DOCK8缺陷患者B细胞IgHV基因高频突变及抗体亲和力研究

Primary immunodeficiency disease (PID) is caused by single gene defect with great importance for study of immune system. As one of the latest featured PID without reports in China so far, DOCK8 defect is characterized as repeated sinopulmonary infections and elevated serum IgE. Absence of DOCK8 expression impairs the generation of secondary antibody response to specific T-D antigens; phenotypically normal CD95+GL7+B220+ GC B cells were readily detected by flow cytometry in homozygous DOCK8 mutants mouse after immunization but their numbers were only 15% of normal; The CD27+memory B cell was almost absent in three DOCK8 mutation patients in our center, all of which indicated the crippled germinal center and B cell late development defect. The abnormal antibody response could account for the functional antibody abnormalities and recurrent sinopulmonary infections observed in DOCK8 defect patients. In this study, we focus on the late development of antibody response including somatic hyper mutation of IgHV gene; B cell subset during the development in germinal center; tilters and affinity of specific antibody in DOCK8 patient with Tetanus and Streptococcus pneumoniae immunization, Aiming to establish gene analysis means for diagnosis; to investigate the relationhip between clinic, gene mutation and immunologic phenotype; to explore in which step the DOC8 mutation affecting the development of memory B cells; to provide preliminary basis to further exploring the late developmental mechanisms of B cells in germinal center and specific IVIG replacement therapy for DOCK8 defect patients as well as bonemarrow transplantation.

原发性免疫缺陷病（primary immunodeficiency disease，PID）作为单基因遗传病对免疫功能研究意义重大，DOCK8缺陷为新近发现的PID，国内尚无报道。该病表现为反复窦肺感染及血清高IgE。DOCK8缺陷鼠对TD抗原再次免疫特异抗体滴度降低、生发中心B细胞减少；结合前期我们已确诊3例患者并发现其记忆B细胞缺乏，提示DOCK8缺陷可引起B细胞在生发中心晚期发育异常。由此本课题拟对患者进行疫苗接种，探索B细胞IgHV基因高频突变、特异性抗体亲和力、持久性及B细胞增值、凋亡等晚期发育相关事件。由此建立我国DOCK8缺陷患者初筛及分子分析手段；探索其临床、基因型、免疫表型的关系；初步明确DOCK8缺陷患者体液免疫记忆异常是否由IgHV基因高频突变及相应特异性抗体滴度及亲和力异常等引起；为进一步探索DOCK8基因与体液免疫记忆关系及特异性IVIG替代治疗该病提供初步依据。

原发性免疫缺陷病（primary immunodeficiency disease，PID）作为单基因遗传病对免疫功能研究意义重大，DOCK8缺陷为新近发现的PID，国内尚无报道。该病表现为反复窦肺感染及血清高IgE。DOCK8缺陷鼠对TD抗原再次免疫特异抗体滴度降低、生发中心B细胞减少；结合前期我们已确诊3例患者并发现其记忆B细胞缺乏，提示DOCK8缺陷可引起B细胞在生发中心晚期发育异常。由此本课题首先在国内建立DOCK8缺陷患者基因分析手段及蛋白快速诊断手段，为我国该病的诊断提供了可靠手段。通过免疫表型的探讨，探明我国DOCK8缺陷患者主要由大段缺失引起，且患者有B记忆细胞降低、NK细胞毒功能降低等免疫学异常，提示DOCK8缺陷引起反复感染临床表型的潜在免疫学机制。利用既往患者进行了破伤风和流感嗜血杆菌疫苗接种，检测DOCK8缺陷患者产生的针对二者的抗体亲和力，发现DOCK8缺陷患者产生的抗体亲和力降低，提示抗体亲和力成熟障碍，为临床IVIG治疗提供了实验基础。同时探讨了B细胞IgHV基因高频突变发现DOCK8缺陷患者IgHV基因辛普生指数、香农-威纳指数及D50指数并无明显降低，但IGHV5取用率明显降低，IGHV1-2_IGHJ3、IGHV3-NL1_IGHJ2、IGHV7-4-1_IGHJ4降低，IGHV1-58_IGHJ6、IGHV2-5_IGHJ6、IGHV3-53_IGHJ5、IGHV4-39_IGHJ4、IGHV4-61_IGHJ4取用率增高。DOCK8缺陷患者IgHV区域带负电荷的氨基酸较正常对照降低，证实DOCK8缺陷确实影响IgHV基因高频突变，也可能是造成其抗体亲和力降低的直接原因。由于生发中心Tfh是辅助抗体应答的主要细胞，后续已经开始探索DOCK8缺陷是否影响Tfh对B细胞的辅助，以探明DOCK8缺陷患者抗体缺陷的根本机制。