Expression of macrophage inflammatory protein-3alpha, stromal cell-derived factor-1, and B-cell-attracting chemokine-1 identifies the tonsil crypt as an attractive site for B cells.

The expression of 3 lymphoid chemokines-macrophage inflammatory protein-3alpha (MIP-3alpha), stromal cell-derived factor-1 (SDF-1), and B-cell-attracting chemokine-1 (BCA-1)-in the tonsil and the possible correlation between their sites of expression and B-cell localization within this tissue were studied. The results show that all 3 chemokines are produced in the crypts but differ by the nature of the cells that produce them and their location within the crypt. SDF-1 and MIP-3alpha are produced by epithelial cells, but their secretion is mutually exclusive. Both MIP-3alpha- and SDF-1-expressing cells are in close contact with memory B cells. By contrast, BCA-1-producing cells in the crypt are not epithelial and form clusters colocalized with plasma cells. Altogether, these data suggest that the chemokines produced in the tonsillar crypt may (1) attract memory B cells to antigen and (2) recruit and retain plasma cells and memory B cells within the supportive epithelial microenvironment of the crypt. (Blood. 2001;97:3992-3994)

Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells.

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1beta-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.

B-cell memory and the persistence of antibody responses.

Antigens such as viral envelope proteins and bacterial exotoxins induce responses which result in the production of neutralizing antibody. These responses persist for years and provide highly efficient defence against reinfection. During these antibody responses a proportion of participating B cells mutate the genes that encode their immunoglobulin variable regions. This can increase the affinity of the antibody, but can also induce autoreactive B cells. Selection mechanisms operate which allow the cells with high affinity for the provoking antigen to persist, while other B cells recruited into the response die.

Mitochondria connects the antigen receptor to effector caspases during B cell receptor-induced apoptosis in normal human B cells.

We have previously reported that CD40 stimulation sensitizes human memory B cells to undergo apoptosis upon subsequent B cell receptor (BCR) ligation. We have proposed that activation stimuli connect the BCR to an apoptotic pathway in mature B cells and that BCR-induced apoptosis of activated B cells could serve a similar function as activation-induced cell death in the mature T cell compartment. Although it has been reported that caspases are activated during this process, the early molecular events that link the Ag receptor to these apoptosis effectors are largely unknown. In this study, we report that acquisition of susceptibility to BCR-induced apoptosis requires entry of memory B cells into the S phase of the cell cycle. We also show that transduction of the death signal via the BCR sequentially proceeds through a caspase-independent and a caspase-dependent phase, which take place upstream and downstream of the mitochondria, respectively. Furthermore, our data indicate that the BCR-induced alterations of the mitochondrial functions are involved in activation of the caspase cascade. We have found both caspases-3 and -9, but not caspase-8, to be involved in the BCR apoptotic pathway, thus supporting the notion that initiation of the caspase cascade could be under the control of the caspase-9/Apaf-1/cytochrome c multimolecular complex. Altogether, our findings establish the mitochondria as the connection point through which the Ag receptor can trigger the executioners of apoptotic cell death in mature B lymphocytes.

Activation sensitizes human memory B cells to B-cell receptor-induced apoptosis.

The outcome of antigen receptor (B-cell receptor; BCR) ligation on B-cell survival can be influenced by multiple parameters. They are linked to the physical properties of the antigen itself, the maturational stage of the cells and the costimuli provided by different components of the innate and acquired immunity. Here we report that apoptosis prevails over stimulation when a BCR agonist is applied to human memory B cells which have been preactivated by CD40 ligand or anti-immunoglobulin antibodies. The susceptibility of activated memory B cells to BCR-induced killing is correlated with their enhanced expression of the transcripts encoding the pro-apoptotic molecules Bax, c-Myc and p53. The BCR-mediated apoptosis of activated memory B cells does not require extensive cross-linking of the antigen receptors and relies neither on engagement of the FcgammaRII nor on the Fas/Fas ligand (Fas-L) system. Our findings suggest that activation stimuli open the BCR-induced apoptotic pathway in memory B cells. Therefore we propose that the concept of activation-induced cell death (AICD), originally described for T cells, also applies to mature B lymphocytes. The functions fulfilled by the AICD of mature B cells in the regulation of B-cell responses are discussed.

Memory B-cell clones and the diversity of their members.

Memory B-cell clones develop from virgin B cells that take up processed antigen, make cognate interaction with primed T cells and then grow in germinal centres. Within the germinal centre the proliferating B cells undergo Ig variable-region mutation and are subsequently selected on their ability to bind antigen held on follicular dendritic cells and then to make cognate interaction with germinal centre T cells. The selected cells emerge as memory B cells or plasmablasts. Although many of the memory B cells and most of the plasma cells emerging from follicles have undergone Ig class switch recombination a substantial minority of the memory B cells have not switched. These non-switched memory cells can be induced to switch on re-exposure to antigen. Affinity maturation following a single immunization ceases as germinal centres wane some 3-4 weeks after immunization - memory cells and antibody production, on the other hand, persist for months and even years.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.

Centrocytes rapidly adopt a memory B cell phenotype on co-culture with autologous germinal centre T cell-enriched preparations.

B cells, after mutating their Ig B-region genes in germinal centres (GC), undergo apoptosis, unless they receive antigen-dependent selection signals. The signals appear to be delivered by GC T cells, require CD40 ligand expression and may induce differentiation to memory cells. Cultured GC B cells are prevented from entering apoptosis by ligating their surface CD40, but the resulting phenotype is not associated with B cells found in vivo. Conversely, GC B cells rapidly adopt a memory B cell phenotype on culture with autologous memory CD4(+) T cells that have been induced to express CD40 ligand transiently. This effect is prevented by blocking CD40 ligand. Naive CD4(+) T cells, induced to express CD40 ligand, do not prevent GC B cells undergoing apoptosis.

A subset of CD4+ memory T cells contains preformed CD40 ligand that is rapidly but transiently expressed on their surface after activation through the T cell receptor complex.

Signaling through surface CD40 is essential for selecting B cells that have mutated their immunoglobulin variable region genes in germinal centers and is an important signal in the early stages of antibody responses to T cell-dependent antigens. It is shown that a subset of CD45RO+, CD4+ T cells isolated from human tonsil contains preformed 30-35-kD ligand for CD40. This is expressed on their surfaces within 5 min of their antigen-receptor complexes interacting with CD3 epsilon antibodies bound to ox erythrocytes. This surface expression does not require de novo protein synthesis and lasts for only 1-2 h. Preformed CD40 ligand (CD40L) was not detected in any CD4+ CD45RA+ T cells, but > 90% of all CD4+ T cells from the tonsil can be induced to express large amounts of CD40L on culture with phorbol myristate acetate and the calcium ionophore ionomycin. This expression of CD40L starts between 1 and 2 h, peaks at 6 h, and remains at a high level for > 20 h. It is totally prevented by adding a concentration of cycloheximide that inhibits CD25 synthesis by these activated cells. While CD3 epsilon antibody bound to ox red cells is a good inducer of surface expression of CD40L, it is a much less potent inducer of CD40L synthesis than phorbol myristate acetate with ionomycin. Immunohistological analysis of tonsil sections shows that cells containing CD40L are located mainly in the outer zone of germinal centers and the margins of the T zones that are rich in dendritic cells (interdigitating cells). The distribution of these cells is consistent with: (a) their interaction in T zones with B cells that have taken up and processed antigen and (b) their involvement in B cell selection in germinal centers.

Isolation and characteristics of tonsil centroblasts with reference to Ig class switching.

Most tonsil B cells have high levels of surface CD44 but this molecule is either expressed at low levels or is absent from germinal centre B cells (GCB). On average 62% of isolated GCB were found to be CD44- and the remainder CD44low. Most CD44- GCB were in cell cycle, indicating that they were centroblasts, while centrocytes, non-dividing GCB, were mainly CD44low. Immunohistological analysis confirms that centrocytes, which are located in the light zone of germinal centres, express low levels of CD44, while centroblasts, cells of the dark zone, are CD44-. While most CD77high GCB are centroblasts and CD77low GCB centrocytes, many centroblasts and centrocytes express intermediate levels of CD77, making this less reliable than CD44 for discriminating between these cells. Most CD44low and CD44- GCB were shown to have undergone Ig switch recombination in vivo. This indicates that switch recombination is independent of the maturation of centroblasts to centrocytes and precedes the signals that induce GCB to differentiate to plasma cells or memory B cells. The average rate of entry of the CD44- GCB fraction to apoptosis on culture at 37 degrees C was faster than that of the total GCB preparation. It is suggested that this may reflect strict stromal-dependence of centroblasts while centrocytes have to survive for long enough to have the chance of receiving antigen-specific selection signals. Inhibition of apoptosis by CD40 mAb with IL-4 or phorbol myristate acetate with ionomycin was similar in the CD44- and CD44low preparations.