Epigenetic Modulation of STAT3 by Histone Deacetylase 6 (HDAC6) Regulates IL-10 Gene Expression and Immune Tolerance Mediated by Antigen-Presenting Cells (APCs)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 519-519 ◽  
Author(s):  
Fengdong Cheng ◽  
Zi Wang ◽  
Hongwei Wang ◽  
Karrune V. Woan ◽  
Eva Sahakian ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cd4 T Cells ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
B Cell Lymphoma ◽  
Gene Promoters ◽  
Enhanced Production ◽  
Stat3 Phosphorylation

Abstract Abstract 519 We have previously shown that the pan-HDAC inhibitors LAQ824 and LBH589 inhibit IL-10 production in APCs, rendering these cells more inflammatory and capable of effectively priming naïve antigen-specific CD4+ T-cells and restoring the responsiveness of tolerant T-cells1. These findings led us to explore which HDAC(s) might be involved in the regulation of IL-10 gene transcription and be the putative target(s) of HDI-mediated IL-10 inhibition. To answer these questions we subjected the macrophage cell line RAW264.7 to shRNA screening using specific shRNAs to knockdown each known HDAC. We found that among all the HDACs, knocking down HDAC6 (HDAC6KD) was associated with a significant decrease in IL-10 mRNA and protein in response to LPS stimulation. Furthermore, HDAC6KD clones display an enhanced expression of the co-stimulatory molecule B7.2. Functionally, HDAC6KD cells were better activators of anti-HA (hemagglutinin-influenza) transgenic CD4+ T cells, leading to significantly enhanced production of IL-2 and IFN-g in response to cognate antigen. More importantly, anti-HA CD4+ anergic T cells isolated from animals bearing HA-expressing A20 B-cell lymphoma regained their ability to produce IL-2 and IFN-g when cultured in vitro with HDAC6KD cells. These results have been confirmed in APCs isolated from HDAC6 knock-out mice and in wild type APCs treated in vitro with isotype-selective HDAC6 inhibitors. Given that HDACs do not bind to DNA and they need to interact with transcription factors to regulate gene expression, we investigate next which transcription factor(s) HDAC6 might be associated with, to regulate IL-10 transcriptional activity. One likely candidate was Stat3, a well-known transcriptional activator of IL-10 gene expression that we have previously shown to play a central role in tolerance induction by APCs2. By co-immunoprecipitation studies we found that HDAC6 indeed interacts physically with Stat3. Of note, knocking down HDAC6 in APCs resulted in absence of Stat3 phosphorylation and decreased recruitment of Stat3 to the IL-10 gene promoter which might explain the inability of HDAC6KD cells to produce IL-10. The additional findings that IL-10 production by HDAC6KD cells was restored when these cells were transfected with a constitutively active mutant version of Stat3 (Stat3c) provides additional support for the important role of HDAC6 upon Stat3 activation. Further confirmation for a concerted regulatory mechanism involving HDAC6 and Stat3 in IL-10 gene regulation was provided by studies using CPA-7, a specific Stat3 inhibitor that disrupts Stat3 recruitment and binding to gene promoters. As expected, a complete abrogation of Stat3 recruitment to the IL-10 gene promoter was observed in CPA-7 treated APCs. Interestingly, such an effect was accompanied by a parallel decrease in HDAC6 recruitment to the IL-10 promoter and inhibition of IL-10 gene transcriptional activity. Taken together, we have shown for the first time that HDAC6 interacts physically with Stat3 and is required for its phosphorylation. Since Stat3 phosphorylation is absolutely necessary for activation of Stat3 target genes, HDAC6 inhibition is an enticing molecular approach to disrupt the Stat3/IL-10 axis and overcome tolerogenic mechanisms in APCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lymphoma endothelium preferentially expresses Tim-3 and facilitates the progression of lymphoma by mediating immune evasion

2010 ◽  
Vol 207 (3) ◽  
pp. 505-520 ◽  
Author(s):  
Xiaoyuan Huang ◽  
Xiangyang Bai ◽  
Yang Cao ◽  
Jingyi Wu ◽  
Mei Huang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
B Cell Lymphoma ◽  
Global Gene Expression ◽  
T Cell Response ◽  
Th1 Polarization

Angiogenesis is increasingly recognized as an important prognosticator associated with the progression of lymphoma and as an attractive target for novel modalities. We report a previously unrecognized mechanism by which lymphoma endothelium facilitates the growth and dissemination of lymphoma by interacting with circulated T cells and suppresses the activation of CD4+ T cells. Global gene expression profiles of microdissected endothelium from lymphoma and reactive lymph nodes revealed that T cell immunoglobulin and mucin domain–containing molecule 3 (Tim-3) was preferentially expressed in lymphoma-derived endothelial cells (ECs). Clinically, the level of Tim-3 in B cell lymphoma endothelium was closely correlated to both dissemination and poor prognosis. In vitro, Tim-3+ ECs modulated T cell response to lymphoma surrogate antigens by suppressing activation of CD4+ T lymphocytes through the activation of the interleukin-6–STAT3 pathway, inhibiting Th1 polarization, and providing protective immunity. In a lymphoma mouse model, Tim-3–expressing ECs promoted the onset, growth, and dissemination of lymphoma by inhibiting activation of CD4+ T cells and Th1 polarization. Our findings strongly argue that the lymphoma endothelium is not only a vessel system but also a functional barrier facilitating the establishment of lymphoma immune tolerance. These findings highlight a novel molecular mechanism that is a potential target for enhancing the efficacy of tumor immunotherapy and controlling metastatic diseases.

JQ1, a Selective Bromodomain Inhibitor, Augment the Immunogenicity of Mantle Cell Lymphoma By Influencing the Expression of PD-L1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 822-822 ◽  
Author(s):  
Hongwei Wang ◽  
Fengdong Cheng ◽  
Jie Cheng ◽  
Alejandro Villagra ◽  
Jianguo Tao ◽  
...  
Keyword(s):  
T Cells ◽  
Mantle Cell Lymphoma ◽  
Cd4 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tumor Bearing ◽  
Stat3 Phosphorylation ◽  
Mantle Cell ◽  
Cognate Antigen

Abstract Mantle cell lymphoma (MCL) is a rare and aggressive form of non-Hodgkin's B-cell lymphoma and represents 4% to 8% of NHL. New therapeutic strategies are under development to improve the clinical outcome of patients with this disease. Recent studies have highlighted the importance of BRD/acetyl-lysine binding in orchestrating molecular interactions in chromatin biology and regulating gene transcription. The Bromodomain (BRD) is a diverse family of evolutionary conserved protein-interaction modules, which recognizes acetylated lysine residues such as those on the N-terminal tails of histones. We have recently determined the functional consequences of inhibiting Bromodomains in MCL. First, we evaluated the effects of JQ1, a selective small-molecule bromodomain inhibitor upon MCL's immunogenicity and antigen-specific CD4+ T-cell responses. In vitro treatment of MCL cells with increasing concentrations of JQ1 resulted in a decreased expression of the tolerogenic molecule PD-L1 as compared to control cells. Second, we evaluated the capabilities of JQ1-treated MCL cells to present cognate antigen to naïve or anergic antigen-specific CD4+ T-cells. We found that treatment of MCL cells with JQ1 enhances their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells and restoring the responsiveness of tolerant T-cells as determined by their increased production of IL-2 and IFN-gamma in response to cognate antigen. More importantly, in vivo studies clearly demonstrated a strong anti-tumor effect in JQ1-treated MCL tumor bearing mice. Mechanistically, we found a decreased STAT3 phosphorylation and c-Myc expression in JQ1-treated MCL cells, which in turn might be responsible for the diminished expression of PDL1 and augmentation of MCL's immunogenicity. Taken together, we have found that MCL cells treated with the Bromodomain specific inhibitor JQ1 are more inflammatory, display lower expression of the immunosuppressive molecule PDL1 and, are capable of restoring the responsiveness of tolerant T-cells. More importantly, JQ1 treatment in vivo inhibits tumor growth in MCL tumor bearing animals. Our studies therefore have unveiled a previously unknown immunological effect of BRD inhibitors and have identified a novel immunotherapeutic approach in MCL. Disclosures No relevant conflicts of interest to declare.

Association of changes in T regulatory cells (Treg) during nivolumab treatment with melanoma outcome.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 3031-3031 ◽  
Author(s):  
Jeffrey S. Weber ◽  
Rupal Ramakrishnan ◽  
Andressa Laino ◽  
Anders E. Berglund ◽  
David Woods
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cd4 T Cells ◽  
T Regulatory Cells ◽  
Mononuclear Cells ◽  
In Vitro Assays ◽  
Peripheral Blood Mononuclear ◽  
Suppressive Function ◽  
Blocking Antibodies

3031 Background: PD-1 blocking antibodies have significant efficacy in the treatment of melanoma; however, many patients fail to respond and resistance mechanisms remain unknown. We addressed the role of Tregs, an immunosuppressive T-cell population, in patient outcome after treatment with nivolumab. Methods: Peripheral blood mononuclear cells (PBMC) were obtained from patients on trials with nivolumab as adjuvant therapy for resected disease or as treatment for metastatic melanoma. To measure suppression, Tregs were flow-sorted from PBMC and evaluated in allogeneic mixed lymphocyte reactions. Tregs and conventional CD4 T-cells were evaluated for gene expression changes by RNA-sequencing. Treg percentages and phosphorylated STAT3 (pSTAT3) expression were evaluated by flow cytometry. The effects of PD-1 blockade with nivolumab were evaluated in vitro using T-cells from baseline patient PBMC samples. Results: Tregs from responding patients or adjuvant patients without evidence of disease (NED) had reduced suppressive function post-nivolumab (p < 0.05), but no changes were observed in relapsing/non-responding patients; their Tregs were more suppressive than NED/responding Tregs (p < 0.001). NED Tregs had unique gene expression changes and associated pathways post-nivolumab compared to relapsing patient Tregs and conventional CD4 T-cells, including up-regulation of proliferation pathways (q < 8e-19) and downregulation of oxidative phosphorylation (q < 7e-5). NED Tregs had upregulation of pSTAT3 expression post-nivolumab (p < 0.05), which was not observed in relapsing patients. Evaluation of Tregs from patients with active disease also showed upregulation of pSTAT3 in responders (p < 0.05) but not non-responders. The relative increase in Treg pSTAT3 was associated with increased overall survival (R2= 0.49, p < 0.05). In vitro assays using PD-1 blocking antibodies recapitulated the increase in pSTAT3 (p < 0.05) and Treg percentages (p < 0.001), which were diminished with the addition of a STAT3 inhibitor (p < 0.01). Conclusions: These results demonstrate previously unknown roles of decreased Treg suppressive function and induction of STAT3 as biomarkers of patient’s outcome to nivolumab therapy.

Sumoylation of CCAAT Enhancer Bnding Protein alpha (C/EBPα) Is Associated with Its Negative Regulatory Activity during Neutrophil Development.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1181-1181
Author(s):  
Arati Khanna-Gupta ◽  
Matthew Silver ◽  
William Hankey ◽  
Hong Sun ◽  
Nancy Berliner
Keyword(s):  
Gene Expression ◽  
Transcriptional Activity ◽  
Myeloid Cells ◽  
Gene Promoter ◽  
Specific Gene ◽  
Gene Promoters ◽  
Post Translational Modification ◽  
Granulocytic Differentiation ◽  
Key Factor ◽  
High Level

Abstract CCAAT enhancer binding protein alpha (C/EBPα) has been shown to be a master regulator of granulopoiesis It is expressed at high levels throughout myeloid differentiation and binds to multiple myeloid-specific gene promoters at different stages of maturation. Mice nullizygous for C/EBPα display a selective early block in granulocytic differentiation. Furthermore, C/EBPα mutations leading to loss of C/EBPα function have been demonstrated in a subset of patients with AML. A complete understanding of the regulation of this key factor during myelopoiesis is therefore critical. Studies from our laboratory have demonstrated that lactoferrin (LF) gene expression in the developing neutrophil is dependent on a C/EBP binding site in the LF gene promoter. Using ChIP analysis of LF non-expressing and expressing cells, we demonstrated that C/EBPα binds to the LF promoter in uninduced myeloid cells, which do not express LF. Induction of differentiation, associated with LF expression, correlates with a loss of C/EBPα binding and a gain of C/EBPε binding coincident with increased C/EBPε expression. Hence, we reasoned that post-translational modification(s) of C/EBPα during neutrophil maturation alters its transcriptional activity, thus altering LF gene expression. C/EBPα was recently shown to be post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) within a region of the C/EBPα protein that can negatively affect transcriptional activity. Sumoylation at K159 is thought to hamper transactivation by preventing association of the SWI/SNF chromatin remodeling complex. We demonstrate that the levels of sumoylated C/EBPα decrease upon neutrophil maturation, and that transactivation of a LF promoter reporter is significantly enhanced by a sumoylation mutant of C/EBPα (K159A). Additionally, in oligonucleotide pull down assays, sumoylated C/EBPα binds to the C/EBP site in the LF promoter in uninduced myeloid cells while loss of sumoylation correlates with loss of C/EBPα binding and LF expression. We therefore conclude that sumoylated C/EBPα is associated with the negative regulation of LF in early myeloid cells. Several C/EBP family members, including shorter isoforms, have negative regulatory functions. C/EBP homologous protein (CHOP/C/EBPζ/gadd 153, CHOP) is one such factor. Transient co-transfection analysis of a LF promoter reporter with expression plasmids for C/EBPα and CHOP demonstrated a steep decline in C/EBPα mediated transactivation. This CHOP-mediated decline in transactivation was specific for C/EBPα, as CHOP induced no significant change in transactivation by C/EBPε. Additionally, EMSA analyses using extracts from 293T cells overexpressing C/EBPα, C/EBPε and CHOP demonstrated that increasing levels of CHOP could remove C/EBPα, but not C/EBPε, from the C/EBP site in the LF promoter. Since CHOP levels increase during neutrophil maturation and CHOP does not recognize C/EBP cis elements, we hypothesize that CHOP sequesters C/EBPα and prevents its binding. We propose the following model: Sumoylated C/EBPα binds the LF promoter in early myeloid cells and inhibits its expression. Upon induction of maturation, levels of CHOP increase, inducing heterodimerization with unsumoylated C/EBPα. Because CHOP-C/EBPα heterodimers will not bind canonical C/EBP binding sites, this decreases C/EBPα binding to the LF promoter. This change in binding dynamics allows C/EBPε to bind the LF promoter, resulting in high level LF expression.

scholarly journals Histone Deacetylase 11 (HDAC11) Interaction with Ikaros Represent a Novel Mechanism of Regulation of Essential Transcriptional Factors in CD4+ T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 864-864
Author(s):  
Jie Chen ◽  
Fengdong Cheng ◽  
David Michael Woods ◽  
Edward Seto ◽  
Alejandro Villagra ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
T Cell ◽  
Histone Deacetylase ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Epigenetic Mechanism ◽  
Gene Promoters ◽  
H3 Acetylation

Abstract Histone deacetylase 11 (HDAC11), the most recently identified histone deacetylase, is the sole member of class IV HDACs [1]. Since its discovery, no biological function was assigned to this HDAC until we demonstrated its central role in negatively regulating IL-10 production in antigen presenting cells (APCs) [2]. More recently, we have found that disruption of HDAC11 in T cells is associated with an enhanced pro-inflammatory cytokine profile and effector molecule production. Furthermore, T-cells lacking HDAC11 were less susceptible to regulatory T-cell (Treg) suppression in vitro, were refractory to tolerance induction in vivo and displayed enhanced allo-reactivity and anti-tumor responses in murine models. Of note, T-cells lacking HDAC11 expressed higher levels of the transcription factors Eomes and Tbet. Conversely, overexpression of HDAC11 in T-cells decreased the expression of both transcription factors. The molecular mechanism(s) by which HDAC11 regulates the expression of these transcription factors have remained unknown. By using chromatin immunoprecipitation (ChIP) assay we found that in resting T-cells HDAC11 is present at the Eomes and Tbet gene promoters where it maintains histone deacetylation, a compacted chromatin and gene repression. Following T-cell stimulation, HDAC11 was largely absent from both promoters, which resulted in increased histone 3 (H3) acetylation and gene transcriptional activity. These findings were confirmed in T-cells isolated from HDAC11 knock out (KO) mice which also displayed an increase in H3 acetylation at the Tbet and Eomes gene promoter regions. Conversely, H3 acetylation was decreased in both gene promoters in T-cells overexpressing HDAC11 as compared to empty-vector transfected cells. Given that HDACs do not bind to DNA, we asked next which transcription factor(s) HDAC11 might be associated with, in order to regulate Tbet and Eomes gene transcriptional activity. In prior studies we have found that HDAC11 form a molecular complex with another member of the HDAC family, HDAC6, which physically interacts with the transcription factor, STAT3 in both the cytoplasmic and nuclear compartments. However, in T-cells no direct interaction of HDAC11 with STAT3 was detected in either compartment. In contrast, we found for the first time that HDAC11 physically associates with Ikaros (Ikzf1), a member of the Ikaros zinc finger transcription factor family that has been previously implicated in the regulation of T-bet gene expression and IFN-g production in T-cells [3-5]. The protein complex HDAC11-Ikaros was mainly detected in the nuclear compartment and both proteins were present at the T-bet gene promoter. Collectively, these results point to the HDAC11-Ikaros complex as a novel epigenetic mechanism of regulation of Tbet and Eomes, transcription factors that are essential for T cell development and function. Disclosures Woods: BMS: Other: Stock; HDAC11: Patents & Royalties: Patent for targeting HDAC11; Lion Biotech: Other: Stock.

scholarly journals Differential transcription directed by discrete gamma interferon promoter elements in naive and memory (effector) CD4 T cells and CD8 T cells.

1997 ◽  
Vol 17 (1) ◽  
pp. 199-208 ◽  
Author(s):  
T M Aune ◽  
L A Penix ◽  
M R Rincón ◽  
R A Flavell
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Transcriptional Activity ◽  
Memory T Cells ◽  
Gamma Interferon ◽  
Ifn Gamma ◽  
Naïve T Cells

Acquisition of the ability to produce gamma interferon (IFN-gamma) is a fundamental property of memory T cells and enables one subset (T helper 1 [TH1]) to deliver its effector functions. To examine regulation of IFN-gamma gene expression in a model system which recapitulates TH1 differentiation, we prepared reporter transgenic mice which express the luciferase gene under the control of proximal and distal regulatory elements (prox.IFN gamma and dist.IFN gamma) from the IFN-gamma promoter. Memory T cells, but not naive T cells, secreted IFN-gamma and expressed both prox.IFN gamma and dist.IFN gamma transcriptional activities. Naive T cells required priming to become producers of IFN-gamma and to direct transcription by these elements. While both CD4+ and CD8+ T cells produced IFN-gamma, only CD4+ T cells expressed prox.IFN gamma transcriptional activity. Induction of transcriptional activity was inhibited by known antagonists of effector T-cell populations. Cyclosporin A inhibited transcriptional activity directed by both elements in effector T cells. Elevated cyclic AMP inhibited transcriptional activity directed by prox.IFN gamma in primed CD4+ T cells but enhanced transcriptional activity directed by dist.IFN gamma in primed CD8+ T cells. Taken together, these data show that prox.IFN gamma and dist.IFN gamma transcriptional activities mirror IFN-gamma gene expression in naive and memory CD4+ T cells but suggest that differences exist in regulation of IFN-gamma gene expression in CD4+ and CD8+ T-cell subsets.

scholarly journals Specific activation of HIV-1 from monocytic reservoir cells by bromodomain inhibitor in humanized mice in vivo

2018 ◽  
Author(s):  
Guangming Li ◽  
Zheng Zhang ◽  
Natalia Reszka-Blanco ◽  
Feng Li ◽  
Liqun Chi ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cd4 T Cells ◽  
Humanized Mice ◽  
Viral Transcription ◽  
Dependent Manner ◽  
Monocytic Cells ◽  
Hiv 1

ABSTRACTThe combination antiretroviral therapy (cART) effectively suppresses HIV-1 infection and enables HIV-infected individuals to live long productive lives. However, the persistence of HIV-1 reservoir cells with latent or low-replicating HIV-1 in patients under cART make HIV-1 infection an incurable disease. Recent studies have focused on the development of strategies such as epigenetic modulators to activate and purge these reservoirs. Bromodomain inhibitors (BETi) are epigenetic modulating compounds able to activate viral transcription in HIV-1 latency cell lines in a positive transcription elongation factor b (P-TEFb)-dependent manner. Little is known about the efficacy of activating HIV-1 reservoir cells under cART by BETi in vivo. In this study, we seek to test the potential of a BETi (I-BET151) in activating HIV-1 reservoir cells under effective cART in humanized mice in vivo. We discover that I-BET151 efficiently activates HIV-1 transcription in monocytic cells, but not in CD4+T cells, during suppressive cART in vivo. We further reveal that HIV-1 proviruses in monocytic cells are more sensitive to I-BET151 treatment than in T cells in vitro. Finally, we demonstrate that I-BET151-activated viral transcription in monocytic cells is dependent on both CDK2 and CDK9, whereas only CDK9 is involved in activation of HIV-1 by I-BET151 in T cells. Our findings indicate a role of myeloid cells in HIV-1 persistence, and highlights the limitation of measuring or targeting T cell reservoirs alone in terms of HIV-1 cure, as well as provides a potential strategy to reactivate monocytic reservoirs during cART.IMPORTANCEIt has been reported the low level of active P-TEFb critically contributes to the maintenance of HIV-1 latency or low-replication in HIV-1 reservoir cells under cART. Bromodomain inhibitors are used to activate HIV-1 replication in vitro but their effect on activation of the HIV-1 resevoirs with cART in vivo is not clear. We found that BETi (I-BET151) treatment reactivated HIV-1 gene expression in humanized mice during suppressive cART. Interestingly, I-BET151 preferentially reactivated HIV-1 gene expression in monocytic cells, but not in CD4 T cells. Furthermore, I-BET151 significantly increased HIV-1 transcription in monocytic cells, but not in latently infected CD4 T cells, via CDK2-dependent mechanisms. Our findings suggest that BETi can preferentially activate monocytic HIV-1 reservoir cells, and a combination of latency reversal agents targeting different cell types and pathways is needed to achieve reactivation of different HIV-1 reservoir cells during cART.

Global Gene Expression Profiles of Activated CD4+CD25high (Regulatory) and CD4+CD25− (Non-Regulatory) T Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3308-3308
Author(s):  
ChaoYan Liu ◽  
Qi-Hong Sun ◽  
Gian Paolo Visentin
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cd4 T Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Platelet Factor ◽  
Homogeneous Population

Abstract Autoreactive T and B cells can be detected in healthy individuals but are normally kept in check by regulatory mechanisms. Among those is an active suppression of naïve T cells by endogenous T regulatory (Tr) cells. Several types of Tr cells exist, including CD4+ T cells which constitutively express the IL-2 receptor α chain (CD25), do not secrete IL-10, and suppress immune responses via direct cell-to-cell interactions. CD4+CD25+ T regulatory cells represent 5%–10% of the endogenous CD4+ T cells subset and are able to suppress CD4+ and CD8+ T cell responses in vitro and in vivo upon TCR ligation. Our recent observation that human platelet factor 4 (PF4; CXCL4) inhibits the proliferative response of human CD4+CD25− T cells, while inducing expansion of CD4+CD25+ Tr cells, and that PF4-induced CD4+CD25+ Tr cells lose their potent suppressor function in vitro, suggests a previously unrecognized role of PF4 in the regulation of immune responsiveness (Liu, et al. J Immunol174:2680–86, 2005). A large body of evidence suggests that human CD4+CD25+ Tr cells share many of the characteristics of murine CD4+CD25+ Tr cells. McHugh et al. (Immunity16:311–23, 2002), have successfully used the microarray approach to identify genes differentially expressed in resting CD4+CD25+ and CD4+CD25− mouse T cells, but with the only exception of a small preliminary report (Pati et al. Ann N Y Acad Sci. 1005:279–83, 2003), little information is available on the gene expression profile of human CD4+CD25+ and CD4+CD25− T cells. We performed global gene expression analysis using oligo-DNA microarrays (CodeLink, Amersham Biosciences) that monitor the expression of whole human genome, to define the gene expression profiles in CD4+CD25+ Tr cells stimulated by anti-CD3 mAb and exposed to PF4. CD4+ T cells were isolated from normal donor’s peripheral blood mononuclear cells by positive selection on magnetic beads (Miltenyi Biotec, Auburn, CA), then labeled with PE-conjugated anti-CD4 and FITC-conjugated anti-CD25 and sorted on a FACStar (BD Biosciences, San Jose, CA) to obtain a homogeneous population of T cells consisting of CD4+CD25+ Tr cells expressing CD25 at high levels (CD4+CD25high) and CD4+CD25− T cells (non-regulatory). Total RNA was extracted from the freshly isolated CD4+CD25high and CD4+CD25− T cells subsets, stimulated with anti-CD3 mAb in the presence or the absence of PF4 for 24 hours. Using this approach, we have identified a little over 100 genes that are differentially expressed, in the presence of PF4, in CD4+CD25+ Tr cells following activation with anti-CD3 mAb. We have focused our attention on about 40 target genes whose increased expression has been validated using real time PCR and, were appropriate, at the protein levels, by flow cytometry or Luminex 100 multiplex cytokine quantification (Table 1). Our data suggest that PF4 modulates proliferation and function of CD4+CD25+ Tr cells by the coordinate increasing expression of a relatively large number of genes, coupled with a further enhanced expression of a limited number of growth promoting genes and the specific silencing of a small subset of negative growth regulatory genes.

scholarly journals Specific ActivationIn Vivoof HIV-1 by a Bromodomain Inhibitor from Monocytic Cells in Humanized Mice under Antiretroviral Therapy

2019 ◽  
Vol 93 (12) ◽  
Author(s):  
Guangming Li ◽  
Zheng Zhang ◽  
Natalia Reszka-Blanco ◽  
Feng Li ◽  
Liqun Chi ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Antiretroviral Therapy ◽  
Cd4 T Cells ◽  
Myeloid Cells ◽  
Humanized Mice ◽  
Monocytic Cells ◽  
Hiv 1

ABSTRACTCombination antiretroviral therapy (cART) effectively suppresses HIV-1 replication and enables HIV‑infected individuals to live long, productive lives. However, the persistence of HIV-1 reservoirs of both T and myeloid cells with latent or low-replicating HIV-1 in patients under cART makes HIV-1 infection an incurable disease. Recent studies have focused on the development of strategies to activate and purge these reservoirs. Bromodomain and extraterminal domain proteins (BETs) are epigenetic readers involved in modulating gene expression. Several bromodomain inhibitors (BETi) are reported to activate viral transcriptionin vitroin HIV-1 latency cell lines in a P-TEFb (CDK9/cyclin T1)-dependent manner. Little is known about BETi efficacy in activating HIV-1 reservoir cells under cARTin vivo. Here we report that a BETi (I-BET151) efficiently activated HIV-1 reservoirs under effective cART in humanized micein vivo. Interestingly, I-BET151 during suppressive cARTin vivoactivated HIV-1 gene expression only in monocytic cells and not in CD4+T cells. We further demonstrate that BETi preferentially enhanced HIV-1 gene expression in monocytic cells rather than in T cells and that whereas CDK9 was involved in activating HIV-1 by I-BET151 in both monocytic and T cells, CDK2 enhanced HIV-1 transcription in monocytic cells but inhibited it in T cells. Our findings reveal a role for CDK2 in differential modulation of HIV-1 gene expression in myeloid cells and in T cells and provide a novel strategy to reactivate monocytic reservoirs with BETi during cART.IMPORTANCEBromodomain inhibitors have been reported to activate HIV-1 transcriptionin vitro, but their effect on activation of HIV-1 reservoirs during cARTin vivois unclear. We found that BETi (I-BET151) treatment reactivated HIV-1 gene expression in humanized mice during suppressive cART. Interestingly, I-BET151 preferentially reactivated HIV-1 gene expression in monocytic cells, but not in CD4 T cells, in cART-treated mice. Furthermore, I-BET151 significantly increased HIV-1 transcription in monocytic cells, but not in HIV-1-infected CD4 T cells, via CDK2-dependent mechanisms. Our findings suggest that BETi can preferentially activate monocytic HIV-1 reservoir cells and that a combination of reservoir activation agents targeting different cell types and pathways is needed to achieve reactivation of different HIV-1 reservoir cells during cART.

scholarly journals Induction of interleukin 12 responsiveness is impaired in anergic T lymphocytes.

1994 ◽  
Vol 179 (3) ◽  
pp. 1065-1070 ◽  
Author(s):  
H Quill ◽  
A Bhandoola ◽  
G Trinchieri ◽  
J Haluskey ◽  
D Peritt
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Proliferative Response ◽  
Interleukin 12 ◽  
T Helper 1 ◽  
Ifn Gamma ◽  
Enhanced Production

The cytokine, interleukin 12 (IL-12), stimulates both natural killer cells and T cells to proliferate and to secrete interferon gamma (IFN-gamma). The T cell proliferative response to IL-12 must be induced and is evident after T cell receptor-mediated stimulation. As reported here, tolerant CD4+ T cells and clones, that are anergic for IL-2 production, are also anergic for induction of the proliferative response to IL-12. Murine T helper 1 clones tolerized in vitro, as well as anergic CD4+ T cells isolated from mice tolerized to the Mls-1a antigen (Ag) in vivo, demonstrated defective induction of proliferation to IL-12 upon restimulation with Ag. IL-12-enhanced production of IFN-gamma was observed in both control and anergic cells after Ag/antigen-presenting cell (APC) activation, although total IFN-gamma secretion by anergic cells was less than that produced by control cells, even in the presence of IL-12. These data indicate that T cell clonal anergy results in profound inhibition of proliferative responses, since the autocrine growth factor, IL-2, is not produced, and the APC-derived cytokine, IL-12, is not an effective stimulus for anergic T cell proliferation.

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