Molecular mechanisms of isotype switch recombination.

小鼠B细胞表达CD38，通过抗CD38抗体连接可诱导细胞增殖并保护细胞凋亡。我们之前的研究表明，用白细胞介素5 （IL-5）和抗小鼠CD38单克隆抗体CS/2刺激小鼠脾B细胞，可诱导IgG1和IgM的产生。本文研究了IL-5和CS/2在种系γ - 1转录本表达中的作用，以及作为μ-γ - 1开关重组副产物形成DNA环的互惠产物的产生。CS/2本身在脾幼稚B细胞中诱导了种系γ - 1转录物的显著表达，而IL-5既不诱导也不增强种系γ - 1的表达。CS/2与IL-5共同培养B细胞后，细胞内的互反产物含量未见增加，这是μ-γ - 1重组的特征；而CS/2与IL-5共同培养B细胞时，细胞内的互反产物含量增加，同时IgG1分泌增多。虽然IL-4本身并不能诱导μ-γ - 1的合成，但CS/2刺激的B细胞中μ-γ - 1的合成更多，与CS/2和IL-5联合，IL-4显著提高了γ - 1的无菌转录和IgG1的产生。这些结果表明，CD38连接仅诱导种系γ - 1转录，而IL-5以独立于IL-4的方式促进μ-γ - 1开关重组和IgG1分泌。虽然在活化的B细胞中IL-5的一些受体后信号传导事件已经被表征，但Janus激酶/信号换能器和激活剂（Jak/Stat）通路在IL-5信号传导中的作用尚未得到彻底的评估。在这项研究中，我们检测了IL-5是否激活了cd38激活的小鼠脾B细胞中的Jak/Stat通路。Stat5a和Stat5b均在IL-5刺激下被激活。证实了Stat5a和Stat5b在IL-5诱导的μ-γ - 1开关重组和IgG1产生中的作用，因为IL-5不作用于cd38刺激的Stat5a^<-/->和Stat5b^<-/->小鼠脾B细胞。在IL-5刺激下，Stat5a^<-/->和Stat5b^<-/-> B细胞中，种系γ - 1转录本CD38和激活诱导胞苷脱氨酶（AID）的表达水平与野生型B细胞相当。因此，Stat5a和Stat5b都是il -5依赖性μ-γ1开关重组所必需的，它们的靶基因可能不是g1和AID基因。Stat5b^<-/-> B细胞的μ-γ - 1开关重组受损，而Stat5a^<-/-> B细胞的μ-γ - 1开关重组受损，部分由IL-4修复，因为在CD38和il -5刺激的B细胞中添加IL-4确实诱导μ-γ - 1开关重组，导致显著的IgG1产生。我们的数据支持这样的观点，即Stat5a和Stat5b不是多余的，而是在B细胞分化的功能上至少部分不同。用5-，6-羧基荧光素二乙酸琥珀酰亚基酯（CFSE）检测B细胞分裂周期数，发现CS/2和IL-5作用5 ~ 6个分裂周期后，出现μ-γ - 1开关重组和表面igg1阳性细胞。Stat5a^<-/->和stat5b^<-/-> B细胞有5 ~ 6个细胞分裂周期，但它们既不能表达μ-γ1开关，也不能表达表面IgG1。少

Mouse B cells express CD38 whose ligation by anti-CD38 antibody induces their proliferation and protection from apoptosis. We previously showed that stimulation of mouse splenic B cells with interleukin 5 (IL-5) together with CS/2, an anti-mouse CD38 monoclonal antibody, induces production of IgG1 and IgM.Here we examined the role of IL-5 and CS/2 in the expression of germline γ1 transcripts and the generation of reciprocal products forming DNA circles as by products of μ-γ1 switch recombination. By itself, CS/2 induced significant expression of germline γ1 transcripts in splenic naive B cells, whereas IL-5 neither induced nor enhanced germline γ1 expression. Increased cellular content of reciprocal product, which is characteristic of μ-γ1 recombination, was not observed after culturing B cells with CS/2, but increased reciprocal product along with high levels of IgG1 secretion was found when B cells were cultured with CS/2 plus IL-5. Although IL-4 did not, by itself, induce μ-γ1 recom … More bination in B cells stimulated with CS/2, in conjunction with CS/2 plus IL-5, IL-4 dramatically enhanced sterile γ1 transcription and IgG1 production. These results demonstrate that CD38 ligation induces only germline γ1 transcription and that IL-5 promotes both μ-γ1 switch recombination and IgG1 secretion in an IL-4 independent manner.Although some post-receptor signaling events of IL-5 in activated B cells have been characterized, the involvement of the Janus kinase/signal transducer and activator (Jak/Stat) pathway in IL-5 signaling has not been thoroughly evaluated. In this study, we examined whether IL-5 activates the Jak/Stat pathway in CD38-activated mouse splenic B cells. Both Stat5a and Stat5b were activated by IL-5 stimulation. The role of Stat5a and Stat5b in IL-5-induced μ-γ1 switch recombination and IgG1 production were documented, as IL-5 did not act on CD38-stimnulated splenic B cells of Stat5a^<-/-> and Stat5b^<-/-> mouse. Expression levels of germline γ1 transcripts to CD38 and of activation-induced cytidine deaminase (AID) in Stat5a^<-/-> and Stat5b^<-/-> B cells upon IL-5 stimulation were comparable to these of wild type B cells. Thus, both Stat5a and Stat5b are essential for at IL-5-dependent μ-γ1 switch recombination, and their targets may not be g1 and AID genes. The impaired μ-γ1 switch recombination by Stat5b^<-/-> B cells, but not by Stat5a^<-/-> B cells, were rescued in part by IL-4, as the addition of IL-4 to the culture of CD38- and IL-5-stimulated B cells did induce μ-γ1 switch recombination leading to significant IgG1 production. Our data support the notion that Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function on B cell differentiation. Analysis of cell division cycle number of B cells, examined by using 5-, 6-carboxyfluorescein diacetate, succinimidyl ester (CFSE), revealed that μ-γ1 switch recombination and surface IgG1-positive cells were observed after 5 to 6 division cycles upon CS/2 and IL-5. Stat5a^<-/-> and stat5b^<-/-> B cells showed 5 to 6 cell division cycles, but they could express neither μ-γ1 switching nor surface IgG1. Less