B cell receptor signal transduction in the GC is short-circuited by high phosphatase activity

Germinal centers (GCs) generate memory B and plasma cells, which are essential for long-lived humoral immunity. GC B cells with high-affinity B cell receptors (BCRs) are selectively expanded. To enable this selection, BCRs of such cells are thought to signal differently from those with lower affinity. We show that, surprisingly, most proliferating GC B cells did not demonstrate active BCR signaling. Rather, spontaneous and induced signaling was limited by increased phosphatase activity. Accordingly, both SH2 domain-containing phosphatase-1 (SHP-1) and SH2 domain-containing inositol 5 phosphatase were hyperphosphorylated in GC cells and remained colocalized with BCRs after ligation. Furthermore, SHP-1 was required for GC maintenance. Intriguingly, GC B cells in the cell-cycle G(2) period regained responsiveness to BCR stimulation. These data have implications for how higher-affinity B cells are selected in the GC.     

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.     

Requirement for RORgamma in thymocyte survival and lymphoid organ development

Most developing thymocytes undergo apoptosis because they cannot interact productively with molecules encoded by the major histocompatibility complex. Here, we show that mice lacking the orphan nuclear hormone receptor RORgamma lose thymic expression of the anti-apoptotic factor Bcl-xL. RORgamma thus regulates the survival of CD4+8+ thymocytes and may control the temporal window during which thymocytes can undergo positive selection. RORgamma was also required for development of lymph nodes and Peyer's patches, but not splenic follicles. In its absence, there was loss of a population of CD3-CD4+CD45+ cells that normally express RORgamma and that are likely early progenitors of lymphoid organs. Hence, RORgamma has critical functions in T cell repertoire selection and lymphoid organogenesis.     

A macrophage-derived factor required by plasmacytomas for survival and proliferation in vitro

Plasmacytoma (PCT) cell lines dependent for proliferation and survival on a factor elaborated by the murine macrophage cell line, P388D1, were established in vitro. Adherent peritoneal cells induced by pristane produced 50-fold greater amounts of this activity in vitro than did resident cells. The molecules responsible for plasmacytoma growth were distinct from a number of characterized factors including interleukin-1, -2, and -3, macrophage colony-stimulating factor, B-cell stimulatory factor-1, B-cell growth factor II, epidermal growth factor, transforming growth factor-beta, and gamma- and beta-interferon, none of which were able to support the growth of the factor-dependent PCT cell lines. These results suggest that PCT growth factor may be a novel factor that has not been previously characterized and, further, that its production is associated with the pristane-induced, chronic peritoneal inflammatory response that precedes plasmacytoma formation.     

In situ imaging of the endogenous CD8 T cell response to infection

Mounting a protective immune response is critically dependent on the orchestrated movement of cells within lymphoid organs. We report here the visualization, using major histocompatability complex class I tetramers, of the CD8-positive (CD8) T cell response in the spleens of mice to Listeria monocytogenes infection. A multistage pathway was revealed that included initial activation at the borders of the B and T cell zones followed by cluster formation with antigenpresenting cells leading to CD8 T cell exit to the red pulp via bridging channels. Strikingly, many memory CD8 T cells localized to the B cell zones and, when challenged, underwent rapid migration to the T cell zones where proliferation occurred, followed by egress via bridging channels in parallel with the primary response. Thus, the ability to track endogenous immune responses has uncovered both distinct and overlapping mechanisms and anatomical locations driving primary and secondary immune responses.     

bcl-x基因在两株鼻咽癌细胞中的转录和表达

目的：研究细胞凋亡相关基因bcl-x在人鼻咽癌高分化上皮细胞株CNE-1和低分化上皮细胞株CNE-2Z中的转录和表达。方法：分别抽提两种细胞的总RNA，通过逆转录PCR(RT-PCR)检测bcl-x基因的转录；同时制备两种细胞的蛋白质样品。经免疫印迹检测bcl-x基因的表达。结果：在两株鼻咽癌细胞中均检测到bcl-xL的转录和表达。但没有bcl-xs的表达。结论：bcl-xL在人鼻咽癌高、低分化上皮细胞株中均有表达。但表达量没有明显差异提示其表达可能与细胞分化程度无关。     

Role of BAX in the apoptotic response to anticancer agents

To assess the role of BAX in drug-induced apoptosis in human colorectal cancer cells, we generated cells that lack functional BAX genes. Such cells were partially resistant to the apoptotic effects of the chemotherapeutic agent 5-fluorouracil, but apoptosis was not abolished. In contrast, the absence of BAX completely abolished the apoptotic response to the chemopreventive agent sulindac and other nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibited the expression of the antiapoptotic protein Bcl-XL, resulting in an altered ratio of BAX to Bcl-XL and subsequent mitochondria-mediated cell death. These results establish an unambiguous role for BAX in apoptotic processes in human epithelial cancers and may have implications for cancer chemoprevention strategies.     

CD8alphaalpha-mediated survival and differentiation of CD8 memory T cell precursors

Memory T cells are long-lived antigen-experienced T cells that are generally accepted to be direct descendants of proliferating primary effector cells. However, the factors that permit selective survival of these T cells are not well established. We show that homodimeric alpha chains of the CD8 molecule (CD8alphaalpha) are transiently induced on a selected subset of CD8alphabeta+ T cells upon antigenic stimulation. These CD8alphaalpha molecules promote the survival and differentiation of activated lymphocytes into memory CD8 T cells. Thus, memory precursors can be identified among primary effector cells and are selected for survival and differentiation by CD8alphaalpha.     

A role for CD40 expression on CD8+ T cells in the generation of CD8+ T cell memory

The delivery of CD4 help to CD8+ T cell responses requires interactions between CD40 and CD40 ligand and is thought to occur through antigen-presenting cell (APC) activation. Here we show that generation of memory CD8+ T cells displaying an enhanced capacity for cell division and cytokine secretion required CD4 help but not CD40 expression by the APCs. Activated CD4+ and CD8+ T cells expressed CD40; and in the absence of this protein, CD8+ T cells were unable to differentiate into memory cells or receive CD4 help. These results suggest that, like B cells, CD8+ T cells receive CD4 help directly through CD40 and that this interaction is fundamental for CD8+ T cell memory generation.     

Involvement of a leukocyte adhesion receptor (LFA-1) in HIV-induced syncytium formation

Cell fusion (syncytium formation) is a major cytopathic effect of infection by human immunodeficiency virus (HIV) and may also represent an important mechanism of CD4+ T-cell depletion in individuals infected with HIV. Syncytium formation requires the interaction of CD4 on the surface of uninfected cells with HIV envelope glycoprotein gp120 expressed on HIV-infected cells. However, several observations suggest that molecules other than CD4 play a role in HIV-induced cell fusion. The leukocyte adhesion receptor LFA-1 is involved in a broad range of leukocyte interactions mediated by diverse receptor-ligand systems including CD4-class II major histocompatibility complex (MHC) molecules. Possible mimicry of class II MHC molecules by gp120 in its interaction with CD4 prompted an examination of the role of LFA-1 in HIV-induced cell fusion. A monoclonal antibody against LFA-1 completely inhibited HIV-induced syncytium formation. The antibody did not block binding of gp120 to CD4. This demonstrates that a molecule other than CD4 is also involved in cell fusion mediated by HIV.     

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.     

Expression of BMP Receptors in Porcine Granulosa Cells （GCs） and Their Regulation by Luteinizing Hormone （LH）

Bone morphogenetic proteins (BMPs) play critical roles in follicle growth and development. BMPs initiate signaling by assembling BMP receptors and activating Smads, which in turn alter expression of target enes. The mechanism underlying the regulation of the expression of BMP receptors and Smads during follicle development in pigs is still unknown. By quantitative real-time PCR, the mRNA expression of BMP receptors and Smads in granulosa cells (GC) was investigated.Cells were obtained from small porcine follicles (SF; <3 mm diameter) and dominant follicles (DF; >6 mm diameter); ActRIA and BMPR2 mRNA levels in DF were significantly higher (P<0.05) than that in SF, whereas BMPRIB, Smad4 and Smad7 expression tended to decrease (P>0.05). The levels of BMPRIA, ActR2, Smadl, Smad5, and Smad8 mRNA did not differ between DFs and SFs. To investigate the effect of LH on BMP receptors in GC, cells obtained from porcine DFs were cultured in medium supplemented with different doses of luteinizing hormone (LH). High doses of LH (4 IU mL-1)significantly decreased the concentration of estradiol (E2) and progesterone (P4) in medium and the expression of Cyp19a1 (P450 aromatase, P450arom) and Cypl lal (cholesterol side-chain cleavage enzyme P450, P450scc), while significantly increased viable cell numbers and up-regulated expression of cyclin dependent kinase-4 (CDK4) and cyclin D2. However,LH had no effect on the expression of BMP receptor genes. Thus, the present study indicates that the expression of members of the BMP signaling pathway in porcine GC is regulated during follicle development and the expression of BMP receptors are not regulated by LH in porcine GCs.     

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.     

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.     

Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration

Phosphoinositide 3-kinases (PI3Ks) regulate fundamental cellular responses such as proliferation, apoptosis, cell motility, and adhesion. Viable gene-targeted mice lacking the p110 catalytic subunit of PI3Kgamma were generated. We show that PI3Kgamma controls thymocyte survival and activation of mature T cells but has no role in the development or function of B cells. PI3Kgamma-deficient neutrophils exhibited severe defects in migration and respiratory burst in response to heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR) agonists and chemotactic agents. PI3Kgamma links GPCR stimulation to the formation of phosphatidylinositol 3,4,5-triphosphate and the activation of protein kinase B, ribosomal protein S6 kinase, and extracellular signal-regulated kinases 1 and 2. Thus, PI3Kgamma regulates thymocyte development, T cell activation, neutrophil migration, and the oxidative burst.     

Regulation of NF-kappaB-dependent lymphocyte activation and development by paracaspase

Paracaspase (MALT1), a member of an evolutionarily conserved superfamily of caspase-like proteins, has been shown to bind and colocalize with the protein Bcl10 in vitro and, because of this association, has been suggested to be involved in the CARMA1-Bcl10 pathway of antigen-induced nuclear factor kappaB (NF-kappaB) activation. We demonstrate that primary T and B lymphocytes from paracaspase-deficient mice are defective in antigen-receptor-induced NF-kappaB activation, cytokine production, and proliferation. Paracaspase acts downstream of Bcl10 to induce NF-kappaB activation and is required for the normal development of B cells, indicating that paracaspase provides the missing link between Bcl10 and activation of the IkappaB kinase complex.     

7个shRNA分子对绵羊BMPR-1B基因的干扰作用分析

为获得有效干扰绵羊BMPR-1B基因的shRNA干扰分子,从绵羊卵巢组织中扩增了1 515 bp BMPR-1B基因全长编码区cDNA序列,添加HA标签序列后,插入Plex-mcs慢病毒质粒,构建Plex-BMPR-1B慢病毒表达载体,转染HEK293细胞,并与2个包装质粒共转染293T细胞进行病毒包装,用获得的重组慢病毒感染HEK293细胞;同时,将7个干扰分子与Pll-LentiLox 3.7载体重组,并与3个包装质粒共转染293T细胞进行病毒包装,获得干扰分子的重组病毒颗粒。最后用干扰分子重组的病毒颗粒感染整合Plex-BMPR-1B的HEK293细胞,进行qRT-PCR和Western blot检测。结果显示:重组BMPR-1B蛋白质在稳定整合Plex-BMPR-1B的HEK293细胞系中获得了表达;研究中设计的7个shRNA分子能抑制绵羊BMPR-1B基因表达水平68.30%～99.86%,其中PLL-BMPR-1B-1306和PLL-BMPR-1B-1475干扰分子的干扰效果最好。