎患者血清免疫球蛋白检测的意义

目的 分析乙型肝炎患者血清免疫球蛋白检测的意义。方法 选取本院2016年8月~2018年3月120例确诊为乙型肝炎的患者作为观察组,对其临床资料进行回顾性分析,同时选取120例健康体检者作为对照组,对比两组受检者血清免疫球蛋白检测结果。结果 观察组IgG、IgM、IgA水平分别为（15.63±2.63）g/L、（2.97±0.98）g/L、（2.97±1.62）mg/L均高于对照组的（11.05±1.08）g/L、（1.52±0.27）g/L、（1.81±0.27）mg/L,差异有统计学意义（P＜0.05）;通过对比120例患者治疗前后相关指标可以发现,治疗好转者IgG、IgM、IgA、TBLI水平均低于病情恶化者,差异有统计学意义（P＜0.05）。结论 对于乙型肝炎患儿患者而言,实施血清免疫球蛋白检测的价值明显,既能对患者病情严重程度加以判断,还能为乙型肝炎的诊断和治疗提供可靠依据。     

Common Patterns of B Cell Perturbation and Expanded V4-34 Immunoglobulin Gene Usage in Autoimmunity and Infection

Features of the lymphocyte population in systemic lupus erythematosus (SLE) include a disordered B cell profile and production of autoantibodies. An additional distinctive perturbation is the over-expression of V4-34-encoded serum immunoglobulins (Ig). A similar rise in V4-34-encoded Ig occurs in normal subjects following infection with c ertain herpesviruses, and is found in Epstein–Barr virus (EBV)-associated infectious mononucleosis (IM). To assess common and distinctive features of B cells in patients with SLE and IM, we compared the B cell profile and V4-34 gene involvement in patients with SLE and IM. B cell profiles from patients with IM paralleled those of patients with SLE, showing a differential loss of naïve and memory B cells and the maintenance of plasmablast/early plasma cells. Class-switched V4-34-encoded IgG from plasmablast/early plasma cells was evident both in patients with SLE and IM and revealed common features of oligoclonal expansions with most having undergone somatic hypermutation. It has been proposed that, in healthy individuals, expression of the V4-34 gene is specifically censored prior to isotype switch as a control on autoreactivity. If so, censoring is bypassed following EBV infection, after which equilibrium is restored. Continuing high serum levels in SLE may arise either by disordered regulation, or by subclinical reactivation of endogenous virus.     

Developmental Origins,Specificities and Immunoglobulin Gene Biases of Murine Ly-1 B Cells

Ly-1 B cells in mouse show numerous phenotypic and functional features that distinguish them from the bulk of IgD^high/Ly-1^? B cells. Their association with autoantibody production and the presence of Ly-1 on a group of murine B lymphomas that also exhibit certain specificities enriched in the normal population has stimulated continuing interest in this population. We have taken two approaches in our investigations of these cells: 1) defining the origins of Ly-1 B cells (the “lineage question”); and 2) studying the expression of particular specificities and associated immunoglobulin V genes enriched in this population. In this review we present the experimental background that supports our current understanding of Ly-1 B cells as the remnant of a fetal B cell differentiation pathway and suggest that the selection of cells from this fetal/neonatal population into the adult long-lived pool results in the over-expression of certain germline-encoded autoreactivities, such as antibody to bromelain-treated mouse red blood cells and intact thymocytes.     

Differential B cell expression of mouse Fc receptor homologs

Five Fc receptor homologs (FcRH1-5) possessing inhibitory and/or activating signaling motifs are differentially expressed during B cell differentiation in humans. In this analysis we describe their three mouse orthologs, moFcRH1, moFcRH2 and moFcRH3. The moFcRH genes are located in a chromosome 3 region that is syntenic with the FcRH locus on human chromosome 1. They encode proteins with 2-5 Ig-like domains that share 20-61% extracellular identity with their human counterparts. One moFcRH1 isoform lacks a transmembrane domain as do both moFcRH2 isoforms. The other moFcRH1 isoform and two moFcRH3 isoforms have transmembrane domains and cytoplasmic ITIM and ITAM-like consensus sequences implying their inhibitory or activating signaling potential. Whereas the moFcRH1 and moFcRH3 orthologs are preferentially expressed at different stages in B cell differentiation, the structurally novel moFcRH2 gene is expressed in non-lymphoid tissues. The highly restricted pattern of moFcRH3 expression suggests this member of the phylogenetically conserved FcRH family may have an important immunoregulatory role in marginal zone B cells.     

Post-Transplantation B Cell Activating Factor and B Cell Recovery before Onset of Chronic Graft-versus-Host Disease

Excessive levels of B cell activating factor (BAFF) are found in patients with active chronic graft-versus-host disease (cGVHD). In mice, BAFF has been shown to be essential for B cell recovery after myeloablation. To assess how BAFF levels relate to transplantation factors and subsequent development of cGVHD, we prospectively monitored 412 patients in the first year after allogeneic peripheral blood or bone marrow hematopoietic stem cell transplantation (HSCT) and censored data at time of cGVHD onset. In patients who did not develop cGVHD, we affirmed a temporal pattern of gradually decreasing BAFF levels as B cell numbers increase after myeloablative conditioning. In contrast, after reduced-intensity conditioning, BAFF levels remained high throughout the first post-HSCT year, suggesting that the degree of myeloablation resulted in delayed B cell recovery associated with persistence of higher BAFF levels. Given that high BAFF/B cell ratios have been associated with active cGVHD, we examined differences in early BAFF/B cell ratios and found significantly different BAFF/B cell ratios at 3 months post-HSCT only after myeloablative conditioning in patients who subsequently developed cGVHD. In addition to HSCT conditioning type, the use of sirolimus was significantly associated with higher BAFF levels after HSCT, and this also was potentially related to lower B cell numbers. Taken together, our results are important for interpreting BAFF measurements in cGVHD biomarker studies.     

Phenotype characteristics of human B cells studied by Epstein-Barr virus infection. I. Immunoglobulin expression on human lymphoblastoid cells established from various lymphoid cells

Forty-seven lymphoblastoid cell lines were established from human fetal lymphoid tissues, cord blood lymphocytes (CBL) and adult peripheral blood lymphocytes (PBL) by Epstein-Barr virus (EBV) infection. Their surface immunoglobulin (sIg), intracytoplasmic immunoglobulin (cIg) expression, and immunoglobulin (Ig) content in the culture supernatant were tested. Expression of sIgM, sIgG and sIgA were predominant on fetus-derived cell lines, while sIgD was the most prominent on CBL-derived cells. Though cIg expression did not vary between cell lines of different origin, Ig content in the culture supernatant differed greatly. Fetus- and CBL-derived cells secreted IgM exclusively, but PBL-derived cells secreted not only IgM, but also IgG and IgA abundantly. These results indicate that the lymphoblastoid cells established by EBV infection reflect the Ig phenotype of the cell from which they originated.     

Inhibition of murine B1 lymphocytes by interleukin-12

B1 cells are a subset of B lymphocytes found in many spectes and are implicated in the development of autoimmunity. B1 cells have previously been shown to be suppressed by the T helper (Th)1 cytokine interferon (IFN)-γ, and to be stimulated by the Th2 cytokines interleukin (IL)-2, IL-4, IL-5 and IL-10. To examine further the interactions of B1 cells and Th1 cells, we have now tested the effects of the Th1 cell-inducing cytokine IL-12 on murine B1 cells. BALB/c mice were immunized with phosphorylcholine conjugated to keyhole limpet hemocyanin (PC-KLH) and simultaneously treated with 1 μg recombinant murine IL-12 for 3 consecutive days. In addition to altering the isotype and idiotype distribution of anti-PC antibodies, IL-12 treatment was found to cause a loss of peritoneal, but not splenic B lymphocytes in immunized mice. B cell depletion required exposure to IL-12 plus antigenic stimulation. Levels of peritoneal B lymphocytes were fully restored by day 45, but the majority of these cells belonged to the B2 subset. Additionally, proliferation of B1 cells in vitro induced by IL-5 was substantially inhibited by IL-12. IL-12 itself had no effect on viable cell recovery of peritoneal cells (PeC) cultured in vitro, but viable cell recovery was significantly decreased in PeC cultured with IL-5 plus IL-12. These results show that IL-12 causes the loss of murine peritoneal B1 cells and suggest that treatment with this cytokine may be useful for disease conditions that involve B1 cell dysfunction.