Regulation of Anti-DNA B Cells in Recombination-activating Gene–deficient Mice

Anti-DNA antibodies are regulated in normal individuals but are found in high concentration in the serum of systemic lupus erythematosus (SLE) patients and the MRL lpr/lpr mouse model of SLE. We previously studied the regulation of anti–double-stranded (ds)DNA and anti–single-stranded (ss)DNA B cells in a nonautoimmune background by generating mice carrying immunoglobulin transgenes coding for anti-DNAs derived from MRL lpr/lpr. Anti-dsDNA B cells undergo receptor editing, but anti-ssDNA B cells seem to be functionally silenced. Here we have investigated how anti-DNA B cells are regulated in recombination- activating gene (RAG)-2^−/− mice. In this setting, anti-dsDNA B cells are eliminated by apoptosis in the bone marrow and anti-ssDNA B cells are partially activated.     

Human Immunoglobulin (Ig)M+IgD+ Peripheral Blood B Cells Expressing the CD27 Cell Surface Antigen Carry Somatically Mutated Variable Region Genes: CD27 as a General Marker for Somatically Mutated (Memory) B Cells

Immunoglobulin (Ig)M⁺IgD⁺ B cells are generally assumed to represent antigen-inexperienced, naive B cells expressing variable (V) region genes without somatic mutations. We report here that human IgM⁺IgD⁺ peripheral blood (PB) B cells expressing the CD27 cell surface antigen carry mutated V genes, in contrast to CD27-negative IgM⁺IgD⁺ B cells. IgM⁺IgD⁺CD27⁺ B cells resemble class-switched and IgM-only memory cells in terms of cell phenotype, and comprise ∼15% of PB B lymphocytes in healthy adults. Moreover, a very small population (<1% of PB B cells) of highly mutated IgD-only B cells was detected, which likely represent the PB counterpart of IgD-only tonsillar germinal center and plasma cells. Overall, the B cell pool in the PB of adults consists of ∼40% mutated memory B cells and 60% unmutated, naive IgD⁺CD27⁻ B cells (including CD5⁺ B cells). In the somatically mutated B cells, V_H region genes carry a two- to threefold higher load of somatic mutation than rearranged V_κ genes. This might be due to an intrinsically lower mutation rate in κ light chain genes compared with heavy chain genes and/or result from κ light chain gene rearrangements in GC B cells. A common feature of the somatically mutated B cell subsets is the expression of the CD27 cell surface antigen which therefore may represent a general marker for memory B cells in humans.     

CIK细胞与HBsAg疫苗联合作用对于转基因乙型肝炎小鼠的影响

目的观察细胞因子激活的杀伤细胞（cytokine induced kill cell, CIK ）与乙型肝炎表面抗原 （Hepatitis B surface antigen, HBsAg ）疫苗联合应用对于乙型肝炎病毒转基因小鼠 （ Hepatitis B virus transgenic mice, HBV-Tg）的治疗作用。方法给予HBV-Tg小鼠腹腔注射CIK细胞及皮下注射HBsAg疫苗,观察外用血中HBVDNA水平变化,流式细胞仪检测T淋巴细胞亚群改变,并用HE染色观察肝脏的组织病理改变。结果给予HBV-Tg小鼠CIK细胞治疗后,其外周血中HBVDNA载量减少,CD3^＋、CD4^＋及CD8^＋细胞增加,CIK细胞与HBsAg疫苗联合作用后,该作用明显加强。结论应用CIK细胞与HBsAg疫苗作用于慢性乙型肝炎小鼠后,两者可协同作用降低外周血中的病毒含量,并增强免疫作用。     

树突状细胞疫苗调节慢性乙型肝炎患者细胞因子诱导的杀伤细胞相关免疫功能的研究

目的研究树突状细胞（DC）疫苗对培养的慢性乙型肝炎（CHB）患者细胞因子诱导的杀伤细胞（CIK）免疫功能的调节作用。方法采用体外细胞培养的方法培养30例CHB患者CIK细胞,分为DC疫苗组和无DC疫苗组,培养14d后用流式细胞术检测各组ClK中CD3’CIM’、CD3’CD8’及CD3’CD56’T细胞的所占比例,ELISA方法检测培养上清中自细胞介素-12（IL-12）、γ干扰素（IFN-1）及白细胞介素-4（IL4）的浓度。结果DC疫苗组CD3^＋CIM^＋、CD3^＋CD8^＋及CD3^＋CD56^＋T细胞所占比例分别为18．27％、64．36％和20．00％,无DC疫苗组则分别为17．79％（P〉0．05）、54．69％（t=4．130,P〈0．01）和13．39％（t=5．601,P〈0．01）。DC疫苗组的CIK培养上清中IL．12、IL4及IFN-1的浓度分别为（177．82±130．06）、（31．774-9．52）、（86．99±56．30）ng/L,无DC疫苗组分别为（80．83±50．15）n∥L（t=3．811,P〈0．01）、（40．33±19．74）ng／L（t=2．141,P〈0．05）和（42．07±19．68）ng／L（t=4．125,P〈0．01）。结论CIK细胞培养中加入DC疫苗进行诱导,增强了所培养CIK细胞的杀伤活性。     

Control of Lytic Function by Mitogen-activated Protein Kinase/Extracellular Regulatory Kinase 2 (ERK2) in a Human Natural Killer Cell Line: Identification of Perforin and Granzyme B Mobilization by Functional ERK2

The signal pathways that control effector function in human natural killer (NK) cells are little known. In this study, we have identified the critical role of the mitogen-activated protein kinase (MAPK) pathway in NK lysis of tumor cells, and this pathway may involve the mobilization of granule components in NK cells upon interaction with sensitive tumor target cells. Evidence was provided by biological, biochemical, and gene transfection methods. NK cell binding to tumor cells for 5 min was sufficient to maximally activate MAPK/extracellular signal–regulatory kinase 2 (ERK2), demonstrated by its tyrosine phosphorylation and by its ability to function as an efficient kinase for myelin basic protein. MAPK activation was achieved in NK cells only after contact with NK-sensitive but not NK-resistant target cells. In immunocytochemical studies, cytoplasmic perforin and granzyme B were both maximally redirected towards the tumor contact zone within 5 min of NK cell contact with tumor cells. A specific MAPK pathway inhibitor, PD098059, could block not only MAPK activation but also redistribution of perforin/granzyme B in NK cells, which occur upon target ligation. PD098059 also interfered with NK lysis of tumor cells in a 5-h ⁵¹Cr-release assay, but had no ability to block NK cell proliferation. Transient transfection studies with wild-type and dominant-negative MAPK/ERK2 genes confirmed the importance of MAPK in NK cell lysis. These results document a pivotal role of MAPK in NK effector function, possibly by its control of movement of lytic granules, and clearly define MAPK involvement in a functional pathway unlinked to cell growth or differentiation.     

多重PCR快速检测弥漫性大B细胞淋巴瘤患者骨髓BCL2/IGH与BCL6/IGH融合基因的临床意义

弥漫性大B细胞淋巴瘤（DLBCL）是最常见的侵袭性非霍奇金淋巴瘤,其临床表现、分子生物学特点都有显著的异质性。本研究旨在探讨多重PCR技术在DLBCL患者骨髓BCL2／IGH及BCL6／IGH等融合基因检测中的临床意义。利用多重巢式PCR技术对80例初治DLBCL患者的骨髓标本进行BCL2／IGH及BCL6／IGH融合基因检测。结果表明,80例患者骨髓标本中携带目的融合基因共12例,阳性率为15％;其中BCL2-IGH阳性患者为6例,BCL6-IGH阳性患者为6例。DLBCL患者携带不同的融合基因具有不同的临床特点。结论：运用多重PCR方法对DLBCL患者进行分子生物学检测具有快速、准确的优点。但需进一步深入研究改善方法,使之对融合基因能做定量或半定量分析,这对于DLBCL患者的诊断、协助分期、预后评估、微小残留病变的估测,指导临床治疗DLBCL有重要意义。