MONOCLONAL ANTI-IGM REGULATION OF HUMAN B CELL FUNCTION

本项目将评估膜IgM：Ligand的影响 人S细胞克隆形成机制的亲和力研究 扩张。研究将评估亲和力的必要条件 MIgM具有三种功能：(1)促有丝分裂转导 T细胞辅助性T细胞缺失时B细胞进入S时相的信号 信号，(2)用于部分激活的信号转换器 B细胞；(3)配体内化的中介物 并呈现给T细胞。这项研究将直接考验 假设扩增所需的T细胞数量 与配体特异性B细胞克隆的数量间接相关 配基亲和力：mIgM相互作用。这些研究应该 加深对亲和力制约因素决定的理解 异体配体的免疫调节潜力 类风湿因子和抗独特型抗体在不同条件下的变化 生理条件，在这样做的时候，应该有助于设计 更理想的B细胞克隆性扩增疫苗。 这项研究将利用一大群小鼠抗人IgM 与mIgM上二价表达表位结合的单抗 广泛的亲和力(Ka=2x105到6x108)。这项工作将会有 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)信号转导潜能的研究 IGM Moab混合物是由于增强了对 MIgM，并评估不同时相是否延长 TI配体诱导B细胞DNA合成所需的信号转导周期 有不同的亲和力要求。这个问题将由In解决 体外培养实验、免疫电子显微镜和细胞 平衡结合分析。(2)评估最小绑定 亲和力为配体诱导的某些S前相现象。 这将涉及Indo 1对细胞内游离钙和 流式细胞仪分析细胞膜上II类MHC和 激活相关分子。(3)确定最小配基 B细胞S时相进入信号的亲和力要求 存在干扰素-γ、白介素4或低分子BCGF。(4)评估 B细胞增殖的最低配基亲和力， 同源T细胞帮助。这将涉及到与摩押一起培养B细胞 Fab片段和小鼠Ig特异性人类T细胞克隆。结果 从这些具有多克隆反应性、亲和力的体外研究 不同的配体应该提供一个统一的解释 MIgM在易化B细胞中的不同功能 细胞克隆性扩增。

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.