A subset of anti-CD21 antibodies promote the rescue of germinal center B cells from apoptosis

Germinal center cells (GCC) are programmed to die by apoptosis unless they receive a positive signal for rescue. The primary signal in vivo is believed to be dependent on interaction with antigen held as immune complexes on follicular dendritic cells (FDC), a subset of which express large amounts of CD23, a low-affinity receptor for IgE. Recombinant soluble CD23 (sCD23) and interleu-kin-1 have been found to potentiate the survival of GCC in vitro. Recently, CD23 was shown to interact specifically with a ligand other than IgE, namely CD21 (CR2/Epstein-Barr virus receptor). In the present study, we show that a subset of anti-CD21 monoclonal antibodies behave similarly to soluble CD23 in their effect on GCC inasmuch as they: (i) diminish the occurrence of apoptosis; (ii) promote a plasmacytoid appearance in rescued cells; (in) up-regulate expression of the Bcl-2 proto-oncogene. These findings indicate that FDC-derived CD23 exerts its effect on GCC via CD21.     

Role of follicular dendritic cells in the apoptosis of germinal center B cells

Follicular dendritic cells (FDCs) provide the most obvious source of antigens, which are essential for the differentiation of GC B cells. It has been reported that most proliferating B cells in germinal centers undergo apoptosis. Quantitative histology shows macrophages with apoptotic debris throughout the germinal center, the highest frequency of these cells being found in the dense FDC network. Based on these findings, we hypothesized that FDC may be involved in an apoptotic pathway of the germinal center B cells. To prove this hypothesis, we performed double immunohistochemical analysis using anti-FDC mAb and peanut agglutinin (PNA), with their respective TUNEL kits. Collated data showed that a great proportion of the apoptotic cells, most of which were positive for PNA, were in close contact with FDC, which indicated an interaction between FDC and B cells in the apoptotic pathway. Further studies using double immunohistochemical staining and FACS analyses demonstrated the expression of Fas-ligand (FasL) in a subset of the FDC. These results suggest that FDC may play a role in the apoptosis of germinal center B cells via Fas-FasL interaction.     

Follicular lymphoma of the skin and superficial soft tissues associated with a prominent follicular dendritic cell proliferation: an unusual pattern which may represent a diagnostic pitfall

We describe two elderly patients with follicular lymphoma (FL) involving the skin and superficial soft tissues, with a striking proliferation of follicular dendritic cells (FDC). In addition, one patient had bone marrow involvement by FL. Histopathologically, the most remarkable feature in both cases seen at low magnification was a striking pallor of the constituent cells, which were arranged in fascicles, whorls, and round islands. The majority of the cells had the typical cytologic features of FDCs. They were intimately intermingled with centroblasts and centrocytes. A large amount of the clear cytoplasm and the pale nuclei of FDCs, which predominated in the tumors, caused the striking overall pallor of the lesions. Small reactive lymphocytes were scattered between the fascicles. A vague follicular growth pattern was seen only focally. The mantle zones were markedly reduced or absent so that the follicles were seen lying unseparated. The close intermixture of the FDCs and the germinal center cells was responsible for the FDCs appearing to be decorated with B-associated marker, and the germinal center cells seemed to be stained to some degree with FDC-markers. The tumor bulk demonstrated a diffuse and strong reaction with CD10, CD20, CD21, CD35, and stained weakly with CD79a. Fascin and CD23 showed only a weak and focal staining pattern. Bcl-2 decorated large centroblasts and small reactive T-cells. The tumor bulk was negative for actin, EMA, cytokeratins, vimentin, desmin, and factor XIIIa. The proliferative index was rather low; MIB-1 mainly decorated large centroblasts. No monoclonal rearrangement of IgH genes was detected. Epstein-Barr virus was not identified. Electron microscopy revealed typical features of FDCs intermingled with germinal center cells. Such cases may represent a diagnostic pitfall, as FDC overgrowth can mask FL and give the neoplasm the appearance of FDC sarcoma/tumor. We believe that, in both cases, the FDC proliferation had a reactive character.     

Germinal center B cells rescued from apoptosis by CD40 ligation or attachment to follicular dendritic cells,but not by engagement of surface immunoglobulin or adhesion receptors,become resistant to CD95-induced apoptosis

Germinal centers (GC) constitute a specialized microenvironment essential for the formation of memory B cells, B cell affinity maturation and isotype switching. Within the GC, the B cells closely interact with follicular dendritic cells (FDC) and T cells, which both provide stimuli to the B cells that prevent their entry into apoptosis and promote their differentiation into memory cells or plasma cells. Cross-linking of B cell immunoglobulin (Ig) receptors by antigen, stimulation of the integrin adhesion molecules LFA-1 and VLA-4 on the B cell through interaction with their counter receptors ICAM-1 and VCAM-1 on the FDC and cross-linking of CD40 on the B cells through interaction with the CD40 ligand (CD40L) on T cells have been shown to prevent entry into apoptosis of GC B cells. Triggering of CD95, on the other hand, has been shown to induce apoptosis. We therefore investigated the interaction between adhesion-mediated signals, Ig, CD40, and CD95. The spontaneous apoptosis of GC B cells was not further increased by adding anti-CD95. However. CD95 stimulation did result in apoptosis of GC B cells in the presence of anti-Ig or adhesion-mediated rescue signals, which indicates that CD95 expressed on GC B cells is functionally active. In contrast, anti-CD95 was unable to induce apoptosis in cells rescued via CD40 stimulation, suggesting an important role for CD40L expressed on GC T cells in apoptosis regulation. We also studied apoptosis of B cells adhering to FDC, and found that B cells that interact with FDC were also rescued from CD95-induced apoptosis. A human CD40.Fcu fusion protein that blocks CD40 ligation failed to inhibit this effect. Our studies therefore indicate that neither CD40, Ig receptors, nor adhesion receptors mediate rescue from apoptosis by FDC.     

Direct evidence that human follicular dendritic cells (FDC) rescue germinal centre B cells from death by apoptosis.

It is supposed that FDC have a pivotal role in the rescue of germinal centre (GC) B lymphocytes from apoptosis. However, formal proof for this hypothesis has not as yet been presented. In the present study FDC and GC B cells were isolated from human tonsils and cultured. When brought into culture FDC and B cells rapidly formed spherical clusters. T cells were not observed inside these clusters. At different time points cultures of FDC and B cells were supravitally stained with Hoechst 33258 or acridine orange and examined by direct observation using fluorescence microscopy. Viable B cells appeared to be profoundly restricted to clusters, whereas cells not taking part in clusters all had an apoptotic appearance. The formation of clusters could be prevented by addition of MoAbs against CD11a (LFA-1 alpha) or CD49d (VLA-4 alpha), resulting in an apoptotic appearance of virtually all B lymphocytes. The present data demonstrate that a physical interaction between FDC and germinal centre B lymphocytes is able to rescue the latter from apoptotic cell death.     

Quantitative analysis of molecules which distinguish functional compartments within germinal centers

Five zones in the secondary follicles of human tonsils are described, which are distinguished by the phenotype of their constituent cells. Moving from the apex to the base of the follicle the zones are termed: follicular mantle, outer zone, apical light zone, basal light zone and dark zone. The dark zone contains proliferating, CD77^high, centroblasts and thin, widely spaced processes of follicular dendritic cells (FDC). The apical and basal light zones on the other hand contain a dense network of FDC which express CD21 and CD54 strongly. The FDC of the apical light zone differ from those of the basal light zone by their high expression of CD23. Centroblasts of the dark zone give rise to non-proliferating centrocytes which move apically through the light zone (Eur. J. Immunol. 1991. 21: 2951). The centrocytes of the basal light zone are more pyroninophilic, more closely-packed and larger than those in the apical light zone. Consequently, by conventional histology the basal light zone appears to be part of the dark zone. The nomenclature adopted, however, adheres to the convention that the dark zone is filled with proliferating centroblasts. Cells undergoing apopotosis were identified both in the dark and light zones, but more than half of these cells were located in the basal light zone. This is consistent with the concept that the progeny of cells which undergo somatic mutation in their immunoglobulin variable region genes in the dark zone migrate to the light zone where they are selected on the basis of their capacity to bind to antigen held on FDC. Cells receiving an antigen-dependent signal survive while those that do not kill themselves by apoptosis. The outer zone does not contain CD23^high cells and in this way is distinguished from the adjacent follicular mantle and apical light zone. It contains small lymphoid cells, blasts and plasmacytoid cells. Many cells of the outer zone express CDw75 strongly. The outer zone also extends as a narrow band around the dark zone. Possible roles of FDC and T cells of the light zone and outer zone in inducing centrocytes to differentiate to memory cells or plasmablasts are discussed.     

Direct cell/cell communication in the lymphoid germinal center: connexin43 gap junctions functionally couple follicular dendritic cells to each other and to B lymphocytes

Direct cell/cell communication occurs through gap junctions (GJ). We mapped GJ expression in secondary lymphoid organs and found, for the first time, a high density of connexin43 (Cx43) GJ in follicular dendritic cells (FDC) in close association with lymphocytes (Krenacs T. and Rosendaal M., J. Histochem. Cytochem. 1995. 43: 1125–1137). In this work, we used a combination of ultrastructural, immunocytochemical, molecular methods, and functional dye transfer experiments to study which germinal center cells are involved in direct cell/cell communication and how GJ expression is regulated during antigen responses. One week after injecting the footpad of mice with 50 μg lysozyme, Cx43 GJ were detected on elongated cells in the paracortex of their popliteal lymph nodes. Repeated challenge led to the formation of secondary follicles with enlarged FDC meshwork full of Cx43 GJ. This positive correlation may reflect an importance for GJ in the pattern formation of FDC and lymphoid follicles. In human tonsil, the density of GJ and FDC was highest in the light zone of germinal centers where the fate of B cells is thought to be decided. Cx43 colocalized with CD21 and CD35 antigens in the vicinity of desmosomal junctions on FDC embracing lymphocytes. Freeze-fracture hallmarks of GJ of 200–400 nm were also found on FDC in the vicinity of desmosomal plaques. Furthermore, Northern blot analysis showed the consistent presence of Cx43 mRNA in human tonsil and spleen. Most Cx43 message was localized in situ to cells with FDC morphology and some to a few germinal center lymphocytes. To investigate functional cell coupling, we set up FDC/B cell cultures from the low density cell fractions of human tonsils. Cx43 plaques associated with lymphocytes were detected both on elongated FDC processes in early cultures (up to 4 h) and in established FDC/B cell clusters (between 4 and 24 h). In early cultures, we injected FDC with Lucifer Yellow, a fluorescent dye which passes through GJ: the dye spread into adjacent FDC and occasionally from FDC into CD19⁺ B cells. Based on these results, we propose that direct cell/cell communication through Cx43 GJ is involved in FDC/FDC and in FDC/B cell interactions. The functionally coupled FDC meshwork may serve as a communication channel synchronizing germinal center events. FDC may also deliver crucial direct signals through GJ involved in the rescue of high-affinity B cell clones from apoptotic cell death.     

Antigenic phenotyping of human follicular dendritic cells isolated from nonmalignant and malignant lymphatic tissue

Follicular dendritic cells (FDC) are located within follicles of secondary lymphoid tissue and in lymph nodes of patients with germinal center cell-derived non-Hodgkin lymphomas. Reliable antigenic phenotyping of FDC within tissue sections has been difficult due to simultaneous labeling of the surrounding germinal center cells. Using an enzyme cocktail to digest human tonsils and cervical lymph nodes with subsequent fractionation by albumin gradient centrifugation, cell isolates containing up to 20% FDC were obtained. This preparation allowed the determination of antigenic phenotype on individual FDC. Molecules expressed by FDC were detected by an isotype-specific immunocytochemical double-labeling procedure, i.e. a monoclonal antibody (mAb) specific for FDC (KiM4 or DRC1) in conjunction with a mAb reactive against an additional antigenic determinant. Nonspecific binding of mAb to immunoglobulin Fc receptors located on FDC membranes was avoided by incubation of cells with human IgG aggregates prior to immunostaining. The results revealed that isolated FDC from these lymphoid tissues express transferrin receptors, the intercellular adhesion molecule 1, class II antigens, the B cell antigens CD20 and CD21, and the myelomonocytic properties CD11b and CD14. Immunoglobulin mu or gamma heavy chains and the B cell antigens CD23 and CD24 are detected on 50% of an isolated FDC population. These FDC are negative for the T helper cell antigen CD4, the B cell cell antigens CD19 and CD22, the immunolobulin alpha and delta chains and the S-100 protein. FDC isolated from lymph nodes of patients with low-grade malignant non-Hodgkin lymphoma, identified by DRC1 or KiM4 mAb, presented the same antigenic profile as seen on FDC from nonmalignant tissue. This suggests that FDC from lymphoma tissue isolated in this manner have the same properties as those found in normal tissue.     

Rewiring of CD40 is necessary for delivery of rescue signals to B cells in germinal centres and subsequent entry into the memory pool

Memory B-cell development is impaired by in vivo blockade of the CD40-CD40 ligand (CD40L) interaction using human Fc immunoglobulin G1 (IgG1)-mouse CD40 fusion protein (CD40-Ig); however, germinal centre (GC) formation is not. We show here that the block in B-cell differentiation in these mice is at the stage of rescue from apoptosis and exit from the GC. Thus, GC from CD40-Ig-treated mice contain a three- to fourfold higher level of apoptotic cells than found in control mice injected with human IgG1 alone. This increase in apoptosis is not caused by a blockade of the CD40L-mediated rescue signal but is the result of an intrinsic defect of GC B cells in CD40-Ig-treated mice to receive rescue signals via CD40. While anti-CD40 stimulation maintained the viability in culture of GC B cells from control mice, it did not rescue GC B cells from CD40-Ig-treated mice. This data is consistent with the notion that a 'rewiring' of the CD40 molecule is induced by CD40 ligation early in the response and is necessary to allow B-cell rescue from apoptosis when they subsequently enter the GC.     

Follicular dendritic cell-signaling molecules required for proliferation and differentiation of GC-B cells

To study the functional role of follicular dendritic cells (FDC) in germinal center (GC)-B cell differentiation, we have established an in vitro experimental model that mimics GC-reactions in vivo. Using this culture system, we have characterized the distinct function of FDC from activated T cells. The primary function of FDC is to prevent apoptosis and support proliferation of GC-B cells, whereas activated T cells induce the differentiation of GC-B cells by providing CD40L and cytokines. Furthermore, we have identified two FDC-signaling molecules that mediate proliferation of GC-B cells in vitro and in vivo.     

Protein tyrosine phosphorylation is mandatory for CD40-mediated rescue of germinal center B cells from apoptosis

Spontaneous apoptosis in germinal center (GC) B cells can be arrested either by engaging cell surface immunoglobulin (Ig) with immobilized ligand or, more effectively, by treatment with soluble monoclonal antibody (mAb) directed against CD40. The present study examines the intracellular signal transduction pathways through which rescue from spontaneous apoptosis is engendered in GC B cells following ligation of surface CD40. Cross-linking the surface CD40 of GC B cells with mAb consistently resulted in enhanced tyrosine phosphorylation on a number of distinct substrates: this process could be blocked, in a dose-dependent fashion, by pre-treating GC B cells with the selective protein tyrosine kinase(s) (PTK) inhibitor, herbimycin A. Moreover, the pattern of phosphorylation on tyrosine observed following treatment with anti-CD40 was remarkably similar to that triggered by polyvalent anti-Ig. By contrast, anti-CD40 failed to stimulate the increase in inositol 1,4,5-trisphosphate and cytosolic free calcium observed in both GC B cells and resting B lymphocytes following ligation of surface Ig. The involvement of the signaling pathways generated in the rescue of GC B cells from apoptosis was studied by using selective inhibitors of PTK and of extracellular and intracellular Ca²⁺. Pre-incubation with the PTK inhibitor herbimycin A (5 μM) abrogated anti-CD40-mediated rescue of GC B cells from apoptosis, while genistein (40 μM) and the tyrphostins AG490 (10 μ M) and AG814 (25 μ M) significantly inhibited this process. Consistent with these results, herbimycin A (5 μM) abolished the expression of the 26 kDa bcl-2 protooncogene product, which confers resistance to apoptosis, normally observed following culture with anti-CD40. The Ca²⁺ chelators BAPTA and EGTA did not significantly affect CD40-promoted rescue. Taken together, these results indicate that CD40 of GC B cells is coupled to functional PTK but not to the phosphatidylinositol signaling pathway and that tyrosine phosphorylation is mandatory for CD40-mediated rescue of GC B cells from apoptosis.     

B cells from a distinct subset of patients with common variable immunodeficiency (CVID) have increased CD95 (Apo-1/fas), diminished CD38 expression, and undergo enhanced apoptosis.

We investigated the role of apoptosis in the differentiation failure of B cells from a selected subpopulation of patients with CVID delineated by B cell surface marker analysis, in vitro IgE response, and molecular markers of B cell VH gene repertoire. These patients had altered display of B cell surface molecules that play a role in apoptosis. The patients' B cells had a 4.5-250-fold increase in CD95 (Apo-1, fas) expression and increased CD95 display on their T cells. CD38, a molecule important in preventing germinal centre B cell apoptosis, was reduced on the patients' B cells. The expression of this molecule was inducible on the CVID lymphocytes with retinoic acid. Increased spontaneous apoptosis in vitro was observed with the patients' B (23%) and T cells (10%) compared with normal cells (13% and 3%, respectively). Stimulation in vitro with IL-4 and CD40 rescued the B cells from apoptosis and allowed for their differentiation. However, IL-4 plus alpha CD40-driven immunoglobulin production was not quantitatively or qualitatively normal. Failure to overcome apoptosis, a normal step in germinal centre B cell development, may be involved in the lack of differentiation seen in this subset of CVID patients.     

Proliferation of germinal center B lymphocytes in vitro by direct membrane contact with follicular dendritic cells.

In secondary lymphoid organs, follicular dendritic cells (FDC) are located within B cell follicles and germinal centers. Through their cytoplasmic extensions they come into contact with a large number of neighboring lymphocytes. Using an enzyme cocktail to digest human tonsils followed by ultracentrifugation on bovine serum albumin gradients, single cell suspensions were obtained. Immunocytochemistry revealed that 7% of the cells were FDC, 5% T cells, and 5% macrophages. The remaining population were B cells with greater than 95% being of the germinal center phenotype (i.e. CD19-positive, CD39/sIgD negative). After 24 h of culture up to 44% of the lymphocytes were found in clusters centered around FDC. At the start of the culture as well as 24 and 72 h later, between 31 and 55% of the B cells within FDC associated clusters were in late G1 to M phase of the cell cycle. In contrast, less than 10% of the B cells not in contact with FDC (i.e. outside the clusters) were in an activated state. Autoradiography revealed that after three days of incubation the rate of proliferation was 26.2 times higher for the lymphocytes involved in cluster formation as compared to those cells not associated with FDC. Furthermore, the number of viable B cells after a 72 h mitogen-free culture period was determined. By adding FDC to these preparations, 31.9% of the lymphocytes were rescued from dying. These data show that FDC provide a microenvironment which can maintain the viability, activation and proliferation of germinal center B cells in vitro.     

CM1 ligation initiates apoptosis in a caspase 8-dependent manner in Ramos cells and in a mitochondria-controlled manner in Raji cells

Burkitt lymphoma (BL) is a tumor with the characteristics of germinal center B cells. We previously reported that the CM1 (centrocyte/-blast marker 1) molecule is expressed only in germinal center B cells, specifically, in a subpopulation of centroblasts and centrocytes. In the present study, we investigated the apoptosis induced by anti-CM1 in the Ramos and Raji human BL cell lines. The Ramos is protected from apoptosis by the crosslinking of sIgM and the calcium ionophore by the ligation of CD40 with anti-CD40 monoclonal antibodies (mAb) or soluble CD40 ligand (sCD40L). In this investigation on the effect of CM1 on apoptosis in BL cell lines, we found that cellular signaling by CM1 induces apoptosis and decreases cell viability, in BL cell lines cultured for 24 hours with protein-G agarose beads conjugated anti-CM1 mAb. Stimulation by CD40 ligated with sCD40L protected Raji cells from CM1-induced apoptosis, but did not protect Ramos cells. Furthermore, after anti-CM1 mAb stimulation, CD95 expression was upregulated and CD40 expression was unaltered or slightly decreased in Ramos cells, whereas CD95 was downregulated and CD40 was slightly upregulated in Raji cells. The engagement of CD40 by sCD40L enhanced CD95 expression, but the level of CM1 expression was unchanged in Ramos. However, sCD40L downregulated both CD95 and CM1 expression in Raji. In addition, the caspase-8 specific inhibitor blocked CM1-induced apoptosis in Ramos cells, but not in Raji cells. Increased mitochondrial membrane permeabilization was observed only in Raji cells. Moreover, the effector caspase inhibitor, z-DEVD, blocked CM1-mediated apoptosis in both cell lines. We found that CM1-induced apoptosis is achieved via different initiation pathways, which are cell-type dependent.     

CD19 regulates B cell maturation, proliferation, and positive selection in the FDC zone of murine splenic germinal centers

Mice with mutations in CD19 Y482/Y513 form germinal centers (GC) but fail to produce high-affinity antibodies. In these mice, GC B cell differentiation, proliferation, and class switching occur but are defective. Altered CD19 signaling results in retention of early GC B cells and reduced proliferation in the follicular dendritic cell (FDC) zone of GC, and causes failure to select for high-affinity mutations. In normal mice, the earliest detectable aggregates of GC B cells are in contact with FDC and IgM+ cells are only found in the FDC zone, further evidence that the FDC zone is the site of initial GC B cell proliferation, differentiation, and class switching. Proliferation in the non-FDC zone and somatic mutation are not dependent on CD19, indicating separate signaling requirements for the two GC compartments, but these CD19-independent GC functions are not sufficient to generate high-affinity antibodies and B cell memory.     

Fas expression and apoptosis in human B cells

Mechanisms of B cell apoptosis are critical in reducing aberrant B cell proliferations such as those that arise in autoimmune disease and in B cell malignancies. The physiologic interaction of CD4+ helper T cells and B lymphocytes has been extensively studied over the past two decades. Although CD4+ T cells are considered primarily to offer positive costimulatory signals for B cell differentiation into active immunoglobulin-secreting cells, recent studies have shown that CD4+ T cells are crucial in downregulating the humoral immune response. In the course of cognate interaction between CD40 ligand (CD40L)-bearing CD4+ T cells and CD40-expressing germinal center B cells, CD40 ligation results in augmented Fas expression at the B cell surface. Like CD40L, Fas ligand is expressed on activated CD4+ Th1 cells and when bound to Fas receptor on the B cell surface, initiates an apoptotic signal in that cell. Thus, CD4+ T cells limit the growth of autologous germinal center B cells by first inducing Fas expression and then instigating a death signal via Fas ligand. In this work, we will consider these observations about CD4+ T-cell-induced, Fas-mediated B cell death in the context of other factors that affect apoptosis in B cells, normal and malignant.     

Germinal center cells express bcl-2 protein after activation by signals which prevent their entry into apoptosis

B cells undergo selection within germinal centers on the basis of their capacity to be activated by antigen held on follicular dendritic cells. Isolated germinal center B cells in culture kill themselves by apoptosis but this is prevented if their receptors for antigen are cross-linked. In this study it is confirmed that almost all germinal center B cells, unlike other B cells, do not express the 25-kDa protein encoded by the bcl-2 oncogene. Cross-linking the surface Ig of isolated germinal center cells causes them to express bcl-2 protein. Two other stimuli which inhibit the entry of germinal center cells to apoptosis result in the expression of bcl-2 protein. These stimuli are: (a) CD40 antibody and (b) recombinant 25-kDa fragment of the CD23 protein plus recombinant interleukin 1 alpha. Respectively, these induce germinal center cells to differentiate to resting B cells or plasmablasts. Dual-fluorescence studies on small lymphocytes confirm the presence of bcl-2 protein in mitochondria but show that this is also present in other extra-nuclear areas. Burkitt lymphoma cells have a phenotype which indicates that they are neoplastic cells of germinal center origin. The expression of bcl-2 protein by Burkitt lymphoma lines was also studied. Burkitt lines which retain the phenotype of fresh Burkitt lymphoma cells can be induced to enter apoptosis on culture with the Ca2+ ionophore ionomycin. These cells were found not to express bcl-2 protein. By contrast, Burkitt lines which have drifted towards a lymphoblastoid cell line phenotype and are resistant to the induction of apoptosis express high levels of the bcl-2 protein. The findings support the concept that the susceptibility of germinal center cells to entering apoptosis is associated with their lack of expression of bcl-2 protein. Aberrant expression of bcl-2 protein by some neoplastic germinal center cells may allow survival in situations where their normal counterparts die.     

CD77: an antigen of germinal center B cells entering apoptosis.

We have previously reported that a neutral glycolipid (globotriosylceramide; Gb3) was specifically expressed on Burkitt's lymphoma cells and on a subset of germinal center tonsillar B lymphocytes. Recently the Gb3 molecule was recognized as a new B cell differentiation antigen and now defines the CD77 cluster. Here we report an extensive phenotypic and functional characterization of the tonsillar CD77+ B lymphocytes. These cells have a low buoyant density and are thus purified using a Percoll gradient. They express various B cell antigens such as CD19, CD20, CD21, CD22 and CD40, as well as the adhesion molecules LFA-1, LFA-3 and CD44. They are positive for surface IgM and negative for surface IgD. Although these results suggest a phenotype of activated B cells, the CD77+ cells are negative for the classical activation antigens: CD23 (the low-affinity Fc receptor for IgE), CD25 [the interleukin (IL) 2 receptor alpha chain] and CD71 (the transferrin receptor). Proliferation and protein synthesis of CD77+ cells was measured after stimulation with a range of mitogens and IL. None of the agents tested are able to induce proliferation and protein synthesis with the exception of a combination of recombinant IL 4 plus anti-CD40 antibody. When examined by electron microscopy, CD77+ B lymphocytes present a morphology similar to that of cells undergoing programmed cell death, also called apoptosis (i.e. chromatin condensation, nuclear fragmentation, membrane blebbing). As shown by direct examination of DNA, these CD77+ cells are indeed in the process of apoptosis. Treatment of the CD77+ cells by recombinant IL 4 and anti-CD40 antibody prevents apoptosis. All these results suggest that the CD77 molecule defines a B lymphocyte maturation pathway, specific for germinal center, where the cells undergo programmed cell death.     

Engagement of CD20 suppresses apoptosis in germinal center B cells

In a screen of 67 monoclonal antibodies (mAb) included in the Blind Panel of B cell antibodies for the 5th International Workshop on Human Leukocyte Differentiation Antigens, only the CD20 mAb – included as a positive control for immunophenotyping studies – was found to suppress the spontaneous apoptosis which occurs in human germinal center (GC) B cells when placed in tissue culture at 37°C. Further detailed study using the 1F5 mAb confirmed this observation, showing that rescue from apoptosis via CD20, while not as efficient as that obtained on ligating CD40, was of similar magnitude to that achieved on engagement of surface immunoglobulin (sIg) by immobilized antibody. Also similar to anti-Ig, the CD20 mAb rescued from apoptosis without priming for the proliferation of GC B cells: this was quite different to its action on resting, non-GC B cells, where it provides a potent priming signal for cell cycle progression in response to IL-4 or anti-CD40. Unlike the survival signal engendered via sIg, CD20 engagement neither mobilized Ca²⁺ from intracellular stores or opening of a Ca²⁺ channel with 1F5, nor did it affect the ability of anti-Ig to open a Ca²⁺ gate in GC B cells. An unexpected feature of CD20-mediated rescue of GC B cells from apoptosis was a failure to turn on Bcl-2 expression.     

Factors modifying survival pathways of germinal center B cells. Glucocorticoids and transforming growth factor-beta, but not cyclosporin A or anti-CD19, block surface immunoglobulin-mediated rescue from apoptosis.

The tendency for germinal center (GC) B cells to enter apoptosis is suppressed on engaging antigen receptor with immobilized anti-immunoglobulin; cross-linking of surface CD40 by monoclonal antibodies provides an additional signal for rescuing GC cells from programmed death. These observations are believed to reflect events that, in vivo, would allow for the selection of centrocytes which have undergone somatic mutation on Ig V-region genes to generate antigen receptor of high affinity. The purpose of the present study was to identify factors capable of modifying the survival pathways of GC cells. Transforming growth factor-beta, at an optimal concentration of 1 ng/ml, was found to inhibit surface immunoglobulin (sIg)-mediated rescue of GC cells but had no influence on survival promoted through CD40. Both routes of rescue were blocked by the glucocorticoid prednisolone at pharmacological concentrations (ID50 = 10(-7) M). Cyclosporin A, an antagonist of sIg-mediated signaling in resting B cells, failed to block rescue of GC cells through either of the receptor-activated pathways. Antibody to CD19--which also suppresses the activation of resting B cells--not only left GC cell rescue undiminished, but rather provided a modest survival signal of its own; interferon-alpha behaved similarly while interferon-gamma failed to influence GC cell survival in either direction.