Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.

Egr2 and 3 maintain anti-tumour responses of exhausted tumour infiltrating CD8 + T cells.

Although T cells can develop into an exhausted state in the tumour microenvironment, tumour infiltrating T cells (TILs) are important to control tumour growth. By analysing single cell RNA-sequencing data from human tumours, we found that the transcription factors Early Growth Response 2 (EGR2) and 3 were highly induced in TILs, but not peripheral CD8 + T cells, in multiple patient cohorts. We found that deficiency of Egr2 and 3 in T cells resulted in enhanced tumour growth and fewer TILs in mouse models. Egr2 is highly expressed together with checkpoint molecules in a proportion of CD8 + TILs and Egr2high cells exhibit better survival and proliferation than Egr2-/-Egr3-/- and Egr2low TILs. Anti-PD-1 treatment increases Egr2 expression in CD8 + TILs and reduces tumour growth, while anti-PD-1 efficacy is abrogated in the absence of Egr2 and 3. Thus, Egr2 and 3 are important for maintaining anti-tumour responses of exhausted CD8 + TILs.

The transcription factors Egr2 and Egr3 are essential for the control of inflammation and antigen-induced proliferation of B and T cells.

Lymphocytes provide optimal responses against pathogens with minimal inflammatory pathology. However, the intrinsic mechanisms regulating these responses are unknown. Here, we report that deletion of both transcription factors Egr2 and Egr3 in lymphocytes resulted in a lethal autoimmune syndrome with excessive serum proinflammatory cytokines but also impaired antigen receptor-induced proliferation of B and T cells. Egr2- and Egr3-defective B and T cells had hyperactive signal transducer and activator of transcription-1 (STAT1) and STAT3 while antigen receptor-induced activation of transcription factor AP-1 was severely impaired. We discovered that Egr2 and/or Egr3 directly induced expression of suppressor of cytokine signaling-1 (SOCS1) and SOCS3, inhibitors of STAT1 and STAT3, and also blocked the function of Batf, an AP-1 inhibitor, in B and T cells. Thus, Egr2 and Egr3 regulate B and T cell function in adaptive immune responses and homeostasis by promoting antigen receptor signaling and controlling inflammation.

Egr2 and 3 Inhibit T-bet-Mediated IFN-γ Production in T Cells.

T-bet is important for differentiation of cytotoxic CD8 and Th1 CD4 T cells. We have discovered that Egr2 and 3 are potent inhibitors of T-bet function in CD4 and CD8 effector T cells. Egr2 and 3 were essential to suppress Th1 differentiation in Th2 and Th17 conditions in vitro and also to control IFN-γ-producing CD4 and CD8 T cells in response to virus infection. Together with Egr2 and 3, T-bet is induced in naive T cells by Ag stimulation, but Egr2 and 3 expression was inhibited by Th1-inducing cytokines. We found that Egr2 and 3 physically interact with the T-box domain of T-bet, blocking T-bet DNA binding and inhibiting T-bet-mediated production of IFN-γ. Thus, Egr2 and 3 are antagonists of T-bet function in effector T cells and are important for the control of inflammatory responses of T cells.

Early Growth Response Genes 2 and 3 Regulate the Expression of Bcl6 and Differentiation of T Follicular Helper Cells.

T follicular helper (Tfh) cells support differentiation of B cells to plasma cells and high affinity antibody production in germinal centers (GCs), and Tfh differentiation requires the function of B cell lymphoma 6 (BCL6). We have now discovered that early growth response gene 2 (EGR2) and EGR3 directly regulate the expression of Bcl6 in Tfh cells, which is required for their function in regulation of GC formation. In the absence of EGR2 and -3, the expression of BCL6 in Tfh cells is defective, leading to impaired differentiation of Tfh cells, resulting in a failure to form GCs following virus infection and defects in production of antiviral antibodies. Enforced expression of BCL6 in EGR2/3-deficient CD4 T cells partially restored Tfh differentiation and GC formation in response to virus infection. Our findings demonstrate a novel function of EGR2/3 that is important for Tfh cell development and Tfh cell-mediated B cell immune responses.

Early growth response gene-2 controls IL-17 expression and Th17 differentiation by negatively regulating Batf.

Early growth response gene (Egr)-2 is important for the maintenance of T cell homeostasis and controls the development of autoimmune disease. However, the underlying mechanisms are unknown. We have now discovered that Egr-2, which is induced by TGF-ß and IL-6, negatively regulates the expression of IL-17, but not IL-2 or IFN-γ, in effector T cells. In the absence of Egr-2, CD4 T cells produce high levels of Th17 cytokines, which renders mice susceptible to experimental autoimmune encephalomyelitis induction. T cells lacking Egr-2 show increased propensity for Th17, but not Th1 or Th2, differentiation. Control of IL-17 expression and Th17 differentiation by Egr-2 is due to inhibition of Batf, a transcription factor that regulates IL-17 expression and Th17 differentiation. Egr-2 interacts with Batf in CD4 T cells and suppresses its interaction with DNA sequences derived from the IL-17 promoter, whereas the activation of STAT3 and expression of retinoic acid-related orphan receptor γt are unchanged in Th17 cells in the absence of Egr-2. Thus, Egr-2 plays an important role to intrinsically control Th17 differentiation. We also found that CD4 T cells from multiple sclerosis patients have reduced expression of Egr-2 and increased expression of IL-17 following stimulation with anti-CD3 in vitro. Collectively, our results demonstrate that Egr-2 is an intrinsic regulator that controls Th17 differentiation by inhibiting Batf activation, which may be important for the control of multiple sclerosis development.

IL-2-engineered nano-APC effectively activates viral antigen-mediated T cell responses from chronic hepatitis B virus-infected patients.

Impaired function of virus-specific T cells resulting from virus persistence is one of the major mechanisms underlying the development of chronic hepatitis B viral infection. Previously, we found that IL-2 can restore the effector function of T cells rendered tolerant by Ag persistence. However, systemic administration of IL-2 induces organ pathology and expansion of T regulatory cells. In this study, we show that nano-APC with engineered HLA alleles and IL-2 deliver peptide-MHC complexes, costimulatory molecules, and IL-2 to Ag-responding T cells, resulting in enhanced expression of CD25 and activation of TCR signaling pathways, while suppressing PD-1 expression on viral-responding CD8 T cells from chronic hepatitis B virus patients. The enhanced activation of CD4 and CD8 T cells induced by IL-2-nano-APC was Ag dependent and IL-2-nano-APC did not affect T regulatory cells. At a size of 500 nm, the nano-APC effectively induce immune synapse formation on Ag-specific T cells and accumulate as free particles in the lymphoid organs. These attributes of IL-2-nano-APC or other bioadjuvant-engineered nano-APC have profound implications for their use as a therapeutic strategy in the treatment of chronic hepatitis B virus infection or other chronic viral diseases.

Egr2 regulation in T cells is mediated through IFNγ/STAT1 and IL-6/STAT3 signalling pathway.

The immune system is a host defence system to protect the body against foreign invaders. T cells are one of the major components of the immune cells and they are essential for immune responses. Early growth response gene (Egr2) in T cells is important for maintaining immune functions of T cells by promoting adaptive immune responses while controlling inflammation and preventing the development of autoimmune diseases. A study by our group demonstrated the function of Egr2 as a checkpoint regulator controlling the proliferation and differentiation of the T cells. In association, Egr2 and 3 play indispensable role in T cell immune response, but the mechanism regulating Egr2 expression in T cells is still unclear. In this study, we analysed the Egr2 expression mechanism in CD4 T cells under antigen stimulation. We found that Egr2 expression is regulated by different cytokines including IL-2 and IL-4, which increased Egr2 induction in activated T cells. However, inflammatory cytokines, including INFγ and IL-6, suppressed Egr2 expression through STAT1 and STAT3 signalling pathway respectively, highlighting a mechanism for tolergenic immune response on T cells.

Egr2 and 3 control inflammation, but maintain homeostasis, of PD-1high memory phenotype CD4 T cells.

The transcription factors Egr2 and 3 are essential for controlling inflammatory autoimmune responses of memory phenotype (MP) CD4 T cells. However, the mechanism is still unclear. We have now found that the Egr2+ subset (PD-1high MP) of MP CD4 T cells expresses high levels of checkpoint molecules (PD-1 and Lag3) and also markers of effector T cells (CXCR3 and ICAM-1). Egr2/3 are not required for PD-1high MP CD4 cell development but mediate a unique transcriptional programme that effectively controls their inflammatory responses, while promoting homeostatic proliferation and adaptive responses. Egr2 negative PD-1high MP CD4 T cells are impaired in homeostatic proliferation and adaptive responses against viral infection but display inflammatory responses to innate stimulation such as IL-12. PD-1high MP CD4 T cells have recently been implicated in rheumatoid arthritis pathogenesis, and we have now found that Egr2 expression is reduced in PD-1high MP CD4 T cells from patients with active rheumatoid arthritis compared with healthy controls. These findings demonstrate that Egr2/3 control the inflammatory responses of PD-1high MP CD4 T cells and maintain their adaptive immune fitness.

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.

Suppressor of cytokine signaling-3 suppresses the ability of activated signal transducer and activator of transcription-3 to stimulate neurite growth in rat primary sensory neurons.

The actions of the neuropoietic cytokines are mediated by the gp130 receptor, which activates several signaling molecules including the transcription factor STAT3 (signal transducer and activator of transcription), which, in turn, is subject to feedback inhibition by SOCS3 (suppressor of cytokine signaling). Activation of the gp130 receptor has been implicated in axonal growth particularly during regeneration, but the specific contribution of STAT3 is the subject of conflicting reports. Measurements of SOCS3 mRNA in rat dorsal root ganglia showed a significant induction in this inhibitory molecule after peripheral nerve injury. The functions of STAT3 and SOCS3 in adult rat primary sensory neurons were investigated in vitro through transduction of lentiviruses yielding a conditionally activated STAT3, native SOCS3, or a mutant SOCS3 with dominant-negative actions. The SOCS3 construct was effective in inhibiting tyrosine phosphorylation of STAT3 in a neuroblastoma cell line and in blocking nuclear accumulation of endogenous STAT3 or of the conditionally activated STAT3 chimera in primary sensory neurons. In such neurons, transduction and activation of STAT3 enhanced neurite growth, transduction with SOCS3 reduced neurite outgrowth, and transduction with mutant SOCS3 enhanced neurite growth, at least under basal conditions. In conclusion, STAT3 signaling is beneficial to axonal growth through activating transcription of unidentified genes, and SOCS3 is detrimental to axonal growth through inhibition of STAT3 and/or other transcription factors.

Egr2 and 3 control adaptive immune responses by temporally uncoupling expansion from T cell differentiation.

Egr2 and 3 are important for maintaining immune homeostasis. Here we define a fundamental function of Egr2 and 3 operating as a checkpoint that controls the transition between clonal expansion and differentiation of effector T cells. Egr2 and 3 deficiency resulted in defective clonal expansion but hyperactivation and excessive differentiation of T cells in response to viral infection. Conversely, sustained Egr2 expression enhanced expansion but severely impaired effector differentiation. Egr2 bound to and controlled the expression of genes regulating proliferation (Myc and Myb) and differentiation repressors (Bcl6, Id3), while repressing transcription factors required for effector function (Zeb2, RORa, RORc, and Bhlhe40). Egr2 and 3 expression in T cells was regulated reciprocally by antigen and IFNγ, providing a mechanism for adjusting proliferation and differentiation of individual T cells. Thus, Egr2 and 3 are upstream regulators of effector CD4 and CD8 T cells that are essential for optimal responses with limited immunopathology.

Modulation of antigen-specific T-cells as immune therapy for chronic infectious diseases and cancer.

T-cell responses are induced by antigen presenting cells (APC) and signals from the microenvironment. Antigen persistence and inflammatory microenvironments in chronic infections and cancer can induce a tolerant state in T-cells resulting in hyporesponsiveness, loss of effector function, and weak biochemical signaling patterns in response to antigen stimulation. Although the mechanisms of T-cell tolerance induced in chronic infection and cancer may differ from those involved in tolerance to self-antigen, the impaired proliferation and production of IL-2 in response to antigen stimulation are hallmarks of all tolerant T cells. In this review, we will summarize the evidence that the immune responses change from non-self to "self"-like in chronic infection and cancer, and will provide an overview of strategies for re-balancing the immune response of antigen-specific T cells in chronic infection and cancer without affecting the homeostasis of the immune system.

Early growth response gene-2 (Egr-2) regulates the development of B and T cells.

BACKGROUND: Understanding of how transcription factors are involved in lymphocyte development still remains a challenge. It has been shown that Egr-2 deficiency results in impaired NKT cell development and defective positive selection of T cells. Here we investigated the development of T, B and NKT cells in Egr-2 transgenic mice and the roles in the regulation of distinct stages of B and T cell development. METHODS AND FINDINGS: The expression of Egr1, 2 and 3 were analysed at different stages of T and B cell development by RT-PCT and results showed that the expression was strictly regulated at different stages. Forced expression of Egr-2 in CD2(+) lymphocytes resulted in a severe reduction of CD4(+)CD8(+) (DP) cells in thymus and pro-B cells in bone marrow, which was associated with reduced expression of Notch1 in ISP thymocytes and Pax5 in pro-B cells, suggesting that retraction of Egr-2 at the ISP and pro-B cell stages is important for the activation of lineage differentiation programs. In contrast to reduction of DP and pro-B cells, Egr-2 enhanced the maturation of DP cells into single positive (SP) T and NKT cells in thymus, and immature B cells into mature B cells in bone marrow. CONCLUSIONS: Our results demonstrate that Egr-2 expressed in restricted stages of lymphocyte development plays a dynamic, but similar role for the development of T, NKT and B cells.

Responses of the nerve cell body to axotomy.

OBJECTIVE: Peripheral nerve injury causes retrograde changes in the damaged neurons, which are beneficial to axonal regeneration. Better understanding of the mechanisms of induction and mediation of these conditioning responses would help to design strategies to invoke stronger regenerative responses in neurons in situations when these responses are inadequate. METHODS: Relevant literature is reviewed. RESULTS: Experimental preparations that measure the influence of peripheral axotomy on regeneration in the central axons of primary sensory neurons are useful to examine mechanisms of conditioning neurons. Despite 4 decades of speculation, the nature of the damage signals from injured nerves that initiate axonal signals to the nerve cell body remains elusive. Members of the family of neuropoietic cytokines are clearly implicated, but what induces them is unknown. Multiple changes in gene regulation in axotomized neurons have been described, and dozens of growth-associated genes have been identified: neurotrophic factors, transcription factors, molecules participating in axonal transport, and molecules active in the growth cone. The mechanisms of interaction of a few regeneration-associated molecules with the signaling cascades that lead to actin and tubulin remodeling at the growth cone are understood in some detail. In animals, viral gene therapy to deliver regeneration-associated genes to neurons or other local measures to induce these genes can improve regeneration. A few pharmacological agents, administered systemically, have small beneficial effects on axonal regeneration. CONCLUSION: Advances in laboratory research have provided knowledge of cell body responses to axotomy with clinical relevance.

SOCS3 suppresses AP-1 transcriptional activity in neuroblastoma cells through inhibition of c-Jun N-terminal kinase.

Transduction and activation of an inducible form of STAT3 (signal transducer and activator of transcription) sufficed to increase VIP (vasoactive intestinal protein) mRNA concentrations in neuroblastoma cells. Overexpression of SOCS3 (suppressor of cytokine signaling) inhibited and mutant SOCS3 (with an inactivating point mutation in amino acid 25) enhanced the induction of VIP mRNA by CNTF (ciliary neurotrophic factor). Because mutant SOCS3 did not augment the increase in STAT transcriptional activity following CNTF stimulation, the enhancement by mutant SOCS3 of the actions of CNTF cannot be attributed to changes in STAT3 signaling. Mutant SOCS3 increased AP-1 (activator protein) transcriptional activity and JNK (c-Jun N-terminal kinase) activity and SOCS3 bound to the scaffolding protein, JNK-interacting protein-1: these observations provide a plausible explanation for the enhancement by mutant SOCS3 of the actions of CNTF. We conclude that endogenous SOCS3 inhibits AP-1 activity through blocking of JNK phosphorylation.

Actions of neuropoietic cytokines and cyclic AMP in regenerative conditioning of rat primary sensory neurons.

A conditioning lesion to peripheral axons of primary sensory neurons accelerates regeneration of their central axons in vivo or neurite outgrowth if the neurons are grown in vitro. Previous evidence has implicated neuropoietic cytokines and also cyclic AMP in regenerative conditioning. In experiments reported here, delivery through a lentivirus vector of ciliary neurotrophic factor to the appropriate dorsal root ganglion in rats was sufficient to mimic the conditioning effect of peripheral nerve injury on the regeneration of dorsal spinal nerve root axons. Regeneration in this experimental preparation was also stimulated by intraganglionic injection of dibutyryl cyclic AMP but the effects of ciliary neurotrophic factor and dibutyryl cyclic AMP were not additive. Dibutyryl cyclic AMP injection into the dorsal root ganglion induced mRNAs for two other neuropoietic cytokines, interleukin-6 and leukemia inhibitory factor and increased the accumulation of phosphorylated STAT3 in neuronal nuclei. The in vitro conditioning action of dibutyryl cyclic AMP was partially blocked by a pharmacological inhibitor of Janus kinase 2, a neuropoietic cytokine signaling molecule. We suggest that the beneficial actions of increased cyclic AMP activity on axonal regeneration of primary sensory neurons are mediated, at least in part, through the induction of neuropoietic cytokine synthesis within the dorsal root ganglion.