The chromatin remodeler Brg1 activates enhancer repertoires to establish B cell identity and modulate cell growth.

Early B cell development is orchestrated by the combined activities of the transcriptional regulators E2A, EBF1, Foxo1 and Ikaros. However, how the genome-wide binding patterns of these regulators are modulated during B lineage development remains to be determined. Here we found that in lymphoid progenitor cells, the chromatin remodeler Brg1 specified the B cell fate. In committed pro-B cells, Brg1 regulated contraction of the locus encoding the immunoglobulin heavy chain (Igh) and controlled expression of the gene encoding the transcription factor c-Myc (Myc) to modulate the expression of genes encoding products that regulate ribosome biogenesis. In committed pro-B cells, Brg1 suppressed a pre-B lineage-specific pattern of gene expression. Finally, we found that Brg1 acted mechanistically to establish B cell fate and modulate cell growth by facilitating access of lineage-specific transcription factors to enhancer repertoires.

c-Myc-induced transcription factor AP4 is required for host protection mediated by CD8+ T cells.

Although the transcription factor c-Myc is essential for the establishment of a metabolically active and proliferative state in T cells after priming, its expression is transient. It remains unknown how T cell activation is maintained after c-Myc expression is downregulated. Here we identified AP4 as the transcription factor that was induced by c-Myc and sustained activation of antigen-specific CD8+ T cells. Despite normal priming, AP4-deficient CD8+ T cells failed to continue transcription of a broad range of c-Myc-dependent targets. Mice lacking AP4 specifically in CD8+ T cells showed enhanced susceptibility to infection with West Nile virus. Genome-wide analysis suggested that many activation-induced genes encoding molecules involved in metabolism were shared targets of c-Myc and AP4. Thus, AP4 maintains c-Myc-initiated cellular activation programs in CD8+ T cells to control microbial infection.

A regulatory role for TGF-ß signaling in the establishment and function of the thymic medulla.

Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-ß in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-ß signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells.

GATA-3 controls the maintenance and proliferation of T cells downstream of TCR and cytokine signaling.

GATA-3 controls T helper type 2 (TH2) differentiation. However, whether GATA-3 regulates the function of mature T cells beyond TH2 determination remains poorly understood. We found that signaling via the T cell antigen receptor (TCR) and cytokine stimulation promoted GATA-3 expression in CD8(+) T cells, which controlled cell proliferation. Although GATA-3-deficient CD8(+) T cells were generated, their peripheral maintenance was impaired, with lower expression of the receptor for interleukin 7 (IL-7R). GATA-3-deficient T cells had defective responses to viral infection and alloantigen. The proto-oncoprotein c-Myc was a critical target of GATA-3 in promoting T cell proliferation. Our study thus demonstrates an essential role for GATA-3 in controlling the maintenance and proliferation of T cells and provides insight into immunoregulation.

The cell-cycle regulator c-Myc is essential for the formation and maintenance of germinal centers.

Germinal centers (GCs) are sites of intense B cell proliferation and are central for T cell-dependent antibody responses. However, the role of c-Myc, a key cell-cycle regulator, in this process has been questioned. Here we identified c-Myc(+) B cell subpopulations in immature and mature GCs and found, by genetic ablation of Myc, that they had indispensable roles in the formation and maintenance of GCs. The identification of these functionally critical cellular subsets has implications for human B cell lymphomagenesis, which originates mostly from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the c-Myc(+) GC subpopulations may be at a particularly high risk for malignant transformation.

Large remodeling of the Myc-induced cell surface proteome in B cells and prostate cells creates new opportunities for immunotherapy.

MYC is a powerful transcription factor overexpressed in many human cancers including B cell and prostate cancers. Antibody therapeutics are exciting opportunities to attack cancers but require knowledge of surface proteins that change due to oncogene expression. To identify how MYC overexpression remodels the cell surface proteome in a cell autologous fashion and in different cell types, we investigated the impact of MYC overexpression on 800 surface proteins in three isogenic model cell lines either of B cell or prostate cell origin engineered to have high or low MYC levels. We found that MYC overexpression resulted in dramatic remodeling (both up- and down-regulation) of the cell surfaceome in a cell type-dependent fashion. We found systematic and large increases in distinct sets of >80 transporters including nucleoside transporters and nutrient transporters making cells more sensitive to toxic nucleoside analogs like cytarabine, commonly used for treating hematological cancers. Paradoxically, MYC overexpression also increased expression of surface proteins driving cell turnover such as TNFRSF10B, also known as death receptor 5, and immune cell attacking signals such as the natural killer cell activating ligand NCR3LG1, also known as B7-H6. We generated recombinant antibodies to these two targets and verified their up-regulation in MYC overexpression cell lines and showed they were sensitive to bispecific T cell engagers (BiTEs). Our studies demonstrate how MYC overexpression leads to dramatic bidirectional remodeling of the surfaceome in a cell type-dependent but functionally convergent fashion and identify surface targets or combinations thereof as possible candidates for cytotoxic metabolite or immunotherapy.

MALT1 protease function in regulatory T cells induces MYC activity to promote mitochondrial function and cellular expansion.

Regulatory T cells (Tregs) are essential for the inhibition of immunity and the maintenance of tissue homeostasis. Signals from the T-cell antigen receptor (TCR) are critical for early Treg development, their expansion, and inhibitory activity. Although TCR-engaged activation of the paracaspase MALT1 is important for these Treg activities, the MALT1 effector pathways in Tregs remain ill-defined. Here, we demonstrate that MALT1 protease activity controls the TCR-induced upregulation of the transcription factor MYC and the subsequent expression of MYC target genes in Tregs. These mechanisms are important for Treg-intrinsic mitochondrial function, optimal respiratory capacity, and homeostatic Treg proliferation. Consistently, conditional deletion of Myc in Tregs results similar to MALT1 inactivation in a lethal autoimmune inflammatory syndrome. Together, these results identify a MALT1 protease-mediated link between TCR signaling in Tregs and MYC control that coordinates metabolism and Treg expansion for the maintenance of immune homeostasis.

Regulation of hematopoietic stem cell differentiation by a single ubiquitin ligase-substrate complex.

Hematopoietic stem cell (HSC) differentiation is regulated by cell-intrinsic and cell-extrinsic cues. In addition to transcriptional regulation, post-translational regulation may also control HSC differentiation. To test this hypothesis, we visualized the ubiquitin-regulated protein stability of a single transcription factor, c-Myc. The stability of c-Myc protein was indicative of HSC quiescence, and c-Myc protein abundance was controlled by the ubiquitin ligase Fbw7. Fine changes in the stability of c-Myc protein regulated the HSC gene-expression signature. Using whole-genome genomic approaches, we identified specific regulators of HSC function directly controlled by c-Myc binding; however, adult HSCs and embryonic stem cells sensed and interpreted c-Myc-regulated gene expression in distinct ways. Our studies show that a ubiquitin ligase-substrate pair can orchestrate the molecular program of HSC differentiation.

IL-7Rαlow CD8+ T Cells from Healthy Individuals Are Anergic with Defective Glycolysis.

Effector memory (EM) CD8+ T cells expressing lower levels of IL-7R α (IL-7Rαlow) from healthy individuals are partly compromised in vitro, but the identity of these cells has remained unclear. In this study, we demonstrate that human IL-7Rαlow EM CD8+ T cells are naturally occurring anergic cells in vivo and impaired in proliferation and IL-2 production but competent in IFN-γ and TNF-α production, a state that can be restored by IL-2 stimulation. IL-7Rαlow EM CD8+ T cells show decreased expression of GATA3 and c-MYC and are defective in metabolic reprogramming toward glycolysis, a process required for the proliferation of T cells. However, IL-7Rαlow EM CD8+ T cells can proliferate with TCR stimulation in the presence of IL-2 and IL-15, suggesting that these cells can be restored to normality or increased activity by inflammatory conditions and may serve as a reservoir for functional immunity.

MYC-driven malignant transformation of mature murine B cells requires inhibition of both intrinsic apoptosis and p53 activity.

Increased expression of the oncogene MYC is a common feature of many B-cell malignancies, however MYC overexpression by itself is not sufficient for transformation, and additional genetic events are required, although the exact nature of these remains unknown. In patients and in transgenic mouse models, oncogenic transformation may occur in B cells at various differentiation stages interacting with complex microenvironments. B-cell oncogenesis often occurs after prolonged periods of time, making it difficult to accurately identify the genetic events required for transformation. An in vitro system, where malignant transformation of primary B cells could be analyzed, would facilitate the identification of genetic events required for transformation. Here, we describe such a system and show that primary murine B cells rapidly become transformed upon forced expression of MYC, in conjunction with simultaneous inhibition of the ARF/p53 axis via overexpression of BMI1, as well as through downregulation of p19ARF or expression of a dominant-negative p53 and suppression of intrinsic apoptosis through overexpression of BCLXL or MCL1. Established tumor cells remained addicted to expression of the lymphoma-inducing genes. In mice, transformed cells rapidly established fatal B-cell lymphomas. Our results suggest that transformation of normal mature B cells into lymphomas can occur as a consequence of three defined events.

Transformation of mature mouse B cells into malignant plasma cells in vitro via introduction of defined genetic elements.

An experimental system where defined alterations in gene function or gene expression levels in primary B cells would result in the development of transformed plasma cells in vitro would be useful in order to facilitate studies of the underlying molecular mechanisms of plasma cell malignancies. Here, such a system is described in which primary murine B cells rapidly become transformed into surface CD138+ , IgM-/low , CD19- IgM-secreting plasma cells as a result of expression of the transcription factors IRF4 and MYC together with simultaneous expression of BMI1, mutated p53 or silencing of p19Arf , and suppression of intrinsic apoptosis through expression of BCLXL. Analysis of gene expression patterns revealed that this combination of transforming genes resulted in expression of a number of genes previously associated with terminally differentiated B cells (plasma cells) and myeloma cells, whereas many genes associated with mature B cells and B-cell lymphomas were not expressed. Upon transplantation, the transformed cells preferentially localized to the bone marrow, presenting features of a plasma cell malignancy of the IgM isotype. The present findings may also be applicable in the development of novel methods for production of monoclonal antibodies.

MYC: Master Regulator of Immune Privilege.

Cancers are often initiated by genetic events that activate proto-oncogenes or inactivate tumor-suppressor genes. These events are also crucial for sustained tumor cell proliferation and survival, a phenomenon described as oncogene addiction. In addition to this cell-intrinsic role, recent evidence indicates that oncogenes also directly regulate immune responses, leading to immunosuppression. Expression of many oncogenes or loss of tumor suppressors induces the expression of immune checkpoints that regulate the immune response, such as PD-L1. We discuss here how oncogenes, and in particular MYC, suppress immune surveillance, and how oncogene-targeted therapies may restore the immune response against tumors.

Id3 Restricts γδ NKT Cell Expansion by Controlling Egr2 and c-Myc Activity.

γδ NKT cells are neonatal-derived γδ T lymphocytes that are grouped together with invariant NKT cells based on their shared innate-like developmental program characterized by the transcription factor PLZF (promyelocytic leukemia zinc finger). Previous studies have demonstrated that the population size of γδ NKT cells is tightly controlled by Id3-mediated inhibition of E-protein activity in mice. However, how E proteins promote γδ NKT cell development and expansion remains to be determined. In this study, we report that the transcription factor Egr2, which also activates PLZF expression in invariant NKT cells, is essential for regulating γδ NKT cell expansion. We observed a higher expression of Egr family genes in γδ NKT cells compared with the conventional γδ T cell population. Loss of function of Id3 caused an expansion of γδ NKT cells, which is accompanied by further upregulation of Egr family genes as well as PLZF. Deletion of Egr2 in Id3-deficient γδ NKT cells prevented cell expansion and blocked PLZF upregulation. We further show that this Egr2-mediated γδ NKT cell expansion is dependent on c-Myc. c-Myc knockdown attenuated the proliferation of Id3-deficient γδ NKT cells, whereas c-Myc overexpression enhanced the proliferation of Id3/Egr2-double-deficient γδ NKT cells. Therefore, our data reveal a regulatory circuit involving Egr2-Id3-E2A, which normally restricts the population size of γδ NKT cells by adjusting Egr2 dosage and c-Myc expression.

Burkitt Leukemia With Precursor B-Cell Immunophenotype and Dual Translocation of t(14;18) and t(8;14) in a Child: Case Report and Review of the Literature.

BACKGROUND: Burkitt leukemia (BL) with the precursor B-cell immunophenotype is a rarely reported condition. The prognosis of such patients is similar to that of classic BL. However, the combination of chromosomal translocations associated with bcl-2 and c-myc rearrangement has a poor prognosis. OBSERVATIONS: An 11-year-old child presented with fever and weakness. Bone marrow aspiration showed morphologically L1 type blasts and flow cytometry analysis was compatible with precursor B-cell immunophenotype. Cytogenetic analysis revealed a combination of t(8;14) and t(14;l8). CONCLUSIONS: The combination of t(8;14) and t(14;l8) can exhibit different immunophenotypical and morphologic features in leukemias.

Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes.

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses.

Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.

A key event required for effective resolution of inflammation is efferocytosis, which is defined as phagocytic removal of apoptotic cells mostly by macrophages acquiring an alternatively activated phenotype (M2). c-Myc has been reported to play a role in alternative activation of human macrophages and is proposed as one of the M2 macrophage markers. We found that M2-like peritoneal macrophages from zymosan A-treated mice exhibited a marked accumulation of Myc-nick, a truncated protein generated by a Calpain-mediated proteolytic cleavage of full-length c-Myc. Further, ectopic expression of Myc-nick in murine bone marrow-derived macrophages promoted the M2 polarization and, consequently, enhanced their efferocytic capability. Notably, Myc-nick-induced efferocytosis was found to be tightly associated with α-tubulin acetylation by K acetyltransferase 2a (Kat2a/Gcn5) activity. These findings suggest Myc-nick as a novel proresolving mediator that has a fundamental function in maintaining homeostasis under inflammatory conditions.-Zhong, X., Lee, H.-N., Kim, S. H., Park, S.-A., Kim, W., Cha, Y.-N., Surh, Y.-J. Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.

Down-regulation of microRNA-451a facilitates the activation and proliferation of CD4+ T cells by targeting Myc in patients with dilated cardiomyopathy.

CD4+ T cells are abnormally activated in patients with dilated cardiomyopathy (DCM) and might be associated with the immunopathogenesis of the disease. However, the underlying mechanisms of CD4+ T cell activation remain largely undefined. Our aim was to investigate whether the dysregulation of microRNAs (miRNAs) was associated with CD4+ T cell activation in DCM. CD4+ T cells from DCM patients showed increased expression levels of CD25 and CD69 and enhanced proliferation in response to anti-CD3/28, indicating an activated state. miRNA profiling analysis of magnetically sorted CD4+ T cells revealed a distinct pattern of miRNA expression in CD4+ T cells from DCM patients compared with controls. The level of miRNA-451a (miR-451a) was significantly decreased in the CD4+ T cells of DCM patients compared with that of the controls. The transfection of T cells with an miR-451a mimic inhibited their activation and proliferation, whereas an miR-451a inhibitor produced the opposite effects. Myc was directly inhibited by miR-451a via interaction with its 3'-UTR, thus identifying it as an miR-451a target in T cells. The knockdown of Myc suppressed the activation and proliferation of T cells, and the expression of Myc was significantly up-regulated at the mRNA level in CD4+ T cells from patients with DCM. A strong inverse correlation was observed between the Myc mRNA expression and miR-451a transcription level. Our data suggest that the down-regulation of miR-451a contributes to the activation and proliferation of CD4+ T cells by targeting the transcription factor Myc in DCM patients and may contribute to the immunopathogenesis of DCM.

Role of c-Myb in the regulation of natural killer cell activity.

The regulation of natural killer (NK) cell activity is an important research goal for the development of immunotherapies. In this study, we identified transcription factors affecting NK cell activity. In particular, we screened transcription factors affected by interleukin-2 (IL-2) and transforming growth factor-beta (TGF-ß) by protein/DNA arrays using primary NK cells. We found that celastrol, a c-Myb inhibitor, inhibited NK-92 cells more strongly than any other inhibitors of transcription factor candidates. In addition, c-Myb and c-Myb-related signaling molecules, e.g., Nemo-like kinase (NLK) and c-Myc, were regulated by the activation status of NK cells, suggesting that c-Myb is a key regulator of NK cell activity. We also found that celastrol inhibits NK-92-cell-mediated cytotoxicity via the downregulation of NKG2D and granzyme B. Knockdown studies also showed that c-Myb is important for NK cell activation. In particular, the knockdown of c-Myb did not significantly affect NK cell proliferation and survival but decreased the secretion of IFN-γ and the cytotoxicity of NK cells. Our data demonstrate that c-Myb plays a critical role in the activation of NK cells and therefore is a therapeutic target for cancer and viral diseases.

Molecular mechanisms of autophagic memory in pathogenic T cells in human arthritis.

T-cell resilience is critical to the immune pathogenesis of human autoimmune arthritis. Autophagy is essential for memory T cell generation and associated with pathogenesis in rheumatoid arthritis (RA). Our aim here was to delineate the role and molecular mechanism of autophagy in resilience and persistence of pathogenic T cells from autoimmune arthritis. We demonstrated "Autophagic memory" as elevated autophagy levels in CD4+ memory T cells compared to CD4+ naive T cells and in Jurkat Human T cell line trained with starvation stress. We then showed increased levels of autophagy in pathogenic CD4+ T cells subsets from autoimmune arthritis patients. Using RNA-sequencing, transcription factor gene regulatory network and methylation analyses we identified MYC as a key regulator of autophagic memory. We validated MYC levels using qPCR and further demonstrated that inhibiting MYC increased autophagy. The present study proposes the novel concept of autophagic memory and suggests that autophagic memory confers metabolic advantage to pathogenic T cells from arthritis and supports its resilience and long term survival. Particularly, suppression of MYC imparted the heightened autophagy levels in pathogenic T cells. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target.