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.     

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.     

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.     

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.     

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 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.     

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.     

Suppression of apoptosis in normal and neoplastic human B lymphocytes by CD40 ligand is independent of Bcl-2 induction

The tendency of isolated germinal center (GC) B cells to undergo apoptosis was suppressed by recombinant cell-bound CD40 ligand (CD40L): after 2 days at 37°C, > 80 % of cells remained viable in the presence of CD40L as compared to < 1 % in control cultures. CD40L sustained a high rate of DNA synthesis in GC cells and was more effective than monoclonal antibody to CD40 in this regard. Group I Burkitt lymphoma (BL) cell lines induced to undergo apoptosis with anti-immunoglobulin or calcium ionophore were also protected by CD40L. In BL cells, this route of rescue was not accompanied by induction of Bcl-2 protein, the expression of which has been linked to hemopoietic cell survival. Bcl-2 was induced in GC cells responding to CD40L, but its appearance was a relatively late event not reaching significant levels over controls until day 2 of culture. Thus induction of Bcl-2 appears to be secondary to the survival signal imparted by CD40L. These findings are discussed in relation to a potential role for CD40L in supporting B cell tumors in vivo and the discovery that the molecular defect in the X-linked Hyper-IgM syndrome is targeted to the CD40L gene.     

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。     

抗人CD38单克隆抗体的制备、鉴定及其功能的初步研究

目的：研制抗人CD38抗原分子的单克隆抗体，进一步研究其生物学功能。方法：采用高表达CD38抗原的Daudi细胞免疫Balb/c小鼠；取其脾细胞与SP2/0细胞融合；用间接免疫荧光法进行杂交瘤筛选；流式细胞术、免疫沉淀法与CD38分子原核表达产物的Western-blot分析鉴定单克隆抗体的特异性。以MTT（四甲偶氨唑盐）法检测单克隆抗体抑制细胞增殖以及介导补体杀伤靶细胞的功能。结果：获得了一株抗人CD38分子的单克隆抗体1C6，流式细胞术显示它具有CD38抗原特异的细胞反应谱，其识别的抗原分子质量为45000u。与CD38分子原核表达产物的Western-blot分析表明，其可特异识别CD38分子胞外结构域。MTT法显示其可介导补体杀伤靶细胞。结论：成功制备了抗人CD38分子的单克隆抗体，并进行了初步的功能研究。     

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.     

Expression of CD5 and CD38 by human CD5− B cells: Requirement for special stimuli

In this study the mode of expression of CD5 by human tonsillar CD5^? B cells after stimulation with different agents was investigated. Resting B cells were separated into CD5⁺ and CD5^? cells and the two cell fractions exposed to phorbol 12-myristate 13-acetate (PMA). CD5^? B cells expressed CD5 and maximum CD5 expression was achieved after approximately 60 h of culture. Based upon the proportions of cells that express CD5 as well as those of the cells surviving in culture, it was calculated that 15–25 % of the total CD5^? B cells were induced to express CD5. Unlike CD5^? B cells, CD5⁺ B cells proliferated vigorously in response to PMA as assessed by [³H] thymidine incorporation and cell cycle analysis in vitro. However, the expression of CD5 by CD5^? B cells was not related to the selective expansion of some CD5⁺ B cells left over as contaminant cells since this occurred in the absence of cell proliferation. Upon exposure to PMA, CD5^? B cells remained in the G0-G1 phases of the cell cycle and did not express the Ki67 antigen or incorporate [³H] thymidine. Furthermore, mitomycin C treatment of the CD5^? B cells did not prevent CD5 expression. Phenotypic studies disclosed that CD5⁺ B cells but not CD5^? B cells expressed CD39. This finding offered the opportunity to carry out an additional control experiment. Separation of the two populations according to the expression of CD39 confirmed the finding obtained by fractionating the cells into CD5⁺ and CD5^? B cells. The cells induced to express CD5 also expressed CD38 that was not detected on resting CD5^? B cells. In this respect, the CD5^? B cells that converted into CD5⁺ cells (inducible CD5⁺ B cells) resembled the cells from the CD5⁺ B cell fractions that up-regulated CD5 and also expressed CD38 upon exposure to PMA alone. Another example of coordinate expression of these two antigens was the finding that exposure to PMA in the presence of recombinant interleukin-4 (rIL-4) resulted in inhibition of the expression of CD5 and CD38. Although virtually all of the tonsillar CD5^? B cells expressed the CD69 activation marker, no cells other than those co-expressing CD5 and CD38 were induced to express CD5 by PMA alone. Resting CD5^? B cells failed to express CD5 and/or CD38 when cultured with PMA in the presence of EL4 T cells and IL-4-free T cell supernatants. Although this combination of stimuli induced a vigorous cell proliferation, the failure to express CD5 and CD38 was not related to cell cycling, since mitomicyn C-treated CD5^? B cells also failed to express CD5 or CD38 when exposed to PMA in the presence of EL4 cells with or without T cell supernatants. Thus, exposure to T cells alone was sufficient to down-regulate CD5 and CD38 expression. Collectively, the above findings indicate that mature CD5^? B cells can follow distinct pathways of differentiation depending upon the nature of the stimuli encountered, and that CD5 expression may identify a special B cell subset or a particular stage of B cell differentiation.     

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.     

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.     

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.     

Establishment of a monoclonal antibody to plasma cells: a comparison with CD38 and PCA-1

A new monoclonal antibody which recognizes plasma cells was developed by utilizing two myeloma cell lines, KMS12PE (12PE) and KMS12BM (12BM). established from the pleural of fusion and bone marrow, respectively, of the same patient. Since I2BM expresses CD20, CD38, and PCA-I antigens, while 12PE has lost CD20. 12PE is considered to be phenotypically more mature than 12BM. The 12PE cells were used to immunize a BALB/c mouse and a MoAb was produced which was more reactive to 12PE than to 12BM. Thus, a clone, D2, was obtained. On Western blotting, D2 detected a single band of 54 kD under both reduced and non-reduced conditions. This antigen was not detected by Western blotting in peripheral blood lymphocytes that had been stimulated with pokeweed mitogen (PWM) for 7 days or in those not so stimulated. On flow cytometry, D2 detected a myeloma cell line, RPMI 8226. Another myeloma cell line, U266, was negative for D2 antigen. Staining various cell lines by D2 and other antiplasma cell antibodies, PCA-1 and CD38, showed that D2 is distinct from PCA-1 and CD38. The fresh myeloma cells of 14 myeloma patients were stained by D2 and for other plasma cell antigens. D2 strongly stained three samples obtained from patients with clinically aggressive myeloma, while CD38 stained all cases except one. PCA-1 was positive in nine samples and negative in five. PCA-1 expression was observed in plasma cells obtained from pleural effusion and peripheral blood, while PCA-1-negative cases were not found in such samples, suggesting that PCA-I expression was related to extramedullary invasion. The morphology of the myeloma cells, classified according to Greipp's criteria, showed that there was no correlation between plasma cell antigen expression and plasma cell morphology. Analysis of D2 antigen expression should provide more information about the heterogeneity of myeloma cells.     

p38 MAPK介导E1A激活基因阻遏子蛋白调控人血管内皮细胞凋亡

目的: 研究E1A激活基因阻遏子蛋白(CREG)在依托泊苷(VP-16) 诱导的人脐静脉内皮细胞 (HUVECs) 凋亡中的作用及其调控机制。方法: 将体外培养的原代HUVECs用pLNCX-CREG和pLXSN-shRNA-CREG反转录病毒真核表达载体分别转染,通过G418 (800 mg/L) 和嘌呤霉素 (2.5 mg/L) 筛选分别获得CREG过表达组HUVECs (H-C) 和CREG低表达组HUVECs (H-S)。在H-C、HUVECs (H) 和H-S 3种细胞模型中加入凋亡诱导剂VP-16 (100 μmol/L,6 h),用TUNEL法和流式细胞术检测细胞的凋亡情况;用Western blotting检测细胞中磷酸化p38 MAPK (p-p38 MAPK) 的蛋白表达量;应用p38特异性抑制剂SB203580 (20 μmol/L) 预处理H-S细胞后,检测VP-16刺激诱导的细胞凋亡情况。结果: HUVECs经pLNCX-CREG和pLXSN-shRNA-CREG反转录病毒真核表达载体转染获得稳定的细胞克隆。经 Western blotting证实,与HUVECs对照比较,H-C组细胞CREG表达明显增加至160%,而H-S组细胞中的CREG表达下降约70%。加入凋亡诱导剂VP-16 (100 μmol/L, 6 h) 后,TUNEL和流式双荧光染色检测结果均提示H-S中的细胞凋亡率明显升高,而H-C组细胞凋亡率较HUVECs对照组明显降低,两者具有显著差异。进一步应用Western blotting分析显示经VP-16刺激后,H-S细胞中p-p38蛋白表达较HUVECs和H-C组细胞显著增加;在H-S中添加SB203580 (20 μmol/L) 后,VP-16诱导的细胞凋亡现象被显著抑制。结论: CREG 过表达可能通过抑制p38 MAPK的激活阻遏VP-16诱导的HUVECs凋亡。     

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.     

激动型CD40单克隆抗体体外抑制结肠癌细胞增殖的实验研究

目的 探讨激动型CD40单克隆抗体在体外对结肠癌细胞增殖的抑制作用.方法 树突状细胞(DCs)经结肠癌冻融抗原致敏后予以不同条件激活,分为激动型CD40单克隆抗体组、阴性对照组及肿瘤坏死因子-α(TNF-α)阳性对照组,诱导培养至第7天,用流式细胞仪检测各组DCs表面分化相关抗原CD80、CD83、CD86和HLA-DR的表达,酶联免疫吸附测定法检测DCs培养液上清中白细胞介素-12(IL-12)的质量浓度,噻唑蓝比色法检测DCs体外刺激T淋巴细胞增殖的能力,进而检测DCs所诱导的肿瘤特异性细胞毒性T淋巴细胞(CTL)对人结肠癌细胞HCT116的杀伤作用.结果 与阴性对照组相比,激动型CD40单克隆抗体组活化的DCs表面抗原CD80、CD83、CD86和HLA-DR的表达率均显著升高(均P＜0.05),DCs上清中IL-12的质量浓度亦显著升高((716.80±53.43) pg/ml比(405.51±12.17) pg/ml,P＜0.05),活化的DCs具有更强的刺激T淋巴细胞增殖的能力(刺激指数2.006 2±0.438 3比1.365 0±0.209 8,P＜0.05),活化的DCs所诱导的CTL对HCT116细胞具有更强的杀伤作用(抑制率(66.08±0.41)％比(46.60±1.10)％,P＜0.05);而与TNF-α阳性对照组相比,其差异均无统计学意义(均P＞0.05).结论 激动型CD40单克隆抗体在体外可促进DCs的活化与成熟,进而诱导肿瘤特异性CTL的产生,从而抑制人结肠癌细胞HCT116的增殖.     

Lymphotoxin α1β2 expression on B cells is required for follicular dendritic cell activation during the germinal center response

CD19‐deficient mice were used as a model to study follicular dendritic cell (FDC) activation because these mice have normal numbers of FDC‐containing primary follicles, but lack the ability to activate FDCs or form GCs. It was hypothesized that CD19 expression is necessary for B‐cell activation and upregulation of membrane lymphotoxin (mLT) expression, which promotes FDC activation. Using VCAM‐1 and FcγRII/III as FDC activation markers, it was determined that the adoptive transfer of CD19⁺ wild‐type B cells into CD19‐deficient hosts rescued GC formation and FDC activation, demonstrating that CD19 expression on B cells is required for FDC activation. In contrast, CD19⁺ donor B cells lacking mLT were unable to induce VCAM‐1 expression on FDCs, furthermore FcγRII/III upregulation was impaired in FDCs stimulated with mLT‐deficient B cells. VCAM‐1 expression on FDCs, but not FcγRII/III, was rescued when CD19‐deficient B cells expressing transgenic mLT were cotransferred into recipient mice with CD19⁺, mLT‐deficient B cells, suggesting that FDC activation requires the CD19‐dependent upregulation of mLT on activated B cells. Collectively, these data demonstrate that activated B cells are responsible for the initiation of FDC activation resulting in a microenvironment supportive of GC development and maintenance.