Failure to down-regulate Bcl-2 protein in thymic germinal center B cells in myasthenia gravis

The most unusual characteristic of myasthenia gravis (MG) is that the thymus has germinal centers (GC). Cultured thymic lymphocytes from MG patients spontaneously produce anti-acetylcholine receptor antibodies, indicating that autoreactive B cells have escaped negative selection. To investigate the underlying mechanism, we examined the expression of the apoptosis-related protein Bcl-2 in GC B cells (defined as CD19⁺ CD38⁺ cells) in the thymus in 14 MG patients using three-color flow cytometry. GC in MG patients did not show the normal down-regulation of Bcl-2 (the frequency of Bcl-2⁺ GC B cells in the MG thymus and in control tonsils 54.3 ± 16.2% versus 20.6 ± 8.0%; mean ± SD, p < 0.0001). In contrast, Bcl-2 in GC in the mediastinal lymph nodes from four patients was down-regulated to a relatively normal level. Using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method to detect DNA fragmentation in situ, the frequency of TUNEL⁺ cells in GC in the MG thymus was lower than in control tonsils. These results suggest that autoreactive B cells which normally undergo apoptosis in GC may survive because of Bcl-2 up-regulation in this unusual location.     

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.     

Langerhans cells promote early germinal center formation in response to Leishmania‐derived cutaneous antigens

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen‐primed CD4⁺ follicular helper T cells (T_FH). A tight and stable formation of T_FH/B cell conjugates is required for cytokine‐driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of T_FH/B cell conjugates is crucial for B‐cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for T_FH‐cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin⁺ DC subsets in vivo, we show that the functionality of T_FH/B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC‐depleted mice show a reduction in somatic hypermutation in B cells isolated from T_FH/B cell conjugates and markedly reduced GC reactions within skin‐draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B‐cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.     

Antigen receptor-induced apoptosis of human germinal center B cells is targeted to a centrocytic subset

The outcome of the signals transduced through the B cell antigen receptor (BCR) depends both on their maturational stage and on the extent of receptor cross-linking. It is established that the BCR-mediated apoptosis of immature B cells represents an important mechanism for tolerance induction in the pre-immune B cell compartment. We show here that mature germinal center (GC) B cells can re-acquire sensitivity to BCR-induced cell death following CD40 ligation. In contrast, neither virgin nor memory B cells become susceptible to antigen receptor-triggered apoptosis upon CD40 stimulation, suggesting that this phenomenon may play a role in the shaping of the mature B cell repertoire in GC. Our data reveal that the death signal evoked through the BCR does not involve the Fcγ receptors, does not operate through the Fas/Fas ligand system, and can be blocked by interleukin-4. Finally, we found that the acquisition of sensitivity to the death-promoting effect of anti-Ig antibodies in CD40-stimulated GC B cell cultures correlates with the induction of a centrocytic phenotype. We propose that negative regulatory signals via the BCR may delete somatically mutated centrocytes with self-reactivity.     

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.     

Plasma cell differentiation during the germinal center reaction

Germinal centers (GCs) are formed in secondary lymphoid tissues upon immunization with T‐dependent antigens. In GCs, somatic hypermutation generates B cells with increased antibody affinity and these high‐affinity B cells preferentially differentiate into plasma cells, which home to bone marrow and confer long‐lived humoral immunity. Recent studies have shed new light on the cellular and molecular basis for initiating the transition from a GC B cell to a plasma cell. Here, we review recent progress in our understanding of how plasma cell generation during the GC reaction is regulated for inducing effective long‐term protective immunity and for preventing harmful autoimmunity.     

Disparate peptide-dependent thymic selection outcomes in beta2M-deficient mice versus TAP-1-deficient mice: implications for repertoire formation

Fetal thymic organ cultures of N15-transgenic RAG-2-/- H-2b mice on normal, beta-2 microglobulin (beta2M)-/- or transporter associated with antigen processing (rAP-1)-/- MHCl-deficient backgrounds were used to examine differentiation of thymocytes bearing a TCR specific for a viral peptide bound to H-2Kb. Strong agonists mediate negative selection in all mice whereas weak agonists are positively selecting in beta2MW-/- mice but negatively selecting on TAP-1-/- or normal backgrounds. Very weak agonists and very weak antagonists are generally without effect in beta2M-/- mice yet foster differentiation in TAP-1-/- animals. The 20-40-fold reduction in beta2M4-/- thymic H-2Kb surface expression suggests that the avidity of the TCR for peptide-MHCI accounts for these differences, consistent with effects of TCR density and individual thymic-peptide abundance in peptide-MHC complexes. TCR-self-MHC interaction dominates Kb-based selection, subtly modulated by peptides as revealed by X-ray crystallography.     

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.     

Swiprosin‐1/EFhd2 limits germinal center responses and humoral type 2 immunity

Activated B cells are selected for in germinal centers by regulation of their apoptosis. The Ca²⁺‐binding cytoskeletal adaptor protein Swiprosin‐1/EFhd2 (EFhd2) can promote apoptosis in activated B cells. We therefore hypothesized that EFhd2 might limit humoral immunity by repressing both the germinal center reaction and the expected enhancement of immune responses in the absence of EFhd2. Here, we established EFhd2^?/? mice on a C57BL/6 background, which revealed normal B‐ and T‐cell development, basal Ab levels, and T‐cell independent type 1, and T‐cell independent type 2 responses. However, T cell‐dependent immunization with sheep red blood cells and infection with the helminth Nippostrongylus brasiliensis (N.b) increased production of antibodies of multiple isotypes, as well as germinal center formation in EFhd2^?/? mice. In addition, serum IgE levels and numbers of IgE⁺ plasma cells were strongly increased in EFhd2^?/? mice, both after primary as well as after secondary N.b infection. Finally, mixed bone marrow chimeras unraveled an EFhd2‐dependent B cell‐intrinsic contribution to increased IgE plasma cell numbers in N.b‐infected mice. Hence, we established a role for EFhd2 as a negative regulator of germinal center‐dependent humoral type 2 immunity, with implications for the generation of IgE.     

A roadmap for thymic epithelial cell development

Thymic epithelial cells (TEC) are essential components of the thymus that guide and control the development and TCR repertoire selection of T cells. Previously, TEC have been considered as postmitotic cells that, once generated during ontogeny, were maintained in their mature state. Recently, it has become clear that TEC can be generated from common or committed medullary and cortical TEC progenitor cells in ontogeny, that stages of immature and mature TEC are phenotypically separable, and that TEC undergo a rapid turnover in a matter of a few weeks. All of these findings strongly suggest that in the adult thymus mature TEC are constantly regenerated from a pool of stem or progenitor cells, a view that renders the thymus structure potentially much more dynamic than previously thought. However, the identity of “thymus stem cells” is elusive, and developmental stages of TEC development are only beginning to be elucidated.     

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。     

Antigen targeting reveals splenic CD169+ macrophages as promoters of germinal center B‐cell responses

Ag delivery to specific APCs is an attractive approach in developing strategies for vaccination. CD169⁺ macrophages in the marginal zone of the spleen represent a suitable target for delivery of Ag because of their strategic location, which is optimal for the capture of blood‐borne Ag and their close proximity to B cells and T cells in the white pulp. Here we show that Ag targeting to CD169⁺ macrophages in mice resulted in strong, isotype‐switched, high‐affinity Ab production and the preferential induction and long‐term persistence of Ag‐specific GC B cells and follicular Th cells. In agreement with these observations, CD169⁺ macrophages retained intact Ag, induced cognate activation of B cells, and increased expression of costimulatory molecules upon activation. In addition, macrophages were required for the production of cytokines that promote B‐cell responses. Our results identify CD169⁺ macrophages as promoters of high‐affinity humoral immune responses and emphasize the value of CD169 as target for Ag delivery to improve vaccine responses.