Differentiation and apoptosis of human germinal center B-lymphocytes

An in vitro experimental model was developed to characterize the cellular and molecular factors that regulate germinal center (GC)B-cell differentiation and apoptosis. In the culture system that sustains the GC-B-cell survival, CD40L stimulation is essential for GC-B-cell proliferation and differentiation in the presence of 1L-2, IL-4, and IL-10. IL-2 and IL-4 promote proliferation of GC-B-cells, whereas IL-10 is required for generation of plasma cells. Generation of memory B cells requires CD40L, IL-2, IL-4, but not IL-10. There are two mechanisms that cause apoptosis. In the early stage, spontaneous apoptosis occurs in the absence of CD40 stimulation. Following CD40L stimulation, Fas-mediated apoptosis operates to eliminate GC-B-cells, unless activated GC-B-cells encounter a second signal via B-cell Ig receptors. Physiological significance of these findings is discussed.     

Identification of a tonsil IgD+ B cell subset with phenotypical and functional characteristics of germinal center B cells

We have identified and isolated a subpopulation of IgD⁺ B cells (IgD⁺ CD38⁺ B cells) from human tonsils which expresses the germinal center (GC)-associated surface markers CD10, CD38, CD75, CD77 and CD95/Fas. The heterogeneity of expression of several markers on IgD⁺ CD38⁺ B cells suggests that this population can be further subdivided into two discrete subtypes. On a functional basis, IgD⁺ CD38⁺ B cells behave as GC B cells as they rapidly and spontaneously undergo apoptosis in vitro and cannot be stimulated to synthesize DNA upon cross-linking of the antigen receptor. However, in contrast with most GC B cells, IgD⁺ CD38⁺ B cells have not completed Ig class switching since they predominantly secrete IgM following stimulation in vitro and lack surface expression of secondary isotypes. Immunoenzymatic staining performed on tonsil tissue sections revealed that IgD⁺ CD38⁺ B cells are located in two distinct histological structures: within the GC of a few classical secondary follicles, in which they appear as scattered cells, and within rare atypical GC, homogeneously constituted of IgD⁺ B cells. Taken together, our findings indicate that IgD⁺ CD38⁺ B cells constitute a novel subset of GC B cells. The possibility that these cells could represent an early stage of the follicular reaction or be generated in response to certain bacterial carbohydrate antigens is discussed.     

Identification of a novel subpopulation of germinal center B cells characterized by expression of IgD and CD70

CD27, which belongs to the tumor necrosis-factor receptor family, is expressed on germinal center (GC) but not on naive B cells, suggesting an important function of this molecule in the regulation of the GC reaction. We described here the expression of CD70, which is the ligand for CD27. We observed that in most tonsils, CD70 is only expressed on part of the IgD^?, CD38^? B cell population, which have been described as memory B cells. However, in 10 % of the tonsils tested, CD70⁺ IgD⁺ GC were found. The CD70⁺ GC B cells were small cells that also expressed CD44 and CD39, but were CD10^? and CD38^?, suggesting that they represent very recent immigrants that are in the process of forming a GC. The concordant expression of CD27 and its ligand CD70 on this primordial subset of GC B cells suggests an important role for CD27/CD70 interaction at this stage of GC formation.     

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.     

IL-10 interrupts memory B cell expansion in the germinal center by inducing differentiation into plasma cells

Germinal center (GC) B cells undergo proliferation, somatic hypermutation and isotype switching in the course of differentiation into plasma cells to produce high-affinity antibodies. To understand the molecular mechanism regulating the expansion of memory B cells and the termination of expansion by differentiation into plasma cells, we investigated the effect of interleukin-2 (IL-2), IL-4, IL-10 and CD40 ligand (CD40L) on the differentiation of GC B cells in the defined culture system containing a follicular dendritic cell (FDC)-like cell line. IL-2, IL-4 and CD40L are required for the optimum proliferation and differentiation of GC B cells. When IL-10 was added to this culture condition, CD20⁺ CD38⁺ GC B cells sequentially differentiated into CD20⁺ CD38⁻ memory B cells and then CD20⁻ CD38⁺ plasma cells. In the absence of IL-10, the resulting CD20⁺ CD38⁻ memory B cells continued to proliferate and retained its phenotype. The proliferation of memory B cells was interrupted by addition of IL-10 which induced the differentiation into plasma cells. The expression of CD80 and CD86 was up-regulated in the memory B cells, compared to naive B cells and plasma cells. The identity of memory B cells generated in vitro from GC B cells was further substantiated since memory B cells generated in vivo displayed the identical pattern of proliferation and differentiation under the same culture condition. These results highlight the potent role of GCT helper cells in the expansion and differentiation of memory B cells by regulating different cytokine production.     

CD40 ligand signals optimize T helper cell cytokine production: role in Th2 development and induction of germinal centers

The role of CD40 in the development of germinal centers (GC) is not simply to initiate the B cell response, as rudimentary GC can develop in CD40^−/− mice that are injected with CD40-immunoglobulin (Ig) fusion protein. This indicates that CD40 ligand (CD40L) transduces a signal to T cells that is important in the process. In this study we have used an in vitro model of GC development to investigate the role of CD40L, cytokines and other co-stimuli. The model involves the specific induction of an H-2E transgene in GC B cells (in Sma58 mice). We find that Th2 cytokines together with Ig and CD40 cross-linking are the most efficient means of induction of the GC phenotype. Although IL-4 plays some inductive role, it is not the sole active ingredient in the mix of cytokines made by Th2 cells. Our studies on primary T cells and T cell clones activated in the absence of CD40 on antigen-presenting cells or CD40L on T cells indicate that the CD40L co-stimulus does not directly bias the response to Th2 cells, as previously reported, but that it augments terminal effector T cell differentiation or the level of secretory activity. However, both in vitro and in vivo, the CD40L co-stimulus is crucially important for Th2 development as in its absence IL-4 production is suboptimal and does not compete with a larger, more rapid IFN-γ response.     

CD38 signaling by agonistic monoclonal antibody prevents apoptosis of human germinal center B cells

The present study demonstrates that an agonistic anti-CD38 monoclonal antibody (mAb) (IB4) is capable of preventing apoptosis of human tonsillar germinal center (GC) B cells as measured by either morphological methods on Giemsa-stained cytospin preparations or flow cytometry on propidium iodidestained cells. Two other anti-CD38 mAb (Leu-17 and OKT10) consistently failed to prevent apoptosis in the same cells, even when tested over a wide range of concentrations. Furthermore, exposure of GC B cells to IB4 mAb up-regulates the bcl-2 proto-oncogene product in a manner similar to that observed with CD40 ligand (CD40L). The ability of IB4 mAb to prevent apoptosis of GC B cells was inferior to that of both anti-CD40 mAb and CD40L. No synergistic or additive effects were observed when IB4 mAb was used together with CD40L. Unlike anti-CD40 mAb or CD40L, IB4 mAb neither induced a proliferation of GC B cells nor increased their proliferative response to anti-CD40, CD40L or recombinant interleukin-4, used alone or in combination. The present results are consistent with the recent findings on either the feature of the CD38 molecules to deliver activation signals and on the mechanisms of selection of B cells that operates in the GC.     

IL-2和IL-4联合诱导B细胞死亡受体CCR3的表达及其功能研究

目的　研究在IL 2和IL 4作用下 ,趋化性细胞因子受体CCR3在人生发中心 (germinalcenter,GC)B细胞上的表达及其功能特性。方法　采用流式细胞术检测人GCB细胞上CCR3表达和在CCR3配体eotaxin作用下B细胞的凋亡 ,实时定量RT PCR和Northernblot法检测GCB细胞内CCR3mRNA的表达 ,淋巴细胞趋化和黏附试验检测B细胞的趋化和黏附能力。结果　人GCB细胞极低表达趋化性细胞因子受体CCR3,经IL 2和IL 4作用后 ,GCB细胞高表达CCR3,但此时CCR3不能在其配体作用下诱导GCB细胞的趋化和黏附功能 ,而是诱导GCB细胞凋亡。结论　IL 2和IL 4联合诱导人GCB细胞CCR3表达 ,CCR3可能具有死亡受体的功能。     

Human T lymphocytes bind to germinal centers of human tonsils via integrin α4/VCAM-1 and LFA-1/ICAM−1 and −2

Binding of T lymphocytes within the different compartments of the secondary lymphoid organs is crucial for the function of the cellular and the humoral immune response. It is still not known which adhesion molecules guide T cells to the distinct areas of the lymphoid microenvironment. In the current study an in situ adhesion assay was used to define the receptors for binding of T cells to human tonsils. The T cell lines Jurkat and MOLT-4 and normal, activated T cells were found to bind exclusively to germinal centers. Jurkat cells used the receptor pair integrin-α4 (VLA-4α)/VCAM-1, whereas activated MOLT-4 cells and normal T cells bound via both adhesion pathways, namely via integrin-α4/VCAM-1 and LFA-1/ICAM-1 and -2. It is suggested that these adhesion mechanisms are involved in the migration of T cells into the germinal centers of secondary lymphoid organs and that they influence the selection of B cells by apoptosis.     

Germinal center derived malignant lymphoma in cystadenolymphoma

Summary Two cases of malignant non-Hodgkin's lymphoma arising in an yAlbrecht-Arzt-tumour are reported. In the first case a centroblastic-centrocytic lymphoma in a palatinal cystadenolymphoma of a 64-year-old female is described. In the other case a centroblastic lymphoma developed in an Albrecht-Arzt-tumor of the submandibular region in an 82-year-old man. The occurence of a high-grade malignant lymphoma in cystadenolymphoma has not been reported in the literature so far.     

A missing link in thymic dendritic cell development

Negative selection in the thymus prevents the generation of self-reactive T cells through the deletion of thymocytes with high affinity for self-antigens. Within the thymus, self-antigens are presented by thymic epithelial cells and DCs. Both cell types can mediate negative selection, although the relative contribution of each cell type remains elusive. Similar to DCs of other lymphoid organs, thymic DCs come in different flavors. Over the past years, various lines of evidence have emerged that either favor a common origin for some thymic DCs and thymocytes or, conversely, indicate the existence of separate intrathymic T lineage and DC precursors. In this issue of the European Journal of Immunology, a study reports the identification of an intrathymic DC precursor that is likely to be unrelated to the earliest physiological T-cell progenitors. Thus this intrathymic DC precursor may constitute a "missing link" between extrathymic DC precursor-types, which are able to generate DCs in secondary lymphoid organs and intrathymic DCs, and supports the notion that intrathymic DCs and thymocytes arise from different precursors.     

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.     

Local thymic caspase-9 inhibition improves survival during polymicrobial sepsis in mice

Caspase-9 is believed to play an essential role in sepsis-induced lymphocyte apoptosis. The aim of this study was therefore to evaluate its contribution within the caspase-dependent apoptosis pathway in a murine model of polymicrobial sepsis. Local injections of Z-LEHD-fmk, a specific caspase-9 inhibitor, into thymi of septic mice led to the complete inhibition of caspase-9, decreased apoptosis of resident tissue cells, and, in addition, reduced further downstream caspase-3 activity. In contrast to its systemic administration, only local injections improved the overall survival of septic mice. However, local injections of a pancaspase inhibitor (Z-VAD-fmk) did not improve survival, although caspase-3 activity was reduced to a similar degree as by the administration of Z-LEHD-fmk. These results indicate that local apoptosis of lymphatic tissue in polymicrobial sepsis is processed dependent of caspase-9 and suggests alternative caspase-dependent beneficial effects, which may determine a positive outcome.     

Re-expression of RAG-1 and RAG-2 genes and evidence for secondary rearrangements in human germinal center B lymphocytes

V(D)J recombination occurs in immature B cells within primary lymphoid organs. However, recent evidence demonstrated that the recombination activating genes RAG-1 and RAG-2 can also be expressed in murine germinal centers (GC) where they can mediate secondary rearrangements. This finding raises a number of interesting questions, the most important of which is what is the physiological role, if any, of secondary immunoglobulin (Ig) gene rearrangements. In the present report, we provide evidence that human GC B cells that have lost surface immunoglobulin re-express RAG-1 and RAG-2, suggesting that they may be able to undergo Ig rearrangement. Furthermore, we describe two mature B cell clones in which secondary rearrangements have possibly occurred, resulting in light chain replacement. The two clones carry both κ and λ light chains productively rearranged, but fail to express the κ chain on the cell surface due to a stop codon acquired by somatic mutation. Interestingly, the analysis of the extent of somatic mutations accumulated by the two light chains might suggest that the λ chain could have been acquired through a secondary rearrangement. Taken together, these data suggest that secondary Ig gene rearrangements leading to replacement may occur in human GC and may contribute to the peripheral B cell repertoire.     

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.     

Accumulation of somatic hypermutation and antigen-driven selection in rapidly cycling surface Ig+ germinal center (GC) B cells which occupy GC at a high frequency during the primary antihapten response in mice

Well-developed germinal centers (GC) contain rapidly dividing surface immunoglobulin-negative (sIg^-) B cells (centroblasts), and most of their progeny are sIg⁺ B cells (centrocytes) in a resting state. It has been predicted that somatic hypermutation occurs in centroblasts, whereas antigen-driven selection takes place in centrocytes. The present analysis indicates that murine GC B cells bearing sIg with specificity for an immunizing antigen are in a rapidly cycling state and increase exponentially in number to occupy spleen GC at high frequency during the 1st week after primary immunization; however, the number of these cells is significantly reduced in the 2nd week of immunization. During that period, these proliferating sIg⁺ GC B cells accumulate somatic hypermutations with nucleotide exchanges indicative of affinity maturation. These sIg⁺ GC B cells co-express B7-2, ICAM-1, and LFA-1, and have potent antigen-presenting activity which results in T cell activation in vitro. These observations indicate that the sIg⁺ GC B cells accumulate somatic hypermutations and undergo antigen-driven selection through proliferation, probably upon activation by T cells. This sIg⁺ GC B cell population may represent cell cycling centrocytes; however, the possibility that these may represent centroblasts undergoing re-expression of sIg could not be excluded.     

胸腺增龄性萎缩分子机制研究进展

胸腺是哺乳动物重要的中枢免疫器官,是决定和制约免疫系统老化的关键.胸腺作为T 细胞分化、发育、成熟重要场所,随着增龄表现出明显萎缩的现象进而影响到T细胞的发育、增殖和输出,从而造成外周淋巴组织中T细胞的质与量的异常,这样就不可避免地降低了免疫系统对外来的抗原应答性,导致病原体清除延迟,增加了老年人罹患癌症、自身免疫性疾...     

Thymic function in young/old chimeras: substantial thymic T cell regenerative capacity despite irreversible age-associated thymic involution

Age-associated thymic involution results in a diminished capacity to regenerate T cell populations, although the magnitude of this effect is unknown. In this report, thymic function was studied in aged vs.young adult mice after lethal irradiation and administration of T cell-depleted bone marrow (BM) from young mice. Abnormalities observed in aged thymi (reduced thymocyte numbers, histologic abnormalities) were not reversed by administration of young BM via bone marrow transplantation (BMT), but agend thymi displayed a normal thymocyte subset distribution and appropriately deleted Mls-reactive T cells after BMT. Aged BMT recipients regenerated significantly reduced numbers of splenic T cells compared to young recipients and showed increased peripheral expansion of thymic emigrants since a higher proportion of BM-derived T cells expressed a memory phenotype in aged vs.young BMT recipients. Because peripheral expansion of thymic emigrants could substantially increase the number of thymic progeny present in the spleen, we sought to measure thymic T cell regenerative capacity after BMT in a setting devoid of peripheral expansion. To do this, TCR-transgenic (Tg⁺) T cell-depleted BM was administered to aged and young recipients lacking antigen specific for the Tg⁺ TCR. Aged recipients regenerated approximately 50 % of the TCR Tg⁺ cells regenerated in young BMT recipients, providing evidence that even very aged thymi retain the capacity to regenerate significant numbers of mature T cell progeny. Therefore, thymic function is reduced with aged but it is not lost, suggesting that therapeutic approaches to enhance thymic function may be successful even in very aged hosts.