A single-round of antigen receptor signaling programs naïve B cells to receive T cell help. Mus musculus

To simulate transient B cell activation that is the likely initiator of T-dependent responses, we examined the molecular and functional consequences of a single-round of immunoglobulin M (IgM) signaling. This form of activation triggered early cytosolic signaling and transcription factor NF-kB activation indistinguishably from conventional continuous IgM cross-linking, but did not induce G1 progression. However, single-round IgM signaling changed the expression of chemokine and chemokine receptor genes implicated in initiating T-dependent responses, as well as accentuated responsiveness to CD40 signaling. Several features of single-round IgM signaling in vitro were recapitulated in B cells after short-term exposure to antigen in vivo. We propose that transient BCR signals prime B cells to receive T cell help by increasing the probability of B-T encounter and creating a cellular environment that is hyper-responsive to CD40 signaling. Overall design: Primary B lymphocytes were isolated using Auto-MACS (Miltenyi Biotec) by negative selection. B cell purity was 90-95% based on flow cytometric analysis with CD19 staining. Purified B cells (2x10^6/ml) were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 55nM beta-mercaptoethanol, 2mM L-glutamine and 100IU penicillin and 100ug/ml streptomycin at 37degrees C. For pulsed anti-IgM treatment experiments, B cells were incubated with 10ug/ml goat anti-mouse IgM F(ab’)2 (Jackson ImmunoResearch Laboratories) at 4 degrees C for 30 min. Unbound anti-IgM was removed from the medium by washing and centrifuging the cells at 4 degrees C. The cells were resuspended in chilled complete medium and shifted to 37 degrees C by placing in an incubator or in water-bath. For continuous anti-IgM treatment experiments, B cells were stimulated with 10ug/ml anti-IgM at 4 degrees C for 30 min, then incubated at 37 degrees C.

为模拟作为T细胞依赖性免疫应答潜在起始诱因的瞬时B细胞活化，我们探究了单轮免疫球蛋白M（IgM）信号转导的分子与功能效应。该活化模式触发的早期胞质信号转导与转录因子核因子κB（NF-κB）活化，与常规持续IgM交联并无显著差异，但并未诱导G1期进程推进。不过，单轮IgM信号转导改变了与T细胞依赖性应答起始相关的趋化因子及趋化因子受体基因的表达，同时增强了细胞对CD40信号转导的应答敏感性。单轮IgM信号转导在体外呈现的若干特征，在体内经短期抗原暴露后的B细胞中得到了重现。我们提出，瞬时B细胞受体（BCR）信号可通过提升B细胞与T细胞相遇的概率，并构建对CD40信号高应答的细胞微环境，使B细胞做好接收T细胞辅助的准备。

总体实验设计：采用Auto-MACS（Miltenyi Biotec）阴性分选法分离原代B淋巴细胞。经CD19染色的流式细胞术分析显示，B细胞纯度为90%~95%。将纯化后的B细胞（浓度为2×10^6/ml）置于RPMI 1640培养基中培养，该培养基添加有10%热灭活胎牛血清（FBS）、55nM β-巯基乙醇、2mM L-谷氨酰胺、100IU/ml青霉素及100μg/ml链霉素，培养温度为37℃。针对脉冲式抗IgM处理实验：将B细胞与10μg/ml山羊抗小鼠IgM F(ab’)2片段（杰克逊免疫研究实验室）于4℃下孵育30分钟。随后通过4℃下离心洗涤细胞，移除未结合的抗IgM。将细胞重悬于预冷的完全培养基中，再转移至孵箱或水浴锅中升温至37℃。针对持续抗IgM处理实验：将B细胞与10μg/ml抗IgM于4℃下孵育30分钟，随后转移至37℃下培养。