人BAFF-R基因启动子活性的初步研究

目的以B淋巴细胞刺激因子受体BAFF-R基因5'上游区序列为研究对象,初步鉴定、分析该基因的启动子所在区域。方法克隆BAFF-R基因5'侧翼区1823 bp序列,构建8个含有不同长度启动子序列的荧光素酶报告基因表达质粒pGL3-B1～B8,将这8个序列缺失重组质粒与psv-β-gal质粒共转染细胞,检测荧光素酶的相对活性,确定启动子所在区域。结果8个重组质粒中pGL3-B2、pGL3-B3、pGL3-B5、pGL3-B6启动子的活性较低;pGL3-B7启动子的活性最强,pGL3-B8启动子活性最低。结论BAFF-R基因5'端-288～-430、-712～-868和-1420～-1562三个区段可能存在转录沉默子元件,-617～-712和-1277～-1420区段存在转录增强子元件,BAFF-R基因的核心启动子可能位于-1420～+261区域。     

人B细胞活化因子基因启动子活性研究

为探索与自身免疫病发病密切相关的B细胞活化因子(BAFF)基因高水平转录的启动序列及IFN-γ等炎性细胞因子对其活性的影响,以人BAFF基因转录起始点上游-1 349 bp至-329 bp的片段为靶序列,取长度不等的片段作为启动子与含氯霉素乙酰基转移酶(CAT)报告基因的质粒组成5个重组体,脂质体转染法转染上述重组体至人骨髓白血病细胞株:HL-60,CAT-ELISA测定细胞CAT表达水平以比较各重组体的启动子活性;同时加入细胞因子IL-10、IFN-γ、IL-4、重组人BAFF蛋白(rBAFF),以测定其对BAFF启动序列的影响。结果表明,-1 349~-329 bp、-1 349~-743 bp序列具有强启动调控活性,而-1 349~-1 099 bp片段启动活性最低。细胞因子IFN-γ、IL-10和rBAFF均能在一定程度上调人BAFF基因的启动活性,IL-4则一定程度地抑制BAFF基因的启动活性。研究提示,人BAFF基因-1349~-743 bp片段是进一步研究BAFF基因转录相关DNA结合蛋白的重要调控靶序列。细胞因子IL-10、IFN-γ、IL-4、rBAFF可以一定程度的在转录水平影响BAFF合成和分泌。     

异基因骨髓源间充质干细胞对BXSB小鼠T、B细胞的影响

目的：探讨异基因骨髓源间充质干细胞（Bonemarrow derived mesenchymal stem cells，BMSCs）对BXSB小鼠T、B细胞增殖、活化及功能成熟等方面的影响。方法：采用。H．TdR掺入法、FACS法、EIJSA等方法检测BMSCs对BXSB小鼠T、B细胞的影响。结果：BALB／c小鼠的BMSCs在不影响BXSB小鼠T细胞诱导活化的基础上抑制其诱导增殖；可降低由ConA诱导的CD4^＋IL^＋细胞的数量，而提高由ConA诱导的CD4^＋IFN-γ^＋细胞数量。对于B细胞，BALB／c小鼠的BMSCs可以抑制其增殖、活化及IgG的分泌。另外，BALB／c小鼠的BMSCs可以抑制BXSB小鼠B细胞上CIMOL的异位表达。结论：异基因BMSCs对自身免疫病小鼠的T、B细胞有一定的调节作用。     

逆转录病毒介导的小鼠IL-23基因在鼠高转移性乳腺癌细胞系MA-891中的表达

目的：建立表达小鼠IL-23基因的鼠乳腺癌细胞系IL-23／MA-891，探讨外源性IL-23基因对MA-891细胞生长及其生物学性状的影响。方法：将插入IL-23基因的质粒，应用逆转录病毒载体LXSN经感染皿（ecotropic）和PA317（amphotropic）两种包装细胞包装，经G418筛选获得表达IL-23分子的PA317阳性细胞克隆，用IL-23／PA317培养上清转染MA-891细胞，获得表达IL-23的IL-23／MA-891细胞。分别用RT-PCR法、ELISA法和免疫组化染色法分析IL-23／MA-891细胞表达IL-23的mRNA和蛋白的水平，筛选出高表达IL-23的IL-23／MA-891细胞克隆用于下层研究中；用流式细胞仪检测MA-891细胞中MI-ICⅠ、MI-ICⅡ、CD80、CD86以及FAS蛋白的表达、细胞周期变化及细胞凋亡情况；用ELISA法检测IL-23／MA-891细胞培养上清诱导脾细胞分泌IFN-γ的能力，用MTT比色法检测细胞增殖反应。结果：外源性IL-23基因转染的小鼠乳腺癌细胞系IL-23／MA-891，在mRNA水平和蛋白水平均可获得稳定表达；IL-23／MA-891细胞与LXSN／MA-891和MA-891比较，细胞周期及细胞凋亡率无显著性差异（P〉0．05），H-2Kb（MHCⅠ类分子）、Ⅰ—Ab（MHCⅡ／类分子）、CD80、CD86以及FAS蛋白的表达水平无显著性差异（P〉0．05）；IL-23／MA-891细胞的增殖反应与LXSN／MA-891和MA-891细胞相比虽有所降低，但无显著性差异（P〉0．05）。然而，IL-23／MA-891细胞的培养上清可明显促进小鼠脾细胞分泌IFN-γ（P〈0．01）。结论：建立的稳定表达IL-23的IL-23／MA-891细胞，具有较强的免疫学调节活性，其生物学形状与MA-891和LXSN／MA-891细胞相比较没有明显差异，但可进一步用于肿瘤相关免疫基因治疗的研究。     

Control of B-cell maturation in mice. I. Increased B-cell maturation in vitro by bone marrow protected during whole body irradiation.

The possibility of a homeostatic control on the production of B cells was studied in CBA mice following whole body irradiation (750 rads). Bone marrow cells from femurs shielded from irradiation were taken at 24 h and the number of surface immunoglobulin positive cells assessed with a fluorescence-activated cell sorter after 24 h in vitro. The cells from the irradiated shielded mice showed greater absolute number of 'bright' B cells with a high density of surface immunoglobulin (mean increase 60%--100%) than cells from control unirradiated mice. These bright B cells did not incorporate (3H) thymidine in vitro and treatment with hydroxyurea (an inhibitor of DNA synthesis) did not prevent their increase. It was concluded that the increased number of bright B cells in vitro arose from augmented maturation or differentiation and not from a proliferative process.     

Differential signalling during B-cell maturation

The molecular mechanism by which the antigen receptors (BCR) on B cells can elicit differential maturation state-specific responses is one of the central problems in B-cell differentiation yet to be resolved. Indeed, many of the early signalling events detected following BCR ligation, such as activation of protein tyrosine kinases (PTK), phospholipase C (PLC), phosphoinositide-3-kinase (PI 3K), protein kinase C (PKC) and the RasMAPK (mitogen activating protein kinase) signalling cascades are observed throughout B-cell maturation. However, it is becoming clear that the differential functional responses of these BCR-coupled signals observed during B-cell maturation are dependent on a number of parameters including signal strength and duration, subcellular localisation of the signal, maturation-restricted expression of downstream signalling effector elements/isoforms and modulation of signal by co-receptors. Thus, the combined signature of BCR signalling is likely to dictate the functional response and act as a developmental checkpoint for B-cell maturation.     

Chromosomal localization of the gene for human B-cell antigen CD40

CD40 is a surface glycoprotein expressed on all human B lymphocytes and plays an important role in B-cell development, growth, and differentiation. Anti-CD40 monoclonal antibodies cause isotype switching in B cells treated with IL-4. CD40 is a member of a family of proteins that include low-affinity nerve growth factor receptor, TNF receptor, and the antigen Fas. The ligand for CD40 had been recently identified and has been assigned to the X chromosome. Using a panel of human-rodent somatic cell hybrids, we now show that CD40 maps to human chromosome 20.     

T- and B-cell epitopes in the secreted Mycobacterium bovis antigen MPB70 in mice

MPB70 is a soluble secreted protein highly expressed in Mycobacterium bovis and strains of bacille Calmette-Guérin (BCG); as such, it is a candidate for subunit and DNA vaccines against tuberculosis. MPB70 was screened for T-cell epitopes in four different inbred mouse strains. Major histocompatibility complex (MHC) H-2b-expressing mice (C57BL/6) secreted interferon-gamma (IFN-gamma) after stimulation with peptides from the regions 1-20, 41-50, 81-110, 121-150 and 161-193 of the MPB70 sequence. H-2db mouse (B6D2) splenocytes secreted IFN-gamma after stimulation with some of the same peptides, whereas H-2d mice (BALB/c and DBA/2) did not secrete IFN-gamma upon stimulation with the peptides. Sera from H-2db mice immunized with native MPB70 in incomplete Freund's adjuvant (IFA), mpb70 DNA or live BCG Moreau were found to contain antibodies against the native MPB70 antigen. H-2db mice immunized with native MPB70 in IFA exhibited high titres of peptide-reactive immunoglobulin G1 (IgG1) antibodies, whereas DNA-immunized mice reacted with IgG2a antibodies against some of the same peptides. As some of the epitopes recognized by mouse T and B cells have previously been found to stimulate immune responses in humans, cattle and rabbits, we conclude that these epitopes may be good general epitopes for the stimulation of T- and B-cell responses and candidates for a DNA vaccine with a broad applicability.     

MHC-restricted B-cell antigen presentation in memory B-cell maintenance and differentiation

Memory B-cell development, maintenance, and differentiation have been believed to be tightly regulated by T cells through major histocompatibility complex (MHC)-II-restricted cognate interaction. However, recent studies have indicated that memory B cells are a heterogeneous population with various ontogeny and with derivations from T-cell-independent as well as T-cell-dependent response. In addition, several studies highlight T-cell-independent maintenance and differentiation of memory B cells by innate polyclonal stimuli associated with Toll-like-receptors (TLRs). Thus, both T-cell-dependent and T-cell-independent mechanisms with possibly different physiological functions are required for maximal memory B-cell maintenance and differentiation. In this review, we discuss the mechanisms of memory B-cell maintenance and differentiation by re-examining the requirement for MHC-II-restricted B-cell antigen presentation based on our recent study in a mouse model that lacks MHC-II on memory B cells. We propose that MHC-II-restricted memory B-cell maintenance and differentiation are the indispensable mechanisms by which we achieve health and the maximum quality of memory response with specific antibody production, while preventing plasma cell differentiation from self-reactive memory B cells under the control of T-helper cells and possibly regulatory T cells.     

Studies on B-cell memory. I. Generation and exhaustion of B-cell memory by thymus-dependent antigen in T-cell depleted mice.

Mice depleted of T cells by adult thymectomy, X-irradiation and reconstitution with syngeneic bone marrow cells, either untreated or treated with anti-Thy-1 serum and complement, were immunized intensively with alum-precipitated bovine serum albumin (AP-BSA) along with or without bacterial lipopolysaccharide (LPS), but no significant anti-BSA antibody response was detected. Priming of the T-cell depleted mice, however, either by a single injection of AP-BSA plus LPS or by multiple injections of AP-BSA without LPS, resulted in the generation of immunological memory. A single injection of AP-BSA without LPS was ineffective. The memory required the aid of syngeneic T cells to be recalled by the challenge with AP-BSA plus LPS. On the other hand, multiple injections of AP-BSA plus LPS did not cause the generation of memory and the response of these mice to the challenge was lower than that of unprimed control mice. These results suggest that (1) the anti-BSA response is highly dependent on the helper function of T cells, (2) the degree of T-cell requirement for the memory generation is very low, and (3) priming with too much strong stimulation in the absence of functional T cells leads to the suppression or abortion of previously generated immunological memory.     

DRC antigen expression in B-cell lymphomas

DRC (dendritic reticulum cell) antigen expression was studied in 38 cases of B-cell lymphomas including follicular lymphoma. The results of this study showed DRC-1 to be expressed in 1/3 of small lymphocytic; 3/3 of mantle zone lymphoma (MZL); 10/10 of follicular, small cleaved; 6/7 of follicular, mixed; 1/2 of follicular, large cell lymphomas. However, DRC-1 was not expressed in any of diffuse, small cleaved (0/6) and diffuse, large cell (0/6). Although S-100 protein was positive in the majority of these DRC-1-positive cases on the paraffin embedded specimens, positive nodules were less intense and smaller in number compared with those of DRC-1 on frozen tissue specimens. These results suggest that in case of small lymphocytic lymphoma found to be positive for DRC-1, the networks of the DRCs are expressed in the pseudofollicular proliferation centers. This study also suggests that the networks of the DRCs newly appear accompanying the neoplastic growth rather than originating from residual germinal centers, and that neoplastic small cleaved cells play a major role in inducing the DRCs in the positive cases of follicular lymphoma, MZL, and small lymphocytic lymphoma.     

Interleukin-5 regulates genes involved in B-cell terminal maturation

Interleukin (IL)-5 induces CD38-activated splenic B cells to differentiate into immunoglobulin M-secreting cells and undergo micro to gamma 1 class switch recombination (CSR) at the DNA level, resulting in immunoglobulin G1 (IgG1) production. Interestingly, IL-4, a well-known IgG1-inducing factor does not induce immunoglobulin production or micro to gamma 1 CSR in CD38-activated B cells. In the present study, we implemented complementary DNA microarrays to investigate the contribution of IL-5-induced gene expression in CD38-stimulated B cells to immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR. IL-5 and IL-4 stimulation of CD38-activated B cells induced the expression of 418 and 289 genes, respectively, that consisted of several clusters. Surprisingly, IL-5-inducible 78 genes were redundantly regulated by IL-4. IL-5 and IL-4 also suppressed the gene expression of 319 and 325 genes, respectively, 97 of which were overlapped. Genes critically regulated by IL-5 include immunoglobulin-related genes such as J chain and immunoglobulinkappa, and genes involved in B-cell maturation such as BCL6, activation-induced cytidine deaminase (Aid) and B lymphocyte-induced maturation protein-1 (Blimp-1) and tend to be induced slowly after IL-5 stimulation. Intriguingly, among genes, the retroviral induction of Blimp-1 and Aid in CD38-activated B cells could induce IL-4-dependent maturation to Syndecan-1+ antibody-secreting cells and micro to gamma 1 CSR, respectively, in CD38-activated B cells. Taken together, preferential Aid and Blimp-1 expression plays a critical role in IL-5-induced immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR in CD38-activated B cells.