TLR7/8激动剂R848体外诱导记忆B细胞分化抗原特异性浆细胞的方法研究

Establishment and evaluation of TLR7/8 agonist R848 induced differentiation of memory B lymphocytes into antigen-specific plasma cells

目的为了从PBMCs获得病原体特异性浆细胞,我们建立了TLR7/8激动剂R848体外诱导记忆B淋巴细胞分化浆细胞的方法。方法使用TLR7/8激动剂R848联合细胞因子组合,诱导接种乙肝疫苗(3年及以上)的健康志愿者来源的PBMCs,进行体外活化和分化浆细胞。分别利用ELISA和ELISPOT检测培养液上清中总IgG、HBsAg特异性IgG以及总抗体分泌细胞(ASCs)和HBsAg抗原特异性ASCs。结果成功建立R848体外诱导PBMCs中记忆B淋巴细胞分化浆细胞的方法。R848(使用质量浓度在1~2.5μg/ml)仅与IL-2联合使用,最早在第5天即可检测到特异性ASCs。结论通过该方法可以从患者恢复期少量(仅需5~10 ml)的外周血中获得抗原特异性抗体分泌细胞用于分析和分选,为中和抗体的制备提供方法。

To obtain pathogen-specific plasma cells from the peripheral blood mononuclear cells(PBMCs),we established the method for inducing memory B lymphocytes to differentiation into plasma cells using TLR7/8 agonist R848 in vitro.PBMCs were isolated from peripheral blood of healthy donors with hepatitis B vaccination(more than3 years)and then activated by R848 in combination with different cytokines for 6 days in vitro.The total IgG and HBsAg-specific IgG in the culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA);total antibody-secreting cells(ASCs)and HBsAg-specific ASCs were assessed by enzyme-linked immunospot test(ELISPOT)in PBMCs.We successfully established a method for inducing B lymphocytes to proliferate and differentiate into plasma cells from PBMCs using R848 in vitro.When R848(using a concentration of 1-2.5μg/ml)was used in combination with IL-2 alone,ASCs could be detected within the early 5 th day.The results showed that only 5-10 ml of peripheral blood is needed to induce enough antigen-specific antibody-secreting cells for analysis and sorting of cells.In the study,we established a method for differentiation of memory B cells into Ag-specific plasma cells from PBMC in vitro,which might provide effective method for the preparation of neutralizing antibodies.