Effector Transcription Factors Research Articles

Loss of the Transfer RNA Wobble Uridine-Modifying Enzyme Elp3 Delays T Cell Cycle Entry and Impairs T Follicular Helper Cell Responses through Deregulation of Atf4.

The activation of T cells is accompanied by intensive posttranscriptional remodeling of their proteome. We observed that protein expression of enzymes that modify wobble uridine in specific tRNAs, namely elongator subunit 3 (Elp3) and cytosolic thiouridylase (Ctu)2, increased in the course of T cell activation. To investigate the role of these tRNA epitranscriptomic modifiers in T cell biology, we generated mice deficient for Elp3 in T cells. We show that deletion of Elp3 has discrete effects on T cells. In vitro, Elp3-deficient naive CD4+ T cells polarize normally but are delayed in entering the first cell cycle following activation. In vivo, different models of immunization revealed that Elp3-deficient T cells display reduced expansion, resulting in functional impairment of T follicular helper (TFH) responses, but not of other CD4+ effector T cell responses. Transcriptomic analyses identified a progressive overactivation of the stress-responsive transcription factor Atf4 in Elp3-deficient T cells. Overexpression of Atf4 in wild-type T cells phenocopies the effect of Elp3 loss on T cell cycle entry and TFH cell responses. Reciprocally, partial silencing of Atf4 or deletion of its downstream effector transcription factor Chop rescues TFH responses of Elp3-deficient T cells. Together, our results reveal that specific epitranscriptomic tRNA modifications contribute to T cell cycle entry and promote optimal TFH responses.

The Wnt Effector TCF7l2 Promotes Oligodendroglial Differentiation by Repressing Autocrine BMP4-Mediated Signaling.

Promoting oligodendrocyte (OL) differentiation represents a promising option for remyelination therapy for treating the demyelinating disease multiple sclerosis (MS). The Wnt effector transcription factor 7-like 2 (TCF7l2) was upregulated in MS lesions and had been proposed to inhibit OL differentiation. Recent data suggest the opposite yet underlying mechanisms remain elusive. Here, we unravel a previously unappreciated function of TCF7l2 in controlling autocrine bone morphogenetic protein (BMP)4-mediated signaling. Disrupting TCF7l2 in mice of both sexes results in oligodendroglial-specific BMP4 upregulation and canonical BMP4 signaling activation in vivo Mechanistically, TCF7l2 binds to Bmp4 gene regulatory element and directly represses its transcriptional activity. Functionally, enforced TCF7l2 expression promotes OL differentiation by reducing autocrine BMP4 secretion and dampening BMP4 signaling. Importantly, compound genetic disruption demonstrates that oligodendroglial-specific BMP4 deletion rescues arrested OL differentiation elicited by TCF7l2 disruption in vivo Collectively, our study reveals a novel connection between TCF7l2 and BMP4 in oligodendroglial lineage and provides new insights into augmenting TCF7l2 for promoting remyelination in demyelinating disorders such as MS.SIGNIFICANCE STATEMENT Incomplete or failed myelin repairs, primarily resulting from the arrested differentiation of myelin-forming oligodendrocytes (OLs) from oligodendroglial progenitor cells, is one of the major reasons for neurologic progression in people affected by multiple sclerosis (MS). Using in vitro culture systems and in vivo animal models, this study unraveled a previously unrecognized autocrine regulation of bone morphogenetic protein (BMP)4-mediated signaling by the Wnt effector transcription factor 7-like 2 (TCF7l2). We showed for the first time that TCF7l2 promotes oligodendroglial differentiation by repressing BMP4-mediated activity, which is dysregulated in MS lesions. Our study suggests that elevating TCF7l2 expression may be possible in overcoming arrested oligodendroglial differentiation as observed in MS patients.

Phase 1 Study of on Site Manufactured Anti-CD19 CAR-T Cells: Responses in Subjects with Rapidly Progressive Refractory Lymphomas

Phase 1 Study of on Site Manufactured Anti-CD19 CAR-T Cells: Responses in Subjects with Rapidly Progressive Refractory Lymphomas

Abstract 209: Metastatic colonization in the perivascular niche: A mechanotransduction perspective

Abstract Metastases are responsible for majority cancer related deaths. Disseminated tumor cells (DTCs) gain a diverse array of metastatic traits in order to adapt to and survive within different microenvironments, making them very difficult to therapeutically target. Remarkably, regardless of the secondary organ, DTCs survive and thrive in perivascular niches. Using xenograft and syngeneic models of metastasis in mouse, we found that upon extravasation, neuronal cell adhesion molecule L1 (L1CAM) expressed by DTCs engages the vascular basal lamina in order to amplify integrin β1 and integrin linked kinase (ILK) signaling in DTCs. ILK promotes actin polymerization for cell spreading and subsequent activation of mechanotransduction effector transcription factors YAP and MRTF. We found that activation of YAP and MRTF coincides with DTCs ability to dislodge resident pericytes from the perivascular niche and is an essential step in the metastatic colonization regardless of dormancy state. Therefore, these results provided us a unifying molecular mechanism that governs cancer progression from metastatic seeding to overt colonization. Ongoing studies will determine how mechanotransduction and pericyte-DTC interactions can be manipulated to prevent metastatic colonization by potentiating the immune response and exposing their biophysical vulnerabilities. Citation Format: Ekrem Emrah Er, Maria Tello-Lafoz, Jing Hu, Morgan Huse, Joan Massagué. Metastatic colonization in the perivascular niche: A mechanotransduction perspective [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 209.

Protein phosphatase 1 of Leishmania donovani exhibits conserved catalytic residues and pro-inflammatory response

Protein phosphorylation, governed by kinases and phosphatases, plays a pivotal role in enormous cellular signaling pathways. Although PPP family of serine/threonine phosphatases have been involved in multiplication and growth of trypanosomatid parasites, but comprehensive knowledge is still very limited. In the present study, protein phosphatase 1 from Leishmania donovani (LdPP1) was purified to homogeneity and its structural attributes were explored employing CD and fluorescence spectroscopy as well as bioinformatics methods. The CD analysis revealed an appropriate secondary structure with α-helices content outnumbering the β-sheets, whereas intrinsic fluorescence study depicted about the buried positioning of tryptophan residues. The three-dimensional structure of LdPP1, determined by homology modeling, displayed all the characteristic features including similar position of metal as well as inhibitor binding site corresponding to the known PP1 structures. Furthermore, ELISA and qRT-PCR results showed that LdPP1 elicit the pro-inflammatory cytokines TNF-α and IL-6 at translated and transcriptional levels in THP1 macrophages. Subsequently, immune effector molecule nitric oxide and transcription factor NF-κB production was also found to be increased upon LdPP1 stimulation. Altogether, this is the first report on PPP phosphatase of trypanosomatid parasite that represents the structural highlights along with protein-mediated immunomodulation in human macrophages.

Trogocytosis-Mediated Intracellular Signaling in CD4+ T Cells Drives TH2-Associated Effector Cytokine Production and Differentiation.

CD4+ T cells have been observed to acquire APC-derived membrane and membrane-associated molecules through trogocytosis in diverse immune settings. Despite this, the consequences of trogocytosis on the recipient T cell remain largely unknown. We previously reported that trogocytosed molecules on CD4+ T cells engage their respective surface receptors, leading to sustained TCR signaling and survival after APC removal. Using peptide-pulsed bone marrow-derived dendritic cells and transfected murine fibroblasts expressing antigenic MHC:peptide complexes as APC, we show that trogocytosis-positive CD4+ T cells display effector cytokines and transcription factor expression consistent with a TH2 phenotype. In vitro-polarized TH2 cells were found to be more efficient at performing trogocytosis than TH1 or nonpolarized CD4+ cells, whereas subsequent trogocytosis-mediated signaling induced TH2 differentiation in polarized TH1 and nonpolarized cells. Trogocytosis-positive CD4+ T cells generated in vivo also display a TH2 phenotype in both TCR-transgenic and wild-type models. These findings suggest that trogocytosis-mediated signaling impacts CD4+ T cell differentiation and effector cytokine production and may play a role in augmenting or shaping a TH2-dominant immune response.

The Role of WNT Signaling in Mature T Cells: T Cell Factor Is Coming Home.

T cell factor, the effector transcription factor of the WNT signaling pathway, was so named because of the primary observation that it is indispensable for T cell development in the thymus. Since this discovery, the role of this signaling pathway has been extensively studied in T cell development, hematopoiesis, and stem cells; however, its functional role in mature T cells has remained relatively underinvestigated. Over the last few years, various studies have demonstrated that T cell factor can directly influence T cell function and the differentiation of Th1, Th2, Th17, regulatory T cell, follicular helper CD4+ T cell subsets, and CD8+ memory T cells. In this paper, we discuss the molecular mechanisms underlying these observations and place them in the general context of immune responses. Furthermore, we explore the implications and limitations of these findings for WNT manipulation as a therapeutic approach for treating immune-related diseases.

EFFECTOR OF TRANSCRIPTION factors are novel plant-specific regulators associated with genomic DNA methylation in Arabidopsis.

Summary Plant‐specific EFFECTORS OF TRANSCRIPTION (ET) are characterised by a variable number of highly conserved ET repeats, which are involved in zinc and DNA binding. In addition, ETs share a GIY‐YIG domain, involved in DNA nicking activity. It was hypothesised that ETs might act as epigenetic regulators.Here, methylome, transcriptome and phenotypic analyses were performed to investigate the role of ET factors and their involvement in DNA methylation in Arabidopsis thaliana.Comparative DNA methylation and transcriptome analyses in flowers and seedlings of et mutants revealed ET‐specific differentially expressed genes and mostly independently characteristic, ET‐specific differentially methylated regions. Loss of ET function results in pleiotropic developmental defects.The accumulation of cyclobutane pyrimidine dimers after ultraviolet stress in et mutants suggests an ET function in DNA repair.

Abstract LB-111: Precise regulation of CAR signaling prevents and reverses CAR T cell exhaustion

Abstract In patients with chronic viral infection or cancer, continuous antigen exposure results in T cell exhaustion, which is characterized by sustained co-expression of multiple inhibitory receptors (ex. PD-1, TIM-3, LAG-3) and a hierarchical loss of effector function. Human T cells expressing a high-affinity anti-GD2 chimeric antigen receptor (CAR, HA.28z) develop phenotypic and functional hallmarks of exhaustion due to clustering of surface CAR and tonic signaling in the absence of antigen. In the present study, we exploited the HA.28z CAR as a model to characterize human T cell exhaustion and assess its potential for reversibility. CyTOF analyses of dysfunctional HA.28z CAR T cells demonstrated a distinct phenotype in which canonical exhaustion markers, T cell differentiation markers, and effector transcription factors were differentially expressed compared to CD19.28z CAR T cells that do not tonically signal. To interrogate the reversibility of T cell exhaustion, we engineered a regulatable HA.28z CAR by fusing a destabilization domain to the C-terminus of the CAR that rapidly induces CAR protein degradation. Addition of a small molecule stabilizes the protein and results in CAR surface expression. Using this model, we modulated the duration and intensity of tonic signaling by precisely modulating CAR surface expression. We hypothesized that transient cessation of tonic signaling would allow exhausted CAR T cells to recover and regain effector function. Following onset of exhaustion, elimination of CAR surface expression for 4 days resulted in a profound reversal of the exhausted phenotype. CyTOF analyses of reinvigorated cells indicated enhanced T cell memory formation, diminished surface marker expression of multiple exhaustion markers (ex. PD-1, TIM-3, LAG-3, CTLA-4, CD39) and reduced expression of transcription factors T-bet and Blimp-1. Furthermore, RNA sequencing of exhausted and reinvigorated HA.28z CAR T cells revealed a global molecular reprogramming upon removal of tonic CAR signaling, suggesting that CAR T cell exhaustion is functionally reversible. Upon CAR re-expression and co-culture with tumor antigen, reinvigorated HA.28z CAR T cells exhibited augmented killing and cytokine secretion compared to exhausted cells that continuously expressed surface CAR during expansion. In vivo studies assessing CAR T cell exhaustion reversibility using this model are currently being evaluated. In summary, tuning CAR surface expression offers a novel strategy to augment both the safety and efficacy of CAR T cell therapy. Moreover, these studies suggest that transient “rest” in T cells experiencing chronic antigen stimulation may be one mechanism by which exhaustion is prevented or reversed. Citation Format: Evan W. Weber, Rachel C. Lynn, Meena Malipatlolla, Elena Sotillo, Peng Xu, Crystal L. Mackall. Precise regulation of CAR signaling prevents and reverses CAR T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-111.

Embryonic Fibroblasts Promote Antitumor Cytotoxic Effects of CD8+ T Cells.

Adoptive CD8+ T cell therapy has emerged as an important modality for the treatment of cancers. However, the significant drawback of transfused T cells is their poor survival and functionality in response to tumors. To overcome this limitation, an important consideration is exploring a culture condition to generate superior antitumor cytotoxic T lymphocytes (CTLs) for adoptive therapy. Here, we provide a novel approach to generate potent CTL clones in mouse embryonic fibroblast-conditioned medium (MEF-CM). We found CTLs derived with MEF-CM have higher potential in long-term persistence in tumor bearing and non-tumor-bearing mice. Importantly, adoptive transfer of MEF-CM-cultured CTLs dramatically regressed tumor growth and prolonged mice survival. Characterization of MEF-CM-cultured CTLs (effector molecules, phenotypes, and transcription factors) suggests that MEF-CM enhances the effector functions of CD8+ T cells in part by the upregulation of the T-box transcription factor eomesodermin. Consequently, MEF-CM enhances the intrinsic qualities of effector CD8+ T cells to augment antitumor immunity.

Innate Lymphoid Cells are Dysregulated after Intestinal Transplantation and Play a Critical Role in Regulating Allograft Homeostasis

The recent discovery of innate lymphoid cells (ILCs) has revealed their importance in intestinal homeostasis; however their role in intestinal transplantation (ITx) has yet to be defined. We hypothesized that ILC subsets are dysregulated in the lamina propria following ITx thus affecting intestinal homeostasis and intestinal stem cell (ISC) regeneration. Intestinal samples were obtained from patients immediately post ITx and at several time points within the first 6 months as well as from stable patients over 6 months post-op without acute complications. Lamina propria cells were isolated and immunophenotyped. Viable lineage negative cells were identified as group 1 and 3 ILCs by expression of CD56, NKp44, CD117, and CD127. Graft infiltrating lymphocytes were flow sorted and rtPCR was performed to investigate effector cytokine and transcription factor profiles. First we compared a group of stable patients with samples from fresh ITx recipients taken immediately after transplant (day 0). Critically, we found that the pro-inflammatory IFN-g-producing CD127+ ILC1 subset (p=0.492) was preserved in fresh ITx and comparable to stable patients. Importantly, however, we found that the pro-homeostatic IL-22-producing NCR+ ILC3 population was absent in fresh allografts when compared to stable recipients (p=0.019). We then hypothesized that the observed ILC1-ILC3 dysequilibrium resolves over time. This was tested by prospective immunomonitoring of ITx patients from the day of transplant onwards utilizing flow cytometry, immunohistochemistry, and rtPCR. Intriguingly, we found that the CD127+ ILC1 population overall remained stable while there was a recovery of the total NCR+ population and ILC3 in complication-free ITx patients after 4–8 weeks post ITx (p=0.025). These results demonstrate that the ILC dysbalance seen post-ITx resolves with the recovery of protective ILC3. This has a potentially profound impact on allograft homeostasis post-ITx as we further found that the recovery of NCR+ cells and ILC3 correlated with significantly enhanced expression levels of LGR5, a marker for IL-22-dependent ISC, by postoperative week 4-8 (p=0.003).In summary, this study first demonstrates a dangerous dysbalance between the pro-inflammatory ILC1s and the protective ILC3s within the first few weeks following ITx, when patients experience most complications. This dysregulation resolves over time with the regeneration of NCR+ ILC3s that target ISCs for allograft recovery.

Mechanisms of action and therapeutic potential of proinsulin C-peptide

C-peptide generated by proinsulin proteolysis is not only a chaperone for insulin in β-cells, but also a signaling molecule that regulates many physiological and biochemical processes via specific C-peptide receptors. Regulatory effects of C-peptide are tissue-specific and largely depend on the physiological state of an organism, C-peptide concentration, and its ability to form complexes. In type 1 diabetes mellitus, which is characterized by a C-peptide deficiency, C-peptide replacement therapy prevents the development of inflammation in vascular endothelial cells, whereas C-peptide excess, in contrast, reveals its pro-inflammatory properties. By affecting a number of effector proteins and transcription factors in the retinal pigment epithelium, C-peptide prevents both blood–retinal barrier damage and retinal neovascularization, which makes it one of the most promising drugs to treat and prevent diabetic retinopathy. C-peptide has been established to be involved in the regulation of synthesis and secretion of adipokines, which is indicative of its role in controlling energy homeostasis. Considering the efficiency of C-peptide in the treatment of diabetic nephropathy, neuropathy and retinopathy, C-peptide medicinal forms are now being developed. Among these forms, of greatest interest are C-peptide analogs modified by polyethylene glycol, C-peptide complexes with zinc cations and albumin, and C-terminal fragments of C-peptide comparable by their biological activity to a full-length molecule.

Dual Targeting of Bromodomain and Extraterminal Domain Proteins, and WNT or MAPK Signaling, Inhibits c-MYC Expression and Proliferation of Colorectal Cancer Cells.

Inhibitors of the bromodomain and extraterminal domain (BET) protein family attenuate the proliferation of several tumor cell lines. These effects are mediated, at least in part, through repression of c-MYC. In colorectal cancer, overexpression of c-MYC due to hyperactive WNT/β-catenin/TCF signaling is a key driver of tumor progression; however, effective strategies to target this oncogene remain elusive. Here, we investigated the effect of BET inhibitors (BETi) on colorectal cancer cell proliferation and c-MYC expression. Treatment of 20 colorectal cancer cell lines with the BETi JQ1 identified a subset of highly sensitive lines. JQ1 sensitivity was higher in cell lines with microsatellite instability but was not associated with the CpG island methylator phenotype, c-MYC expression or amplification status, BET protein expression, or mutation status of TP53, KRAS/BRAF, or PIK3CA/PTEN Conversely, JQ1 sensitivity correlated significantly with the magnitude of c-MYC mRNA and protein repression. JQ1-mediated c-MYC repression was not due to generalized attenuation of β-catenin/TCF-mediated transcription, as JQ1 had minimal effects on other β-catenin/TCF target genes or β-catenin/TCF reporter activity. BETi preferentially target super-enhancer-regulated genes, and a super-enhancer in c-MYC was recently identified in HCT116 cells to which BRD4 and effector transcription factors of the WNT/β-catenin/TCF and MEK/ERK pathways are recruited. Combined targeting of c-MYC with JQ1 and inhibitors of these pathways additively repressed c-MYC and proliferation of HCT116 cells. These findings demonstrate that BETi downregulate c-MYC expression and inhibit colorectal cancer cell proliferation and identify strategies for enhancing the effects of BETi on c-MYC repression by combinatorial targeting the c-MYC super-enhancer. Mol Cancer Ther; 15(6); 1217-26. ©2016 AACR.

A Maternal System Initiating the Zygotic Developmental Program through Combinatorial Repression in the Ascidian Embryo.

Maternal factors initiate the zygotic developmental program in animal embryos. In embryos of the chordate, Ciona intestinalis, three maternal factors—Gata.a, β-catenin, and Zic-r.a—are required to establish three domains of gene expression at the 16-cell stage; the animal hemisphere, vegetal hemisphere, and posterior vegetal domains. Here, we show how the maternal factors establish these domains. First, only β-catenin and its effector transcription factor, Tcf7, are required to establish the vegetal hemisphere domain. Second, genes specifically expressed in the posterior vegetal domain have additional repressive cis-elements that antagonize the activity of β-catenin/Tcf7. This antagonizing activity is suppressed by Zic-r.a, which is specifically localized in the posterior vegetal domain and binds to DNA indirectly through the interaction with Tcf7. Third, Gata.a directs specific gene expression in the animal hemisphere domain, because β-catenin/Tcf7 weakens the Gata.a-binding activity for target sites through a physical interaction in the vegetal cells. Thus, repressive regulation through protein-protein interactions among the maternal transcription factors is essential to establish the first distinct domains of gene expression in the chordate embryo.

Developmental Acquisition of Regulomes Underlies Innate Lymphoid Cell Functionality

Innate lymphoid cells (ILCs) play key roles in host defense, barrier integrity, and homeostasis and mirror adaptive CD4(+) T helper (Th) cell subtypes in both usage of effector molecules and transcription factors. To better understand the relationship between ILC subsets and their Th cell counterparts, we measured genome-wide chromatin accessibility. We find that chromatin in proximity to effector genes is selectively accessible in ILCs prior to high-level transcription upon activation. Accessibility of these regions is acquired in a stepwise manner during development and changes little after invitro or invivo activation. Conversely, dramatic chromatin remodeling occurs in naive CD4(+) Tcells during Th cell differentiation using a type-2-infection model. This alteration results in a substantial convergence of Th2 cells toward ILC2 regulomes. Our data indicate extensive sharing of regulatory circuitry across the innate and adaptive compartments of the immune system, in spite of their divergent developing pathways.

Mechanism and Role of SOX2 Repression in Seminoma: Relevance to Human Germline Specification

SummaryHuman male germ cell tumors (GCTs) are derived from primordial germ cells (PGCs). The master pluripotency regulator and neuroectodermal lineage effector transcription factor SOX2 is repressed in PGCs and the seminoma (SEM) subset of GCTs. The mechanism of SOX2 repression and its significance to GC and GCT development currently are not understood. Here, we show that SOX2 repression in SEM-derived TCam-2 cells is mediated by the Polycomb repressive complex (PcG) and the repressive H3K27me3 chromatin mark that are enriched at its promoter. Furthermore, SOX2 repression in TCam-2 cells can be abrogated by recruitment of the constitutively expressed H3K27 demethylase UTX to the SOX2 promoter through retinoid signaling, leading to expression of neuronal and other lineage genes. SOX17 has been shown to initiate human PGC specification, with its target PRDM1 suppressing mesendodermal genes. Our results are consistent with a role for SOX2 repression in normal germline development by suppressing neuroectodermal genes.

SensiPath: computer-aided design of sensing-enabling metabolic pathways.

Genetically-encoded biosensors offer a wide range of opportunities to develop advanced synthetic biology applications. Circuits with the ability of detecting and quantifying intracellular amounts of a compound of interest are central to whole-cell biosensors design for medical and environmental applications, and they also constitute essential parts for the selection and regulation of high-producer strains in metabolic engineering. However, the number of compounds that can be detected through natural mechanisms, like allosteric transcription factors, is limited; expanding the set of detectable compounds is therefore highly desirable. Here, we present the SensiPath web server, accessible at http://sensipath.micalis.fr. SensiPath implements a strategy to enlarge the set of detectable compounds by screening for multi-step enzymatic transformations converting non-detectable compounds into detectable ones. The SensiPath approach is based on the encoding of reactions through signature descriptors to explore sensing-enabling metabolic pathways, which are putative biochemical transformations of the target compound leading to known effectors of transcription factors. In that way, SensiPath enlarges the design space by broadening the potential use of biosensors in synthetic biology applications.

Targeting Mechanisms for Natural Killer Cell Dysfunction in Patients with Multiple Myeloma

Patients with multiple myeloma (MM) have a multi-faceted immune dysfunction with poor NK cell cytotoxic function. We show that NK cells in MM have a disturbed homeostasis, featured by an increased frequency of terminally differentiated CD57+ NK cells and almost complete absence of the less mature CD56hi NK cell subset. MM patient NK cells have reduced levels of killer activating receptors (KAR) [Nkp46, Nkp44 and NKG2D] and increased levels of killer inhibitory receptor (KIR) [KIR2DL1 and KIR2DL3]. Despite expressing normal levels of the NK effector transcription factors Tbet and eomesdermin, and the effector protein perforin, MM patient NK cells demonstrate poor cytotoxic function against myeloma cell lines and lower levels of the cytotoxic proteins granzyme B (gzmB).Elotuzumab (Elo, Bristol Myers Squibb) is a human IgG1 antibody which targets SLAM-F7, a protein highly expressed on myeloma plasma cells and on specific subsets of immune cells. Elo mediates dual activation of NK cells and has shown promising clinical responses when combined with lenalidomide; however the complete mechanism(s) whereby Elo mediates these effects have not been fully described. Thus, this project investigates the Elo mechanisms of action in myeloma. To explore whether MM patient NK cells were capable of ADCC, we examined NK cell subset degranulation (CD107a) and IFN-γ secretion when co-cultured with the SLAM-F7 expressing OPM-2 myeloma cells and Elo. Healthy donor NK cells degranulated in response to Elo-labeled OPM-2 cells, which correlated with loss of the CD56lo CD16+ NK cell subset. In contrast, newly diagnosed (NEW) and refractory/relapsed MM (RRMM) patient NK cells had a decreased response to Elo-labeled OPM-2 cells as the CD56lo CD16+ NKsubset was retained, and they degranulated at a significantly lower level than that observed in healthy donor NK cells. Therefore, all MM patient NK subsets (CD56lo CD16+, CD161+ and CD57+) assessed were low responders to Elo-labeled OPM-2 cells.We then visually examined NK cell killing kinetics of Elo-labeled myeloma target cells using time lapse live video microscopy (TLLVM). NK cells used ADCC to kill OPM-2 cells in the presence of Elo, the killing kinetics of was equivalent to direct killing induced in presence of the human IgG isotype control, the delivery of the lethal hit occurring in under three minutes. Elo also mediated serial killing of OPM-2 cells. Interestingly, Elo did delay the NK cell detachment from dying myeloma plasma cell targets, which results in prolonged signaling to the NK cell and consequently higher levels of secreted cytokines and pro-inflammatory chemokines. Taken together, this data indicates Elo-induces a profound change in the NK-mediated killing kinetics of myeloma cells and that likely recruits other immune effectors to the myeloma killing site.Given that NK cells can also express SLAM-F7, we assessed whether they were likely to be a target for other NK cells when bound to Elo, and induce fratricide. We used killing assays, flow cytometry and TLLVM to show that in the presence of Elo, healthy donor NK cells were capable of NK fratricide under the assay conditions tested. Interestingly, fratricide induced death saw the depletion of the CD56lo CD16+ NKsubset. This data may partially explain why there is a rapid but transient reduction of NK cells from the peripheral blood of MM patients who received single agent Elo.In summary, MM patient NK dysfunction is due to aberrant NK cell homeostasis featured by terminal differentiation of the NK cells to a subset with poor natural cytotoxic function and high KIR expression. In vitro, Elo is less effective at inducing ADCC responses by NK cells from MM patients compared to normal donors. Nonetheless, Elo induces efficient normal donor NK cell killing of myeloma target cells, the killing kinetics is featured by a protracted immune synapse time and secretion of high levels of pro-inflammatory cytokines/chemokines by 24 hours, plus serial killing of myeloma target cells. These data likely explain the single agent efficacy of Elo in RRMM patients. The data also underlines the exciting potential of Elo to induce NK mediated ADCC of myeloma plasma cells, and recruit multiple arms of the immune system to the killing site. To harness its full potential, combination of Elo with other drugs (eg. Lenalidomide and anti-KIR) maybe required to normalize MM patient NK cell function and initiate the immune attack. DisclosuresRobbins:Bristol-Myers Squibb: Employment, Other: shareholder. Neeson:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Investigation of Melanogenic Factors Gene Expression in Human Adult and Neonate Retinal Pigment Epithelium Cell Cultures

Retinal pigment epithelium is responsible for maintaining the structural integrity of the retina by an efficient defense against free radicals, photo-oxidative exposure and light energy. For this purpose the main RPE line of defense is melanosomes. Melanin content of retinal pigment epithelium cells in adults and neonates reveals remarkable variations. In the current study we compared melanogenic factors gene expression levels in human adult and neonate RPE cells in culture. RPE cells from adult and neonate human eye globes were isolated and then cultured in DMEM: F12 (1:1) containing 10% FBS. Expression of RPE65 and Cytokeratin 8/18 markers in isolated cells was confirmed by immunocytochemistry. To evaluate the responsible factors in the pathway of melanin biosynthesis, gene expression of orthodenticle homeobox 2, microphthalmia-associated transcription factor A and H as prominent transcription factors, tyrosinase and dopachrome tautomerase as effective enzymes in the melanin biosynthetic pathway were examined in human neonate derived RPE cells compared to human adult derived RPE cells in culture. According to the Real-Time RT-PCR data, gene expression of MITF-H, OTX2, TYR and DCT in RPE cells of the neonates showed a significant increase compared to the adults. With increasing passage number, gene expression of MITF-H, OTX2, TYR and DCT showed remarkable decline. According to the role of OTX2 and MITF-H as the main transcription factor effectors on the TYR and DCT, restoration of OTX2 and MITF-H gene expression may be retain melanin content in RPE cells.

Genome-wide target analysis of NEUROD2 provides new insights into regulation of cortical projection neuron migration and differentiation.

BackgroundCellular differentiation programs are controlled, to a large extent, by the combinatorial functioning of specific transcription factors. Cortical projection neurons constitute the major excitatory neuron population within the cortex and mediate long distance communication between the cortex and other brain regions. Our understanding of effector transcription factors and their downstream transcriptional programs that direct the differentiation process of cortical projection neurons is far from complete.ResultsIn this study, we carried out a ChIP-Seq (chromatin-immunoprecipitation and sequencing) analysis of NEUROD2, an effector transcription factor expressed in lineages of cortical projection neurons during the peak of cortical excitatory neurogenesis. Our results suggest that during cortical development NEUROD2 targets key genes that are required for Reelin signaling, a major pathway that regulates the migration of neurons from germinal zones to their final layers of residence within the cortex. We also find that NEUROD2 binds to a large set of genes with functions in layer-specific differentiation and in axonal pathfinding of cortical projection neurons.ConclusionsOur analysis of in vivo NEUROD2 target genes offers mechanistic insight into signaling pathways that regulate neuronal migration and axon guidance and identifies genes that are likely to be required for proper cortical development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1882-9) contains supplementary material, which is available to authorized users.