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.

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.

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.

Antibody-secreting cell destiny emerges during the initial stages of B-cell activation.

Upon stimulation, B cells assume heterogeneous cell fates, with only a fraction differentiating into antibody-secreting cells (ASC). Here we investigate B cell fate programming and heterogeneity during ASC differentiation using T cell-independent models. We find that maximal ASC induction requires at least eight cell divisions in vivo, with BLIMP-1 being required for differentiation at division eight. Single cell RNA-sequencing of activated B cells and construction of differentiation trajectories reveal an early cell fate bifurcation. The ASC-destined branch requires induction of IRF4, MYC-target genes, and oxidative phosphorylation, with the loss of CD62L expression serving as a potential early marker of ASC fate commitment. Meanwhile, the non-ASC branch expresses an inflammatory signature, and maintains B cell fate programming. Finally, ASC can be further subseted based on their differential responses to ER-stress, indicating multiple development branch points. Our data thus define the cell division kinetics of B cell differentiation in vivo, and identify the molecular trajectories of B cell fate and ASC formation.

IgM, IgG, and IgA Influenza-Specific Plasma Cells Express Divergent Transcriptomes.

Ab-secreting cells (ASC) or plasma cells are essential components of the humoral immune system. Although Abs of different isotypes have distinct functions, it is not known if the ASC that secrete each isotype are also distinct. ASC downregulate their surface BCR upon differentiation, hindering analyses that couple BCR information to other molecular characteristics. In this study, we developed a methodology using fixation, permeabilization, and intracellular staining coupled with cell sorting and reversal of the cross-links to allow RNA sequencing of isolated cell subsets. Using hemagglutinin and nucleoprotein Ag-specific B cell tetramers and intracellular staining for IgM, IgG, and IgA isotypes, we were able to derive and compare the gene expression programs of ASC subsets that were responding to the same Ags following influenza infection in mice. Intriguingly, whereas a shared ASC signature was identified, each ASC isotype-specific population expressed distinct transcriptional programs controlling cellular homing, metabolism, and potential effector functions. Additionally, we extracted and compared BCR clonotypes and found that each ASC isotype contained a unique, clonally related CDR3 repertoire. In summary, these data reveal specific complexities in the transcriptional programming of Ag-specific ASC populations.

Progressive Upregulation of Oxidative Metabolism Facilitates Plasmablast Differentiation to a T-Independent Antigen.

Transitioning from a metabolically quiescent naive B cell to an antibody-secreting plasmablast requires division-dependent cellular differentiation. Though cell division demands significant ATP and metabolites, the metabolic processes used for ATP synthesis during plasmablast formation are not well described. Here, the metabolic requirements for plasmablast formation were determined. Following T-independent stimulation with lipopolysaccharide, B cells increased expression of the oxidative phosphorylation machinery in a stepwise manner. Such activated B cells have increased capacity to perform oxidative phosphorylation but showed dependency on glycolysis. Plasmablasts displayed higher oxidative metabolism to support antibody secretion, as inhibiting oxidative ATP production resulted in decreased antibody titers. Differentiation by Blimp1 was required for this increase in oxidative metabolism, as Blimp1-deficient cells proliferate but do not upregulate oxidative phosphorylation. Together, these findings identify a shift in metabolic pathways as B cells differentiate, as well as the requirement for increased metabolic potential to support antibody production.

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.

EZH2 Represses the B Cell Transcriptional Program and Regulates Antibody-Secreting Cell Metabolism and Antibody Production.

Epigenetic remodeling is required during B cell differentiation. However, little is known about the direct functions of epigenetic enzymes in Ab-secreting cells (ASC) in vivo. In this study, we examined ASC differentiation independent of T cell help and germinal center reactions using mice with inducible or B cell-specific deletions of Ezh2 Following stimulation with influenza virus or LPS, Ezh2-deficient ASC poorly proliferated and inappropriately maintained expression of inflammatory pathways, B cell-lineage transcription factors, and Blimp-1-repressed genes, leading to fewer and less functional ASC. In the absence of EZH2, genes that normally gained histone H3 lysine 27 trimethylation were dysregulated and exhibited increased chromatin accessibility. Furthermore, EZH2 was also required for maximal Ab secretion by ASC, in part due to reduced mitochondrial respiration, impaired glucose metabolism, and poor expression of the unfolded-protein response pathway. Together, these data demonstrate that EZH2 is essential in facilitating epigenetic changes that regulate ASC fate, function, and metabolism.

Au-ACRAMTU-PEt3 Alters Redox Balance To Inhibit T Cell Proliferation and Function.

Although T cells play a critical role in protection from viruses, bacteria, and tumors, they also cause autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. Unwanted T cell responses during organ transplant, graft-versus-host disease, and allergies are also major clinical problems. Although drugs are available to suppress unwanted immune responses, they have limited efficacy with serious side effects. Thus, new therapeutics limiting T cell activation, proliferation, and function can make an immediate clinical impact. To identify new suppressors of lymphocyte activation, proliferation, and function, we examined the immunosuppressive activity of gold(I) analogs of platinum-acridine antitumor agents. We found that the gold complex Au-ACRAMTU-PEt3 is a potent suppressor of murine and human T cell activation. Preincubation with Au-ACRAMTU-PEt3 suppresses the proliferation of CD4(+) and CD8(+) T cells at a similar concentration as pharmaceutical grade cyclosporine A. Au-ACRAMTU-PEt3 pretreatment decreases the production of IFN-γ, TNF-α, IL-2, and IL-17 by human and murine CD4(+) and CD8(+) T cells. When mice were treated with Au-ACRAMTU-PEt3 during viral infection, the expansion of virus-specific CD8(+) T cells was decreased 10-fold and viral load was elevated. Taken together, these results demonstrate that Au-ACRAMTU-PEt3 has potent immunosuppressive activity that could be used to suppress immune responses during transplantation and autoimmunity.

Gammaherpesvirus latency differentially impacts the generation of primary versus secondary memory CD8+ T cells during subsequent infection.

UNLABELLED: Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were first infected with murine gammaherpesvirus 68 (MHV68), a model of Epstein-Barr virus (EBV) infection, and then after latency was established, they were challenged with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). The initial replication of LCMV was lower in latently infected mice, and the maturation of dendritic cells was abated. Although the number of LCMV-specific effector CD8(+) T cells was not altered, they were skewed to a memory phenotype. In contrast, LCMV-specific effector CD4(+) T cells were increased in latently infected mice compared to those in mice infected solely with LCMV. When the memory phase was reached, latently infected mice had an LCMV-specific memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8(+) T cells had decreased CD27 and increased killer cell lectin-like receptor G1 (KLRG1) expression. Upon secondary challenge, LCMV-specific secondary effector CD8(+) T cells expanded and cleared the infection. However, the LCMV-specific secondary memory CD8(+) T cell pool was decreased in latently infected animals, abrogating the boosting effect normally observed following rechallenge. Taken together, these results demonstrate that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. IMPORTANCE: CD8(+) T cells are critical for the clearance of intracellular pathogens, including viruses, certain bacteria, and tumors. However, current models for memory CD8(+) T cell differentiation are derived from pathogen-free laboratory mice challenged with a single pathogen or vaccine vector. Unlike laboratory animals, all humans are infected with multiple acute and chronic pathogens, including the highly prevalent herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV), and varicella-zoster virus (VZV). The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. We observed that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. These results suggest that unlike pathogen-free laboratory mice, infection or immunization of latently infected humans may result in the generation of T cells with limited potential for long-term protection.