Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens

The long-standing hypothesis that tolerance to self antigens is mediated by either elimination or functional inactivation (anergy) or self-reactive lymphocytes is now accepted, but little is known about the factors responsible for initiating one process rather than the other. In the B-cell lineage, tolerant self-reactive cells persist in the peripheral lymphoid organs of transgenic mice expressing lysozyme and anti-lysozyme immunoglobulin genes, but are eliminated in similar transgenic mice expressing anti-major histocompatibility complex immunoglobulin genes. By modifying the structure of the lysozyme transgene and the isotype of the anti-lysozyme immunoglobulin genes, we demonstrate here that induction of anergy or deletion is not due to differences in antibody affinity or isotype, but to recognition of monomeric or oligomeric soluble antigen versus highly multivalent membrane-bound antigen. Our findings indicate that the degree of receptor crosslinking can have qualitatively distinct signalling consequences for lymphocyte development.     

T cells with receptors for IgD

The role of IgD in the immune response has been elusive, although its predominance on the cell surface suggests a receptor function. We have shown previously that euthymic but not athymic BALB/c mice, injected with IgD before antigen, exhibit enhanced antibody responses which can be transferred by T cells. Isotype-specific T cells have been reported to have both upward and downward immunoregulatory effects. Here we demonstrate the existence of T cells with receptors for IgD, and show that exposure to IgD in vivo or in vitro significantly increases the number of T delta cells in the spleen and lymph nodes but not in the thymus. The kinetics of T delta-cell appearance in vivo parallels that of the immunoenhancing effect which occurs after injection of IgD. These T delta cells are of the Lyt 1+2- T-cell phenotype.     

Breakdown of self-tolerance in anergic B lymphocytes.

Production of autoantibodies, which characterizes most autoimmune diseases, is normally avoided by active elimination or functional inactivation (anergy) of B and T lymphocytes bearing receptors for self antigens. The mechanisms leading to the escape of self-reactive clones from these normal tolerance mechanisms in autoimmune diseases nevertheless remain obscure. Here, we demonstrate that clonal anergy in B lymphocytes is a reversible process, and that silenced self-reactive B cells can be reactivated under particular conditions to give rise to vigorous antibody responses. Reactivation of anergic lymphocytes may explain many examples of transient autoimmune reactions in normal individuals, and may under pathological conditions be important in the development of chronic autoimmune disease.     

重用抗体优良片断的免疫进化算法

基于克隆选择原理与算法,通过分析具体现象阐述了改进克隆选择算法的思想来源,设计了挖掘抗体中优秀决定基因并生成记忆集、封装优秀决定基片段、用变异抗体群中亲和度高的抗体按概率替换记忆抗体群中低亲和度抗体的方法,获得了重用抗体优良片断的克隆选择算法.借鉴强度Pareto进化算法的进化框架,提出了重用抗体优良片断的免疫进化算法.该算法通过克隆选择替代选择、交叉、重组等遗传操作.在一组0/1背包问题上的测试结果表明,所提出的算法可以有效保持种群多样性,获得较高质量的Pareto非劣解集.
     

Three-dimensional structure of an idiotope-anti-idiotope complex.

Serologically detected antigenic determinants unique to an antibody or group of antibodies are called idiotopes. The sum of idiotopes of an antibody constitute its idiotype. Idiotypes have been intensively studied following a hypothesis for the self-regulation of the immune system through a network of idiotype-anti-idiotype interactions. Furthermore, as antigen and anti-idiotypes can competitively bind to idiotype-positive, antigen-specific antibodies, anti-idiotypes may carry an 'internal image' of the external antigen. Here we describe the structure of the complex between the monoclonal anti-lysozyme FabD1.3 and the anti-idiotopic FabE225 at 2.5 A resolution. This complex defines a private idiotope consisting of 13 amino-acid residues, mainly from the complementarity-determining regions of D1.3. Seven of these residues make contacts with the antigen, indicating a significant overlap between idiotope and antigen-combining site. Idiotopic mimicry of the external antigen is not achieved at the molecular level in this example.     

Glycosyl-phosphatidylinositol linkage as a mechanism for cell-surface expression of immunoglobulin D.

The B-cell antigen receptor of the IgM and IgD class is a multimeric complex consisting of the membrane-bound form of the immunoglobulin molecule and two other proteins, Ig-alpha and Ig-beta. The Ig-alpha and Ig-beta proteins form a disulphide-linked alpha/beta heterodimer and are encoded by the mb-1 (ref 9, 10) and B29 genes, respectively. Surface expression of the membrane-bound IgM molecule requires assembly with the alpha/beta heterodimer. The IgD molecule, however, can be expressed on the cell surface in an alpha/beta-dependent and -independent form. We show here that in the alpha/beta-independent form the IgD molecule is anchored in the plasma membrane through a glycosyl-phosphatidylinositol linker. In the presence of the alpha/beta heterodimer, most of the otherwise glycosyl-phosphatidylinositol-linked IgD molecule is expressed on the cell surface as transmembrane proteins.     

Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes

B lymphocytes can be rendered specifically unresponsive to antigen by experimental manipulation in vivo and in vitro, but it remains unclear whether or not natural tolerance involves B-cell tolerance because B cells are controlled by T lymphocytes, and in their absence respond poorly to antigen (reviewed in ref. 7). In addition, autoantibody-producing cells can be found in normal mice and their formation is enhanced by B-cell mitogens such as lipopolysaccharides. We have studied B-cell tolerance in transgenic mice using genes for IgM anti-H-2k MHC class I antibody. In H-2d transgenic mice about 25-50% of the splenic B cells bear membrane immunoglobulin of this specificity, and abundant serum IgM encoded by the transgenes is produced. In contrast, H-2k x H-2d (H-2-d/k) transgenic mice lack B cells bearing the anti-H-2k idiotype and contain no detectable serum anti-H-2k antibody, suggesting that very large numbers of autospecific B cells can be controlled by clonal deletion.     

兔抗人多发性骨髓瘤细胞多克隆抗体的制备与鉴定

制备人多发性骨髓瘤全细胞兔多克隆抗体。用人多发性骨髓瘤细胞系ARH-77全细胞和福氏完全佐剂通过背部皮内多点注射首次免疫新西兰大白兔,第14 d用ARH-77全细胞和福氏不完全佐剂同样剂量加强免疫,第28 d和38 d时再次免疫。第45 d颈动脉采血80 mL,4℃静置过夜,收集血清。然后采用亲和层析系统纯化血清中的多克隆抗体。用酶联免疫吸附试验(ELISA)检测纯化多克隆抗体的效价;免疫印迹法和荧光免疫细胞化学检测纯化多克隆抗体的特异性。结果:ELISA检测多克隆抗体滴度为1∶20 000,免疫印迹检测结果显示该抗体具有较高特异性,荧光免疫细胞化学检测多克隆抗体能够有效的结合细胞表面抗原。实验获得了高效价的特异性的兔抗人骨髓瘤细胞多克隆抗体,为进一步研究多发性骨髓瘤诊断和治疗奠定了基础。     

A role for clonal inactivation in T cell tolerance to Mls-1a

Clonal deletion plays a major part in the maintenance of natural self-tolerance in both normal and transgenic mice. Self antigens that are expressed in the thymus result in the physical elimination of autoreactive thymocytes at a particular stage in their development. For example, the majority V beta 6- and V beta 8.1-bearing T cells that recognize the minor lymphocyte-stimulating antigen, Mls-1a (ref. 10) , are clonally deleted in the thymuses of normal mice and transgenic mice expressing Mls-1a (refs 2, 3, 9). In contrast, a very different mechanism of tolerance involving the functional inactivation, but not elimination, of autoreactive cells, termed clonal inactivation or clonal anergy, has been implicated in some experimentally manipulated systems of tolerance. To test further the mechanisms involved in self-tolerance, we have generated transgenic mice expressing a V beta 8.1 beta chain on greater than 95% of peripheral T cells and have tested tolerance to Mls-1a in these mice. Surprisingly, a significant fraction of the CD4+ peripheral cells that survived deletion were non-responsive in vitro to any stimulus tested. Naturally occurring tolerance to a self antigen expressed in the thymus can thus be mediated by clonal anergy, as well as by clonal deletion.     

Virus-induced autoantibody response to a transgenic viral antigen

The induction of autoantibodies and their possible role in the pathogenesis of autoimmune disease are poorly understood. Involvement of infectious agents has been suspected, but direct evidence is sparse. Whether immunological unresponsiveness to self by antibody-forming B cells is maintained by clonal abortion, clonal anergy or suppression, or how the scenario of interactions between helper T cells, B cells and antigen-presenting cells is distorted in autoantibody responses, is being analysed and widely debated. To evaluate tolerance of neutralizing B-cell responses we used transgenic mice expressing the cell membrane associated glycoprotein (G) of vesicular stomatitis virus (VSV) as self-antigen. We show that autoantibodies to VSV-G cannot be induced by VSV-G in adjuvant or by recombinant vaccinia virus expressing VSV-G, but are triggered by infection with wild-type VSV. The data show that helper T-cell tolerance is crucial in maintenance of B-cell non-reactivity and that cognate T-B recognition is necessary to break tolerance of self-reactive B cells. These results may help to understand mechanisms of virus-induced autoimmunity.     

Three-dimensional structure of an antigen-antibody complex at 6 A resolution

Present understanding of the three-dimensional structure of antibody combining sites is based on X-ray diffraction studies of myeloma immunoglobulins. The structures of the antigen-binding fragment (Fab) complexes of two of these immunoglobulins with small ligands have also been determined. However, there is no crystallographic information concerning the interactions of an antibody with an antigen, nor do we know the precise structure of antigenic determinants on protein molecules. We now report the first structure determination of an antigen-antibody complex at 6 A resolution. The structure of the complex between hen egg-white lysozyme and the Fab of a monoclonal anti-lysozyme antibody (D1.3) shows that the combining site of antibodies is not merely a cleft delineated by the complementarity-determining regions of the variable regions of the light and heavy chains, but is a larger area extending beyond it. A correspondingly large area of the antigen makes close contacts with the antibody, in agreement with the notion of a 'topographical' rather than 'sequential' antigenic determinant. The structural basis of cross-reactivities of an antibody with heterologous antigens and the effect of a single amino acid substitution on antigenic specificity can thus be visualized in the structural model presented here.     

Endogenous antigen tunes the responsiveness of naive B cells but not T cells

In humans, up to 75% of newly generated B cells and about 30% of mature B cells show some degree of autoreactivity. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model B-cell antigen receptor (BCR) transgenic systems have highlighted the critical role of functional unresponsiveness or ‘anergy’. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B-cell tolerance. However, it remains unclear whether the mature diverse B-cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which BCR signalling rapidly and robustly induces green fluorescent protein expression under the control of the Nur77 regulatory region, antigen-dependent and antigen-independent BCR signalling events in vivo during B-cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure, in turn, tunes the responsiveness of BCR signalling in B cells at least partly by downmodulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or anergy exists in the mature B-cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease.     

Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells

One mechanism ensuring self tolerance of T cells is the clonal deletion of thymocytes bearing alpha beta T-cell receptors. The stage of thymocyte development at which the interaction with antigen-presenting cells (APCs) leads to deletion, however, has not been determined directly. Indirect evidence suggests that intrathymic APCs induce deletion of CD4+8+ thymocytes (which die by apoptosis) but deletion at less and more mature developmental stages has also been implied. It is also not clear if clonal elimination of thymocytes can be triggered by peripheral antigens carried on extrathymic APCs migrating through the thymus. Here we show antigen-specific induction of apoptosis in CD4+8+ thymocytes cultured in suspension, by thymic as well as splenic APCs. Thus the recognition of antigen by CD4+8+ thymocytes may lead to deletion, suggesting that this is the central mechanism of tolerance induction, which is not limited by the antigen-presenting ability of the thymic stroma.     

Neonatal T-cell tolerance to minimal immunogenic peptides is caused by clonal inactivation

The mechanisms underlying T-lymphocyte tolerance induced in neonatal mice are still unknown. It is unclear whether the tolerant state is the result of inactivation of T cells on exposure to antigen during development or of active suppression by other T cells specific for the same antigen. To distinguish between these two hypotheses, we have analysed the specificity of tolerance to three cytochrome peptides which differ by only a single amino-acid substitution in the epitope recognized by proliferative T cells. The peptides stimulate proliferative responses which are highly specific with minimal cross-reactivity. As antigen-induced clonal inactivation would address the same cells normally activated by that antigen, the specificity of tolerance should exactly match that of the proliferative response to the antigen, and each cytochrome peptide should induce tolerance to itself alone. Conversely, as T-suppressor (Ts) and T-proliferative (Tp) cells almost invariably seem to recognize distinct, non-overlapping determinants on protein antigens, suppressor-mediated tolerance should not be affected by substitutions in the proliferative T-cell epitope. Tolerance would depend solely on the existence of a shared suppressor determinant, so each cytochrome peptide should induce cross-tolerance to the others. We found that the specificity of tolerance matched that of the proliferative response: each peptide induced tolerance for itself but the response to the variants was unaltered. This result strongly supports the hypothesis of clonal inactivation as an important mechanism in induction of neonatal tolerance.     

基于克隆思维进化算法的机器人足球协作策略

针对足球机器人比赛系统的实时性要求,采用了一种克隆思维进化算法对足球机器人比赛系统的高层策略系统进行优化。克隆思维进化算法集免疫机制与进化机制于一体,在发挥思维进化算法优势的基础上增加了克隆(复制)、克隆重组、克隆变异和克隆选择等算子,既保持了种群的多样性,又提高了算法的收敛速度。足球比赛场上的瞬时信息作为抗原,待选策略作为抗体,二者均采用二进制编码方式。用克隆思维进化算法对抗体群进行优化,实验结果表明,采用该算法能快速找到最佳策略,简化了足球机器人决策系统,提高了决策效率。     

Infection breaks T-cell tolerance.

Clonal deletion or clonal anergy establish tolerance in T cells that bear potentially autoreactive antigen receptors. Here we report that concomitant infection with the nematode Nippostrongylus brasiliensis breaks an established T-cell tolerance induced by injection of mice with Staphylococcus enterotoxin B (SEB). CD4+ T cells from SEB-tolerant mice did not produce either interleukin-2 or interleukin-4 when challenged in vitro with SEB. N. brasiliensis infection of SEB-primed animals resulted in a normal expansion of SEB-tolerant CD4+V beta 8+ T cells in vivo as well as an equivalent increase of SEB-reactive, interleukin-4-producing CD4+V beta 8+ T cells both in SEB-tolerant and in normal animals. Thus, infection with N. brasiliensis circumvented the tolerance established with SEB. Activation of anergic, potentially autoreactive CD4+ T cells by infectious agents seems to be a major pathway for the initiation of autoimmune diseases. Our results suggest that infectious agents may break tolerance in potentially autoreactive CD4+ T cells by activation of alternative reaction pathways.     

Protein kinase Cdelta controls self-antigen-induced B-cell tolerance

Interaction of a B cell expressing self-specific B-cell antigen receptor (BCR) with an auto-antigen results in either clonal deletion or functional inactivation. Both of these processes lead to B-cell tolerance and are essential for the prevention of auto-immune diseases. Whereas clonal deletion results in the death of developing autoreactive B cells, functional inactivation of self-reactive B lymphocytes leads to complex changes in the phenotype of peripheral B cells, described collectively as anergy. Here we demonstrate that deficiency in protein kinase Cdelta (PKC-delta) prevents B-cell tolerance, and allows maturation and terminal differentiation of self-reactive B cells in the presence of the tolerizing antigen. The importance of PKC-delta in B-cell tolerance is further underscored by the appearance of autoreactive anti-DNA and anti-nuclear antibodies in the serum of PKC-delta-deficient mice. As deficiency of PKC-delta does not affect BCR-mediated B-cell activation in vitro and in vivo, our data suggest a selective and essential role of PKC-delta in tolerogenic, but not immunogenic, B-cell responses.     

T-cell tolerance by clonal anergy in transgenic mice with nonlymphoid expression of MHC class II I-E

T-cell reactivity to the class II major histocompatibility complex I-E antigen is associated with T-cell antigen receptors containing the V beta gene segments V beta 17a and V beta 5. Mice expressing I-E with the normal tissue distribution (on B cells, macrophages, dendritic cells and thymic epithelium) induce tolerance to self I-E by clonal deletion in the thymus. By contrast, we find that transgenic INS-I-E mice that express I-E on pancreatic beta-cells, but not in the thymus or peripheral lymphoid organs, are tolerant to I-E but have not deleted V beta 5- and V beta 17a-bearing T cells. Moreover, whereas T-cell populations from nontransgenic mice proliferate in response to receptor crosslinking with V beta 5- and V beta 17a-specific antibodies, T cells from INS-I-E mice do not. Thus, our experiments provide direct evidence that T-cell tolerance by clonal paralysis does occur during normal T-cell development in vivo.     

基于免疫记忆克隆的关联规则挖掘

针对数据关联规则挖掘的不足,提出了一种基于免疫记忆克隆算法的关联规则挖掘方法。算法利用了免疫记忆特性,把挖掘的关联规则存入记忆库,加快了挖掘速度。在克隆扩增过程中,设计了一种基于矢量距的抗体浓度计算方法,保证克隆扩增过程中解的多样性。仿真实验结果表明,现算法具有较快的运行速度,提高了所得关联规则的准确性。