WT mice were either immunized with Alum/HEL-OVA or transfused with HOD RBCs and levels of anti-HEL IgG subtypes were measured using end-point dilution ELISAs. To study the role of STAT6 in IgG class-switching, we first generated and validated novel STAT6 KO mice using CRISPR/cas9 gene editing. STAT6 KO mice were then transfused with HOD RBCs or immunized with Alum/HEL-OVA, and IgG subclasses were quantified by ELISA.
When compared with antibody responses to Alum/HEL-OVA, transfusion of HOD RBCs induced lower levels of IgG1, IgG2b, and IgG2c but similar levels of IgG3. Class switching to most IgG subtypes remained largely unaffected in STAT6 deficient mice in response to HOD RBC transfusion, with the one exception being IgG2b. In contrast, STAT6 deficient mice showed altered levels of all IgG subtypes following Alum vaccination.
Our results show that anti-RBC class-switching occurs via alternate mechanisms when compared with the well-studied immunogen alum vaccination.
方法:WT小鼠用明矾/HEL-OVA免疫或用HOD红细胞输注,并使用终点稀释ELISA测量抗HELIgG亚型的水平。研究STAT6在IgG类别转换中的作用,我们首先使用CRISPR/cas9基因编辑技术产生并验证了新型STAT6KO小鼠.然后用HOD红细胞输注STAT6KO小鼠或用明矾/HEL-OVA免疫,和IgG亚类通过ELISA定量。
结果:当与对明矾/HEL-OVA的抗体反应相比时,输注HOD红细胞诱导较低水平的IgG1,IgG2b,和IgG2c,但IgG3水平相似。在响应HOD红细胞输血的STAT6缺陷型小鼠中,大多数IgG亚型的类别转换在很大程度上不受影响。一个例外是IgG2b。相比之下,STAT6缺陷型小鼠在明矾疫苗接种后显示所有IgG亚型的水平改变。
结论:我们的结果表明,与经过充分研究的免疫原明矾疫苗接种相比,抗RBC类别转换是通过替代机制发生的。