自身免疫疾病相关FcγRIIB-I232T抑制功能缺失的分子机制研究

Aberrant B cell activations is associated with autoimmune diseases such as systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), however the underlying mechanism is incompletely understood. To balance immunoprotection and immunopathology, B cell activation is under strict control by BCR co-receptor FcγRIIB which is the only Fcγ receptor that expressed on B cell and play a key role in inhibiting B cell activation upon antigen-antibody complex stimulation. Our recent work found that transmembrane mutant FcγRIIB-I232T was associated with RA patients in Han Chinese population. This mutant also showed loss-of-function phenotype in departing the synaptic colocalization of BCR microclusters and CD19 microclusters in B cells from SLE patients. It’s our interest to study activation and response of the lymphocytes from autoimmune disease patients. What’s more, to investigate the loss-of-function mechanism of FcγRIIB-I232T, we combined super-resolution imaging, single molecule tracking, computer simulation and nuclear magnetic resonance (NMR) techniques. Based on the observations that slower diffusion mobility of FcγRIIB-I232T, which leaded it to form more nanoclusters smaller than 100 nM and higher phosphorylation level of ITIM in FcγRIIB-I232T cytoplasmic tail, we propose a novel model for the FcγRIIB-I232T loss-of-function phenotype. Due to slow mobility of FcγRIIB-I232T it cannot efficiently catch up the Fc fragment of IgG antibody in the immune complex which was captured by BCR, thus cannot fully inhibit the activation of B cell.

B淋巴细胞的异常活化与自身免疫疾病发生发展密切相关。FcγRIIB是B细胞上唯一表达的Fcγ受体，在抗原抗体复合物介导的B细胞活化中起到关键性抑制作用。我们近期的工作发现FcγRIIB穿膜区点突变I232T与大陆人群类风湿性关节炎易感性正相关，且FcγRIIB-I232T无法阻止狼疮患者B细胞BCR微簇体在免疫突触内与CD19微簇体共定位，而导致此点突变抑制功能丧失的分子机制不完全清楚。活细胞单分子成像实验揭示FcγRIIB-I232T在细胞膜上布朗运动能力显著低于野生型，且易形成100 nm以下的纳簇体。在本计划为期一年的研究中，我们将采用结构模拟及核磁共振光谱分析穿膜区短肽的结构变化结合高精度单分子和超分辨率成像实验，研究当FcγRIIB活化时，由于FcγRIIB-I232T布朗运动能力低无法捕捉免疫复合物中的抗体Fc区，导致其丧失抑制能力的新机制。

FcγRIIB表达于多种免疫细胞表面，严格调控着免疫细胞的活化，其穿膜区单核苷酸多态性FcγRIIB-I232T与系统性红斑狼疮的易感性正相关。我们之前的工作表明当B细胞被抗原抗体复合物活化时，FcγRIIB-I232T相比于FcγRIIB-WT丧失了抑制BCR微簇体和CD19微簇体在B细胞免疫突触内共定位的能力。本研究中我们通过检测FcγRIIB-I232T穿膜区的物理性质改变，从FcγRIIB的穿膜区单次穿膜螺旋拓扑构象及其在细胞质膜上的侧向分子扩散速率的新层面提出了I232T的单核苷酸多态性导致FcγRIIB-I232T抑制功能缺失的新机制。我们发现FcγRIIB-I232T在细胞膜上的侧向扩散速率更慢，并且超分辨率成像结果显示当B细胞被抗原抗体复合物激活早期，FcγRIIB-I232T无法及时聚集于被BCR捕捉的抗原抗体复合物微簇体中。然而，不论是给予FcγRIIB-I232T足够的反应时间与抗原抗体复合物中的抗体Fc区结合或是将FcγRIIB-I232T的穿膜区替换成具有较高扩散速率的CD86分子的穿膜区，均可以一定程度上恢复FcγRIIB-I232T的抑制功能。此外，分子模拟及核磁共振的结果表明FcγRIIB-I232T穿膜区在磷脂双层中呈现更加倾斜的拓扑构象，进而在分子水平解释FcγRIIB-I232T扩散速率为何变慢。综上，我们的研究通过检测FcγRIIB-I232T穿膜区物理性质的改变为其抑制功能缺失提供了新机制，为微观分子物理性质对宏观病理的发生发展的研究提供了新思路，并为FcγRIIB-I232T与系统性红斑性狼疮易感性正相关提供了一定的理论解释，对系统性红斑狼疮疾病的个性化治疗，疾病进展判断及预后提供了参考依据。