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.     

The germinal center response is impaired in the absence of T cell-expressed CXCR5

Germinal centers support the differentiation of memory B cells and long-lived antibody-secreting cells during infection or upon vaccination. Here, we constructed mice with T cells that selectively lack the chemokine receptor CXCR5 to determine if expression of this receptor by T cells is mandatory for germinal center formation and function. In these animals, germinal centers that are properly localized in B cell follicles and contain T cells do form after immunization with a thymus-dependent antigen. However, fewer and smaller germinal centers form, resulting in a significant reduction in the frequency of germinal center B cells. The defect in germinal center formation is paralleled by decreased frequencies of isotype-switched antibody-secreting cells in the spleen and bone marrow and reduced serum concentrations of total and high-affinity hapten-specific IgG1. The results demonstrate that although CXCR5-dependent T cell positioning is important for maximal induction and expansion of germinal centers, stimulation of isotype class switching, and development of antibody-secreting cells that seed the spleen and bone marrow, it is not absolutely required for the formation and function of follicular germinal centers.     

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.     

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.     

Subpopulations of B lymphocytes in germinal centers, II. A germinal center B cell subpopulation expresses sIgD and CD23

To understand B cell development in germinal centers, it is important to delineate the expression of surface antigens among germinal center cells. Because it is unclear whether germinal center cells express common antigens such as sIgD and CD23, we studied their expression among tonsillar lymphocytes with flow cytometry, immunohistochemistry, and in vitro stimulation. Upon studying a large number of tonsils with flow cytometry, we found that occasional tonsils have a very large number of sIgD+ cells among their PNA+ cells. Furthermore, the occasional tonsils with a large number of sIgD+ and PNA+ cells also have many CD23+ cells among their PNA+ cells. Tonsil sections stained immunohistochemically revealed germinal centers containing sIgD+ cells. In addition, PNA- and sIgD+ cells can be induced to express PNA binding sites in vitro without losing the expression of sIgD. Taking these findings together, we conclude that a subpopulation of germinal center B cells coexpresses sIgD and CD23.     

Naive and memory B cells respond differentially to T-dependent signaling but display an equal potential for differentiation toward the centroblast-restricted CD77/globotriaosylceramide phenotype

Resting (CD38(low)) tonsillar B cells differentiate to express the centroblast-restricted CD77/globotriaosylceramide antigen on high-level engagement of CD154. As the CD38(low) population comprises both naive and memory subsets, we wished to compare the propensity of each to develop this germinal center phenotype; particularly as the capacity of memory B cells to re-enter afollicular reaction remains unclear. Resting B lymphocytes were therefore separated into CD27(-)IgA(-)IgG(-) and IgD(-) fractions to generate subsets enriched for naive and memory cells, respectively. Following stimulation via BCR and/or CD40 - surrogate signals for B cells engaged in T-dependent signaling - differences between the two subsets were seen in the kinetics and/or magnitude of responses such as entry into DNA synthesis, induction of the costimulatory molecules CD80 and CD86; up-regulation of CD23, and changes in BCL-6 mRNA expression. Nevertheless, naive and memory cells revealed a nigh identical capacity for acquiring CD77: both appeared equally sensitive in this regard, with high-level CD40 engagement via cell-bound CD154 being required for both subsets to achieve the hallmark centroblast phenotype. These findings suggest that, provided with the opportunity to encounter cell membrane CD154 in abundance, both naive and memory B cells display the potential to be diverted towards a germinal center pathway of differentiation.     

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.     

T-cell subsets in the germinal center

T cells are known to migrate to B-cell-enriched follicles and germinal centers within secondary lymphoid organs to provide help to B cells. Cognate T:B interactions that take place at the T:B border and subsequently within germinal centers are essential for B-cell priming, differentiation into germinal center B cells, and selection of mutated cells into memory B cells or memory plasma cells. In recent years, different stages of maturation within B-cell helper T cells, collectively known as B-follicular helper T (Tfh) cells, as well as heterogeneity amid germinal center T cells are becoming clear. Indeed, germinal centers support not only bona fide Tfh cells but also CD4⁺ and CD8⁺ follicular regulatory T (Tfr) cells that act to suppress germinal center responses and B-cell helper natural killer T cells. There is a growing need for more precise phenotypic and functional distinction of these specialized T-cell subsets. In this review, we summarize current knowledge on the ontogeny, molecular identity, and functional relevance of the various subsets of germinal center T cells.     

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.     

Polyclonal activation of the murine immune system by an antibody to IgD V. Effect on germinal centers

Heterologous anti-mouse delta chain has been shown to induce T-independent polyclonal B cell proliferation by 24 h after injection into mice and a T-dependent polyclonal increase in the number of IgG-secreting cells in spleen and lymph nodes 6 days after injection. The effect of anti-delta on germinal center cells has been difficult to determine, however, since anti-delta-induced blast transformation of mantle zone cells makes it difficult to distinguish these cells from germinal center cells. To clarify the effect of anti-delta on germinal centers, we stained lymph node sections from control and anti-delta-injected mice with peanut agglutinin conjugated to horseradish peroxidase which clearly differentiates the germinal center cells from other large and small lymphoid cells. For the first 5 days after i.v. injection of goat anti-mouse delta, the germinal center volumes did not differ significantly from those of mice injected with control antibodies. On days 6 and 7 after injection, the lymph node germinal center volumes of anti-delta-treated mice increased on average over 10-fold as compared to those of mice injected with an equal amount of control antibody. Thus, while anti-delta antibody directly induced blastogenesis of mantle layer B cells, it appears to have little direct effect on germinal center cells. In contrast, rapid germinal center development is seen in anti-delta-injected mice during the T-dependent phase of B cell activation and occurs simultaneously with the appearance of large numbers of IgG-secreting cells.     

Early ontogeny of germinal center formation in the chicken.

Germinal center formation was studied in the spleen of young chickens immunized in ovo and at the time of hatching. When immunization was performed on day 18 in ovo and on the day of hatching, the first germinal centers were observed at 4 days. This is markedly earlier than in unimmunized chickens, where the first germinal centers appear at the age of 10 days or later. Germinal center formation preceded significant antibody production. The possible role of germinal centers in the generation of immunological memory is discussed in the light of these and earlier observations.     

Immigration of thoracic duct B lymphocytes into established germinal centers in the rat

Immigration of B lymphocytes into established germinal centers in the rat was studied by transferring genetically marked thoracic duct B cells to non-irradiated congenic hosts at various times between 3 days before and 6 days after host immunization. Seven days after host immunization, the distribution of donor B cells to lymph node germinal centers (relative to their distribution to non-germinal center lymph node areas) was measured by two-color flow cytometry in which (a) donor and host B cells were distinguished by their Ig kappa chain allotypes, and (b) germinal center B cells were distinguished by their lack of labeling with the monoclonal antibody HIS22. Thoracic duct B cells from long-term antigen-primed rats were found to immigrate into host germinal centers much better than B cells from unprimed donors. This effect was antigen specific: primed B cells only immigrated well into host germinal centers induced by the priming antigen. Although B cells localized in germinal centers most efficiently when injected before immunization, specifically primed donor B cells injected after immunization were still found to be at least as evenly distributed to germinal centers as to other lymph node areas, whereas unprimed B cells transferred after immunization localized poorly in host germinal centers. These findings are discussed in light of recent suggestions that memory B cell clones are maintained by continued antigenic stimulation within secondary lymphoid follicles.     

Development of antibody diversity in single germinal centers: selective expansion of high-affinity variants

In a T cell-dependent immune response the microenvironment of the germinal center plays a crucial role in the affinity maturation of the antigen-specific immunoglobulins. In order to look at the development of antibody diversity we have isolated single germinal centers and sequenced light chains characteristic of 2-phenyl-oxazolone (phOx)-specific antibodies. Fourteen days after immunization we can demonstrate various stages of intraclonal diversity. There are germinal centers where B cells are practically unmutated, suggesting that in these cases a substantial clonal expansion has taken place prior to the activation of the hypermutation mechanism. In other germinal centers, sequences with a low number of randomly distributed somatic mutations were observed, indicating that these changes have been introduced recently and/or that they fail to generate high-affinity variants and hence provide no basis for affinity selection. Finally, germinal centers are found in which practically all sequences carry the amino acid substitutions characteristic of the high affinity phOx antibodies. In these latter cases the high-affinity variants have been preferentially expanded. We conclude that affinity selection is a process that operates right from the beginning of germinal center development. Those B cells with a relative high affinity for the antigen gain a proliferative advantage over other cells and will dominate the response and these are the cells which will be selected to differentiate into memory cells.     

The ontogeny of germinal centre forming capacity of neonatal rat spleen.

In non-specifically immunized rats, bred under conventional conditions, the first 'spontaneous' germinal centres were observed by 21 days after birth. Deliberate antigenic stimulation led to an earlier appearance of germinal centres in neonatal spleen: immunization with sheep red blood cells as early as 7 days after birth resulted in germinal centre formation in the spleen as observed 7 days later. By that time the first primary follicles could also be observed, in both immunized and non-immunized rats. Although 3-day-old rats upon antigenic stimulation failed to generate germinal centres in their spleen, transfer experiments of 3-day-old spleen cells to lethally X-irradiated syngeneic adult recipients indicated that 3-day-old spleens at least contained all the essential lymphoid elements (B and T cells) needed for germinal centre formation. These results strongly suggest that the failure to induce germinal centres in 3-day-old rats is most probably due to an immaturity of their splenic microenvironment. Immunohistochemical staining of frozen sections of neonatal rat spleen using mAb ED 5 and MRC OX-2 showed that follicular dendritic cells (FDC) were found as soon as primary follicles were found (i.e. by 14 days after birth). The appearance of FDC in neonatal spleens was not influenced by deliberate antigenic stimulation nor by the administration of adult spleen cells. We postulate that, during the development of FDC, a splenic microenvironment is created that allows primary follicle formation and the generation of germinal centres.     

Lymphocyte population kinetics during the development of the immune system. B cell persistence and life-span can be determined by the host environment

We have investigated the kinetic behaviour of LPS reactive Bcells during the development of the immune system. By studyingthe persistence of LPS reactive spleen cells transferred fromadult C57BL/6 donor mice into histocompatible C57BL/10 Sc.CrLPS non-responder mice we have confirmed that B cell populationsobtained from adult donor mice decay rapidly after transferinto adult recipients. In contrast, the same cell populationsafter transfer into neonatal reclpients are able to divide andmaintain their numbers for {small tilde}2-5 weeks in the host'sspleen. In fact, comparison of hosts at different ages showthat with the increasing age of the host the fate of donor Bcells evolves to mimic the behaviour observed upon transferinto adult recipients. Kinetic studies of LPS reactive spleencells obtained from newborn (2 weeks old) C57BL/6 donors aftertransfer into adult C57BL/10 Sc.Cr mice have shown that youngdonor cells were able to keep at constant numbers in the adultenvironment for the first {small tilde}10–15 days aftertransfer and to decay afterwards in the host's spleen at thesame rate as observed upon transfer of spleen cells from adultdonors into adult hosts. These studies provide the first evidencefor the different kinetic behaviour of lymphoid B cell populationsin developing and mature immune systems, confirm that cell persistencecorrelates with cell activation and division, and show thatB cell life-span is also dependent on the host environment.     

How do we avoid developing allergy: modifications of the TH2 response from a B-cell perspective

The synthesis of IgE by B cells occurs at a low rate compared with that of other antibodies, even in allergic subjects. One rate-limiting step is the class switch, by which B lymphocytes switch to produce immunoglobulin epsilon heavy chains rather than micro or gamma heavy chains. We propose an additional rate-limiting step: survival of the B lymphocyte after the switch to IgE. The hypothesis we present here is that the survival of the IgE-switched B cell is compromised, particularly in an active germinal center. Antigenic stimulation in the absence of a danger signal fails to induce a mature germinal center, which allows IgE-switched B cells to escape and mature into plasma cells. Antigenic stimulation in the presence of a danger signal (or under nonhygienic conditions) induces germinal centers, which eliminate IgE-switched B cells. Thus the essence of an allergen is antigenic stimulation in the absence of conditions that generate mature germinal centers. Because germinal centers are important for the generation of B-cell memory, the IgE immune response is characteristically poor in memory (but might be long lasting because of the generation of long-lived plasma cells). In addition to this direct route to IgE, typical for atopic sensitization, another type of T(H)2 response exists. On chronic allergen exposure with the concomitant induction of germinal centers, IgG4-switched B memory cells are induced that are slow to differentiate into plasma cells. These IgG4-switched B memory cells might occasionally undergo a secondary switch to IgE.     

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.