MONOCLONAL ANTI-IGM REGULATION OF HUMAN B CELL FUNCTION

本项目将评估膜IgM：配体的影响 亲和力对人S细胞克隆形成机制的影响 扩张. 这些研究将评估 mIgM有三种功能： (1)促有丝分裂转导子 在缺乏T细胞辅助的情况下，B细胞进入S期的信号 信号，（2）用于部分激活的信号转换器， B细胞，和（3）用于加工的配体内化的介导剂 并呈递给T细胞。 这项研究将直接测试 假设T细胞的数量有助于扩增， 配体特异性B细胞克隆的数量与 配体的亲和力：mIgM相互作用。 研究应 增强我们对决定 外源配体的免疫调节潜力， 类风湿因子和抗独特型抗体， 生理条件，并在这样做，应有助于设计 更适合B细胞克隆扩增的疫苗。 这些研究将利用大量的小鼠抗人IgM抗体， 与mIgM上的生物素表达表位结合的单克隆抗体， 广泛的亲和力范围（Ka = 2x 105至6x 108）。 这项工作将有 四个主要目标：（1）建立明显的T细胞和T细胞 细胞因子非依赖性（TI）信号传导潜力的某些抗- IgM单克隆抗体混合物是由于增强的功能亲和力， mIgM，并评估是否不同阶段的延长 TI配体诱导B细胞DNA合成所需的信号周期 有不同的亲和力要求。 这一问题将在 体外培养实验，免疫电镜，和细胞 平衡结合分析 (2)评估最小绑定 某些前S相现象的配体诱导的亲和力。 这将涉及细胞内游离Ca 2+的Indo 1分析， 流式细胞术分析II类MHC膜表达， 活化相关分子 (3)评估最小配体 信号传导B细胞S期进入的亲和力要求 IFN-γ、IL 4或低MW BCGF的存在。 (4)评价 对B细胞增殖的最小配体亲和力， 同源T细胞帮助。 这将涉及用单克隆抗体培养B细胞 Fab片段和小鼠IG特异性人T细胞克隆。 结果 从这些体外研究中， 不同的配体应该提供一个统一的解释， mIgM在促进B中具有明显不同的功能 细胞克隆扩增

This project will evaluate the impact of membrane IgM:ligand affinity on the mechanism by which human s cells undergo clonal expansion. The studies will assess the affinity requisites for mIgM to function in three capacities: (1) transducer of mitogenic signals for B cell S phase entry in the absence of T cell accessory signals, (2) transducer of signals for the partial activation of B cells, and (3) mediator of ligand internalization for processing and presentation to T cells. The research will directly test the hypothesis that the amount of T cell help required for expansion of ligand-specific B cell clones is indirectly related to the affinity of the ligand:mIgM interaction. The studies should enhance our understanding of the affinity constraints determining the immunoregulatory potential of foreign ligands, autologous rheumatoid factors, and anti-idiotype Abs under varying physiological conditions, and in so doing, should aid in the design of more optimal vaccines for B cell clonal expansion. The studies will utilize a large group of mouse anti-human IgM MoAbs that bind to bivalently expressed epitopes on mIgM with a wide range of affinities (Ka = 2x105 to 6x108). The work will have four major aims: (1) Establish that the pronounced T cell and T cell factor independent (TI) signaling potential of certain anti- IgM MoAb mixtures is due to an enhanced functional affinity for mIgM, and assess whether different phases of the prolonged signaling period needed for TI ligand-induced B cell DNA synthesis have distinct affinity requirements. This will be addressed by in vitro culture experiments, immunoelectron microscopy, and cellular equilibrium binding analyses. (2) Evaluate the minimal binding affinity for ligand induction of certain pre-S phase phenomena. This will involve Indo 1 analysis of intracellular free Ca2+ and FACS analysis of the membrane expression of class II MHC and activation-associated molecules. (3) Assess the minimal ligand affinity requirement for signaling B cell S phase entry in the presence of IFN-gamma, IL4, or low MW BCGF. (4) Evaluate the minimal ligand affinity for B cell proliferation with linked, cognate T cell help. This will involve B cell culture with MoAb Fab fragments and mouse Ig specific human T cell clones. Results from these in vitro studies with polyclonally-reactive, affinity diverse ligands should provide a unifying explanation for the apparently diverse functions attributed to mIgM in facilitating B cell clonal expansion.