RESUMO
Germinal centers (GCs) are specialized microenvironments where antigen-activated B cells undergo proliferation, immunoglobulin (Ig) class switch recombination, somatic hypermutation (SHM), and affinity maturation. Within GCs, follicular dendritic cells (FDCs) are key players in driving these events via direct interaction with GC B cells. Here, we provide in vivo evidence that FDCs express and upregulate Toll-like-receptor (TLR) 4 in situ during germinal center reactions, confirm that their maturation is driven by TLR4, and associate the role of FDC-expressed TLR4 with quantitative and qualitative affects of GC biology. In iterative cycles of predictions by in silico modeling subsequently verified by in vivo experiments, we demonstrated that TLR4 signaling modulates FDC activation, strongly impacting SHM and generation of Ig class-switched high-affinity plasma and memory B cells. Thus, our data place TLR4 in the heart of adaptive humoral immunity, providing further insight into mechanisms driving GCs arising in both health and disease.
Assuntos
Linfócitos B/metabolismo , Células Dendríticas Foliculares/metabolismo , Centro Germinativo/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Anticorpos Bloqueadores , Afinidade de Anticorpos , Antígenos de Diferenciação/biossíntese , Linfócitos B/imunologia , Linfócitos B/patologia , Transplante de Medula Óssea , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Dendríticas Foliculares/patologia , Centro Germinativo/patologia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Memória Imunológica , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Mutação/genética , Quimera por Radiação , Transdução de Sinais/imunologia , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL-17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL-17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL-17F expression improves the efficiency of cell line subcloning processes. IL-17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame.