The transcription factor Interferon regulatory factor 8 (IRF8) is involved in maintaining B cell identity. However, how IRF8 regulates T cell independent B cell responses are not fully characterized. Here, an in vivo CRISPR/Cas9 system was optimized to generate Irf8-deficient murine B cells and used to determine the role of IRF8 in B cells responding to LPS stimulation. Irf8-deficient B cells more readily formed CD138+ plasmablasts in response to LPS with the principal dysregulation occurring at the activated B cell stage. Transcriptional profiling revealed an upregulation of plasma cell associated genes prematurely in activated B cells and a failure to repress the gene expression programs of IRF1 and IRF7 in Irf8-deficient cells. These data expand on the known roles of IRF8 in regulating B cell identity by preventing premature plasma cell formation and highlight how IRF8 helps evolve TLR responses away from the initial activation towards those driving humoral immunity.

The world’s population with obesity is reaching pandemic levels. If current trends continue, it is predicted that there will be 1.5 billion people with obesity by 2030. This projection is alarming due to the association of obesity with numerous diseases including cancer, with recent studies demonstrating a positive association with acute myeloid leukemia (AML) and B cell acute lymphoblastic leukemia (B-ALL). Interestingly, several epidemiological studies suggest the converse relationship may exist in patients with T cell acute lymphoblastic leukemia (T-ALL). To determine the relationship between obesity and T-ALL development, we employed the diet-induced obesity (DIO) murine model and cultured human T-ALL cells in adipocyte-conditioned media (ACM), bone marrow stromal cell-conditioned media, stromal conditioned media (SCM), and unconditioned media to determine the functional impact of increased adiposity on leukemia progression. Whereas only 20% of lean mice transplanted with T-ALL cells survived longer than 3 months post-inoculation, 50%–80% of obese mice with leukemia survived over this same period. Furthermore, culturing human T-ALL cells in ACM resulted in increased histone H3 acetylation (K9/K14/K18/K23/K27) and methylation (K4me3 and K27me3) posttranslational modifications (PTMs), which preceded accelerated cell cycle progression, DNA damage, and cell death. Adipocyte-mediated epigenetic changes in human T-ALL cells were recapitulated with the H3K27 demethylase inhibitor GSK-J4 and the pan-HDAC inhibitor vorinostat. These drugs were also highly cytotoxic to human T-ALL cells at low micromolar concentrations. In summary, our data support epidemiological studies demonstrating that adiposity suppresses T-ALL pathogenesis. We present data demonstrating that T-ALL cell death in adipose-rich microenvironments is induced by epigenetic modifications, which are not tolerated by leukemia cells. Similarly, GSK-J4 and vorinostat treatment induced epigenomic instability and cytotoxicity profiles that phenocopied the responses of human T-ALL cells to ACM, which provides additional support for the use of epigenetic modifying drugs as a treatment option for T-ALL.

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.

B cell differentiation into Ab-secreting plasma cells requires transcriptional, metabolic, and epigenetic remodeling. Histone H3 lysine 27 trimethylation (H3K27me3), a histone modification associated with gene silencing, is dynamically regulated during B cell differentiation. Although several studies have focused on mechanisms involving the gain of this modification in plasmablasts (PB), the role of active demethylation of H3K27me3 by ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and Jumonji domain-containing protein 3 (JMDJ3) during B cell differentiation has not been examined. In this study, this process was assessed using a pharmacological inhibitor of UTX and JMJD3, GSK-J4. Treatment of ex vivo stimulated mouse B cells with GSK-J4 led to an increase in PB frequency without affecting the ability of the newly formed PB to secrete Abs. Consistent with the role of UTX and JMJD3 in promoting gene expression, the majority of differentially expressed were downregulated upon GSK-J4 treatment. GSKJ4–treated cells downregulated genes associated with signaling and P53 pathways. Inhibitor treated cells upregulated genes associated with cell cycle and proliferation, which correlated with an increase in actively proliferating cells. Unexpectedly, a majority of the downregulated transcripts corresponded to genes that in the wild-type setting were genes that gain H3K27me3 and downregulated in PB. Together, our results show that UTX and JMDJ3 are required to restrain B cell differentiation and suggest that they function as a rheostat for H3K27me3 to control this process.

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. ImmunoHorizons, 2022, 6: 872-882.

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.

Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection1. Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1+TCF1+ stem-like CD8+ T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+ T cells that resemble highly functional effector CD8+ T cells seen after an acute viral infection. The generation of these qualitatively superior CD8+ T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1+TCF1+ stem-like CD8+ T cells, also referred to as precursors of exhausted CD8+ T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8+ T cells emerging from the stem-like CD8+ T cells after combination therapy expressed increased levels of the high-affinity IL-2 trimeric (CD25–CD122–CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 has an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer2,3, and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.

Major histocompatibility class II (MHC-II) expression is critical for immune responses and is controlled by the MHC-II transactivator CIITA. CIITA is primarily regulated at the transcriptional level and is expressed from three main promoters with myeloid, lymphoid and interferon (IFN)-γ-treated non-hematopoietic cells using promoters pI, pIII and pIV, respectively. Recent studies in non-hematopoietic cells suggest that a series of distal regulatory elements may be involved in regulating CIITA transcription. To identify distal elements in B cells, a DNase I hypersensitivity screen was performed, revealing a series of potential novel regulatory elements. These elements were analyzed computationally and biochemically. Several regions displayed active histone modifications and/or enhanced expression of a reporter gene. Four of the elements interacted with pIII in B cells. These same four regions were also found to interact with pI in splenic dendritic cells (spDC). Intriguingly, examination of the above interactions in pI-knockout-derived spDC showed a switch to the next available promoter, pIII. Extensive DNA methylation was found at the pI region in B cells, suggesting that this promoter is not accessible in B cells. Thus, CIITA expression is likely mediated in hematopoietic cells by common elements with promoter accessibility having a part in promoter choice.

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.

The cumulative effects of T cell receptor (TCR) signal transduction over extended periods of time influences T cell biology, such as the positive selection of immature thymocytes or the proliferative responses of naive T cells. Naive T cells experience recurrent TCR signaling in response to self-antigens in the steady state. However, how these signals influence the responsiveness of naive CD8 + T cells to subsequent agonist TCR stimulation remains incompletely understood. We investigated how naive CD8 + T cells that experienced relatively low or high levels of TCR signaling in response to self-antigens respond to stimulation with foreign antigens. A transcriptional reporter of Nr4a1 (Nur77-GFP) revealed substantial heterogeneity of the amount of TCR signaling naive CD8 + T cells accumulate in the steady state. Nur77-GFP HI cells exhibited diminished T cell activation and secretion of IFNγ and IL-2 relative to Nur77-GFP LO cells in response to agonist TCR stimulation. Differential gene expression analyses revealed upregulation of genes associated with acutely stimulated T cells in Nur77-GFP HI cells but also increased expression of negative regulators such as the phosphatase Sts1. Responsiveness of Nur77-GFP HI cells to TCR stimulation was partially restored at the level of IFNγ secretion by deficiency of Sts1 or the ubiquitin ligase Cbl-b. Our data suggest that extensive accumulation of TCR signaling during steady state conditions induces a recalibration of the responsiveness of naive CD8 + T cells through gene expression changes and negative regulation, at least in part, dependent on Sts1 and Cbl-b. This cell-intrinsic negative feedback loop may allow the immune system to limit the autoreactive potential of highly self-reactive naive CD8 + T cells.