Identification of EPZ004777 and FG2216 as inhibitors of TGF-ß1 induced Treg cells by screening a library of epigenetic compounds.

AIMS: Regulatory T cells play an essential role in immune tolerance and homeostasis. Their long-term stability depends on epigenetic modifications and identification of small molecule modulators of Treg differentiation therefore has many applications. In this study, we performed a novel functional screen of an epigenetic compound library to identify compounds that can modulate generation of TGF-ß1 induced T regulatory cells. MAIN METHODS: A screening strategy based on IFN-γ ELISA was designed to screen epigenetic compound library. Effect of hit compounds on Treg phenotype and function was assessed by RT-PCR, flow cytometry and suppression assays. TGF-ß signalling proteins were assayed by western blotting. Chromatin Immunoprecipitation (ChIP) assay was used to assess epigenetic modifications at Foxp3 gene locus. KEY FINDINGS: We screened 160 compounds to identify hits capable of reversing TGF-ß induced inhibition of IFN-γ production in activated spleen cells and CD4+ T cells. Two compounds EPZ004777 and FG-2216 consistently reversed TGF-ß1 iTregs in terms of (a) differentiation of naïve T cells into CD4+CD25+Foxp3+Treg cells, (b) Foxp3 target gene expression and (c) Treg suppressive function without affecting TGF-ß downstream signalling. ChIP assay revealed that the compounds were able to reverse - TGF- ß mediated decrease in epigenetic marks H3K27me3 and 5-mC and an increase in epigenetic marks H3K4me3 and H3K27Ac in the promoter and conserved non coding sequence (CNS1) regions of the Foxp3 gene. SIGNIFICANCE: EPZ004777 and FG-2216 have been identified as potent epigenetic modulators that can reverse TGF-ß1 induced T regulatory cells and may be used to treat diverse immune disorders.

TGF-ßR inhibitor SB431542 restores immune suppression induced by regulatory B-T cell axis and decreases tumour burden in murine fibrosarcoma.

The contribution of immune cells in soft tissue sarcomas (STS) is not completely known and understanding their role is very essential for employing immunotherapy strategies. Here, we show that murine fibrosarcoma-conditioned medium promoted total spleen cell proliferation but inhibited T cell responses to mitogenic and allo-antigen-mediated stimulation. This increased proliferation was found to be in B cells resulting in generation of Breg further leading to Treg population. This was found to be the same in vitro and in vivo. The phenotype of these B cells was CD19+CD81+CD27+CD25+PD-L1hi and they secreted both IL-10 and TGF-ß. These tumor evoked Bregs (tBreg), when co-cultured with B depleted T cells, suppressed their proliferation in response to anti-CD3/CD28 stimulation. tBreg-induced suppression of T cell responses was not abrogated by the inhibition or neutralization of IL-10 but by the small molecule inhibitor of TGFß Receptor type I, SB431542. While SB531542 per se was not cytotoxic to tumor cells, administration of SB431542 in tumor-bearing mice (TBM) significantly reduced the tumor burden. In addition, the treatment significantly reduced Treg cells and rescued proliferation of T cells in response to mitogen and allo-antigen. Collectively, our results identify that tumor evoked Breg cells mediate T cell immune suppression through TGFß-mediated pathway and that targeting the Breg-Treg axis can be potentially used as an immunotherapy agent.

Differential radio-adaptive responses in BALB/c and C57BL/6 mice: pivotal role of calcium and nitric oxide signalling.

Purpose: Our earlier studies demonstrated that transient radio-adaptive responses (RAR) in BALB/c mice were due to MAPK hyperactivation. The objective of this study was to determine the time duration of this low dose induced MAPK activation in BALB/c mice and to find out if similar adaptive responses are observed in C57BL/6 mice. Materials and methods: Mice were irradiated with 0.1 Gy priming dose (PD), 2 Gy challenge dose (CD) with an interval of 4 h (P + CD) and radiation induced immunosuppression in splenic lymphocytes was monitored as the endpoint for RAR. Results: Time kinetics following 0.1 Gy demonstrated persistence of MAPK hyperactivation till 48 h. Similar experiments in C57BL/6 mice indicated absence of RAR at 24 h following CD, in spite of MAPK activation which was also confirmed by time kinetics. Therefore, upstream activators of MAPK, viz., reactive oxygen and nitrogen species (ROS, RNS) and calcium levels were estimated. There was increased intracellular calcium (Ca2+) and nitric oxide (NO) in BALB/c and an increase in intracellular ROS in C57BL/6 mice 24 h after PD. Inhibition of NO and calcium chelation abrogated RAR in BALB/c mice. In vitro treatment of spleen cells with combination of NO donor and Ca2+ ionophore mimicked the effect of PD and induced adaptive response after 2 Gy not only in BALB/c but also in C57BL/6 mice confirming their crucial role in RAR. Conclusions: These results suggest that low dose induced differential induction of Ca2+ and NO signaling along with MAPK was responsible for contrasting RAR with respect to immune system of BALB/c and C57BL/6 mice. Abbreviations [3H]-TdR: 3H-methyl-thymidine; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CD: Challenge Dose; CFSE: Carboxy Fluorescein Succinamidyl Ester; on A: Concanavalin A; DAF-FM: 4-amino-5-methylamino-2',7'-difluorescein; DCF-DA: 2',7'-dichlorofluorescein diacetate; DSB: Double Strand Break; ELISA: Enzyme Linked ImmunoSorbent Assay; ERK: Extracellular signal-Regulated protein Kinase; FBS: Fetal Bovine Serum; HIF-1A: Hypoxia-Inducible Factor 1-alpha; LDR: Low Dose Radiation; MAPK: Mitogen Activated Protein Kinase; MAPKK/MKK: MAPK Kinase; MAPKKK: MAPK Kinase Kinase; NO: Nitric Oxide; NOS: Nitric Oxide Synthase; P + CD: Priming + Challenge dose; PBS: Phosphate Buffered Saline; PBST: Phosphate Buffered Saline-Tween 20; PD: Priming Dose; PI3K: Phosphatidyl Inositol 3-Kinase; PKC: Protein Kinase C; RAR: Radio Adaptive Response; RNS: Reactive Nitrogen Species; ROS: Reactive Oxygen Species; RPMI-1640: Roswell Park Memorial Institute-1640 medium; SAPK/JNK: Stress-Activated Protein Kinase/ c-Jun NH2-terminal Kinase; SEM: Standard Error of Mean; SNAP: S-nitro amino penicillamine; TP53: Tumor Protein 53; γ-H2AX: Gamma- H2A histone family member X; Th1: Type 1 helper T cell responses; Th2: Type 2 helper T cell responses.

Involvement of MAPK signalling in radioadaptive response in BALB/c mice exposed to low dose ionizing radiation.

To investigate low dose ionizing radiation (LDIR)-induced adaptive response in lymphocytes of BALB/c mice and to elucidate related molecular mechanisms. Mice were exposed to a priming dose (PD) of 0.1 Gy and challenge dose (CD) of 2 Gy ionizing radiation. Proliferation response to mitogen concanavalin A was assessed using (3)H thymidine incorporation and carboxyfluoresceinsuccinamidylester (CFSE) dye dilution. Early activation markers were assessed by flow cytometry, cytokines by ELISA, DNA damage by comet assay and mitogen activated protein kinase (MAPK) signaling by Western blotting. Radioadaptive response was observed in lymphocytes of mice exposed to PD prior to CD of ionizing radiation in terms of DNA damage, early activation markers CD69, CD71, cytokines IL-2, IFN-γ as well as proliferation. This effect was transient and observed 24 h after CD and not after 0 h or 72 h. Hyper activation of MAPK signaling pathways in lymphocytes from LDIR-exposed mice and abrogation by ERK and p38 inhibitors suggests the involvement of MAPK signaling in radioadaptive response. Our study demonstrates that LDIR-induced transient adaptive response was due to hyper activation of MAPK signaling. Our findings contribute towards the understanding of LDIR-induced adaptive response.