(c) Selected gene expression from modules M3 and M13

(c) Selected gene expression from modules M3 and M13. To explore the gene expression profile of the cells in more detail, PGCNA was applied. functional properties for the resultant plasma cells. Moreover, using the new culture conditions we demonstrate that alternative promoter choice regulating the expression of the master transcription factor Blimp-1/Prdm1 can be observed; when the canonical B cell promoter for is deleted differentiating B cells exhibit flexibility in the choice of promoter, dictated by the initiating stimulus, with preferential maintenance of expression following exposure to TLR ligation. Thus, our novel system provides a readily tractable model for furthering our understanding of plasma cell biology. Introduction Following antigenic encounter, B cells alter their physiological state and initiate a differentiation process that ultimately produces antibody secreting cells (ASCs). The activation of B cells can be separated into two pathways based on the type of antigen and whether it requires the presence of T cells to elicit an immune response. T RASAL1 cell independent antigens such as bacterial lipopolysaccharides and polymeric proteins stimulate B lymphocytes through toll-like receptor (TLR) activation or B cell receptor (BCR) crosslinking. In contrast, a T cell dependent response arises from B cells binding to a protein antigen, followed by cognate interaction with follicular helper T cells (TFH). Initially, an extrafollicular pathway produces short-lived antibody secreting plasmablasts with moderate affinity. Secondary signals such as CD40 engagement are vital to the formation of germinal centers where somatic hypermutation leads to higher affinity antibodies 1C3 . Consequently, thousands of memory B cells and plasma cells emerge, providing a range of antigen-specific effector cell types 4,5 . ASC is a broad term that encompasses both short-lived, cycling plasmablasts and long-lived quiescent plasma cells that reside in specialized niches, primarily within the bone marrow 6 . During recent years, optimization of human B cell differentiation conditions has resulted in the generation of long-lived plasma cells, capable of surviving in culture for extended periods of time 7C10 . This has resulted in the ability to sequentially explore and manipulate each stage of differentiation. To the best of our knowledge, a murine counterpart system does not exist. There are many variations of murine ASC generation 11 , but the most commonly described technique uses the toll-like receptor 4 (TLR4) agonist, bacterial lipopolysaccharide (LPS). To enhance B cell activation, T cell help can be included in the form of co-stimulatory proteins (CD40L) and cytokines (IL-4 and IL-5) 12 . These approaches are effective for modelling partially differentiated, short-lived plasmablasts, but fail to generate terminally differentiated plasma cells that can be sustained as a quiescent population ASCs stimulated using CD40L, IL-4 and IL-5 for 5 days AM966 was as different from fully mature plasma cells as it was from activated B cells 13 . The inability to generate long-lived plasma cells means that studies of normal murine plasma cell biology are limited in an setting. AM966 An example of this is the ability to track the relationships between signaling pathways and transcriptional control in a continuously differentiating population. AM966 A gene for which this may be particularly relevant is encoding the transcription factor Blimp-1 is essential for the changes in gene expression that accompany the generation of ASCs 14 . Previous work has suggested that transcription of in ASCs is strictly governed by a promoter initiating in exon 1A 15 , which is maintained in a primed accessible state in na?ve B cells 16 . To address this issue, we have adapted two model systems used for human 9 and murine 17 B cell differentiation. Following additional optimization, two methods of plasma cell generation using LPS-induced TLR signaling or antibody-induced BCR signaling in conjunction with CD40L and cytokines were achieved. The plasma cells generated in each condition were validated to ensure characteristics such as quiescence and phenotypic markers along with gene expression profile were consistent with murine plasma cells. Using our newly developed models, we demonstrate that can be generated in the absence of exon 1A, giving rise AM966 to plasma cells in culture, despite a reduction in the levels of this key transcript and an altered gene expression profile in exon 1A deleted cells. Materials and Methods B cell isolation Spleen tissue was harvested from sex- and age-matched C57BL/6, Blimp-1-Venus 18 or generated time-course data and any batch effects removed using ComBat (R SVA package) 24 before visualization with MDS and GENE-E. Details of PGCNA network analysis have been described elsewhere 25 . Briefly, Spearman rank correlations were calculated for all gene pairs and correlation matrices clustered using the fast unfolding of communities in large networks algorithm (version 0.3). The overlap of the modules of correlated genes between the networks at the gene and signature level was assessed using a hypergeometric test and visualized as heatmap using the Broad GENE-E package (https://software.broadinstitute.org/GENE-E/). Data were hierarchically clustered (Pearson correlations and average AM966 linkage and signature enrichments were filtered to FDR.