Epigenetic tuning of PD-1 expression improves exhausted T cell function and viral control.

PD-1 is a key negative regulator of CD8+ T cell activation and is highly expressed by exhausted T cells in cancer and chronic viral infection. Although PD-1 blockade can improve viral and tumor control, physiological PD-1 expression prevents immunopathology and improves memory formation. The mechanisms driving high PD-1 expression in exhaustion are not well understood and could be critical to disentangling its beneficial and detrimental effects. Here, we functionally interrogated the epigenetic regulation of PD-1 using a mouse model with deletion of an exhaustion-specific PD-1 enhancer. Enhancer deletion exclusively alters PD-1 expression in CD8+ T cells in chronic infection, creating a 'sweet spot' of intermediate expression where T cell function is optimized compared to wild-type and Pdcd1-knockout cells. This permits improved control of chronic infection without additional immunopathology. Together, these results demonstrate that tuning PD-1 via epigenetic editing can reduce CD8+ T cell dysfunction while avoiding excess immunopathology.

Environmental cues regulate epigenetic reprogramming of airway-resident memory CD8+ T cells.

Tissue-resident memory T cells (TRM cells) are critical for cellular immunity to respiratory pathogens and reside in both the airways and the interstitium. In the present study, we found that the airway environment drove transcriptional and epigenetic changes that specifically regulated the cytolytic functions of airway TRM cells and promoted apoptosis due to amino acid starvation and activation of the integrated stress response. Comparison of airway TRM cells and splenic effector-memory T cells transferred into the airways indicated that the environment was necessary to activate these pathways, but did not induce TRM cell lineage reprogramming. Importantly, activation of the integrated stress response was reversed in airway TRM cells placed in a nutrient-rich environment. Our data defined the genetic programs of distinct lung TRM cell populations and show that local environmental cues altered airway TRM cells to limit cytolytic function and promote cell death, which ultimately leads to fewer TRM cells in the lung.

A transcriptionally distinct subset of influenza-specific effector memory B cells predicts long-lived antibody responses to vaccination in humans.

Seasonal influenza vaccination elicits hemagglutinin (HA)-specific memory B (Bmem) cells, and although multiple Bmem cell populations have been characterized, considerable heterogeneity exists. We found that HA-specific human Bmem cells differed in the expression of surface marker FcRL5 and transcriptional factor T-bet. FcRL5+T-bet+ Bmem cells were transcriptionally similar to effector-like memory cells, while T-betnegFcRL5neg Bmem cells exhibited stem-like central memory properties. FcRL5+ Bmem cells did not express plasma-cell-commitment factors but did express transcriptional, epigenetic, metabolic, and functional programs that poised these cells for antibody production. Accordingly, HA+ T-bet+ Bmem cells at day 7 post-vaccination expressed intracellular immunoglobulin, and tonsil-derived FcRL5+ Bmem cells differentiated more rapidly into antibody-secreting cells (ASCs) in vitro. The T-bet+ Bmem cell response positively correlated with long-lived humoral immunity, and clonotypes from T-bet+ Bmem cells were represented in the secondary ASC response to repeat vaccination, suggesting that this effector-like population predicts influenza vaccine durability and recall potential.

Epigenetic programming underpins B cell dysfunction in human SLE.

Systemic lupus erythematosus (SLE) is characterized by the expansion of extrafollicular pathogenic B cells derived from newly activated naive cells. Although these cells express distinct markers, their epigenetic architecture and how it contributes to SLE remain poorly understood. To address this, we determined the DNA methylomes, chromatin accessibility profiles and transcriptomes from five human B cell subsets, including a newly defined effector B cell subset, from subjects with SLE and healthy controls. Our data define a differentiation hierarchy for the subsets and elucidate the epigenetic and transcriptional differences between effector and memory B cells. Importantly, an SLE molecular signature was already established in resting naive cells and was dominated by enrichment of accessible chromatin in motifs for AP-1 and EGR transcription factors. Together, these factors acted in synergy with T-BET to shape the epigenome of expanded SLE effector B cell subsets. Thus, our data define the molecular foundation of pathogenic B cell dysfunction in SLE.

Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

Signaling through the Inhibitory Fc Receptor FcγRIIB Induces CD8+ T Cell Apoptosis to Limit T Cell Immunity.

Effector CD8+ T cells are important mediators of adaptive immunity, and receptor-ligand interactions that regulate their survival may have therapeutic potential. Here, we identified a subset of effector CD8+ T cells that expressed the inhibitory fragment crystallizable (Fc) receptor FcγRIIB following activation and multiple rounds of division. CD8+ T cell-intrinsic genetic deletion of Fcgr2b increased CD8+ effector T cell accumulation, resulting in accelerated graft rejection and decreased tumor volume in mouse models. Immunoglobulin G (IgG) antibody was not required for FcγRIIB-mediated control of CD8+ T cell immunity, and instead, the immunosuppressive cytokine fibrinogen-like 2 (Fgl2) was a functional ligand for FcγRIIB on CD8+ T cells. Fgl2 induced caspase-3/7-mediated apoptosis in Fcgr2b+, but not Fcgr2b-/-, CD8+ T cells. Increased expression of FcγRIIB correlated with freedom from rejection following withdrawal from immunosuppression in a clinical trial of kidney transplant recipients. Together, these findings demonstrate a cell-intrinsic coinhibitory function of FcγRIIB in regulating CD8+ T cell immunity.

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.

T-bet Transcription Factor Promotes Antibody-Secreting Cell Differentiation by Limiting the Inflammatory Effects of IFN-γ on B Cells.

Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses.

Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5- CD11c+ cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets.

Epigenetic gene regulation in plasma cells.

Humoral immunity provides protection from pathogenic infection and is mediated by antibodies following the differentiation of naive B cells (nBs) to antibody-secreting cells (ASCs). This process requires substantial epigenetic and transcriptional rewiring to ultimately repress the nB program and replace it with one conducive to ASC physiology and function. Notably, these reprogramming events occur within the framework of cell division. Efforts to understand the relationship of cell division with reprogramming and ASC differentiation in vivo have uncovered the timing and scope of reprogramming, as well as key factors that influence these events. Herein, we discuss the unique physiology of ASC and how nBs undergo epigenetic and genome architectural reorganization to acquire the necessary functions to support antibody production. We also discuss the stage-wise manner in which reprogramming occurs across cell divisions and how key molecular determinants can influence B cell fate outcomes.

Persistent Antigen Harbored by Alveolar Macrophages Enhances the Maintenance of Lung-Resident Memory CD8+ T Cells.

Lung tissue-resident memory T cells are crucial mediators of cellular immunity against respiratory viruses; however, their gradual decline hinders the development of T cell-based vaccines against respiratory pathogens. Recently, studies using adenovirus (Ad)-based vaccine vectors have shown that the number of protective lung-resident CD8+ TRMs can be maintained long term. In this article, we show that immunization of mice with a replication-deficient Ad serotype 5 expressing influenza (A/Puerto Rico/8/34) nucleoprotein (AdNP) generates a long-lived lung TRM pool that is transcriptionally indistinct from those generated during a primary influenza infection. In addition, we demonstrate that CD4+ T cells contribute to the long-term maintenance of AdNP-induced CD8+ TRMs. Using a lineage tracing approach, we identify alveolar macrophages as a cell source of persistent NP Ag after immunization with AdNP. Importantly, depletion of alveolar macrophages after AdNP immunization resulted in significantly reduced numbers of NP-specific CD8+ TRMs in the lungs and airways. Combined, our results provide further insight to the mechanisms governing the enhanced longevity of Ag-specific CD8+ lung TRMs observed after immunization with recombinant Ad.

H3K27me3 Demethylase UTX Restrains Plasma Cell Formation.

B cell differentiation is associated with substantial transcriptional, metabolic, and epigenetic remodeling, including redistribution of histone 3 lysine 27 trimethylation (H3K27me3), which is associated with a repressive chromatin state and gene silencing. Although the role of the methyltransferase EZH2 (Enhancer of zeste homolog 2) in B cell fate decisions has been well established, it is not known whether H3K27me3 demethylation is equally important. In this study, we showed that simultaneous genetic deletion of the two H3K27 demethylases UTX and JMJD3 (double-knockout [Utx fl/fl Jmjd3 fl/fl Cd19 cre/+] [dKO]) led to a significant increase in plasma cell (PC) formation after stimulation with the T cell-independent Ags LPS and NP-Ficoll. This phenotype occurred in a UTX-dependent manner as UTX single-knockout mice, but not JMJD3 single-knockout mice, mimicked the dKO. Although UTX- and JMJD3-deficient marginal zone B cells showed increased proliferation, dKO follicular B cells also showed increased PC formation. PCs from dKO mice upregulated genes associated with oxidative phosphorylation and exhibited increased spare respiratory capacity. Mechanistically, deletion of Utx and Jmjd3 resulted in higher levels of H3K27me3 at proapoptotic genes and resulted in reduced apoptosis of dKO PCs in vivo. Furthermore, UTX regulated chromatin accessibility at regions containing ETS and IFN regulatory factor (IRF) transcription factor family motifs, including motifs of known repressors of PC fate. Taken together, these data demonstrate that the H3K27me3 demethylases restrain B cell differentiation.

Somatic Diversification of Rearranged Antibody Gene Segments by Intra- and Interchromosomal Templated Mutagenesis.

The ability of the humoral immune system to generate Abs capable of specifically binding a myriad of Ags is critically dependent on the somatic hypermutation program. This program induces both templated mutations (i.e., gene conversion) and untemplated mutations. In humans, somatic hypermutation is widely believed to result in untemplated point mutations. In this study, we demonstrate detection of large-scale templated events that occur in human memory B cells and circulating plasmablasts. We find that such mutations are templated intrachromosomally from IGHV genes and interchromosomally from IGHV pseudogenes as well as other homologous regions unrelated to IGHV genes. These same donor regions are used in multiple individuals, and they predominantly originate from chromosomes 14, 15, and 16. In addition, we find that exogenous sequences placed at the IgH locus, such as LAIR1, undergo templated mutagenesis and that homology appears to be the major determinant for donor choice. Furthermore, we find that donor tracts originate from areas in proximity with open chromatin, which are transcriptionally active, and are found in spatial proximity with the IgH locus during the germinal center reaction. These donor sequences are inserted into the Ig gene segment in association with overlapping activation-induced cytidine deaminase hotspots. Taken together, these studies suggest that diversity generated during the germinal center response is driven by untemplated point mutations as well as templated mutagenesis using local and distant regions of the genome.

Transcription factor T-bet represses expression of the inhibitory receptor PD-1 and sustains virus-specific CD8+ T cell responses during chronic infection.

T cell exhaustion has a major role in failure to control chronic infection. High expression of inhibitory receptors, including PD-1, and the inability to sustain functional T cell responses contribute to exhaustion. However, the transcriptional control of these processes remains unclear. Here we demonstrate that the transcription factor T-bet regulated the exhaustion of CD8(+) T cells and the expression of inhibitory receptors. T-bet directly repressed transcription of the gene encoding PD-1 and resulted in lower expression of other inhibitory receptors. Although a greater abundance of T-bet promoted terminal differentiation after acute infection, high T-bet expression sustained exhausted CD8(+) T cells and repressed the expression of inhibitory receptors during chronic viral infection. Persistent antigenic stimulation caused downregulation of T-bet, which resulted in more severe exhaustion of CD8(+) T cells. Our observations suggest therapeutic opportunities involving higher T-bet expression during chronic infection.

Conserved Epigenetic Programming and Enhanced Heme Metabolism Drive Memory B Cell Reactivation.

Memory B cells (MBCs) have enhanced capabilities to differentiate to plasma cells and generate a rapid burst of Abs upon secondary stimulation. To determine if MBCs harbor an epigenetic landscape that contributes to increased differentiation potential, we derived the chromatin accessibility and transcriptomes of influenza-specific IgM and IgG MBCs compared with naive cells. MBCs possessed an accessible chromatin architecture surrounding plasma cell-specific genes, as well as altered expression of transcription factors and genes encoding cell cycle, chemotaxis, and signal transduction processes. Intriguingly, this MBC signature was conserved between humans and mice. MBCs of both species possessed a heightened heme signature compared with naive cells. Differentiation in the presence of hemin enhanced oxidative phosphorylation metabolism and MBC differentiation into Ab-secreting plasma cells. Thus, these data define conserved MBC transcriptional and epigenetic signatures that include a central role for heme and multiple other pathways in augmenting MBC reactivation potential.

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.

An IRF4-MYC-mTORC1 Integrated Pathway Controls Cell Growth and the Proliferative Capacity of Activated B Cells during B Cell Differentiation In Vivo.

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.

Understanding and measuring human B-cell tolerance and its breakdown in autoimmune disease.

The maintenance of immunological tolerance of B lymphocytes is a complex and critical process that must be implemented as to avoid the detrimental development of autoreactivity and possible autoimmunity. Murine models have been invaluable to elucidate many of the key components in B-cell tolerance; however, translation to human homeostatic and pathogenic immune states can be difficult to assess. Functional autoreactive, flow cytometric, and single-cell cloning assays have proven to be critical in deciphering breaks in B-cell tolerance within autoimmunity; however, newer approaches to assess human B-cell tolerance may prove to be vital in the further exploration of underlying tolerance defects. In this review, we supply a comprehensive overview of human immune tolerance checkpoints with associated mechanisms of enforcement, and highlight current and future methodologies which are likely to benefit future studies into the mechanisms that become defective in human autoimmune conditions.

Transcriptomic and epigenomic dynamics associated with development of human iPSC-derived GABAergic interneurons.

GABAergic interneurons (GINs) are a heterogeneous population of inhibitory neurons that collectively contribute to the maintenance of normal neuronal excitability and network activity. Identification of the genetic regulatory elements and transcription factors that contribute toward GIN function may provide new insight into the pathways underlying proper GIN activity while also indicating potential therapeutic targets for GIN-associated disorders, such as schizophrenia and epilepsy. In this study, we examined the temporal changes in gene expression and chromatin accessibility during GIN development by performing transcriptomic and epigenomic analyses on human induced pluripotent stem cell-derived neurons at 22, 50 and 78 days (D) post-differentiation. We observed 13 221 differentially accessible regions (DARs) of chromatin that associate with temporal changes in gene expression at D78 and D50, relative to D22. We also classified families of transcription factors that are increasingly enriched at DARs during differentiation, indicating regulatory networks that likely drive GIN development. Collectively, these data provide a resource for examining the molecular networks regulating GIN functionality.