Asymmetric segregation of polarized antigen on B cell division shapes presentation capacity

During the activation of humoral immune responses, B cells acquire antigen for subsequent presentation to cognate T cells. Here we show that after mouse B cells accumulate antigen, it is maintained in a polarized distribution for extended periods in vivo. Using high-throughput imaging flow cytometry, we observed that this polarization is preserved during B cell division, promoting asymmetric antigen segregation among progeny. Antigen inheritance correlates with the ability of progeny to activate T cells: Daughter cells receiving larger antigen stores exhibit a prolonged capacity to present antigen, which renders them more effective in competing for T cell help. The generation of progeny with differential capacities for antigen presentation may have implications for somatic hypermutation and class switching during affinity maturation and as B cells commit to effector cell fates.     

A virus-encoded "superantigen" in a retrovirus-induced immunodeficiency syndrome of mice.

The development of an immunodeficiency syndrome of mice caused by a replication-defective murine leukemia virus (MuLV) is paradoxically associated with a rapid activation and proliferation of CD4+ T cells that are dependent on the presence of B cells. The responses of normal spleen cells to B cell lines that express the defective virus indicated that these lines express a cell surface determinant that shares "superantigenic" properties with some microbial antigens and Mls-like self antigens. This antigen elicited a potent proliferative response that was dependent on the presence of CD4+ T cells and was associated with selective expansion of cells bearing V beta 5. This response was markedly inhibited by a monoclonal antibody specific for the MuLV gag-encoded p30 antigen.     

Strategies for enhancing DNA vaccine potency by targeting antigen-presenting cells

The potency of DNA vaccines to stimulate the immune response, especially the T-cell immune response against viral infections and tumors, depends mainly on the ability of antigen-presenting cells to process and present DNA-encoded antigens. Targeting the specific antigen to antigen-presenting cells is believed to be a crucial step for eliciting the T-cell response. Many strategies for enhancing DNA vaccine potency by targeting antigen-presenting cells have been developed. In this article, we generally introduce a T cell immune system and review some strategies which have been recently developed for enhancing DNA vaccine potency.     

The minimal number of class II MHC-antigen complexes needed for T cell activation

Major histocompatibility complex (MHC) molecules are exposed to large quantities of self and nonself antigens. It is not known what fraction of MHC molecules needs to be occupied by antigen to induce a T cell response. A quantitative study of naturally processed antigen indicated that T cells could be activated when only 0.03 percent of the total I-Ed purified from antigen-presenting cells (APCs) was occupied with antigen. B cells and macrophages processed hen egg lysozyme (HEL) with different efficiencies, but similar degrees of occupancy were required for T cell stimulation. Higher occupancy was needed for I-Ed-transfected L cells, possibly reflecting the requirement for other accessory molecules for efficient APC-T cell interaction.     

装载鸡传染性支气管炎病毒S1蛋白重组HD11细胞源外体的构建

为了给禽类巨噬细胞源外体在免疫应答的功能研究和家禽外体载体疫苗的研发奠定基础,本研究以鸡传染性支气管炎病毒(Infectious bronchins virus, IBV)S1蛋白作为模式抗原,通过慢病毒表达系统技术实现在HD11细胞中稳定表达S1蛋白,再利用超速离心从细胞培养物上清中分离纯化得到外体。蛋白免疫印分析、电镜与粒径分析检测等结果表明,在该重组HD11细胞分泌的外体中可检测到稳定的S1蛋白表达,显示成功构建了装载IBV S1蛋白的重组外体。     

Production of immunoglobulin isotypes by Ly-1+ B cells in viable motheaten and normal mice

Almost all B cells in autoimmune mice with the viable motheaten (mev) mutation express the Ly-1 cell surface antigen, which marks a minor population of B cells constituting a separate lineage in normal mice. Immunoglobulins primarily of the M and G3 classes, which in both normal and mev mice contain high levels of lambda light chain, are produced in excess in mev mice. These and other observations suggest that the development of B cells that express Ly-1 is regulated independently from the development of B cells that do not express Ly-1. B cells bearing the Ly-1 surface antigen may play specialized roles in the normal immune system and in autoimmunity by regulating other B cells via lymphokines, by producing antibodies to self and certain foreign antigens, and by preferentially secreting immunoglobulin M and immunoglobulin G3.     

Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells

In contrast to na?ve T cells that recognize short antigen-derived peptides displayed by specialized antigen-presenting cells, immunoglobulin receptors of B lymphocytes primarily recognize intact proteins. How and where within a lymph node such unprocessed antigens become available for na?ve B cell recognition is not clear. We used two-photon intravital imaging to show that, after exiting high-endothelial venules and before entry into lymph node follicles, B cells survey locally concentrated dendritic cells. Engagement of the B cell receptor by the dendritic cell (DC)-associated antigen leads to lymphocyte calcium signaling, migration arrest, antigen acquisition, and extrafollicular accumulation. These findings suggest a possible role for antigen-specific B-DC interactions in promoting T cell-dependent antibody responses in vivo.     

Methylation of an immediate-early inducible gene as a mechanism for B cell tolerance induction

Stage-specific gene regulation is important in determining cell function during development. Immature B cells expressing membrane-bound immunoglobulin M (mIgM) are sensitive to antigen-induced tolerance, whereas mature B cells are activated by antigen. Previous studies have established an association between Egr-1 gene induction and antigen receptor (mIgM)-mediated activation of mature B cells. Here it is shown that the immature B cell line WEHI-231 and tolerance-sensitive bone marrow-derived B cells do not express Egr-1. It is further shown that lack of inducible expression in these cells is due to specific methylation of the Egr-1 gene. Thus, covalent inactivation of an activation-associated gene may explain tolerance sensitivity at specific stages of B cell development.     

Maintenance of serological memory by polyclonal activation of human memory B cells

Production of antibodies can last for a lifetime, through mechanisms that remain poorly understood. Here, we show that human memory B lymphocytes proliferate and differentiate into plasma cells in response to polyclonal stimuli, such as bystander T cell help and CpG DNA. Furthermore, plasma cells secreting antibodies to recall antigens are produced in vivo at levels proportional to the frequency of specific memory B cells, even several years after antigenic stimulation. Although antigen boosting leads to a transient increase in specific antibody levels, ongoing polyclonal activation of memory B cells offers a means to maintain serological memory for a human lifetime.     

Antigens and antibodies as cell phenotypes

The paradoxical features of transplantation specificity-its strict genetic control in transfers of tissue from strain to strain as compared with its malleability on tissue passage in foreign immunological environments where the host does not reject the implant (F(1) hybrid passage, tolerance actively acquired by immature hosts, and so on)-present a challenge to genetic interpretation. The attempt is made in this article to show parallels between this behavior and such changes as the transformation of serotypes in Paramecium, in which the activity of genetic units becomes fixed as a cytoplasmic state-a cellular heredity persistent under specified environmental conditions but capable of change to an alternative state-while the genetic structure of the cell remains constant. The reactions appear to differ from those in the Paramecium case in that the diverse loci control a mosaic of different specificities, which change relatively independently of each other, in contrast to mutual exclusion of cytoplasmic states influenced by the different loci in Paramecium. The process of antibody formation is considered as a change in cellular phenotype from the same point of view. The primary response in the stem cells of the lymphoid tissues is interpretable as the establishment of a new cytoplasmic state in response to a nuclear stimulus by the foreign antigen. For the secondary response, the suggestion is made that a reaction of antigen with cellular antibody at the surface of stem cells exhibiting the primary response serves as the stimulus for specific proliferation of antibody-forming clones of cells. A parallel is drawn with the fertilization reaction, specifically with regard to the initiation of cleavage in eggs by antisera to them. Finally, a general chromosomal mechanism is sought for these phenomena, on the basis of activities of specific chromosome regions in response to special developmental stimuli, such as the disproportionate local synthesis of deoxyribonucleic acid demonstrated in the giant chromosomes of the Diptera. By a correlation of such activities with the nucleocytoplasmic system of ribonucleic acid granules on membranes, a possible mechanism appears for the formation, in response to environmental stimuli, of cytoplasmic states which might supply the persistent pattern required for this type of cell heredity. The analogies made, it is believed, provide a framework for the design of test experiments.     

Monoclonal antibody-mediated tumor regression by induction of apoptosis

To characterize cell surface molecules involved in control of growth of malignant lymphocytes, monoclonal antibodies were raised against the human B lymphoblast cell line SKW6.4. One monoclonal antibody, anti-APO-1, reacted with a 52-kilodalton antigen (APO-1) on a set of activated human lymphocytes, on malignant human lymphocyte lines, and on some patient-derived leukemic cells. Nanogram quantities of anti-APO-1 completely blocked proliferation of cells bearing APO-1 in vitro in a manner characteristic of a process called programmed cell death or apoptosis. Cell death was preceded by changes in cell morphology and fragmentation of DNA. This process was distinct from antibody- and complement-dependent cell lysis and was mediated by the antibody alone. A single intravenous injection of anti-APO-1 into nu/nu mice carrying a xenotransplant of a human B cell tumor induced regression of this tumor within a few days. Histological thin sections of the regressing tumor showed that anti-APO-1 was able to induce apoptosis in vivo. Thus, induction of apoptosis as a consequence of a signal mediated through cell surface molecules like APO-1 may be a useful therapeutic approach in treatment of malignancy.     

Imaging of germinal center selection events during affinity maturation

The germinal center (GC) is an important site for the generation and selection of B cells bearing high-affinity antibodies, yet GC cell migration and interaction dynamics have not been directly observed. Using two-photon microscopy of mouse lymph nodes, we revealed that GC B cells are highly motile and extend long cell processes. They transited between GC dark and light zones and divided in both regions, although these B cells resided for only several hours in the light zone where antigen is displayed. GC B cells formed few stable contacts with GC T cells despite frequent encounters, and T cells were seen to carry dead B cell blebs. On the basis of these observations, we propose a model in which competition for T cell help plays a more dominant role in the selection of GC B cells than previously appreciated.     

Contingent genetic regulatory events in T lymphocyte activation

Interaction of antigen in the proper histocompatibility context with the T lymphocyte antigen receptor leads to an orderly series of events resulting in morphologic change, proliferation, and the acquisition of immunologic function. In most T lymphocytes two signals are required to initiate this process, one supplied by the antigen receptor and the other by accessory cells or agents that activate protein kinase C. Recently, DNA sequences have been identified that act as response elements for one or the other of the two signals, but do not respond to both signals. The fact that these sequences lie within the control regions of the same genes suggests that signals originating from separate cell membrane receptors are integrated at the level of the responsive gene. The view is put forth that these signals initiate a contingent series of gene activations that bring about proliferation and impart immunologic function.     

Evolutionary and somatic selection of the antibody repertoire in the mouse

The repertoire of antibody variable (V) regions has been subject to evolutionary selection, affecting both the diversity of V region genes in the germline and their expression in the B lymphocyte population and its subsets. In ontogeny, contact with an antigen leads to the expansion of B cells expressing antibodies complementary to it. In a defined phase of B cell differentiation, new sets of V regions are generated from the existing repertoire through somatic hypermutation. Cells carrying advantageous antibody mutants are selected into the memory compartment and produce a stable secondary response upon reexposure to the antigen.     

T cell-independent rescue of B lymphocytes from peripheral immune tolerance

Autoimmunity arises when immune tolerance to specific self-antigens is broken. The mechanisms leading to such a failure remain poorly understood. One hypothesis proposes that infectious agents or antigens can break B or T lymphocyte self-tolerance by expressing epitopes that mimic self. Using a transgenic immunoglobulin model, we show that challenge with self-mimicking foreign antigen rescues B cells from peripheral tolerance independent of T cell help, resulting in the accumulation of self-reactive cells in the lymph nodes and secretion of immunoglobulins that bind to a liver-expressed self-antigen. Therefore, our studies reveal a potentially important mechanism by which B lymphocytes can escape self-tolerance.     

Rheumatoid factor secretion from human Leu-1+ B cells

A human B cell subpopulation identifiable by the expression of the cell surface antigen Leu-1 (CD5) is responsible for most of the immunoglobulin M rheumatoid factor secreted in vitro after the cells are stimulated with Staphylococcus aureus. The ability of B cells bearing the Leu-1 marker (Leu-1+) to secrete rheumatoid factor is present early in development and extends to adulthood, since Leu-1+ B cells from cord blood and from peripheral blood lymphocytes of both normal adults and patients with certain autoimmune conditions secrete rheumatoid factor in comparable amounts. The neonatal enrichment of Leu-1+ B cells, the presence of Leu-1+ B cells in increased frequencies in patients with autoimmune disease, and the involvement of Leu-1+ B cells in autoantibody secretion suggest both developmental and functional homologies between this human B cell subpopulation and the murine Ly-1 B cell subpopulation.     

Promotion of B cell immune responses via an alum-induced myeloid cell population

Exposure of na?ve B cells to the cytokine interleukin-4 (IL-4) and/or antigen leads to a state of "priming," in which subsequent aggregation of major histocompatibility complex class II molecules induces the mobilization of calcium ions and cell proliferation. However, it is not clear how critical this priming is for immune responses or how it is normally induced in vivo. Injection of mice with the commonly used adjuvant alum led to priming of splenic B cells and to the accumulation in the spleen of a previously unknown population of IL-4-producing, Gr1+ cells. These cells and IL-4 were both required for in vivo priming and expansion of antigen-specific B cells, as well as for optimal production of antibody. These studies reveal a key role for a previously unknown accessory myeloid cell population in the generation of humoral immune responses.     

Tet-on系统诱导猿猴病毒40T在CHO细胞中的表达

四环素诱导表达系统（Tet-off／Tet-on系统）是比较成熟的真核生物基因诱导表达系统之一,具有高效、无毒、严密开／关功能的特点。猿猴病毒40T（SV40T）是一种病毒癌蛋白,其与肿瘤抑制蛋白p53和Rb结合,并使之失活,从而消除它们抑制细胞生长的功能,使细胞分裂加速,形成肿瘤。利用Tet-on系统首先稳定筛选获得了表达Tet-on系统调节元件rtTA的阳性细胞CHO-pTet-on,再通过稳定筛选又成功得到导入其反应元件的双阳性细胞CHO-pTet-on-pTRE2-SV40T-Hyg,经强力霉素诱导表达了目的基因SV40T,建立了Tet-on基因诱导表达系统的细胞诱导表达研究平台。     

TCR-induced transmembrane signaling by peptide/MHC class II via associated Ig-alpha/beta dimers

Previous findings suggest that during cognate T cell-B cell interactions, major histocompatability complex (MHC) class II molecules transduce signals, leading to Src-family kinase activation, Ca2+ mobilization, and proliferation. Here, we show that antigen stimulation of resting B cells induces MHC class II molecules to associate with Immunoglobulin (Ig)-alpha/Ig-beta (CD79a/CD79b) heterodimers, which function as signal transducers upon MHC class II aggregation by the T cell receptor (TCR). The B cell receptor (BCR) and MHC class II/Ig-alpha/Ig-beta are distinct complexes, yet class II-associated Ig-alpha/beta appears to be derived from BCR. Hence, Ig-alpha/beta are used in a sequential fashion for transduction of antigen and cognate T cell help signals.