IL-6/STAT3 Signaling Axis Enhances and Prolongs Pdcd1 Expression in Murine CD8 T Cells.

CD8 cytotoxic T cells are a potent line of defense against invading pathogens. To aid in curtailing aberrant immune responses, the activation status of CD8 T cells is highly regulated. One mechanism in which CD8 T cell responses are dampened is via signaling through the immune-inhibitory receptor Programmed Cell Death Protein-1, encoded by Pdcd1. Pdcd1 expression is regulated through engagement of the TCR, as well as by signaling from extracellular cytokines. Understanding such pathways has influenced the development of numerous clinical treatments. In this study, we showed that signals from the cytokine IL-6 enhanced Pdcd1 expression when paired with TCR stimulation in murine CD8 T cells. Mechanistically, signals from IL-6 were propagated through activation of the transcription factor STAT3, resulting in IL-6-dependent binding of STAT3 to Pdcd1 cis-regulatory elements. Intriguingly, IL-6 stimulation overcame B Lymphocyte Maturation Protein 1-mediated epigenetic repression of Pdcd1, which resulted in a transcriptionally permissive landscape marked by heightened histone acetylation. Furthermore, in vivo-activated CD8 T cells derived from lymphocytic choriomeningitis virus infection required STAT3 for optimal Programmed Cell Death Protein-1 surface expression. Importantly, STAT3 was the only member of the STAT family present at Pdcd1 regulatory elements in lymphocytic choriomeningitis virus Ag-specific CD8 T cells. Collectively, these data define mechanisms by which the IL-6/STAT3 signaling axis can enhance and prolong Pdcd1 expression in murine CD8 T cells.

The Murine MHC Class II Super Enhancer IA/IE-SE Contains a Functionally Redundant CTCF-Binding Component and a Novel Element Critical for Maximal Expression.

In both humans and mice, CTCF-binding elements form a series of interacting loops across the MHC class II (MHC-II) locus, and CTCF is required for maximal MHC-II gene expression. In humans, a CTCF-bound chromatin insulator termed XL9 and a super enhancer (SE) DR/DQ-SE situated in the intergenic region between HLA-DRB1 and HLA-DQA1 play critical roles in regulating MHC-II expression. In this study, we identify a similar SE, termed IA/IE-SE, located between H2-Eb1 and H2-Aa of the mouse that contains a CTCF site (C15) and a novel region of high histone H3K27 acetylation. A genetic knockout of C15 was created and its role on MHC-II expression tested on immune cells. We found that C15 deletion did not alter MHC-II expression in B cells, macrophages, and macrophages treated with IFN-γ because of functional redundancy of the remaining MHC-II CTCF sites. Surprisingly, embryonic fibroblasts derived from C15-deleted mice failed to induce MHC-II gene expression in response to IFN-γ, suggesting that at least in this developmental lineage, C15 was required. Examination of the three-dimensional interactions with C15 and the H2-Eb1 and H2-Aa promoters identified interactions within the novel region of high histone acetylation within the IA/IE-SE (termed N1) that contains a PU.1 binding site. CRISPR/Cas9 deletion of N1 altered chromatin interactions across the locus and resulted in reduced MHC-II expression. Together, these data demonstrate the functional redundancy of the MHC-II CTCF elements and identify a functionally conserved SE that is critical for maximal expression of MHC-II genes.

PD-1 Expression during Acute Infection Is Repressed through an LSD1-Blimp-1 Axis.

During prolonged exposure to Ags, such as chronic viral infections, sustained TCR signaling can result in T cell exhaustion mediated in part by expression of programmed cell death-1 (PD-1) encoded by the Pdcd1 gene. In this study, dynamic changes in histone H3K4 modifications at the Pdcd1 locus during ex vivo and in vivo activation of CD8 T cells suggested a potential role for the histone H3 lysine 4 demethylase LSD1 in regulating PD-1 expression. CD8 T cells lacking LSD1 expressed higher levels of Pdcd1 mRNA following ex vivo stimulation as well as increased surface levels of PD-1 during acute, but not chronic, infection with lymphocytic choriomeningitis virus (LCMV). Blimp-1, a known repressor of PD-1, recruited LSD1 to the Pdcd1 gene during acute, but not chronic, LCMV infection. Loss of DNA methylation at Pdcd1's promoter-proximal regulatory regions is highly correlated with its expression. However, following acute LCMV infection, in which PD-1 expression levels return to near baseline, LSD1-deficient CD8 T cells failed to remethylate the Pdcd1 locus to the levels of wild-type cells. Finally, in a murine melanoma model, the frequency of PD-1-expressing tumor-infiltrating LSD1-deficient CD8 T cells was greater than in wild type. Thus, LSD1 is recruited to the Pdcd1 locus by Blimp-1, downregulates PD-1 expression by facilitating the removal of activating histone marks, and is important for remethylation of the locus. Together, these data provide insight into the complex regulatory mechanisms governing T cell immunity and regulation of a critical T cell checkpoint gene.

B cell activation and plasma cell differentiation are inhibited by de novo DNA methylation.

B cells provide humoral immunity by differentiating into antibody-secreting plasma cells, a process that requires cellular division and is linked to DNA hypomethylation. Conversely, little is known about how de novo deposition of DNA methylation affects B cell fate and function. Here we show that genetic deletion of the de novo DNA methyltransferases Dnmt3a and Dnmt3b (Dnmt3-deficient) in mouse B cells results in normal B cell development and maturation, but increased cell activation and expansion of the germinal center B cell and plasma cell populations upon immunization. Gene expression is mostly unaltered in naive and germinal center B cells, but dysregulated in Dnmt3-deficient plasma cells. Differences in gene expression are proximal to Dnmt3-dependent DNA methylation and chromatin changes, both of which coincide with E2A and PU.1-IRF composite-binding motifs. Thus, de novo DNA methylation limits B cell activation, represses the plasma cell chromatin state, and regulates plasma cell differentiation.

Plasma cell differentiation is controlled by multiple cell division-coupled epigenetic programs.

The genomic loci associated with B cell differentiation that are subject to transcriptional and epigenetic regulation in vivo are not well defined, leaving a gap in our understanding of the development of humoral immune responses. Here, using an in vivo T cell independent B cell differentiation model, we define a cellular division-dependent cis-regulatory element road map using ATAC-seq. Chromatin accessibility changes correlate with gene expression and reveal the reprogramming of transcriptional networks and the genes they regulate at specific cell divisions. A subset of genes in naive B cells display accessible promoters in the absence of transcription and are marked by H3K27me3, an EZH2 catalyzed repressive modification. Such genes encode regulators of cell division and metabolism and include the essential plasma cell transcription factor Blimp-1. Chemical inhibition of EZH2 results in enhanced plasma cell formation, increased expression of the above gene set, and premature expression of Blimp-1 ex vivo. These data provide insights into cell-division coupled epigenetic and transcriptional processes that program plasma cells.

Cutting Edge: Chromatin Accessibility Programs CD8 T Cell Memory.

CD8 T cell memory is characterized by rapid recall of effector function, increased proliferation, and reduced activation requirements. Despite the extensive functional characterization, the molecular mechanisms that facilitate these enhanced properties are not well characterized. In this study, the assay for transposase-accessible chromatin sequencing was employed to map the cis-regulatory elements in CD8 T cells responding to acute and chronic lymphocytic choriomeningitis virus infections. Integration of chromatin accessibility profiles with gene expression data identified unique regulatory modules that were enriched for distinct combinations of transcription factor-binding motifs. Memory CD8 T cells displayed a chromatin accessibility structure that was absent from other acute and exhausted cells types and included key effector and proliferative genes. Stimulation of memory cells revealed enhanced transcription of "memory-primed" genes compared with naive cells. Thus, memory CD8 T cells display a preprogrammed chromatin accessibility profile and maintain a molecular history of cis-element usage, thereby reducing the steps necessary to revive effector functions.

Conserved Region C Functions To Regulate PD-1 Expression and Subsequent CD8 T Cell Memory.

Expression of programmed death 1 (PD-1) on CD8 T cells promotes T cell exhaustion during chronic Ag exposure. During acute infections, PD-1 is transiently expressed and has the potential to modulate CD8 T cell memory formation. Conserved region C (CR-C), a promoter proximal cis-regulatory element that is critical to PD-1 expression in vitro, responds to NFATc1, FoxO1, and/or NF-κB signaling pathways. Here, a CR-C knockout mouse was established to determine its role on PD-1 expression and the corresponding effects on T cell function in vivo. Deletion of CR-C decreased PD-1 expression on CD4 T cells and Ag-specific CD8 T cells during acute and chronic lymphocytic choriomeningitis virus challenges, but did not affect the ability to clear an infection. Following acute lymphocytic choriomeningitis virus infection, memory CD8 T cells in the CR-C knockout mouse were formed in greater numbers, were more functional, and were more effective at responding to a melanoma tumor than wild-type memory cells. These data implicate a critical role for CR-C in governing PD-1 expression, and a subsequent role in guiding CD8 T cell differentiation. The data suggest the possibility that titrating PD-1 expression during CD8 T cell activation could have important ramifications in vaccine development and clinical care.

Plasma cell differentiation is coupled to division-dependent DNA hypomethylation and gene regulation.

The epigenetic processes that regulate antibody-secreting plasma cells are not well understood. Here, analysis of plasma cell differentiation revealed DNA hypomethylation of 10% of CpG loci that were overrepresented at enhancers. Inhibition of DNA methylation enhanced plasma cell commitment in a cell-division-dependent manner. Analysis of B cells differentiating in vivo stratified by cell division revealed a fivefold increase in mRNA transcription coupled to DNA hypomethylation. Demethylation occurred first at binding motifs for the transcription factors NF-κB and AP-1 and later at those for the transcription factors IRF and Oct-2 and was coincident with activation and differentiation gene-expression programs in a cell-division-dependent manner. These data provide mechanistic insight into cell-division-coupled transcriptional and epigenetic reprogramming and suggest that DNA hypomethylation reflects the cis-regulatory history of plasma cell differentiation.

Genetic and Epigenetic Regulation of PD-1 Expression.

The inhibitory immune receptor programmed cell death-1 (PD-1) is intricately regulated. In T cells, PD-1 is expressed in response to most immune challenges, but it is rapidly downregulated in acute settings, allowing for normal immune responses. On chronically stimulated Ag-specific T cells, PD-1 expression remains high, leading to an impaired response to stimuli. Ab blockade of PD-1 interactions during chronic Ag settings partially restores immune function and is now used clinically to treat a variety of devastating cancers. Understanding the regulation of PD-1 expression may be useful for developing novel immune-based therapies. In this review, the molecular mechanisms that drive dynamic PD-1 expression during acute and chronic antigenic stimuli are discussed. An array of cis-DNA elements, transcription factors, and epigenetic components, including DNA methylation and histone modifications, control PD-1 expression. The interplay between these regulators fine-tunes PD-1 expression in different inflammatory environments and across numerous cell types to modulate immune responses.

NF-κB regulates PD-1 expression in macrophages.

Programmed cell death-1 (PD-1) is responsible for T cell exhaustion during chronic viral infections and is expressed on a variety of immune cells following activation. Despite its importance, the mechanisms that regulate PD-1 in cell types other than CD8 T cells are poorly defined. In this study, the molecular mechanisms for inducing PD-1 expression in CD4 T cells, macrophages, and B cells were explored. In CD4 T cells, PD-1 induction following TCR stimulation required NFAT, as the calcineurin/NFAT pathway inhibitor cyclosporin A was able to block PD-1 induction in a manner similar to that seen in CD8 T cells. In contrast, LPS but not PMA and ionomycin stimulation was able to induce PD-1 expression in macrophages in a manner insensitive to cyclosporin A-mediated inhibition. B cells could use both pathways, although the levels of PD-1 expression were highest with PMA and ionomycin. An NF-κB binding site located upstream of the gene in conserved region C was required for NF-κB-dependent PD-1 gene activation in macrophages. Chromatin immunoprecipitation showed NF-κB p65 binding to this region following stimulation of macrophages with LPS. PD-1 induction was associated with histone modifications characteristic of accessible chromatin; however, in contrast to CD8 T cells, conserved region B in macrophages did not lose CpG methylation upon stimulation and PD-1 expression. The linkage of TLR/NF-κB signaling to the induction of PD-1 suggests the possibility of an opportunistic advantage to microbial infections in manipulating immune inhibitory responses.