Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells

After immunization with T cell-dependent antigens, the high-affinity B cells selected in germinal centers differentiate into memory B cells or long-lived antibody-forming cells. However, a role for germinal centers in development of these B lineage cells is still controversial. We show here that Bcl6-deficient B cells, which cannot develop germinal centers, differentiated into IgM and IgG1 memory B cells in the spleen but barely differentiated into long-lived IgG1 antibody-forming cells in the bone marrow. Mutation in the V-heavy gene was null in these memory B cells. Therefore, Bcl6 and germinal center formation are essential for somatic hypermutation, and generation of memory B cells can occur independently of germinal center formation, somatic hypermutation, and Ig class switching.     

Bursal and postbursal stem cells in chicken. Functional characteristics

Cyclophosphamide (CY)-treated or surgically bursectomized, CY-treated 3-day old chicks were injected with bursa or bone marrow cells from donors of different ages. Cell recipients and donors were isogeneic at the major histocompatibility locus. Antibody responses to sheep red blood cells and Brucella abortus, and microscopic morphology of spleen and bursa were assessed 5–6 weeks after the cell transplantation. Relative weight of the bursa was found to be a reliable indicator of the restoration of bursal structure. The results indicate that stem cells or progenitor cells for the B cell line in chicken can be divided into a bursal stem cell and a postbursal stem cell. Both of these cell types are effective in restoring antibody formation of CY-treated chickens. Bursal stem cells restore the bursal morphology; they are not capable of further maturation without the influence of the bursal microenvironment. This influence is not effected by bursa in a cell impermeable diffusion chamber; actual contact with the bursal stroma is necessary. Bursal stem cells are also capable of restoring the formation of germinal centers in the spleen of CY-treated chickens. Postbursal stem cells do not restore bursal structure and they do not need the bursal micro-environment for further maturation. They also have no clear effect on the formation of germinal centers in the spleen. Bursal stem cells are found in the bursa during the first few weeks after hatching. Postbursal stem cells start to appear in the involuting bursa and at the same time, in the bone marrow also. They are found as the majority of cells in the bursa and bone marrow after at least the 10th week following hatching. Early postbursal stem cells have already passed the education given by the bursa, but have not yet totally lost their capacity to induce germinal centers in the spleen. They restore germinal center formation even in surgically bursectomized recipients, demonstrating that presence of bursal follicles is not necessary for the production of germinal centers. The findings are discussed to stress the significance of two equally important factors in the development of immunity: the lymphoid cells themselves and, at each stage ontogeny, the proper microenvironment for their further function and maturation.     

B cell memory and the role of apoptosis in its formation

B cell memory consists of quiescent memory B cell and bone marrow plasma cell populations, generated in germinal centers during immune responses to T cell dependent antigens. The regulation of cell survival, both within germinal centers and in the maintenance of the effector cells generated in this response, is central to the qualitative and quantitative regulation of memory. In spite of this, the pro- and anti-apoptotic molecules that control survival in these peri-antigenic B cell immune compartments are poorly defined. In this review, we discuss the current perception of the main apoptotic regulators of germinal center B cell, plasma cell, and memory B cell survival during the formation, affinity maturation and maintenance of immunological memory.     

CXCR5-deficient mice develop functional germinal centers in the splenic T cell zone

The chemokine receptor CXCR5 is thought to be essential for the migration of B cells into the network of follicular dendritic cells in the spleen. However, as shown here, B cells and follicular dendritic cells do co-localize, albeit aberrantly, even in the absence of CXCR5. In mice lacking CXCR5 both cell types are found in a broad ring around the sinuses of the marginal zones. Upon immunization with the T cell-dependent antigen 2-phenyl-oxazolone, ectopic germinal centers develop in the periarteriolar lymphocyte sheath. A network of follicular dendritic cells forms in the vicinity of the central arteriole within which the antigen-activated B cells proliferate. The analysis of the expressed V gene repertoire revealed that during B cell proliferation, hypermutation is activated and V region genes accumulate somatic mutations. The pattern of somatic mutations suggests that affinity selection may occur. This analysis confirms that in CXCR5-deficient mice, the organization of splenic primary follicles is severely impaired. However, within the T cell zone a micro-environment is built up, which provides all requirements needed for the affinity maturation to take place.     

Dissecting CXCR5+ T cell populations--on the quest for a better understanding of B cell help during T dependent antibody responses

High affinity antibody responses against protein antigens critically depend on T cell help during the germinal center reaction. So called follicular helper T cells (T(FH)), present in the germinal centers of human tonsils, have been characterised by the expression of CXCR5; however, only subgroups of the heterogeneous CXCR5 T cell population strongly support antibody production. A paper in this issue of the European Journal of Immunology demonstrates that high expression of the inducible co-stimulator (ICOS) molecule, rather than CD57, correlates with the follicular helper function. This result represents another step towards a better understanding of the complexity of the different T cell subpopulations participating in the germinal center reaction.     

Tetraspanins in the immune response against cancer

The SLAM (CD150) family receptors are leukocyte cell-surface glycoproteins involved in leukocyte activation. These molecules and their adaptor protein SAP contribute to the effective germinal center formation, generation of high-affinity antibody-secreting plasma cells, and memory B cells, thereby facilitating long-term humoral immune response. Multi-color flow cytometric analysis was performed to determine the expression of CD48 (SLAMF2), CD84 (SLAMF5), CD150 (SLAM or SLAMF1), CD229 (Ly9 or SLAMF3), CD244 (2B4 or SLAMF4), CD319 (CRACC, CS1, or SLAMF7), and CD352 (NTB-A or SLAMF6) on human cell lines and B-cell subsets. The following subsets were assessed: pro-B, pre-B, immature-B, and mature-B cells from bone marrow; transitional and B1/B2 subsets from peripheral blood; and na?ve, pre-germinal center, germinal center, memory, plasmablasts, and plasma cells from tonsil and spleen. All receptors were expressed on B cells, with the exception of CD244. SLAM family molecules were widely distributed during B-cell development, maturation and terminal differentiation into plasmablasts and plasma cells, but their expression among various B-cell subsets differed significantly. Such heterogeneous expression patterns suggest that SLAM molecules play an essential and non-redundant role in the control of humoral immune responses.     

Human B‐cell memory is shaped by age‐ and tissue‐specific T‐independent and GC‐dependent events

Switched and IgM memory B cells execute different and noninterchangeable functions. We studied memory B cells in children of different ages, in peripheral blood and spleen and compared them with those of children born asplenic or unable to build germinal centers. We show that, whereas switched memory B cells are mostly generated in the germinal centers at all ages, IgM memory B cells can be distinct in three types with different developmental history. Innate IgM memory B cells, the largest pool in infants, are generated in the spleen by a germinal center‐independent mechanism. With age, if the spleen is present and germinal centers are functional, innate IgM memory B cells are remodelled and accumulate somatic mutations. The third type of IgM memory B cell is a by‐product of the germinal center reaction. Our data suggest that the B‐cell memory developmental program is implemented during the first 5–6 years of life.     

Bursal and postbursal cells in chicken. Occurrence of postbursal cells in bone marrow, thymus and spleen

To study the postbursal nature of lymphoid cells found in the bone marrow, thymus and spleen, their capacity for a long-term restoration of bursa-dependent immune functions and of bursal and splenic morphology was evaluated using cyclophosphamide-treated chicks as cell recipients. The results reveal that postbursal cells appear in significant numbers, first in the spleen and then in the bone marrow and thymus. This observation is interpreted to indicate that migration of postbursal cells from the bursa to bone marrow and thymus most probably occurs through the spleen. The findings also indicate that in the development of the B cell line an early postbursal cell and a mature postbursal cell can be distinguished. Both of these cell types are capable of further function without the influence of a bursal microenvironment. The mature postbursal cell does not have a capacity for formation of germinal centers in significant numbers in the spleen, but the early postbursal cell still shares this potential with its precursor, the bursal stem cell. They are also distinguishable by a slight proliferation of the early postbursal cells in the bursa when transferred into cyclophosphamide-treated newly-hatched recipients. Both of these cell types represent distinct entities in the stepwise development of the B cell line.     

Multiple routes to B-cell memory

B-cell memory describes the populations of cells that provide long-term humoral immunity: long-lived antibody-secreting plasma cells that reside mainly in the bone marrow and memory B cells. Interestingly, the memory B-cell population is heterogenous, although the importance of this heterogeneity has been unclear. Recent studies have investigated the formation and function of memory in different settings. In particular, T-independent memory-like cells and T-dependent (TD) IgM memory B cells qualitatively differ from canonical TD class-switched memory B cells; however, these studies suggest that IgM memory cells preserve the memory population over long periods of time. These subsets are evocative of the evolution of the humoral immune response, with memory-like cells appearing before acquisition of germinal centers, suggesting that there are multiple pathways to producing B-cell memory.     

CXCR5 is critically involved in progression of lupus through regulation of B cell and double‐negative T cell trafficking

The recruitment of immune cells to sites of tissue inflammation is orchestrated by chemokine/chemokine receptor networks. Among these, the CXCL13/CXCR5 axis is thought to be involved critically in systemic lupus erythematosus (SLE) and lupus nephritis pathogenesis. Beyond B cell abnormalities, another hallmark of SLE disease is the occurrence of aberrant T cell responses. In particular, double‐negative (DN) T cells are expanded in the peripheral blood of patients with SLE and in lupus‐prone mice. DN T cells induce immunoglobulin production, secrete proinflammatory cytokines and infiltrate inflamed tissue, including kidneys. We aimed to investigate how CXCR5 deficiency changes immune cell trafficking in murine lupus. We therefore crossed CXCR5^–/– mice with B6/lpr mice, a well‐established murine lupus model. B cell numbers and B cellular immune responses were diminished in CXCR5‐deficient B6/lpr mice. In addition, we observed reduced accumulation of DN T cells in spleen and lymph nodes, paralleled by reduced splenomegaly and lymphadenopathy. In‐vivo migration assays revealed reduced migration of CXCR5‐deficient DN T cells into lymph nodes, and ex‐vivo‐activated CXCR5‐deficient DN T cells failed to infiltrate kidneys of recipients. Moreover, DN T cells and B cells of CXCR5‐deficient B6/lpr mice failed to migrate towards CXCL13 in vitro. We propose that CXCR5 is involved critically in B cell trafficking and germinal cell (GC) formation in murine lupus and in guiding pathogenic DN T cells into lymphoid organs and kidneys, and we therefore describe new pathomechanisms for the CXCL13/CXCR5 axis in SLE.     

Inflamed kidneys of NZB / W mice are a major site for the homeostasis of plasma cells

(NZB x NZW)F1 (NZB / W) mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hypergammaglobulinaemia and inflammation of the kidneys. It is known that large numbers of lymphocytes infiltrate the kidneys of these mice. Here, we compare the roles of bone marrow, spleen and inflamed kidneys of NZB / W mice in the activation of B cells and the persistence of antibody-secreting cells (ASC). ASC are present in the kidneys of NZB / W mice with full-blown disease, as many as in the spleen and bone marrow. The specificity of the ASC in the inflamed kidneys is not restricted to self-antigens. After immunization of NZB / W mice with ovalbumin (OVA) the OVA-specific ASC are found initially in the spleen. Weeks later, OVA-specific ASC are found in high numbers in the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal center phenotype (B220(+) / PNA(+)) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB / W mice. Germinal centers could not be detected in the kidneys, but in the spleen, and plasma cells appear to be scattered over the tissue. These data suggest that in autoimmune NZB / W mice, plasma cells generated in immune reactions of secondary lymphoid organs, later accumulate and persist in the inflamed kidneys, were they enhance the local concentrations of Ab and immunocomplexes. These experiments identify the inflamed kidneys of NZB / W mice as a site of prime relevance for the homeostasis of plasma cells, irrespective of their specificity.     

Germinal centers and the B cell system. VIII. Functional characteristics and cell surface markers of germinal center cell subsets differing in density and in sedimentation velocity

Germinal center cells from the rabbit appendix were fractionated by velocity sedimentation and isopycnic gradient centrifugation. Subsets were analysed with respect to cell size and surface markers, and were functionally characterized by testing the capacities for primary antibody synthesis, memory cell production, and formation of new germinal centers in an autologous transfer system. The migratory behaviour of the germinal center cell subsets within the spleen of homologous recipients was also studied using autoradiography. Both cell fractionation methods yielded a separation of large and small cells. Surface immunoglobulin and C3 receptors were equally expressed on germinal center cells differing in size and density. The different subsets were also equally capable in giving rise to IgM-antibody-forming cells and memory cells upon antigenic stimulation. Furthermore, large germinal centers were newly formed in the spleen of the recipients, irrespective of the cell subset injected. It was concluded that the results do not support the hypothesis that, inside germinal centers, the differentiation of large lymphoid cells (centro-blasts) into small centrocytes also implies a maturation process. Subsets of germinal center cells, however, showed a different and characteristic migratory behaviour; while small cells migrated preferentially to the corona of lymphocytes in spleen follicles, large, light cells showed an affinity for the germinal center area. We postulate that, upon stimulation, immature B cells develop an affinity for the germinal center microenvironment, to participate in a germinal center reaction.     

CD4-deficient T helper cells are capable of supporting somatic hypermutation and affinity maturation of germinal center B cells

Collaborative interactions between B lymphocytes and CD4+ helper T cells are necessary for the induction of Ab responses to most protein Ag and for the generation of memory B cells in germinal centers. To study the role of the CD4 molecule in the germinal center response and in the development of B cell memory, we have investigated T helper function in the initiation and maturation of humoral immunity in CD4-deficient mice. In the absence of CD4+ T cells, immunization with thymus-dependent Ag was able to induce germinal center formation and Ig somatic hypermutation. In addition, Ag-driven affinity maturation and development of B cell memory were largely intact in CD4-deficient mice. Thus, CD4-deficient T helper cells are able to collaborate with Ag-activated B cells to elicit the germinal center reaction, switch on the mutational machinery, and deliver signals necessary for B cell memory development.     

B7h-expressing dendritic cells and plasma B cells mediate distinct outcomes of ICOS costimulation in T cell-dependent antibody responses

ABSTRACT: BACKGROUND: The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. RESULTS: We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naive and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a corresponding increase in the concentration of antigen-specific high affinity serum IgG antibodies of all isotypes, without affecting the number of responding germinal center B cells. In contrast, ICOS costimulation mediated by dendritic cells in DC-B7hTg mice contributed to germinal center formation and selectively increased IgG2a production without affecting the overall magnitude of antibody responses. CONCLUSIONS: Using transgenic mice with lineage-restricted B7h expression, we have revealed distinct roles of ICOS costimulation mediated by dendritic cells and B cells in the regulation of T celldependent antibody responses.     

Long-lived plasma cells in immunity and immunopathology

Following tetanus vaccination, a wave of antibody-secreting cells appear in the peripheral blood composed of vaccine-specific, migratory plasmablasts and plasma cells secreting antibodies specific for other antigens. The latter probably were tissue resident plasma cells formed in earlier immune responses that are mobilized due to competition with the newly formed tetanus-specific plasmablasts. Newly formed plasma cells secreting antibodies specific for a particular antigen/vaccine are accommodated in the bone marrow likely at the global expense of the pre-existing long-lived plasma cell population providing humoral memory for other antigens. Plasmablasts but not mature plasma cells are attracted by the ligands for the chemokine receptors CXCR4 and CXCR3. While CXCR4 and its cognate ligand is important for plasma cell homing to the bone marrow, CXCR3 and its ligand IP10 are likely to be involved in attracting them to inflamed tissue. In NZB/W mice, a model for systemic lupus, long-lived autoreactive plasma cells are present not only in bone marrow, but also in inflamed tissues and spleen. Autoreactive plasma cells in the spleen are present long before the onset of the disease, suggesting that these cells contribute to induction of immunopathology.     

Sialyl LewisX carbohydrate is expressed differentially during avian lymphoid cell development

Sialyl LewisX is a carbohydrate moiety involved in the regulation of white blood cell adhesion to endothelial cells. In this work, we have studied the expression, localization and function of sialyl LewisX carbohydrate on maturing B and T cells and the stroma of avian bursa, thymus and spleen as well as the role of sialyl LewisX in the generation of immune response and formation of germinal centers in the spleen. The expression of sialyl LewisX on bursal B cells decreases during the embryonic period, while on T cells it remains at a stable low level throughout their thymic development. Immediately after hatching, by which time the expression of sialyl LewisX on bursal B cells has already decreased, the stromal microenvironment of the bursa starts to express it. After hatching, sialyl LewisX is localized exclusively in the medullae of the follicles and the number of sialyl LewisX-positive follicles declines gradually during the following weeks. Thymic stroma does not express sialyl LewisX at any stage of development. In spleen, sialyl LewisX is expressed on a number of B and T cells as well as on other cell types in a way that seems not to be restricted to any structurally defined area. The cells or stroma of the germinal centers, however, do not express sialyl LewisX. When given simultaneously with an immunizing agent, anti-sialyl LewisX monoclonal antibody reduced the formation of germinal centers and suppressed antibody response in young birds. These results demonstrate that sialyl LewisX-bearing cell surface molecules are required during the generation of immune responses. They also suggest a role for sialyl LewisX in B cell maturation.