Cell division is an essential component of B cell differentiation to Ab-secreting plasma cells, with critical reprogramming occurring during the initial stages of B cell activation. However, a complete understanding of the factors that coordinate early reprogramming events in vivo remain to be determined. In this study, we examined the initial reprogramming by IRF4 in activated B cells using an adoptive transfer system and mice with a B cell–specific deletion of IRF4. IRF4-deficient B cells responding to influenza, 4-hydroxy-3-nitrophenylacetyl–Ficoll, and LPS divided but stalled during the proliferative response. Gene expression profiling of IRF4-deficient B cells at discrete divisions revealed IRF4 was critical for inducing MYC target genes, oxidative phosphorylation, and glycolysis. Moreover, IRF4-deficient B cells maintained an inflammatory gene expression signature. Complementary chromatin accessibility analyses established a hierarchy of IRF4 activity and identified networks of dysregulated transcription factor families in IRF4-deficient B cells, including E-box binding bHLH family members. Indeed, B cells lacking IRF4 failed to fully induce Myc after stimulation and displayed aberrant cell cycle distribution. Furthermore, IRF4-deficient B cells showed reduced mTORC1 activity and failed to initiate the B cell activation unfolded protein response and grow in cell size. Myc overexpression in IRF4-deficient cells was sufficient to overcome the cell growth defect. Together, these data reveal an IRF4–MYC–mTORC1 relationship critical for controlling cell growth and the proliferative response during B cell differentiation.

Human memory T cells (MTC) are poised to rapidly respond to antigen re-exposure. Here, we derived the transcriptional and epigenetic programs of resting and ex vivo activated, circulating CD4+ and CD8+ MTC subsets. A progressive gradient of gene expression from naïve to TCM to TEM is observed, which is accompanied by corresponding changes in chromatin accessibility. Transcriptional changes suggest adaptations of metabolism that are reflected in altered metabolic capacity. Other differences involve regulatory modalities comprised of discrete accessible chromatin patterns, transcription factor binding motif enrichment, and evidence of epigenetic priming. Basic-helix-loop-helix factor motifs for AHR and HIF1A distinguish subsets and predict transcription networks to sense environmental changes. Following stimulation, primed accessible chromatin correlate with an augmentation of MTC gene expression as well as effector transcription factor gene expression. These results identify coordinated epigenetic remodeling, metabolic, and transcriptional changes that enable MTC subsets to ultimately respond to antigen re-encounters more efficiently.

Cell division is an essential component of B cell differentiation to antibody-secreting plasma cells, with critical reprogramming occurring during the initial stages of B cell activation. However, a complete understanding of the factors that coordinate early reprogramming events in vivo remain to be determined. In this study, we examined the initial reprogramming by IRF4 in activated B cells using an adoptive transfer system and mice with a B cell-specific deletion of IRF4. IRF4-deficient B cells responding to influenza, NP-Ficoll and LPS divided, but stalled during the proliferative response. Gene expression profiling of IRF4-deficient B cells at discrete divisions revealed IRF4 was critical for inducing MYC target genes, oxidative phosphorylation, and glycolysis. Moreover, IRF4-deficient B cells maintained an inflammatory gene expression signature. Complementary chromatin accessibility analyses established a hierarchy of IRF4 activity and identified networks of dysregulated transcription factor families in IRF4-deficient B cells, including E-box binding bHLH family members. Indeed, B cells lacking IRF4 failed to fully induce Myc after stimulation and displayed aberrant cell cycle distribution. Furthermore, IRF4-deficient B cells showed reduced mTORC1 activity and failed to initiate the B cell-activation unfolded protein response and grow in cell size. Myc overexpression in IRF4-deficient was sufficient to overcome the cell growth defect. Together, these data reveal an IRF4-MYC-mTORC1 relationship critical for controlling cell growth and the proliferative response during B cell differentiation.

Memory T cells are a diverse set of cells that respond to immunologic challenge more quickly and with greater efficacy than their naïve counterparts. Their potential in the resting and stimulated state has not been explored for each of the subsets. To define their capabilities, RNA-seq and ATAC-seq data assessing the transcriptome and chromatin accessibility potential of both resting and activated human memory T cells was analyzed. Clustering gene expression modules identified a core set of genes highly expressed in both resting central memory and effector memory cells (but not in naïve or terminally differentiated effector cells). Memory subsets also expressed unique gene modules enabling specialized functions such as cell cytotoxicity, migration, or self-renewal. Subset differences in gene expression were reflected in differentiated chromatin accessibility and enhanced variation around binding motifs for key T cell differentiation transcription factors such as Tbet, EOMES, and LEF1. Upon activation memory T cells showed substantial rapid upregulation of genes including cytokines which were absent in activated naïve T cells. These differences in gene expression from naïve T cells were often preceded by areas of open chromatin in a resting memory state, suggesting mechanisms of epigenetic priming for rapid recall or augmentation of effector T cell function in memory. In summary, memory T cell subsets are distinct in both resting gene expression functionalities and differentiated epigenetic landscape—both of which enable unique, rapid, and effective response to antigen.

Programed Cell death (PD-1), encoded by Pdcd1, is an immune inhibitory receptor expressed on the surface of lymphocytes. Surface PD-1 expression is amongst its highest in a subset of CD4+ T cells called T follicular helper cells (TFH). TFH cells are pivotal for optimal germinal center formation and subsequent generation of pathogen- and vaccine-induced antibodies by B cells. Despite the clear connection to human health, the majority of previous work has centered on elucidating the mechanisms surrounding murine Pdcd1 regulation. Surprisingly, there is almost nothing known about how human Pdcd1 (hPdcd1) is regulated! Here we seek to close this gap in knowledge by identifying the cis-, trans-, and epigenetic pathways that regulate hPdcd1 in TFH cells. To this end, human TFH cells were sourced from both discarded tonsil tissue as well as from a recently described model to generate ex vivo human TFH cells through treatment with the cytokines Activin A, IL-12 and TGFβ. RNA-sequencing demonstrated that ex vivo TFH cells exhibit characteristics of bona-fide tonsillar TFH cells, including increased expression of PD-1. Furthermore, ATAC-seq and H3K27Ac Hi-ChIP experiments revealed the presence of 7 conserved accessible regions within the hPdcd1 locus that exhibit unique looping characteristics and potential enhancer properties. Functionality of each regulatory element was further defined through promoter reporter assays. Additionally, PAGE-RANK analysis elucidated transcription factors that may play a role in the induction of hPdcd1 expression. Collectively, these findings begin to unearth the mechanisms that control hPdcd1, informing the design of future therapeutics aimed at precisely manipulating human PD-1 expression.