Maintenance of B-cell memory by long-lived cells generated from proliferating precursors

A basic feature of T-cell dependent antibody responses is the generation of memory: on a second contact with an antigen a secondary response is produced in which somatically mutated antibodies with increased affinity are synthesized. Memory can persist for long periods of time. This has classically been ascribed to the generation of long-lived memory B cells. However, it is also possible that persisting antigen, on which memory may depend, maintains a population of cycling memory cells under continuous selection or continuously recruits newly generated B cells into the memory B-cell compartment. To discriminate between these mechanisms we have now directly analysed the proliferative activity in the memory B-cell compartment of the mouse by measuring bromodeoxyuridine incorporation in vivo. We show that after an initial phase of extensive proliferation after primary immunization, memory cells can persist in the organism for extended periods of time in the absence of cell division.     

内耳免疫反应诱导Bcl-2和Bax表达研究

探讨内耳免疫反应过程是否引起细胞凋亡以及Bcl-2和Bax的表达情况。选用雌性白色豚鼠16只，随机分为实验组和对照组各8只，以钥孔虫戚血蓝蛋白（KLH）全身免疫后，实验组以相同抗原进行内耳免疫，对照组内耳注射等量的磷酸盐缓冲生理盐水（PBS），在内耳免疫5d后处死动物，取内耳免疫侧耳蜗制作石蜡切片。通过免疫组化检测内耳Bcl-2和Bax的表达。结果表明，内耳免疫反应可以诱导细胞凋亡的发生以及Bcl-2和Bax蛋白在其中起了重要的调节作用。     

SAP is required for generating long-term humoral immunity

Long-lived plasma cells and memory B cells are the primary cellular components of long-term humoral immunity and as such are vitally important for the protection afforded by most vaccines. The SAP gene has been identified as the genetic locus responsible for X-linked lymphoproliferative disease, a fatal immunodeficiency. Mutations in SAP have also been identified in some cases of severe common variable immunodeficiency disease. The underlying cellular basis of this genetic disorder remains unclear. We have used a SAP knockout mouse model system to explore the role of SAP in immune responses. Here we report that mice lacking expression of SAP generate strong acute IgG antibody responses after viral infection, but show a near complete absence of virus-specific long-lived plasma cells and memory B cells, despite the presence of virus-specific memory CD4+ T cells. Adoptive transfer experiments show that SAP-deficient B cells are normal and the defect is in CD4+ T cells. Thus, SAP has a crucial role in CD4+ T-cell function: it is essential for late B-cell help and the development of long-term humoral immunity but is not required for early B-cell help and class switching.     

Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death

The t(14; 18) chromosomal translocation of human follicular B-cell lymphoma juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus. The bcl-2 immunoglobulin fusion gene is markedly deregulated resulting in inappropriately elevated levels of bcl-2 RNA and protein. Transgenic mice bearing a bcl-2 immunoglobulin minigene demonstrate a polyclonal expansion of resting yet responsive IgM-IgD B cells which display prolonged cell survival but no increase in cell cycling. Moreover, deregulated bcl-2 extends the survival of certain haematopoietic cell lines following growth-factor deprivation. By using immunolocalization studies we now demonstrate that Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular mass 25,000 (25k). Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line. Thus, Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division.     

Memory B-cell persistence is independent of persisting immunizing antigen

Immunological memory in the antibody system is generated in T-cell-dependent responses and carried by long-lived memory B cells that recognize antigen by high-affinity antibodies. But it remains controversial whether these B cells represent true 'memory' cells (that is, their maintenance is independent of the immunizing antigen), or whether they are a product of a chronic immune response driven by the immunizing antigen, which can be retained in the organism for extended time periods on the surface of specialized antigen-presenting cells (follicular dendritic cells). Cell transfer experiments provided evidence in favour of a role of the immunizing antigen; however, analysis of memory cells in intact animals, which showed that these cells are mostly resting and can persist in the absence of detectable T-cell help or follicular dendritic cells, argued against it. Here we show, by using a genetic switch mediated by Cre recombinase, that memory B cells switching their antibody specificity away from the immunizing antigen are indeed maintained in the animal over long periods of time, similar to cells retaining their original antigen-binding specificity.     

Progression from lymphoid hyperplasia to high-grade malignant lymphoma in mice transgenic for the t(14; 18)

Follicular lymphoma, the most common human lymphoma, characteristically has a t(14; 18) interchromosomal translocation. It is typically an indolent disease comprised of small resting B cells, but frequently develops into a high-grade lymphoma. The t(14; 18) translocates the Bcl-2 gene, generating a deregulated Bcl-2-immunoglobulin fusion gene. Bcl-2 is a novel inner mitochondrial membrane protein that extends the survival of certain cells by blocking programmed cell death. To determine the oncogenic potential of the t(14; 18) translocation, we produced transgenic mice bearing a Bcl-2-immunoglobulin minigene that structurally mimicked the t(14; 18). An indolent follicular hyperplasia in these transgenic mice progressed to a malignant diffuse large-cell lymphoma. The long latency, progression from polyclonal to monoclonal disease, and histological conversion, are all suggestive of secondary changes. Half of the immunoblastic high-grade lymphomas had a rearranged c-myc gene. Our transgenic mice provide an animal model for tumour progression in t(14; 18) lymphoma and show that prolonged B-cell life increases tumour incidence.     

CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death

The 'help' provided by CD4+ T lymphocytes during the priming of CD8+ T lymphocytes confers a key feature of immune memory: the capacity for autonomous secondary expansion following re-encounter with antigen. Once primed in the presence of CD4+ T cells, 'helped' CD8+ T cells acquire the ability to undergo a second round of clonal expansion upon restimulation in the absence of T-cell help. 'Helpless' CD8+ T cells that are primed in the absence of CD4+ T cells, in contrast, can mediate effector functions such as cytotoxicity and cytokine secretion upon restimulation, but do not undergo a second round of clonal expansion. These disparate responses have features of being 'programmed', that is, guided by signals that are transmitted to naive CD8+ T cells during priming, which encode specific fates for their clonal progeny. Here we explore the instructional programme that governs the secondary response of CD8+ T cells and find that helpless cells undergo death by activation-induced cell death upon secondary stimulation. This death is mediated by tumour-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Regulation of Trail expression can therefore account for the role of CD4+ T cells in the generation of CD8+ T cell memory and represents a novel mechanism for controlling adaptive immune responses.     

Modelling T-cell memory by genetic marking of memory T cells in vivo.

Immunological memory is the ability of the immune system to respond with enhanced vigour to pathogens that have been encountered in the past. Following infection or immunization, most effector T cells undergo apoptotic cell death, but a small fraction of these cells, proportional to the early antigen load and initial clonal burst size, persist in the host as a stable pool of memory T cells. The existence of immunological memory has been recognized for over 2,000 years, but our understanding of this phenomenon is limited, primarily because memory lymphocytes cannot be unequivocally identified as they lack specific, permanent markers. Here we have developed a transgenic mouse model system whereby memory T cells and their precursors can be irreversibly marked with a reporter gene and thus can be unambiguously identified. Adoptive transfer of marked CD8+ T cells specific for lymphocytic choriomeningitis virus protected naive recipients following viral challenge, demonstrating that we have marked memory T cells. We also show that cytotoxic effector lymphocytes that develop into memory T cells can be identified in the primary response.     

Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity

Protective T-cell memory has long been thought to reside in blood and lymph nodes, but recently the concept of immune memory in peripheral tissues mediated by resident memory T (T(RM)) cells has been proposed. Here we show in mice that localized vaccinia virus (VACV) skin infection generates long-lived non-recirculating CD8(+) skin T(RM) cells that reside within the entire skin. These skin T(RM) cells are potent effector cells, and are superior to circulating central memory T (T(CM)) cells at providing rapid long-term protection against cutaneous re-infection. We find that CD8(+) T cells are rapidly recruited to skin after acute VACV infection. CD8(+) T-cell recruitment to skin is independent of CD4(+) T cells and interferon-γ, but requires the expression of E- and P-selectin ligands by CD8(+) T cells. Using parabiotic mice, we further show that circulating CD8(+) T(CM) and CD8(+) skin T(RM) cells are both generated after skin infection; however, CD8(+) T(CM) cells recirculate between blood and lymph nodes whereas T(RM) cells remain in the skin. Cutaneous CD8(+) T(RM) cells produce effector cytokines and persist for at least 6 months after infection. Mice with CD8(+) skin T(RM) cells rapidly cleared a subsequent re-infection with VACV whereas mice with circulating T(CM) but no skin T(RM) cells showed greatly impaired viral clearance, indicating that T(RM) cells provide superior protection. Finally, we show that T(RM) cells generated as a result of localized VACV skin infection reside not only in the site of infection, but also populate the entire skin surface and remain present for many months. Repeated re-infections lead to progressive accumulation of highly protective T(RM) cells in non-involved skin. These findings have important implications for our understanding of protective immune memory at epithelial interfaces with the environment, and suggest novel strategies for vaccines that protect against tissue tropic organisms.     

Treatment of PC-3 cells with ultrasound combined with microbubbles induces distinct alterations in the expression of Bcl-2 and Bax

The objective of the present study was to investigate whether ultrasound combined with microbubbles induces apoptotic cell death in androgen-independent prostate cancer cells and to identify the probable mechanism. We used ultrasound in continuous wave mode with a frequency of 21 kHz and a spatial-average temporal-average intensity of 46 mW/cm². Ultrasound combined with microbubbles (200 μL SonoVue?) was used to treat androgen-independent human prostate cancer PC-3 cells for 30 s. PC-3 cells were divided into three groups: the control group, the ultrasound group and the ultrasound combined with microbubbles group. Immediately after treatment, trypan blue exclusion was used to assess cell viability. Cell apoptosis at 24 h after treatment was measured using transmission electron microscopy and flow cytometry. Western blotting was used to evaluate the expression of the apoptosis-related proteins, Bcl-2 and Bax. Ultrasound combined with microbubbles had a minimal effect on the viability of PC-3 cells and induced minimal levels of cell lysis. The level of apoptosis in PC-3 cells induced by this modality was significantly higher than in controls (12.77 ± 0.31% vs. 2.56 ± 0.22%, P<0.01). Treatment with ultrasound combined with microbubbles increased the expression of Bax, a pro-apoptotic protein, and decreased the expression of Bcl-2, an anti-apoptotic protein. It was concluded that ultrasound combined with microbubbles induces apoptotic cell death in human prostate cancer PC-3 cells through down-regulation of Bcl-2 and up-regulation of Bax.     

Apoptotic cell death induced by c-myc is inhibited by bcl-2.

Apoptosis is a form of physiological cell death, characterized by chromatin condensation, cytoplasmic blebbing and DNA fragmentation, which often depends on RNA and protein synthesis by the dying cell. The c-myc proto-oncogene, usually implicated in cell transformation, differentiation and cell-cycle progression also has a central role in some forms of apoptosis. These opposing roles of myc in cell growth and death require that other gene products dictate the outcome of c-Myc expression on a cell. A candidate for such a modifying gene is bcl-2, whose product prolongs cell survival and blocks apoptosis in some systems. Here we demonstrate that Bcl-2 prevents apoptotic death induced by c-Myc, provide a mechanism whereby cells can express c-Myc without undergoing apoptosis, and give a possible explanation for the ability of Bcl-2 to synergize with c-Myc in cell transformation.     

Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death

Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. The mitochondrial permeability transition (mPT) leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mPT pore is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. Here we generated mice lacking Ppif and mice overexpressing cyclophilin D in the heart. Ppif null mice are protected from ischaemia/reperfusion-induced cell death in vivo, whereas cyclophilin D-overexpressing mice show mitochondrial swelling and spontaneous cell death. Mitochondria isolated from the livers, hearts and brains of Ppif null mice are resistant to mitochondrial swelling and permeability transition in vitro. Moreover, primary hepatocytes and fibroblasts isolated from Ppif null mice are largely protected from Ca2+-overload and oxidative stress-induced cell death. However, Bcl-2 family member-induced cell death does not depend on cyclophilin D, and Ppif null fibroblasts are not protected from staurosporine or tumour-necrosis factor-alpha-induced death. Thus, cyclophilin D and the mitochondrial permeability transition are required for mediating Ca2+- and oxidative damage-induced cell death, but not Bcl-2 family member-regulated death.     

CD8<Superscript>+</Superscript> T cell response mediates the therapeutic effects of oncolytic adenovirus in an immunocompetent mouse model

The role of anti-tumor immune responses in oncolytic adenoviral therapy has not been well studied due to lack of efficacious tu- mor model in immunocompetent mice.Here,we evaluated the contributions of immune components to the therapeutic effects of oncolytic adenoviruse in an immunocompetent murine tumor model permissive for infection and replication of adenovirus.We found that CD8+T cells were critical mediator for antitumor efficacy by oncolytic adenovirus.Intratumoral viral therapy induced intensive infiltration of CD8+T cells in tumor,increased tumor-specific IFN-?(interferon-?)production and CTL(cytotoxic T lymphocyte)activity of lymphocytes,and generated a long-term tumor-specific immune memory.Boosting CD8+T cell responses by agonistic anti-4-1BB(cluster differentiation 137,CD137)antibody showed synergistic anticancer effects with oncolytic viro- therapy.Our results provide insight into antitumor mechanisms of oncolytic adenovirus in addition to their direct oncolytic effect.     

An inhibitor of Bcl-2 family proteins induces regression of solid tumours

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.     

Regulation of B-cell survival by BAFF-dependent PKCdelta-mediated nuclear signalling

Approximately 65% of B cells generated in human bone marrow are potentially harmful autoreactive B cells. Most of these cells are clonally deleted in the bone marrow, while those autoreactive B cells that escape to the periphery are anergized or perish before becoming mature B cells. Escape of self-reactive B cells from tolerance permits production of pathogenic auto-antibodies; recent studies suggest that extended B lymphocyte survival is a cause of autoimmune disease in mice and humans. Here we report a mechanism for the regulation of peripheral B-cell survival by serine/threonine protein kinase Cdelta (PKCdelta): spontaneous death of resting B cells is regulated by nuclear localization of PKCdelta that contributes to phosphorylation of histone H2B at serine 14 (S14-H2B). We show that treatment of B cells with the potent B-cell survival factor BAFF ('B-cell-activating factor belonging to the TNF family') prevents nuclear accumulation of PKCdelta. Our data suggest the existence of a previously unknown BAFF-induced and PKCdelta-mediated nuclear signalling pathway which regulates B-cell survival.     

Germinal centre B cells: antigen specificity and changes in heavy chain class expression

Germinal centres are histologically defined aggregates of blast cells that occur in B-cell areas of lymphoid tissues after antigenic stimulation. They are believed to be associated with the development of B-cell memory and plasma cell (especially secondary, IgG and IgA) responses. Recent studies of murine lymphoid tissues have defined cell-surface markers that distinguish germinal centre B cells from other mature B cells, permitting their identification and characterization in cell suspensions. Here we have used these markers to define and study germinal centre cells in lympho nodes, and have found that they constitute a unique population of B cells which (1) arises in response to antigenic stimulation, (2) contains nearly all of the demonstrably antigen-specific B cells in the stimulated organ, (3) bears surface IgM after primary stimulation and (4) as a population, demonstrates isotype switching to a predominant population, demonstrates isotype switching to a predominant surface IgG phenotype after secondary stimulation with specific surface IgG phenotype after secondary stimulation with specific antigen. These findings demonstrate that germinal centres are a major site of proliferation and differentiation of antigen-specific B cells in vivo, and suggest that the germinal centre microenvironment may have an important role in heavy chain class switching during B-cell responses.     

Molecular characterization of single memory B cells

Primary antigenic exposure results in an initial antibody response and the T cell-dependent induction of B-cell memory. Memory B-cell differentiation is characterized by somatic hypermutation in antibody variable region genes (V) and selection of B cells expressing high-affinity variants of this antigen receptor. Despite our current understanding of B-cell memory, the origin of memory B cells and the regulation of their differentiation remain elusive. This is largely due to the difficulties in observing and purifying this minor component of the immunized spleen. Further, molecular characterization of memory B cells requires hybridoma formation which restricts analyses to only those clones capable of fusion and does not allow isolation of cells in a normal physiological state. We have therefore developed a unique system which allows isolation and unambiguous enumeration of IgG1+ memory B cells, based on six-parameter flow cytometry, secretion of antibody in clonal cultures and analysis of clonally expressed V genes using the polymerase chain reaction. Here we report that single IgG1+ antigen-binding B cells from an early secondary immune response proliferate in lipopolysaccharide-driven microcultures and produce antigen-specific IgG1 antibodies. Individual B-cell clones in these cultures express somatically mutated heavy chain V genes, confirming their designation as memory B cells. Although isolated memory B cells undergo extensive proliferation in vitro, V gene sequence analysis of their individual progeny shows that further hypermutation does not occur.     

Can B cells turn on virgin T cells?

The first event in the initiation of an immune response is the capture and presentation of antigen to T cells. Such presentation involves two distinct steps: (1) display of the antigen, which requires uptake, processing and re-expression of the antigen in association with MHC molecules on the presenting cell surface; and (2) triggering, in which the presenting cell provides signals leading to the activation of the responding T cell. Two sorts of cells can capture antigens, the 'professional' antigen-presenting cells (APCs) such as dendritic cells and macrophages, and the B cells. Both types of cells can display antigens and the APCs are known to be able to trigger resting T cells. But despite in vitro evidence that certain B-cell types can reactivate previously-activated T cells, it is not yet clear whether a B cell can initiate an immune response by providing the signals necessary to activate a resting T cell. We reasoned that resting B cells should not have this capacity because of the problems this would present with tolerance to self idiotypes. By exploiting the unique properties of the avian haematopoietic system, we have examined the presenting capacity of B cells in vivo and found that resting B cells are indeed unable to activate resting T cells.     

Lymphokine-induced IgM secretion by clones of neoplastic B cells

The induction of antibody secretion by B cells requires T-cell-derived factors1-5. Such factors have been described1,2,6-12 but the precise relationship among these various factors is not clear, and it has been difficult to demonstrate that these factors act directly on the B cell and do not exert their effect via T cells or macrophages. In this report we describe the direct induction of IgM synthesis and secretion in cloned lines of long-term tissue culture adapted neoplastic B cells (BCL1) by T-cell supernatants from phorbol-12-myristate 13-acetate (PMA)-induced EL-4 cells or concanavalin A (Con A)-induced 7.1.1a cells5,9. We have termed this activity BCDFmu (B-cell differentiation factor for IgM). The supernatants containing BCDFmu induce activated and neoplastic B cells to secrete IgM5 and the factor responsible is distinct from BCGF13, interleukin-2 (IL-2)5, the classical T-cell replacing factor (TRF) described by Schimpl and Wecker5, and immune interferon (IFN gamma)5.     

Oxysterols direct immune cell migration via EBI2

Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.