B cell antigen receptor specificity and surface density together determine B-1 versus B-2 cell development.

Mice expressing the immunoglobulin (Ig) heavy (H) chain variable (V) region from a rearranged V(H)12 gene inserted into the IgH locus generate predominantly B-1 cells, whereas expression of two other V(H) region transgenes (V(H)B1-8 and V(H)glD42) leads to the almost exclusive generation of conventional, or B-2, cells. To determine the developmental potential of B cells bearing two distinct B cell antigen receptors (BCRs), one favoring B-1 and the other favoring B-2 cell development, we crossed V(H)12 insertion mice with mice bearing either V(H)B1-8 or V(H)glD42. B cells coexpressing V(H)12 and one of the other V(H) genes are readily detected in the double IgH insertion mice, and are of the B-2 phenotype. In mice coexpressing V(H)12, V(H)B1-8 and a transgenic kappa chain able to pair with both H chains, double H chain-expressing B-2 cells, and B-1 cells that have lost V(H)B1-8 are generated, whereas V(H)B1-8 single producers are undetectable. These data suggest that B-1 but not B-2 cells are selected by antigenic stimuli in whose delivery BCR specificity and surface density are of critical importance.     

Positive selection of anti-thy-1 autoreactive B-1 cells and natural serum autoantibody production independent from bone marrow B cell development

A natural serum autoantibody specific for the Thy-1 glycoprotein (anti-Thy-1 autoantibody [ATA]) is produced by B-1 cells that are positively selected by self-antigen. Here, using ATA micro kappa transgenic mice we show that cells with this B cell receptor are negatively selected during bone marrow (BM) development. In a Thy-1 null environment, BM ATA B cells progress to a normal follicular stage in spleen. However, in a self-antigen-positive environment, development is arrested at an immature stage in the spleen, concomitant with induction of CD5. Such cells are tolerant and short-lived, different from B-1. Nonetheless, ATA-positive selection was evident by self-antigen-dependent high serum ATA production, comprising approximately 90% of serum immunoglobulin M in ATA micro kappa mice. Splenectomy did not eliminate ATA production and transfer of tolerant splenic B cells did not induce it. These findings demonstrate that B-1 positive selection, resulting in the production of natural serum ATA, arises independently from the major pathway of BM B cell development and selection.     

The cytoplasmic domains of immunoglobulin (Ig) alpha and Ig beta can independently induce the precursor B cell transition and allelic exclusion

In mature B cells, signals transduced through membrane immunoglobulin (Ig) produce cellular activation, yet the same receptor can also mediate deletion and silencing of autoreactive B cells. In addition, Ig expression during the antigen-independent phase of B cell development regulates the precursor B (pre-B) cell transition and allelic exclusion. To account for the diverse regulatory functions induced by membrane Ig, it has been proposed that individual receptor components have independent physiologic activities. Here we establish a role for Ig alpha in the pre-B cell transition and allelic exclusion. We find that the cytoplasmic domain of Ig alpha contains sufficient information to trigger both of these antigen-independent events. Direct comparisons of the cytoplasmic domains of Ig alpha and Ig beta show that the two are indistinguishable in the induction of the pre-B cell transition and allelic exclusion. Our experiments suggest that, despite the reported differences in certain biochemical assays, Ig alpha and Ig beta have redundant functions in the developing B cell.     

Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice

The pre-B cell receptor consists of immunoglobulin (Ig) mu heavy chains and surrogate light chain, i.e., the VpreB and lambda5 proteins. To analyze the role of the two VpreB proteins, mice lacking the VpreB1 and VpreB2 genes were generated. VpreB1(-/-) VpreB2(-/-) mice were impaired in their B cell development at the transition from pre-BI to large pre-BII cells. Pre-BII cells did not expand by proliferation, consequently 40-fold less small pre-BII and immature B cells were found in bone marrow, and the generation of immature and mature conventional B cells in spleen appeared reduced. In addition, only low numbers of B-1a cells were detected in the peritoneum. Surprisingly, Ig heavy chain allelic exclusion was still active, apparently ruling out a signaling role of a VpreB1/VpreB2-containing receptor in this process.     

乙型肝炎外周血Th1/Th2、Tc1/Tc2表达失衡的临床意义

目的探讨Th1/Th2、Tc1/Tc2细胞因子在乙型肝炎中的表达及临床意义。方法采用流式细胞术分析乙型肝炎患者外周血不同T淋巴细胞亚群中细胞因子(IFN-γ、IL-4)的表达,分别比较急慢性乙型肝炎以及慢性轻、中、重度肝炎IFN-γ、IL-4表达水平的变化。结果慢性肝炎Th2、Tc2细胞高于急性肝炎(P<0.01;P<0.05);相对于轻度组,中度Th1、Tc1以及重度组Th1、Tc1均有升高(P<0.01),相对于中度组,重度组Th1、Tc1亦有增高(P<0.05;P<0.01)。结论乙型肝炎外周血Th2、Tc2与炎症慢性化有关;随着慢性乙型肝炎活动程度的加剧Th1、Tc1细胞逐渐增多。     

原发性干燥综合征患者外周血Th17细胞的检测及意义

目的探讨Th17细胞原发性干燥综合征（pSS）患者外周血中表达水平及意义。方法分离pSS患者和健康者外周血单个核细胞（PBMC）,磁珠阴选CD4＋T细胞,然后加佛波酯/离子霉素,经过固定及透膜处理后进行细胞内染色,流式细胞术（FCM）检测CD4＋T细胞内白细胞介素17（IL-17）水平。结果免疫磁珠阴选CD4＋T细胞纯度〉90%。pSS患者Th17细胞水平（2.14±0.91）显著高于正常对照组（0.56±0.21）,P〈0.01;pSS患者TH17细胞水平与ANA滴度、Anti-SSA、Anti-SSB、C3、C4无显著相关性（P〉0.05）。结论 pSS患者外周血中TH17细胞表达增高,Th17细胞表达异常增高可能是pSS发病的重要环节。     

调节性B细胞在类风湿性关节炎中的表达及其与疾病严重程度的相关性分析

目的检测类风湿关节炎(RA)患者外周血IL-10~+CD19~+调节性B细胞(Breg)的表达情况,并分析其与患者疾病活动程度的相关性。方法选择40例活动期RA患者为RA组,30例体检健康者作为对照组(HCs),抽取外周血并分离单个核细胞(PBMC),以CpG寡脱氧核苷酸2006(CpG ODN 2006)和佛波醇酯(PMA)进行体外共培养,流式细胞术分析作用前后Breg的表达情况,并与疾病严重程度指标血沉(ESR)、C反应蛋白(CRP)和关节活动度评分(DAS28)作相关性分析。结果体外诱导前RA组和HCs组PBMC中Breg的表达比例分别为1.92%(1.58%,2.56%)和2.04%(1.73%,2.93%),差异无统计学意义(P=0.258);经体外培养诱导后,Breg表达显著升高,RA组为13.00%(9.75%,14.85%),HCs组为9.12%(6.83%,10.22%),差异有统计学意义(P0.001)。对RA组Breg与ESR、CRP、DAS28进行Spearman相关性分析发现,Breg与DAS28呈负相关关系(P=0.002),而与ESR和CRP无显著相关(P0.05)。结论诱导后Breg在RA患者中显著升高,并且与RA活动性呈负相关关系,可能在RA的发生、发展中具有重要的免疫调节作用。     

B7共刺激分子在八种人类恶性血液病细胞系中的表达及意义

目的：探讨B7共刺激分子在人类恶性血液病细胞系中的表达及意义。方法：用质粒DNA的扩增、脂质体介导法转基因，流式细胞术、RT－PCR等方法检测U937、K562、CEM、DOUDI、GM、PEER、Jurkat、Raji等细胞转基因前后B7－1分子的表达。结果：U937、K562、CEM、DOUDI、GM、PEER、Jurkat、Raji等8种恶性血液病细胞株均表达或高表达HLA抗原和B7－2分子，低表达或不表达B7－1分子，转基因后细胞株B7分子的表达明显增强，转B7－1基因后肿瘤细胞刺激T细胞表达高水平的IL－2 mRNA。结论：大多数人类恶性血液病细胞株均不表达或低表达B7分子，提示B7－1可能在肿瘤免疫中起主要作用。     

the relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes. III. Expression of a single predominant isotype on primed and unprimed B cells

We determined whether primed and unprimed B cells in the spleen of (BALB/c × C57BL/Ka)F(1) mice contain subpopulations that express a predominant surface Ig isotype. Spleen cells were stained for surface isotypes and sorted on the fluorescence-activated cell sorter (FACS) in order to obtain B cells bearing predominantly IgM (μp cells), IgD (δp cells), or IgG (γp cells). Each population was assayed for its capacity to restore the adoptive primary and secondary anti-bovine serum albumin (BSA) antibody response in irradiated syngeneic recipients. In addition, the adoptive response restored by isotype-predominant cells was compared to that restored by isotype- positive cells (B cells bearing a given surface isotype alone or in combination with others). The experimental results show that μp cells restore the adoptive primary and secondary IgM and IgG responses to BSA, and γP cells restore only the primary and secondary IgG response. Δp Cells restored the adoptive secondary IgG response, but failed to restore the adoptive primary response at the cell doses tested. ΓP Cells but not δp cells suppressed the IgM response of the μ(+) and δ(+) cells. The contribution of isotype-predominant cells to both the adoptive primary and secondary anti-BSA response was smaller than that of B cells bearing a combination of surface isotypes. Differences in the Ig isotype pattern expressed on the surface of primed and unprimed B cells are discussed.     

非小细胞肺癌患者外周血调节性T细胞表达及意义

目的探讨CD4+CD25+,CD8+CD28-调节性T细胞及其亚群表型CD45RO在非小细胞肺癌(non-small cell lung cancer,NSCLC)患者外周血中表达及其与NSCLC临床病理及手术的关系。方法采用流式细胞仪检测40例NSCLC患者和20例对照外周血中CD4+CD25+,CD8+CD28-,CD8+CD28+T细胞亚群比率,并对其亚群表型CD45RO进行检测。结果 NSCLC组CD4+CD25+,CD4+CD25+CD45RO+,CD8+CD28-细胞亚群比率较对照组明显升高(P<0.05);CD8+CD28+细胞亚群比率较对照组明显降低(P<0.01);NSCLC组手术后CD4+CD25+细胞亚群比率较术前明显降低(P<0.01),CD8+CD28+细胞亚群比率较术前明显升高(P<0.01);外周血调节性T细胞水平与NSCLC临床病理无相关性(P>0.05)。结论 CD4+CD25+,CD8+CD28-和CD8+CD28+T细胞亚群比率的改变可能与肺癌免疫耐受和抗肿瘤能力下降有关;手术治疗可下调患者机体的肿瘤免疫耐受,改善患者抗肿瘤免疫功能。     

Stimulated mobilization of monocyte Mac-1 and p150,95 adhesion proteins from an intracellular vesicular compartment to the cell surface.

Monocytes were stimulated to increase their cell surface quantity of leukocyte adhesion proteins p150,95 and Mac-1 by the chemoattractant formyl-methionyl-leucyl-phenylalanine, or other mediators such as platelet-derived growth factor, tumor necrosis factor, C5a, and leukotriene B4. Dose-response curves indicated variations in the sensitivity of monocytes and granulocytes to these mediators. These increases were independent of protein synthesis and half-maximal at 2 min. Human alveolar and murine peritoneal macrophages, cells that had previously diapedised, could not be induced to upregulate Mac-1 or p150,95. Detergent permeabilization studies in monocytes indicated that these proteins were stored in internal latent pools, which were reduced upon stimulation. Electron microscopy utilizing rabbit antiserum against p150,95 revealed these proteins on the plasma membrane, and in intracellular vesicles and peroxidase negative granules. Together with other functional studies, these findings suggest that the mobilization of Mac-1 and p150,95 from an intracellular compartment to the plasma membrane regulates the monocyte's ability to adhere and diapedese.     

人参皂苷对烫伤脓毒症大鼠CD19细胞和NK细胞的影响

目的：探讨人参皂苷对烫伤后脓毒症大鼠CD19细胞（B淋巴细胞）和自然杀伤细胞（NK细胞）的影响及其作用机制。方法：健康雄性SD大鼠66只被随机分为脓毒症组（n-24）、人参皂苷组（n-24）和对照组（n-18）；将大鼠用沸水致背部30％总体表面积Ⅲ度烫伤，烫伤后12h经腹腔注射内毒素（5mg／kg）予以“二次打击”，制成烫伤脓毒症模型。各组分别于“二次打击”后12、24和72h活杀大鼠，用流式细胞仪检测大鼠外周血中CD19细胞和NK细胞占淋巴细胞的百分比。结果：“二次打击”后大鼠外周血中CD19细胞和NK细胞活性均显著下降，此过程随时间延长而加重，各时间点与对照组比较差异均有显著性（P均〈O．01）；内毒素打击后24h和72h人参皂苷组CD19细胞和NK细胞占淋巴细胞百分比均显著高于脓毒症组（P均〈O．01）。结论：烫伤和内毒素“二次打击”后外周血中CD19细胞和NK细胞活性显著下降，人参皂苷可有效恢复其功能，显著改善烫伤脓毒症时细胞免疫功能受抑状态。     

CD39+CD73+B细胞亚群在原发性胆汁性胆管炎患者中的变化及其临床价值分析

目的:分析原发性胆汁性胆管炎(PBC)患者外周血CD39^+CD73^+B细胞亚群在B细胞中所占比例的变化,及其与PBC的临床指标间的关系。方法:收集江苏苏州吴江区和常熟地区PBC患者60例,健康人对照者40例,以流式细胞术分析所有研究对象外周血CD39^+CD73^+B细胞在B细胞中的比例,多重微珠流式荧光法定量分析PBC患者抗线粒体抗体M2(AMA-M2)水平,全自动生化分析仪检测肝功能各项指标。统计分析CD39^+CD73^+B细胞比例的组间差异及其与自身抗体和肝脏酶指标之间的关系。结果:PBC患者CD39^+CD73^+B细胞的比例较对照组显著降低[55.6%(44.9%,60.5%)vs 73.0%(69.8%,74.9%),P<0.001]。PBC组CD39^+CD73^+B细胞比例与AMA-M2呈明显的负相关关系(r=-0.349,P=0.027)。抗糖蛋白210抗体(anti-GP210)阳性患者CD39^+CD73^+B细胞比例较anti-GP210阴性患者明显降低[44.6%(38.2%,49.9%)vs 58.7%(49.8%,63.8%),P<0.001],抗核蛋体100抗体(anti-SP100)阳性和阴性患者间CD39^+CD73^+B细胞比例差异无统计学意义[58.6%(47.9%,62.7%)vs 51.6%(44.7%,60.4%),P=0.292]。结论:PBC患者外周血CD39^+CD73^+B细胞比例显著降低,且与特异性自身抗体存在显著相关性,提示该群B细胞在PBC发病过程中可能发挥重要作用。     

溃疡性结肠炎患者组织中细胞表面趋化因子受体1、2的表达

目的:探讨细胞表面趋化因子受体1(CXCR1)和细胞表面趋化因子受体2(CXCR2)在溃疡性结肠炎(UC)患者组织中的表达和分布,了解其与UC发病的关系。方法:应用免疫组织化学SP法检测25例UC患者(其中活动期患者15例,缓解期患者10例)和10例结肠息肉患者(对照组)肠道活检组织中CXCR1和CXCR2的表达,对结果进行统计学分析。结果:UC活动期组中CXCR1的表达明显高于UC缓解期组和对照组,差异具有统计学意义,UC缓解期组与对照组之间的表达差异无统计学意义,CXCR1主要表达于浸润的炎性细胞和肠上皮细胞表面;CXCR2在3组均未见明显表达,组间差异无统计学意义。结论:IL-8主要通过CXCR1介导炎性细胞的浸润及炎症反应,CXCR1可能参与了上皮细胞对IL-8的反应,拮抗CXCR1可能为UC的治疗提供一种新的方法。     

A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein.

Dendritic cells (DCs) are known to activate naive T cells to become effective helper cells. In addition, recent evidence suggests that DCs may influence naive B cells during the initial priming of antibody responses. In this study, using three-color confocal microscopy and three-dimensional immunohistograms, we have observed that in the first few days after a primary immunization, cells with dendritic morphology progressively localize within primary B cell follicles. These cells were identified by their ability to bind a fusion protein consisting of the terminal cysteine-rich portion of the mouse mannose receptor and the Fc portion of human immunoglobulin (Ig)G1 (CR-Fc). In situ, these CR-Fc binding cells express major histocompatibility complex class II, sialoadhesin, and CD11c and are negative for other markers identifying the myeloid DC lineage, such as (CD11b), macrophages (F4/80), follicular DCs (FDC-M2), B cells (B220), and T cells (CD4). Using CR-Fc binding capacity and flow cytometry, the cells were purified from the draining lymph nodes of mice 24 h after immunization. When injected into naive mice, these cells were able to prime T cells as well as induce production of antigen-specific IgM and IgG1. Furthermore, they produced significantly more of the lymphocyte chemoattractant, macrophage inflammatory protein (MIP)-1alpha, than isolated interdigitating cells. Taken together, these results provide evidence that a subset of DCs enters primary follicles, armed with the capacity to attract and provide antigenic stimulation for T and B lymphocytes.     

Comparison of the deep immune profiling of B cell subsets between healthy adults and Sjögren’s syndrome

ObjectivesDetailed analysis targeting B cell subgroups was considered crucial in monitoring autoimmune diseases and treatment responses. Thus, precisely describing the phenotypes of B cell differentiation and their variation in primary Sjögren’s syndrome (pSS) is particularly needed.MethodsTo characterize the proportions and absolute counts of B cell subsets, peripheral blood from 114 healthy adults of China (age range: 19–73 years) and 55 patients with pSS were performed by flow cytometry and CD19, CD20, CD24, CD27, CD38 and IgD were used as surface markers to identify B cell mature process. Age- and gender-stratified analyses were then carried out to improve the interpretation of B cell subsets.ResultsThe assessments from healthy adults showed that the proportion of naive B cells presented a significant increase with age. A reversal trend was noted that the percentage of B10 decreased markedly with age. In addition, analysis based on gender showed that the relative percentage and number of naive B cells were higher in females than in males whereas the proportions of switched memory B cells and B10 cells were decreased in female. Patients with pSS exhibited a significant expansion in naïve B cells and unswitched memory B cells, accompanied with decreased switched memory B cells and B10 cells, which were identified to be associated with autoantibody production.ConclusionsOur study presented a reliable analysis by flow cytometry to cover the principal B cell subtypes. These different stages of B lymphocytes may have implications for evaluating the activation of pSS and other autoimmune diseases and treatment efficacy.

KEY MESSAGES

• B cell subsets play a pivotal role in the pathogenesis of primary Sjögren’s syndrome (pSS) and other autoimmune diseases. A practical and accurate flow cytometry method to profile B cell phenotypes in peripheral blood of healthy adults is especially essential.
• Additionally, we presented reliable reference ranges for B cell subsets in regards to the local population. Age- and gender-related analyses are available to better understand their influence in immune status and treatment outcome.
• The distribution of B-cell subsets is found substantially altered in patients with pSS, bringing novel avenues for pSS research in the future.