关注天然自身抗体

大然自身抗体（ｎａｔｕｒａｌ ａｕｔｏａｎｔｉｂｏｄｙ，ＮＡＡ）是指在没有任何抗原主动免疫的情况下，正常机体内天然产生并存在的针对一种或多种自身抗原的免疫球蛋白。历史上人们对ＮＡＡ的认识经历了一个复杂而矛盾的过程，从一开始把所有的自身抗体一概看作是“可怕的自身毒素（ｈｏｒｒｏｒ ａｕｔｏｔｏｘｉｃｕｓ）”，到发现并证实了 ＮＡＡ在正常人体内的存在，目前已逐步认识到ＮＡＡ具有一系列重要的生物学功能，是机体大然免疫的重要组成部分。作为生理机制的ＮＡＡ正在引起学术界的高度关注，有关ＮＡＡ的研究论著数量也…     

正常人血清中IgG类抗角蛋白自身抗体的免疫印迹分析

抗角蛋白自身抗体是正常人天然自身抗体的一部分。本研究用一组角蛋白对正常人血清中的ＩｇＧ 类抗角蛋白抗体进行了免疫印迹分析。２１ 份正常人血清均可识别至少一种角蛋白成分， 可区分出１４ 种反应性模式。３ 份合并血清的反应性模式高度均一。结果表明， 血清抗角蛋白抗体普遍存在， 但在人群中呈高度异质性； 不同个体血清可互补。角蛋白 抗角蛋白系统可能是天然自身抗体研究的一个理想模式系统。     

正常人IgG类天然抗角蛋白自身抗体反应特性分析

目的:分析纯化前后正常人血清IgG类抗角蛋白自身抗体对角蛋白的反应型式,探讨血清非IgG成分对自身AKautoAbs结合特性的影响。方法:用间接ELISA检测正常人血清IgG类AKautoAbs纯化前后的滴度,Westernblot检测全血清和纯化IgG对自身角蛋白的反应特征。结果:纯化前不同个体血清中IgG类AKautoAb的滴度差异很大,反应型式高度异质;纯化后IgG的AKautoAb滴度较纯化前明显升高。Westernblot分析显示纯化后的IgG类AKautoAb识别的角蛋白成分条带增加,反应性增强,且个体之间的反应型式趋于一致。结论:血清中IgG类AKautoAbs的反应型式受一些非IgG成分不同程度的影响;个体之间高度一致的反应型式提示产生IgG类AKautoAbs的B细胞在发育过程中可能受正常机体一组自身抗原(角蛋白)的选择,而与机体接触的外来抗原无关。     

天然抗角蛋白单克隆抗体3B4的噬菌体呈现的scFv的构建及表达

目的:构建并表达噬菌体呈现的天然抗角蛋白单克隆抗体(mAb)3B4的单链抗体(scFv),并测定其活性。方法:分别以pMD18TmAb3B4VH和pMD18TmAb3B4VL为模板进行PCR,扩增mAb3B4的VH和VL基因,然后将其依次插入噬菌体表达载体pscMH中。经DNA序列测定正确后,转化大肠杆菌,用辅助噬菌体VCSM13超感染诱导表达scFv;用ELISA检测其与亲本mAb3B4的抗原结合活性的改变。结果:对扩增的mAb3B4的VH和VL基因进行测序,结果表明VH基因与原序列完全一致,VL基因在骨架区FR1有1个碱基发生突变。用VCSM13超感染后能检测到scFv的表达,其与亲本一样具有多反应性,但抗原结合模式不完全相同。结论:成功地构建并表达了天然抗角蛋白mAb3B4噬菌体呈现的单链抗体,表达的scFv与mAb3B4的抗原结合活性有一定的差异,为探讨天然自身抗体的抗原结合模式奠定了基础。     

多发性硬化症与自身反应性B细胞

多发性硬化（Multiple sclerosis，MS），一种中枢神经系统（Central nervous system，CNS）脱髓鞘病，其病因至今尚未真正明确。长期以来，从免疫学角度而言，MS一直被认为是CD4^＋Th1细胞所介导的自身免疫病，这主要是基于对实验性变态反应性脑脊髓炎（ Experimental allergic encephalomyelitis, EAE）的研究而得出的结论。然而近年来，越来越多的科学证据对这一传统理论提出挑战。存在于MS患者体内的自身抗体可能经旁路活化、分子模拟、表位扩展等机制活化的B细胞而产生，并且B细胞和自身抗体可通过多种途径影响着MS发病和病程转归。     

类风湿性关节炎与自身反应性B细胞

类风湿性关节炎(Rheumatoid Arthritis,RA)是一类以人体肢端关节滑膜中炎症细胞浸润为主要表现的自身免疫性疾病,其病因至今尚未真正明确.从免疫学角度研究发现:除了免疫复合物(炎性沉淀物)在关节囊液中起着一定的作用外,其患者体内的自身抗体与RA存在着密切关联.尽管目前仍有较为明确的证据表明RA是以T细胞--特别是Th1介导为主:这是由于依赖于胶原诱导关节炎(CIA,Collagen-induced arthritis)小鼠模型研究所得的结论.然而,新近的实验研究和临床免疫干预的进展使人们重新认识到B细胞是如何受自身抗原的诱导、激活、增殖和分泌自身抗体导致了RA发病以及这些自身抗体所累及的患者临床病理变化的本质,无疑将类风湿性关节炎与自身反应性B细胞的关联性重新提到一新的重要地位.毫无疑问:患者体内自身反应性B细胞和其产生的自身抗体在RA的发病中起到关键作用.患者关节滑液中浸润着高表达IL-13受体的B细胞,它们所分泌的一系列细胞因子发挥着特殊作用.值得特别注意的是IL-13可以作用于B细胞(诱导B细胞高表达CD23,促进幼稚B细胞的增殖和分化直至这种自身反应性B细胞克隆的异常扩增),越发突显自身反应性B细胞协同自身反应性T细胞介导RA发病的重要性,同时人们将更加关注其独特的免疫病理学机制对RA的诊断、进展和疾病预后的重要意义.本文将着重综述和讨论类风湿性关节炎与自身反应性B细胞的关联性的最新进展,旨在重新评估自身反应性B细胞和其分泌的自身抗体,包括一些细胞因子特别是IL-13介导的免疫失衡在类风湿性关节炎中的重要作用.     

不同年龄正常人血清抗角蛋白自身抗体水平的分析及意义

目的 分析不同年龄段正常人的血清抗角蛋白自身抗体（anti-keratin autoantibody,AK auto Ab)的水平与年龄的相关性,并探讨AK auto Ab产生的可能机制。方法 以人角蛋白进行包被,采用间接ELISA法检测新生儿脐带血、幼儿、成年人及老年人血清IgM与IgG类AK auto Ab的水平,并进行Kruskal-Wallis秩和检验分析。结果 脐带血IgM和IgG类AK auto Ab的水平,均明显低于其它年龄组（P＜0．05）;而幼儿脐带血及成年人、老年人血清IgM和IgG类AK auto Ab的水平无明显差异（P＞0．05）。结论 出生时AK auto Ab即存在但水平较低,幼年期AK auto Ab的水平迅速增加,以后各年龄段基本保持稳定。     

家兔抗角蛋白自身抗体独特型抗体的产生及临床意义

目的 观察长期注射大剂量同种异体抗角蛋白自身抗体（AKautoAb)后,家兔血清中针对抗角蛋白自身抗体（AKautoAb)独特型抗体的产生,方法 肌肉注射亲和层析纯化的AKautoAb(5mg/kg),隔日1次,连续90d,用ELISA检测血清中的抗独特型抗体,结果 注射AKautoAb的家兔产生了针对AKautoAbF(ab′）2片段的抗独特型抗体,于给药4wk血清抗体滴度达高峰,以后逐渐降低,结论 长期注射大剂量同种异体AKautoAb的家兔可产生免疫耐受性。     

作为生理机制的天然自身抗体

近20 年来的研究表明,正常人和动物体内普遍存在着众多的天然自身抗体(naturalautoantibody, NAA) , 作为机体生理性自身免疫(normalautoimmunity) 的主要组成部分。本文就NAA 的产生机理、特性、生理功能及临床意义等方面作了综述介绍, 强调了NAA 在维持机体自身稳定方面的重要性。     

细胞角蛋白与肝脏疾病

细胞角蛋白在肝细胞和胆管细胞的不同表型与细胞功能密切相关。通过胚胎肝组织发生过程中角蛋白表型的变化和肿瘤性肝细胞不同成份角蛋白表达的异质性研究，提示成年人肝脏可能贮备有多潜能干细胞。此外，细胞角蛋白作为细胞系标记物，其病理学的改变是肝脏疾病的参考指标。     

Autoreactive B-1 B cells: constraints on natural autoantibody B cell antigen receptors

B-1 B-cells constitute a distinctive population of cells that are enriched for self-reactive B cell receptors (BCRs). These BCRs are encoded by a restricted set of heavy and light chains, including heavy chains that lack nontemplated nucleotide additions at the V-D and D-J joining regions. One prototype natural autoantibody produced by B-1 B cells binds to a cryptic determinant exposed on senescent red blood cells that includes the phosphatidylcholine (PtC) moiety. The V(H)11Vkappa9 BCR, which accounts for a large fraction of the anti-PtC specificity, is underrepresented in other B-cell populations, including newly formed B cells in bone marrow, and the transitional B cells, follicular B cells, and marginal zone B cells in spleen. Previous work has shown that V(H)11 heavy chains pair ineffectively with surrogate light chain (SLC) and so do not promote development in bone marrow, but instead allow fetal liver maturation because of a fetal preference for weaker pre-BCR signaling. Such inefficient SLC pairing constitutes one constraint on the maturation of B cells containing V(H)11 rearrangements that biases their generation to fetal development. Here, we examine another possible bottleneck to the B1 cell repertoire: light chain pairing with V(H)11 heavy chain, finding very significant preferences.     

B cell superantigens: a microbe's answer to innate-like B cells and natural antibodies

Marginal zone B cells and B-1 cells have been termed innate-like B cells as they express limited repertoires that play special roles in immune defenses against common infections. These B cells are the sources of natural antibodies and are capable of highly accelerated clonal responses that help counter blood-borne infections. We have characterized a class of microbial product with highly adapted binding interactions with host immunoglobulins/B cell receptors (BCRs), which enable the targeting of large supra-clonal sets of B cells for activation-associated apoptotic death. In recent studies, we have shown that all B cells with V region-targeted BCRs are susceptible. However, compared to follicular B cells, in vivo exposure preferentially causes innate-like B cells to undergo induced death with subsequent long-lasting supra-clonal depletion and immune tolerance. Based on these properties, it is likely that B cell superantigens influence the pathogenesis of some common infections, but also may provide novel therapeutic opportunities to treat B cell neoplastic and autoimmune diseases.     

Development of B cells producing natural autoantibodies to thymocytes and senescent erythrocytes

Natural antibodies produced by CD5⁺ B-1 B cells include those with specificity for senescent erythrocytes (anti-BrMRBC, anti-PtC) and for thymocytes (anti-thymocyte autoantibody, ATA). Here we describe work from our laboratories studying two prototypic examples, V_H11V9-encoded anti-BrMRBC and V_H3609V21c-encoded ATA. Using V_H11- transgenic mice, we discovered that certain natural autoantibodies utilize V_H genes that are selected against in bone marrow B cell development, but not fetal liver, effectively restricting their generation to fetal/neonatal life. Studies with ATA- transgenic mice demonstrated a critical requirement for self antigen in the accumulation of B cells with this specificity and for the production of high levels of serum ATA. Finally, analysis of B cell development in ATA- transgenic mice revealed two distinct responses by B cells to expression of this B cell receptor (BCR): most developing B cells in spleen of adult mice were blocked at an immature stage and only escaped apoptosis by editing their BCR to eliminate the ATA specificity; nevertheless, high levels of serum ATA were observed, indicating that some B cells differentiated to antibody-forming cells without altering their specificity. Thus, our studies reveal mechanisms for restricting the generation of B cells producing natural autoantibodies, demonstrate a key positive selection step in their development, and show that most developing B cells in adult mice bearing such specificities fail to reach a mature stage.     

Invariant natural killer T cells balance B cell immunity

Invariant natural killer T (iNKT) cells mediate rapid immune responses which bridge the gap between innate and adaptive responses to pathogens while also providing key regulation to maintain immune homeostasis. Both types of important iNKT immune responses are mediated through interactions with innate and adaptive B cells. As such, iNKT cells sit at the decision‐making fulcrum between regulating inflammatory or autoreactive B cells and supporting protective or regulatory B cell populations. iNKT cells interpret the signals in their environment to set the tone for subsequent adaptive responses, with outcomes ranging from getting licensed to maintain homeostasis as an iNKT regulatory cell (iNKT_reg) or being activated to become an iNKT follicular helper (iNKT_FH) cell supporting pathogen‐specific effector B cells. Here we review iNKT and B cell cooperation across the spectrum of immune outcomes, including during allergy and autoimmune disease, tumor surveillance and immunotherapy, or pathogen defense and vaccine responses. Because of their key role as influencers, iNKT cells provide a valuable target for therapeutic interventions. Understanding the nature of the interactions between iNKT and B cells will enable the development of clinical interventions to strategically target regulatory iNKT and B cell populations or inflammatory ones, depending on the circumstance.     

Severe impairment of B cell function in lpr/lpr mice expressing transgenic Fas selectively on B cells.

Transgenic lpr/lpr mice expressing functional Fas selectively on B cells were produced in an attempt to elucidate the role of Fas on B cells in the regulation of autoantibody production. The homozygous lpr/lpr mice carrying the transgene did not produce anti-double-stranded DNA antibodies throughout their lives, whereas the development of abnormal lpr T cells (double negative, B220(+)) was not suppressed. Further analyses, however, revealed that the expression of the transgenic Fas on B cells of lpr/lpr homozygous mice resulted in severe impairment of the B cell function. The defect was characterized by a decrease in the number of mature peripheral B cells, a reduction in the serum Ig level and the total failure of B cells to mount antibody responses to stimulations of T-dependent as well as T-independent antigens. Such a defect was prominent only when the transgene was expressed on the lpr/lpr homozygous background. On the contrary, B cells of the transgenic lpr/lpr mice were shown to be capable of producing Ig when stimulated with anti-CD40 in the presence of IL-4 and IL-5. Furthermore, lpr/lpr T cells showed enhanced non-specific cytolytic activity. These observations suggested that the observed B cell defect was probably attributable to the destruction of activated B cells expressing transgenic Fas by aggressive lpr/lpr T cells.     

B cell subsets in postmenopausal women and the effect of hormone replacement therapy

Objectives: In elderly subjects the capacity for antibody production is depressed. This immunosenescence state of humoral immunity is associated with the occurrence of autoimmune disorders involving CD5⁺ B (B-1) cells. Since estrogen is capable of stimulating the production of autoantibodies, this sex steroid hormone may be a contributing cause of the higher incidence of autoimmune diseases in women. In the present study, B cell subsets in women during the postmenopausal period was determined. The effect of hormone replacement therapy (HRT) on B cell subsets was examined to establish whether the administration of gonadal hormones influence humoral immunity in postmenopausal women. Methods: Forty six untreated pre- and postmenopausal women and 39 women on HRT were studied. The proportion of B-1 (CD5⁺) and conventional CD5⁻ B (B-2) lymphocytes was determined by two-color flow cytometry. Serum autoantibodies to a nuclear antigen and to interleukin (IL)-1 were measured by immunofluorescence and by radioimmunoassay, respectively. Thirteen women were examined prospectively before and during HRT. Results: In late postmenopausal women (≥30 years postmenopausal period), the proportion of B-2 cells was significantly reduced (p<0.01) compared to those of premenopausal and perimenopausal women. HRT induced a significant (p<0.01) increase in the percentage of B-2 cells, while that of B-1 cells remained unchanged. HRT did not affect autoantibody production. Conclusion: HRT may retard the progress of immunosenescence by increasing the production of B-2 cells. Moreover, HRT appears not to increase the risk of autoimmune diseases developing in postmenopausal women.     

Manipulation of the glycan-specific natural antibody repertoire for immunotherapy

Natural immunoglobulin derived from innate-like B lymphocytes plays important roles in the suppression of inflammatory responses and represents a promising therapeutic target in a growing number of allergic and autoimmune diseases. These antibodies are commonly autoreactive and incorporate evolutionarily conserved specificities, including certain glycan-specific antibodies. Despite this conservation, exposure to bacterial polysaccharides during innate-like B lymphocyte development, through either natural exposure or immunization, induces significant changes in clonal representation within the glycan-reactive B cell pool. Glycan-reactive natural antibodies (NAbs) have been reported to play protective and pathogenic roles in autoimmune and inflammatory diseases. An understanding of the composition and functions of a healthy glycan-reactive NAb repertoire is therefore paramount. A more thorough understanding of NAb repertoire development holds promise for the design of both biological diagnostics and therapies. In this article, we review the development and functions of NAbs and examine three glycan specificities, represented in the innate-like B cell pool, to illustrate the complex roles environmental antigens play in NAb repertoire development. We also discuss the implications of increased clonal plasticity of the innate-like B cell repertoire during neonatal and perinatal periods, and the prospect of targeting B cell development with interventional therapies and correct defects in this important arm of the adaptive immune system.     

The ontogeny of B cells in the thymus of normal, CD3 epsilon knockout (KO), RAG-2 KO and IL-7 transgenic mice.

The ontogeny of thymic B cells was determined by three-color flow cytometry and the presence or absence of B cell progenitors confirmed by cell culture experiments. In the thymus of young normal mice, CD117(+), B220(low) pro- and pre-B cells are present but disappear with age. B220(low), CD5(+), B-1 B cells are present in the thymus of older animals following the appearance of similar cells in the peritoneal cavity and blood. In CD3 epsilon gene-deleted mice, the phenotypic progression and number of thymic B cells remains unaltered, showing that blocking T cell development does not automatically result in an increase of thymic B lymphopoiesis. Pro-B cells in RAG-2 knockout mice are found in the fetal and neonatal blood, spleen and thymus, but with increasing age are only found in the bone marrow. B lymphopoiesis in adult IL-7 transgenic mice is dramatically altered with CD117(+) pro- and pre-B cells present in spleen, lymph node and blood. In the thymus of adult IL-7 transgenic mice, the fraction of CD117(+) thymic B cells is significantly increased. These results show that in the steady state, the phenotype of thymic B cells is critically dependent on both mouse age and the phenotype of circulating B cells.     

Putative role of marginal zone B cells in pathophysiological processes

The maintenance of inner integrity of an organism is founded on the proper performance of two immunity branches, innate and adaptive immune responses. Recently, it became apparent that subset of splenic B cells named marginal zone B cells (MZB cells) exhibits unique developmental and functional features that bridge these two immunity branches. Strategically positioned at the site where blood and lymph are filtered, MZB cells represent a population of sentinels that rapidly proliferate and differentiate into IgM plasmablast cells when encountered with blood-borne, thymus-independent (TI) Ags. Moreover, MZB cells have intrinsic capability to induce potent CD4⁺ helper T cell response and cytokine production upon stimulation with soluble antigens. Due to their ability to overcome a time gap prior the establishment of the full adaptive response towards pathogens, MZB cells connect and direct innate and adaptive immunity. An additional interesting characteristic of MZB cells is capacity to function as regulatory cells in autoimmune processes. MZB cells may also contribute to the control of autoimmunity via the induction of tolerance by apoptotic cells. Importantly, in the clear association with inflammation and autoimmunity, MZB cells may transform into MALT lymphoma, representing a concurrence point for the infection, immunity and malignancy. This paper presents an insight into the complex biology of marginal zone B cells and their role in intertwining and directing innate and adaptive immune processes at the physiological and pathological level.