Dysfunctional natural killer cells can be reprogrammed to regain anti-tumor activity.

Natural killer (NK) cells are critical to the innate immune system, as they recognize antigens without prior sensitization, and contribute to the control and clearance of viral infections and cancer. However, a significant proportion of NK cells in mice and humans do not express classical inhibitory receptors during their education process and are rendered naturally "anergic", i.e., exhibiting reduced effector functions. The molecular events leading to NK cell anergy as well as their relation to those underlying NK cell exhaustion that arises from overstimulation in chronic conditions, remain unknown. Here, we characterize the "anergic" phenotype and demonstrate functional, transcriptional, and phenotypic similarities to the "exhausted" state in tumor-infiltrating NK cells. Furthermore, we identify zinc finger transcription factor Egr2 and diacylglycerol kinase DGKα as common negative regulators controlling NK cell dysfunction. Finally, experiments in a 3D organotypic spheroid culture model and an in vivo tumor model suggest that a nanoparticle-based delivery platform can reprogram these dysfunctional natural killer cell populations in their native microenvironment. This approach may become clinically relevant for the development of novel anti-tumor immunotherapeutic strategies.

Uncontrolled CD21low age-associated and B1 B cell accumulation caused by failure of an EGR2/3 tolerance checkpoint.

CD21low age-associated or atypical memory B cells are autoantibody enriched and poised for plasma cell differentiation. These cells overaccumulate in chronic infections, autoimmune disease, and immunodeficiency, posing the question of what checkpoints normally oppose their accumulation. Here, we reveal a critical role for paralogous calcium-NFAT-regulated transcription factors EGR2 and EGR3 that are induced in self-reactive B cells. CD21low and B1 B cells lacking EGR2 and EGR3 accumulate and circulate in young mice in numbers 10- to 20-fold greater than normal and overexpress a large set of EGR2 ChIP-seq target genes, including known drivers of plasma cell differentiation. Most follicular B cells constitutively express Egr2 proportionally to surface IgM downregulation by self-antigens, and EGR2/3 deficiency abolishes this cardinal feature of B cell anergy. These results explain the cardinal features of B cell anergy, define a key transcriptional checkpoint repressing CD21low B cell formation, and inform how NFATC1 or EGR2 mutations promote B1 cell-derived chronic lymphocytic leukemias.

The Clusters of Transcription Factors NFATC2, STAT5, GATA2, AP1, RUNX1 and EGR2 Binding Sites at the Induced Il13 Enhancers Mediate Il13 Gene Transcription in Response to Antigenic Stimulation.

IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.

Early growth response genes 2 and 3 induced by AP-1 and NF-κB modulate TGF-ß1 transcription in NK1.1- CD4+ NKG2D+ T cells.

NK1.1- CD4+ NKG2D+ T cells are a subpopulation of regulatory T cells that downregulate the functions of CD4+ T, CD8+ T, natural killer (NK) cells, and macrophages through TGF-ß1 production. Early growth response genes 2 (Egr2) and 3 (Egr3) maintain immune homeostasis by modulating T lymphocyte development, inhibiting effector T cell function, and promoting the induction of regulatory T cells. Whether Egr2 and Egr3 directly regulate TGF-ß1 transcription in NK1.1- CD4+ NKG2D+ T cells remains elusive. The expression levels of Egr2 and Egr3 were higher in NK1.1- CD4+ NKG2D+ T cells than in NK1.1- CD4+ NKG2D- T cells. Egr2 and Egr3 expression were remarkably increased after stimulating NK1.1- CD4+ NKG2D+ T cells with sRAE or α-CD3/sRAE. The ectopic expression of Egr2 or Egr3 resulted in the enhancement of TGF-ß1 expression, while knockdown of Egr2 or Egr3 led to the decreased expression of TGF-ß1 in NK1.1- CD4+ NKG2D+ T cells. Egr2 and Egr3 directly bound with the TGF-ß1 promoter as demonstrated by the electrophoretic mobility shift assay and dual-luciferase gene reporter assay. Furthermore, the Egr2 and Egr3 expression of NK1.1- CD4+ NKG2D+ T cells could be induced by the AP-1 and NF-κB transcriptional factors, but had no involvement with the activation of NF-AT and STAT3. In conclusion, Egr2 and Egr3 induced by AP-1 and NF-κB directly initiate TGF-ß1 transcription in NK1.1- CD4+ NKG2D+ T cells. This study indicates that manipulating Egr2 and Egr3 expression would potentiate or alleviate the regulatory function of NK1.1- CD4+ NKG2D+ T cells and this strategy could be used in the therapy for patients with autoimmune diseases or tumor.

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.

Transcription factors early growth response gene (Egr) 2 and 3 control inflammatory responses of tolerant T cells.

INTRODUCTION: Impaired proliferation and production of IL2 are the hallmarks of experimental T cell tolerance. However, in most autoimmune diseases, auto-reactive T cells do not display hyper proliferation, but inflammatory phenotypes. METHODS: We have now demonstrated that the transcription factors Egr2 and 3 are important for the control of inflammatory cytokine production by tolerant T cells, but not for tolerance induction. RESULTS: In the absence of Egr2 and 3, T cell tolerance, as measured by impaired proliferation and production of IL2, can still be induced, but tolerant T cells produced high levels of inflammatory cytokines. Egr2 and 3 regulate expression of differentiation repressors and directly inhibit T-bet function in T cells. Indeed, decreased expression of differentiation repressors, such as Id3 and Tcf1, and increased expression of inflammatory transcription factors, such as RORγt and Bhlhe40 were found in Egr2/3 deficient T cells under tolerogenic conditions. In addition, T-bet was co-expressed with Egr2 in tolerant T cells and Egr2/3 defects leads to production of high levels of IFNγ in tolerant T cells. CONCLUSIONS: Our findings demonstrated that despite impaired proliferation and IL2 production, tolerant T cells can display inflammatory responses in response to antigen stimulation and this is controlled at least partly by Egr2 and 3.

The Lysine Acetyltransferase GCN5 Is Required for iNKT Cell Development through EGR2 Acetylation.

The development of CD1d-restricted invariant natural killer T (iNKT) cells, a population that is critical for both innate and adaptive immunity, is regulated by multiple transcription factors, but the molecular mechanisms underlying how the transcriptional activation of these factors are regulated during iNKT development remain largely unknown. We found that the histone acetyltransferase general control non-derepressible 5 (GCN5) is essential for iNKT cell development during the maturation stage. GCN5 deficiency blocked iNKT cell development in a cell-intrinsic manner. At the molecular level, GCN5 is a specific lysine acetyltransferase of early growth responsive gene 2 (EGR2), a transcription factor required for iNKT cell development. GCN5-mediated acetylation positively regulated EGR2 transcriptional activity, and both genetic and pharmacological GCN5 suppression specifically inhibited the transcription of EGR2 target genes in iNKT cells, including Runx1, promyelocytic leukemia zinc finger protein (PLZF), interleukin (IL)-2Rb, and T-bet. Therefore, our study revealed GCN5-mediated EGR2 acetylation as a molecular mechanism that regulates iNKT development.

Enhanced suppression of polyclonal CD8+25+ regulatory T cells via exosomal arming of antigen-specific peptide/MHC complexes.

Compared with CD4+25+ regulatory T cells (Tregs), the mechanisms for natural, polyclonal CD8+25+ Treg immune suppression have been significantly less studied. We previously showed that polyclonal T cells can acquire antigen-specific targeting activity through arming with exosomal peptide-MHC (pMHC). In this study, we assessed the suppressive effect of CD8+25+ Tregs or CD8+25+ Tregs armed with ovalbumin (OVA)-specific exosomes on other immune cells and OVA-specific dendritic cell (DCOVA)-stimulated antitumor immunity. We demonstrate that CD8+25+ Tregs inhibit T cell proliferation in vitro in a cell contact-dependent fashion but independent of the expression of immunosuppressive IL-10, TGF-ß, and CTLA-4. CD8+25+ Tregs anergize naïve T cells upon stimulation by up-regulating T cell anergy-associated Egr2 and down-regulating IL-2 production. Tregs also anergize DCs by preventing DC maturation through the down-regulation of Iab, CD80, CD86, and inflammatory cytokines, leading to defects in T cell stimulation. Moreover, CD8+25+ Tregs inhibit CTLs through inducing CTL death via perforin-mediated apoptosis and through reducing effector CTL cytotoxic activity via down-regulating CTL perforin-production and degranulation. In addition, we show that CD8+25+ Tregs suppress DCOVA-stimulated CTL responses in priming and effector phases and inhibit immunity against OVA-expressing CCLOVA lung cancer. Remarkably, polyclonal CD8+25+ Tregs armed with OVA-specific exosomal pMHC class-II (pMHC-II), or pMHC class-I (pMHC-I) complexes exert their enhanced inhibition of CTL responses in the priming and the effector phases, respectively. Taken together, our investigation reveals that assigning antigen specificity to nonspecific polyclonal CD8+25+ Tregs for enhanced immune suppression can be achieved through exosomal pMHC arming. This principle may have a great effect on Treg-mediated immunotherapy of autoimmune diseases.

Interleukin 27 (IL-27) Alleviates Bone Loss in Estrogen-deficient Conditions by Induction of Early Growth Response-2 Gene.

A growing understanding of the bone remodeling process suggests that inflammation significantly contributes to the pathogenesis of osteoporosis. T cells and various cytokines contribute majorly to the estrogen deficiency-induced bone loss. Recent studies have identified the IL-12 cytokine family as consisting of pro-inflammatory IL-12 and IL-23 and the anti-inflammatory IL-27 and IL-35 cytokines. IL-27 exerts protective effects in autoimmune diseases like experimental autoimmune encephalomyelitis; however, its role in the pathogenesis of osteoporosis remains to be determined. In this report, we study the effect of IL-27 supplementation on ovariectomized estrogen-deficient mice on various immune and skeletal parameters. IL-27 treatment in ovariectomized mice suppressed Th17 cell differentiation by inhibiting transcription factor RORγt. Supplementation of IL-27 activates Egr-2 to induce IL-10 producing Tr1 cells. IL-27 treatment prevented the loss of trabecular micro-architecture and preserved cortical bone parameters. IL-27 also inhibited osteoblast apoptosis through increased Egr-2 expression, which induces anti-apoptotic factors like MCL-1. IL-27 suppressed osteoclastogenesis in an Egr-2-dependent manner that up-regulates Id2, the repressor of the receptor activator of nuclear factor-κB ligand-mediated osteoclastogenesis. Additionally, these results were corroborated in female osteoporotic subjects where we found decreased serum IL-27 levels along with reduced Egr-2 expression. Our study forms a strong basis for using humanized IL-27 toward the treatment of post-menopausal osteoporosis.

The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αß transgenic T cells (24αß T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αß T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.

Tuberous sclerosis 1 promotes invariant NKT cell anergy and inhibits invariant NKT cell-mediated antitumor immunity.

Development of effective immune therapies for cancer patients requires better understanding of hurdles that prevent the generation of effective antitumor immune responses. Administration of α-galactosylceramide (α-GalCer) in animals enhances antitumor immunity via activation of the invariant NKT (iNKT) cells. However, repeated injections of α-GalCer result in long-term unresponsiveness or anergy of iNKT cells, severely limiting its efficacy in tumor eradication. The mechanisms leading to iNKT cell anergy remain poorly understood. We report in this study that the tuberous sclerosis 1 (TSC1), a negative regulator of mTOR signaling, plays a crucial role in iNKT cell anergy. Deficiency of TSC1 in iNKT cells results in resistance to α-GalCer-induced anergy, manifested by increased expansion of and cytokine production by iNKT cells in response to secondary Ag stimulation. It is correlated with impaired upregulation of programmed death-1, Egr2, and Grail. Moreover, TSC1-deficient iNKT cells display enhanced antitumor immunity in a melanoma lung metastasis model. Our data suggest targeting TSC1/2 as a strategy for boosting antitumor immune therapy.

Early growth response-2 signaling mediates immunomodulatory effects of human multipotential stromal cells.

While most studies have suggested multipotential stromal cell or mesenchymal stem cell (MSC) therapies are useful for immune-mediated diseases, MSCs' immunomodulatory effects were not entirely reproduced in some studies, indicating the necessity to determine the underlying mechanism of MSCs' effects on immune response regulation to maximize their immunomodulatory effects. We have identified the transcription factor early growth response gene-2 (EGR2) as a novel molecular switch regulating known immunomodulatory molecules in human MSCs. EGR2 binds to the promoter regions of these genes, interleukin-6 (IL6), leukemia inhibitory factor (LIF), indoleamine dioxygenase-1 (IDO1), and cyclooxygenase-2/prostaglandin-endoperoxide synthase 2 (COX2/PTGS2), and siRNA against EGR2 was shown to downregulate these genes and reduce the production of prostaglandin E2, an immunomodulatory mediator produced downstream of COX2/PTGS2. Moreover, EGR2 knockdown restores T-lymphocyte proliferation reduced by MSC coculture. Therefore, EGR2 is a potential target for the optimization of immunomodulatory properties of MSC-based therapies.

Leptin up-regulates TLR2 in human monocytes.

The adipokine leptin elicits changes in the expression of the activation markers CD40 and CD69 in PBMCs and DCs, yet its effect on PRRs remains to be elucidated. Serum leptin concentrations are elevated in obesity and T2DM, which are both diseases associated with a proinflammatory state. We therefore investigated a possible role for leptin in monocyte TLR and CD14 expression. Leptin increased TLR2 cell-surface and mRNA expression in THP-1 and primary human monocytes. In contrast, leptin had no effect on monocyte TLR4 expression in THP-1 or primary monocytes. CD14 cell-surface and mRNA expression were increased after leptin stimulation in THP-1 monocytes. However, no change in cell-surface CD14 expression was observed after leptin treatment in primary human monocytes. Leptin also up-regulated the expression of PU.1 and EGR2, transcription factors involved in myeloid cell differentiation. Additionally, leptin potentiated Escherichia coli and Porphyromonas gingivalis LPS-induced TNF-α secretion in THP-1 monocytes. In conclusion, we show that leptin and LPS differentially influence monocyte phenotype and demonstrate, for the first time, a regulatory effect of leptin on the monocyte expression of TLR2. Leptin-stimulated TLR2 expression may potentiate innate immunity and inflammation in conditions of hyperleptinemia, such as obesity and T2DM.

A role for Ly108 in the induction of promyelocytic zinc finger transcription factor in developing thymocytes.

The promyelocytic zinc finger transcription factor (PLZF) is required for the development of activated phenotypes in NKT and other innate T lymphocytes. Although strong TCR stimulation has been implicated in the induction of PLZF, the factors regulating PLZF expression are incompletely understood. We show in this study that costimulation of preselection double-positive thymocytes through the signaling lymphocyte activation molecule family receptor Ly108 markedly enhanced PLZF expression compared with that induced by TCR stimulation alone. Costimulation with Ly108 increased expression of early growth response protein (Egr)-2 and binding of Egr-2 to the promoter of Zbtb16, which encodes PLZF, and resulted in PLZF levels similar to those seen in NKT cells. In contrast, costimulation with anti-CD28 failed to enhance Egr-2 binding and Zbtb16 expression. Moreover, mice lacking Ly108 showed decreased numbers of PLZF-expressing CD4(+) T cells. Together, these results support a potential role for Ly108 in the induction of PLZF.

The transcription factor NFAT exhibits signal memory during serial T cell interactions with antigen-presenting cells.

Interactions with antigen-presenting cells (APCs) interrupt T cell migration through tissues and trigger signaling pathways that converge on the activation of transcriptional regulators, including nuclear factor of activated T cells (NFAT), which control T cell function and differentiation. Both stable and unstable modes of cognate T cell-APC interactions have been observed in vivo, but the functional significance of unstable, serial contacts has remained unclear. Here we used multiphoton intravital microscopy in lymph nodes and tumors to show that while NFAT nuclear import was fast (t(1/2 max)∼1 min), nuclear export was slow (t(1/2)∼20 min) in T cells. During delayed export, nuclear NFAT constituted a short-term imprint of transient TCR signals and remained transcriptionally active for the T cell tolerance gene Egr2, but not for the effector gene Ifng, which required continuous TCR triggering for expression. This provides a potential mechanistic basis for the observation that a predominance of unstable APC interactions correlates with the induction of T cell tolerance.

Transcriptional regulator early growth response gene 2 (Egr2) is required for T cell anergy in vitro and in vivo.

T cell receptor engagement in the absence of costimulation results in a hyporesponsive state termed anergy. Understanding the transcriptional regulation of other T cell differentiation states has provided critical information regarding the biology of T cell regulation in vivo. However, the transcriptional regulation of T cell anergy has been poorly understood. Using the key anergy target gene diacylglycerol kinase (DGK) α as a focal point, we identified early growth response gene 2 (Egr2) as a central transcription factor that regulates the anergic state. Conditional Egr2 deletion in peripheral T cells abolishes induced expression of DGK-α and other anergy genes and restores Ras/MAPK signaling, IL-2 production, and proliferation upon attempted anergy induction. Using superantigen- and tumor-induced anergy models, we found that Egr2 is necessary for anergy induction in vivo. Collectively, our results implicate Egr2 as an essential transcriptional regulator of the T cell anergy program.

Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR signaling.

Interactions driven by the T cell antigen receptor (TCR) determine the lineage fate of CD4(+)CD8(+) thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we found that TCR-induced transcription factors Egr2 and Egr1 had higher and more-prolonged expression in precursors of the natural killer T (NKT) than in cells of conventional lineages. Chromatin immunoprecipitation followed by deep sequencing showed that Egr2 directly bound and activated the promoter of Zbtb16, which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the promoter of Il2rb, which encodes the interleukin 2 (IL-2) receptor ß-chain, and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that persistent higher expression of Egr2 specifies the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to 'instruct' a thymic lineage.

Deltex1 is a target of the transcription factor NFAT that promotes T cell anergy.

The molecular process underlying T cell anergy is incompletely understood. Deltex1 (DTX1) is a Notch target with unknown physiological function. Here we show that Dtx1 was a transcription target of nuclear factor of activated T cells (NFAT) and participated in T cell anergy. DTX1 protein was upregulated during T cell anergy, and transgenic expression of Dtx1 attenuated T cell activation. DTX1 inhibited T cell activation by both E3-dependent and E3-independent mechanisms. In addition, DTX1 suppressed T cell activation in the absence of its Notch-binding domain. Importantly, DTX1 regulated the expression of two anergy-associated molecules, growth arrest and DNA-damage-inducible 45 beta (Gadd45 beta) and Cbl-b. DTX1 interacted with early growth response 2 (Egr-2) for optimum expression of Cbl-b. Furthermore, deficiency of DTX1 augmented T cell activation, conferred resistance to anergy induction, enhanced autoantibody generation, and increased inflammation. DTX1 therefore represents a component downstream of calcium-NFAT signaling that regulates T cell anergy.

Egr2 is required for Bcl-2 induction during positive selection.

The repertoire of TCR specificities is established by a selection process in the thymus, during which precursor survival and maturation is dictated by the nature of the TCR signals. The differences in signals that determine whether precursors will survive and mature or be induced to die remain poorly understood. Among the molecular effectors involved in executing the differentiation process initiated by TCR-ligand interactions is a family of Zn-finger transcription factors termed early growth response genes (Egr). Indeed, ablation of the Egr1 gene impairs ligand-induced maturation (positive selection) but not ligand-induced deletion (negative selection). The partial impairment of positive selection by Egr1 deficiency is not enhanced by simultaneous deletion of another Egr family member, Egr3. Accordingly, we asked whether this results from compensation by another family member, Egr2. In this manuscript, we demonstrate that deletion of Egr2 impairs positive selection of both CD4 and CD8 single-positive thymocytes. Interestingly, many of the genes involved in positive selection and T cell differentiation are up-regulated normally in the Egr2-deficient thymocytes. However, Bcl-2 up-regulation is not sustained during late stages of positive selection. This defect is at least partially responsible for the developmental blockade in Egr2-deficient thymocytes, as enforced expression of Bcl-2 rescues T cell development in Egr2(-/-) thymocytes. Taken together, these data suggest that Egr2 plays a central role in the up-regulation of the survival molecule Bcl-2 during positive selection.