A gene regulatory network armature for T lymphocyte specification

Choice of a T lymphoid fate by hematopoietic progenitor cells depends on sustained Notch–Delta signaling combined with tightly regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification, tests of the short-term Notch dependence of these gene expression changes, and analyses of the effects of overexpression of two essential transcription factors, namely PU.1 and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T cell precursors progress from primitive multipotency to T lineage commitment. Our analyses reveal separate contributions of Notch signaling, GATA-3 activity, and down-regulation of PU.1. Using BioTapestry (www.BioTapestry.org), the results have been assembled into a draft gene regulatory network for the specification of T cell precursors and the choice of T as opposed to myeloid/dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfi1 against Egr-2 and of TCF-1 against PU.1 as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose dependence of GATA-3 effects, the gene-specific modulation of PU.1 activity based on Notch activity, the lack of direct opposition between PU.1 and GATA-3, and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression

GATA-3 Dose-Dependent Checkpoints in Early T Cell Commitment

GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4CD8 (double-negative [DN]) stages 1–3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3–deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3–deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro–T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment

Differential regulation of GLUT1 activity in human corneal limbal epithelial cells and fibroblasts

The corneal epithelial tissue is a layer of rapidly growing cells that are highly glycolytic and express GLUT1 as the major glucose transporter. It has been shown that GLUT1 in L929 fibroblast cells and other cell lines can be acutely activated by a variety agents. However, the acute regulation of glucose uptake in corneal cells has not been systematically investigated. Therefore, we examined glucose uptake in an immortalized human corneal-limbal epithelial (HCLE) cell line and compared it to glucose uptake in L929 fibroblast cells, a cell line where glucose uptake has been well characterized. We report that the expression of GLUT1 in HCLE cells is 6.6-fold higher than in L929 fibroblast cells, but the HCLE cells have a 25-fold higher basal rate of glucose uptake. Treatment with agents that interfere with mitochondrial metabolism, such as sodium azide and berberine, activate glucose uptake in L929 cells over 3-fold, but have no effect on glucose uptake HCLE cells. Also, agents known to react with thiols, such cinnamaldehyde, phenyarsine oxide and nitroxyl stimulate glucose uptake in L929 cells 3-4-fold, but actually inhibit glucose uptake in HCLE cells. These data suggest that in the fast growing HCLE cells, GLUT1 is expressed at a higher concentration and is already highly activated at basal conditions. These data support a model for the acute activation of GLUT1 that suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond within GLUT1 itself. © 2012 Elsevier Masson SAS. All rights reserved

Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16

Multipotent progenitor cells confirm their T cell–lineage identity in the CD4^–CD8^– double-negative (DN) pro-T cell DN2 stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomics analysis revealed that Bcl11b associated with multiple cofactors and that its direct action was needed to recruit those cofactors to selective target sites. Regions near functionally regulated target genes showed enrichment for those sites of Bcl11b-dependent recruitment of cofactors, and deletion of individual cofactors relieved the repression of many genes normally repressed by Bcl11b. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via the transcription inhibitor Id2 (encoded by Id2) and transcription factor PLZF (encoded by Zbtb16); Id2 and Zbtb16 were directly repressed by Bcl11b, and Id2 and PLZF controlled distinct alternative programs. Thus, our study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials

Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment

During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation

PSCA-targeted BPX-601 CAR T cells with pharmacological activation by rimiducid in metastatic pancreatic and prostate cancer: A phase 1 dose escalation trial

Here we report results of a phase 1 multi-institutional, open-label, dose-escalation trial (NCT02744287) of BPX-601, an investigational autologous PSCA-directed GoCAR-T® cell product containing an inducible MyD88/CD40 ON-switch responsive to the activating dimerizer rimiducid, in patients with metastatic pancreatic (mPDAC) or castration-resistant prostate cancer (mCRPC). Primary objectives were to evaluate safety and tolerability and determine the recommended phase 2 dose/schedule (RP2D). Secondary objectives included the assessment of efficacy and characterization of the pharmacokinetics of rimiducid. Thirty-three patients received BPX-601 with or without rimiducid, 24 patients with mPDAC and 9 with mCRPC. Two dose-limiting toxicities and two treatment-related deaths occurred in the highest-dose mCRPC cohort, after which the study was terminated, without determination of the RP2D. Two mCRPC patients experienced partial responses (one unconfirmed), and 56% of mCRPC patients achieved ≥50% reduction in prostate-specific antigen. BPX-601 cell expansion, long-term persistence in peripheral blood, and tumor infiltration were observed. Rimiducid increased circulating inflammatory cytokines/chemokines consistent with GoCAR-T® cell activation. These results suggest that pharmacological activation of GoCAR-T® cells is feasible and may offer a promising avenue to control chimeric antigen receptor-T cell activity with continued dose-optimization to improve tolerability

Homeostatic Proliferation Fails to Efficiently Reactivate HIV-1 Latently Infected Central Memory CD4+ T Cells

Homeostatic proliferation ensures the longevity of central memory T-cells by inducing cell proliferation in the absence of cellular differentiation or activation. This process is governed mainly by IL-7. Central memory T-cells can also be stimulated via engagement of the T-cell receptor, leading to cell proliferation but also activation and differentiation. Using an in vitro model of HIV-1 latency, we have examined in detail the effects of homeostatic proliferation on latently infected central memory T cells. We have also used antigenic stimulation via anti-CD3/anti-CD28 antibodies and established a comparison with a homeostatic proliferation stimulus, to evaluate potential differences in how either treatment affects the dynamics of latent virus populations. First, we show that homeostatic proliferation, as induced by a combination of IL-2 plus IL-7, leads to partial reactivation of latent HIV-1 but is unable to reduce the size of the reservoir in vitro. Second, latently infected cells are able to homeostatically proliferate in the absence of viral reactivation or cell differentiation. These results indicate that IL-2 plus IL-7 may induce a detrimental effect by favoring the maintenance of the latent HIV-1 reservoir. On the other hand, antigenic stimulation efficiently reactivated latent HIV-1 in cultured central memory cells and led to depletion of the latently infected cells via virus-induced cell death

A Canadian Critical Care Trials Group project in collaboration with the international forum for acute care trialists - Collaborative H1N1 Adjuvant Treatment pilot trial (CHAT): study protocol and design of a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Swine origin influenza A/H1N1 infection (H1N1) emerged in early 2009 and rapidly spread to humans. For most infected individuals, symptoms were mild and self-limited; however, a small number developed a more severe clinical syndrome characterized by profound respiratory failure with hospital mortality ranging from 10 to 30%. While supportive care and neuraminidase inhibitors are the main treatment for influenza, data from observational and interventional studies suggest that the course of influenza can be favorably influenced by agents not classically considered as influenza treatments. Multiple observational studies have suggested that HMGCoA reductase inhibitors (statins) can exert a class effect in attenuating inflammation. The Collaborative H1N1 Adjuvant Treatment (CHAT) Pilot Trial sought to investigate the feasibility of conducting a trial during a global pandemic in critically ill patients with H1N1 with the goal of informing the design of a larger trial powered to determine impact of statins on important outcomes.</p> <p>Methods/Design</p> <p>A multi-national, pilot randomized controlled trial (RCT) of once daily enteral rosuvastatin versus matched placebo administered for 14 days for the treatment of critically ill patients with suspected, probable or confirmed H1N1 infection. We propose to randomize 80 critically ill adults with a moderate to high index of suspicion for H1N1 infection who require mechanical ventilation and have received antiviral therapy for ≤ 72 hours. Site investigators, research coordinators and clinical pharmacists will be blinded to treatment assignment. Only research pharmacy staff will be aware of treatment assignment. We propose several approaches to informed consent including a priori consent from the substitute decision maker (SDM), waived and deferred consent. The primary outcome of the CHAT trial is the proportion of eligible patients enrolled in the study. Secondary outcomes will evaluate adherence to medication administration regimens, the proportion of primary and secondary endpoints collected, the number of patients receiving open-label statins, consent withdrawals and the effect of approved consent models on recruitment rates.</p> <p>Discussion</p> <p>Several aspects of study design including the need to include central randomization, preserve allocation concealment, ensure study blinding compare to a matched placebo and the use novel consent models pose challenges to investigators conducting pandemic research. Moreover, study implementation requires that trial design be pragmatic and initiated in a short time period amidst uncertainty regarding the scope and duration of the pandemic.</p> <p>Trial Registration Number</p> <p><a href="http://www.controlled-trials.com/ISRCTN45190901">ISRCTN45190901</a></p

Activation of Latent HIV Using Drug-Loaded Nanoparticles

Antiretroviral therapy is currently only capable of controlling HIV replication rather than completely eradicating virus from patients. This is due in part to the establishment of a latent virus reservoir in resting CD4+ T cells, which persists even in the presence of HAART. It is thought that forced activation of latently infected cells could induce virus production, allowing targeting of the cell by the immune response. A variety of molecules are able to stimulate HIV from latency. However no tested purging strategy has proven capable of eliminating the infection completely or preventing viral rebound if therapy is stopped. Hence novel latency activation approaches are required. Nanoparticles can offer several advantages over more traditional drug delivery methods, including improved drug solubility, stability, and the ability to simultaneously target multiple different molecules to particular cell or tissue types. Here we describe the development of a novel lipid nanoparticle with the protein kinase C activator bryostatin-2 incorporated (LNP-Bry). These particles can target and activate primary human CD4+ T-cells and stimulate latent virus production from human T-cell lines in vitro and from latently infected cells in a humanized mouse model ex vivo. This activation was synergistically enhanced by the HDAC inhibitor sodium butyrate. Furthermore, LNP-Bry can also be loaded with the protease inhibitor nelfinavir (LNP-Bry-Nel), producing a particle capable of both activating latent virus and inhibiting viral spread. Taken together these data demonstrate the ability of nanotechnological approaches to provide improved methods for activating latent HIV and provide key proof-of-principle experiments showing how novel delivery systems may enhance future HIV therapy

Fluvastatin synergistically enhances the antiproliferative effect of gemcitabine in human pancreatic cancer MIAPaCa-2 cells

The new combination between the nucleoside analogue gemcitabine and the cholesterol-lowering drug fluvastatin was investigated in vitro and in vivo on the human pancreatic tumour cell line MIAPaCa-2. The present study demonstrates that fluvastatin inhibits proliferation, induces apoptosis in pancreatic cancer cells harbouring a p21ras mutation at codon 12 and synergistically potentiates the cytotoxic effect of gemcitabine. The pharmacologic activities of fluvastatin are prevented by administration of mevalonic acid, suggesting that the shown inhibition of geranyl-geranylation and farnesylation of cellular proteins, including p21rhoA and p21ras, plays a major role in its anticancer effect. Fluvastatin treatment also indirectly inhibits the phosphorylation of p42ERK2/mitogen-activated protein kinase, the cellular effector of ras and other signal transduction peptides. Moreover, fluvastatin administration significantly increases the expression of the deoxycytidine kinase, the enzyme required for the activation of gemcitabine, and simultaneously reduces the 5′-nucleotidase, responsible for deactivation of gemcitabine, suggesting a possible additional role of these enzymes in the enhanced cytotoxic activity of gemcitabine. Finally, a significant in vivo antitumour effect on MIAPaCa-2 xenografts was observed with the simultaneous combination of fluvastatin and gemcitabine, resulting in an almost complete suppression and a marked delay in relapse of tumour growth. In conclusion, the combination of fluvastatin and gemcitabine is an effective cytotoxic, proapoptotic treatment in vitro and in vivo against MIAPaCa-2 cells by a mechanism of action mediated, at least in part, by the inhibition of p21ras and rhoA prenylation. The obtained experimental findings might constitute the basis for a novel translational research in humans