Intracellular Trafficking and Signaling Of The B-cell Antigen Receptor

B细胞激活是由抗原与B细胞受体(BCR)结合而启动的，触发信号级联反应，导致与B细胞激活相关的各种基因的转录。在信号传递开始后，抗原结合的BCR进入细胞并运输到含有MHC II类分子的细胞内，在那里抗原被蛋白分解，所产生的多肽与MHC II类分子结合，最终表达在B细胞表面，允许与抗原特异性辅助T细胞相互作用。我们确定，BCR信号也触发了内细胞室的重组，将含有Toll样受体的内小体招募到BCR进入的自噬体室。我们现在了解，BCR在进入细胞时继续发出信号，BCR在细胞内的正确贩运及其招募TLR取决于这些信号。这个项目的目标是了解BCR信号级联中的离散步骤发生在哪里，以及信号的空间和时间组织如何调节抗原与BCR结合的结果。将特别关注BCR与细胞内TLR的相互作用以及这些相互作用的结果。 在过去的一年里，我们表征了TLR9激动剂CpG对BCR抗原处理和呈递的影响。我们现在认识到，体内T细胞依赖的抗体反应中的关键事件依赖于抗原特异性的T细胞-B细胞相互作用。T细胞依赖的抗体反应发生在T细胞/B细胞交界处的次级淋巴器官，并依赖于抗原致敏的Th细胞通过B细胞表面的MHC-II类多肽复合体获得了T滤泡辅助细胞(TFH)的某些特征与活化的抗原特异性B细胞的稳定相互作用。结果，B细胞被充分激活和增殖，TFH细胞完全分化。B细胞要么进入生发中心(GC)，要么分化为短期浆细胞(PC)或非GC非依赖性记忆B细胞(MBCs)，这在一定程度上取决于B细胞-TfH细胞相互作用的质量。在GC内，B细胞在进入发生抗原依赖选择的GC亮区之前，在GC暗区中增殖并经历体细胞的超突变。选择依赖于B细胞捕获、处理抗原并将其呈递给TFH细胞的能力，这一事件最终导致GC B细胞分化为长期存活的MBCs和PC。我们发现，TLR9激动剂CpG可以消除B细胞处理抗原并将抗原呈递给抗原特异性辅助T细胞的能力。当在CpG存在的情况下将抗原提供给B细胞时，BCR启动了早期信号事件，涉及几个早期激酶和适配器的磷酸化，BCR被内吞到细胞内，类似于B细胞对单独的Ag的反应。然而，在CpG存在的情况下，BCR的运输被失调，BCR没有到达抗原加工区，并且用复合体特异性抗体检测到的多肽-MHC复合体没有出现在B细胞表面。当与抗原特异的CD4+T细胞一起培养时，抗原特异的B细胞在CpG存在的情况下不能激活T细胞对抗原的反应。使用一种新的方法，使我们能够观察和量化从膜片上提取抗原的B细胞，我们确定CpG极大地减少了B细胞通过BCR捕获的抗原量。根据这些结果，我们得出结论，CpG的存在导致了B细胞与Ag相遇的结果的改变，强烈地促进了B细胞的增殖和分化，但代价是与辅助性T细胞的Ag依赖相互作用。

B cell activation is initiated by the binding of the antigen to the B cell receptor (BCR), triggering signal cascades that result in the transcription of a variety of genes associated with B cell activation. Following the initiation of signaling the antigen-bound BCR enters the cell and trafficks to specialized MHC class II-containing intracellular compartments where the antigen is proteolytically cleaved and the resulting peptides bound to MHC class II molecules that are ultimately expressed on the B cell surface allowing for interaction with antigen-specific helper T cells. We determined that BCR signaling also triggers reorganization of the endocytic compartments, recruiting endosomes containing toll-like receptors to autophagosome compartments into which the BCR trafficks. We now understand that the BCR continues to signal as it enters the cell and that the correct intracellular trafficking of the BCR and its recruitment of the TLRs depend on these signals. The goal of this project is to understand where discrete steps in the BCR signaling cascade occur and how the spatial and temporal organization of signaling regulates the outcome of antigen binding to the BCR. Particular focus will be on the interaction of the BCR with the intracellular TLRs and on the outcome of these interactions. Over the last year we characterized the effect of the TLR9 agonist CpG on BCR antigen processing and presentation. We now appreciate that key events in T cell-dependent Ab responses in vivo are dependent on antigen-specific T cell-B cell interactions. The initiation of T cell-dependent Ab responses occurs in secondary lymphoid organs at the T cell/B cell border and is dependent on the stable interaction of antigen-primed Th cells that have acquired some of the features of T follicular helper cells (Tfh) with activated antigen-specific B cell through MHC-class II peptide complexes presented on the B cell surface. As a result B cells become fully activated and proliferate and Tfh cells fully differentiate. Dependent, in part, on the quality of the B cell-Tfh cells interaction, B cells will either enter germinal centers (GCs) or differentiate into either short-lived plasma cells (PCs) or GC-independent memory B cells (MBCs). Within the GC B cells proliferate and undergo somatic hypermutation in the GC dark zone prior to entering the GC light zone where antigen-dependent selection occurs. Selection is dependent on the ability of B cells to capture, process and present Ag to Tfh cells an event that ultimately results in the differentiation of GC B cells to long-lived MBCs and PCs. We discovered that the TLR9 agonist, CpG, ablated the ability of B cells to process and present antigen to antigen-specific helper T cells. When Ag was provided to B cells in the presence of CpG the BCR initiated early signaling events involving phosphorylation of several early kinases and adaptors and the BCR was endocytosed into the cell similarly to the response of B cells to Ag alone. However, in the presence of CpG, BCR trafficking was dysregulated, the BCR did not reach Ag processing compartments and peptide-MHC complexes, detected by complex-specific antibodies, did not appear on the B cell surface. When placed in culture with Ag-specific CD4+ T cells Ag-specific B cells were unable to activate T cells in response to Ag in the presence of CpG. Using a new methodology that allowed us to view and quantify B cells pulling antigen from membrane sheets we determined that CpG greatly diminished the amount of antigen captured by B cells through the BCR. We conclude from these results that the presence of CpG results in a switch in the outcome of B cell encounter with Ag, strongly promoting proliferation and differentiation of the B cells at the expense of Ag-dependent interactions with helper T cells.