Functional Cross-talk among Cytokines, T-Cell Receptor, and Glucocorticoid Receptor Transcriptional Activity and Action

Abstract CCR6 is a chemokine receptor expressed on Th17 cells and regulatory T cells that is induced by T-cell priming with certain cytokines, but how its expression and stability are regulated at the molecular level is largely unknown. Here, we identified and characterized a noncoding region of the human CCR6 locus that displayed unmethylated CpG motifs (differentially methylated region [DMR]) selectively in CCR6+ lymphocytes. CCR6 expression on circulating CD4+ T cells was stable on cytokine-induced proliferation but partially down-regulated on T-cell receptor stimulation. However, CCR6 down-regulation was mostly transient, and the DMR within the CCR6 locus remained demethylated. Notably, in vitro induction of CCR6 expression with cytokines in T-cell receptor-activated naive CD4+ T cells was not associated with a demethylated DMR and resulted in unstable CCR6 expression. Conversely, treatment with the DNA methylation inhibitor 5′-azacytidine induced demethylation of the DMR and led to increased and stable CCR6 expression. Finally, when cloned into a reporter gene plasmid, the DMR displayed transcriptional activity in memory T cells that was suppressed by DNA methylation. In summary, we have identified a noncoding region of the human CCR6 gene with methylation-sensitive transcriptional activity in CCR6+ T cells that controls stable CCR6 expression via epigenetic mechanisms.

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Regulation of β1Integrin-Mediated Adhesion by T Cell Receptor Signaling Involves ZAP-70 but Differs from Signaling Events That Regulate Transcriptional Activity

The Journal of Immunology ◽

10.4049/jimmunol.165.9.4941 ◽

2000 ◽

Vol 165 (9) ◽

pp. 4941-4949 ◽

Cited By ~ 32

Author(s):

Jennifer A. Epler ◽

Rugao Liu ◽

Heekyoung Chung ◽

Nadine C. Ottoson ◽

Yoji Shimizu

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Transcriptional Activity ◽

Cell Receptor ◽

Receptor Signaling ◽

T Cell Receptor Signaling ◽

Signaling Events ◽

Cell Receptor Signaling

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c-Myb and core-binding factor/PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor delta enhancer.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3090 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3090-3099 ◽

Cited By ~ 76

Author(s):

C Hernandez-Munain ◽

M S Krangel

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Transcriptional Activity ◽

Cell Receptor ◽

Core Binding Factor ◽

Composite Surface ◽

The Core ◽

Human T Cell ◽

Binding Factor ◽

Two Factors

A T-cell-specific transcriptional enhancer lies within the J delta 3-C delta intron of the human T-cell receptor delta gene. We have previously shown that a 30-bp element, denoted delta E3, acts as the minimal TCR delta enhancer and that within delta E3, adjacent and precisely spaced binding sites for core-binding factor (CBF/PEBP2) and c-Myb are essential for transcriptional activity. These data suggested that CBF/PEBP2 and c-Myb synergize to mediate transcriptional activity but did not establish the molecular basis for synergy. In this study, we have examined in detail the binding of CBF/PEBP2 and c-Myb to delta E3. We found that CBF/PEBP2 and c-Myb could simultaneously occupy the core site and one of two overlapping Myb sites within delta E3. However, equilibrium binding and kinetic dissociation experiments suggest that the two factors bind to delta E3 independently, rather than cooperatively. This was found to be true by using isoforms of these factors present in extracts of transfected COS-7 cells, as well as the natural factors present in nuclear extracts of the Jurkat T-cell line. We further showed that CBF/PEBP2 and c-Myb provide unique transactivation functions, since the core-Myb combination cannot be substituted by dimerized core or Myb sites. We propose that spatially precise synergy between CBF/PEBP2 and c-Myb may result from the ability of the two factors to form a composite surface that makes unique and stereospecific contacts with one or more additional components of the transcriptional machinery.

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“Designer” Cells for Cellular Immunotherapy: Zinc Finger Nuclease (ZFN) Stimulated Targeted Recombination for the Simultaneous Disruption of the Endogenous Glucocorticoid Receptor and Site-Specific Addition of the IL13-Zetakine.

Blood ◽

10.1182/blood.v108.11.3705.3705 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3705-3705

Author(s):

Andreas Reik ◽

Michael C. Holmes ◽

Fyodor D. Urnov ◽

Yuanyue Zhou ◽

Matthew Mendel ◽

...

Keyword(s):

T Cell ◽

Glucocorticoid Receptor ◽

T Cell Receptor ◽

Zinc Finger ◽

High Efficiency ◽

Cell Receptor ◽

Cellular Immunotherapy ◽

Genomic Locus ◽

Promising Therapeutic Approach

Abstract Immunotherapy utilizing genetically-modified cytolytic T-lymphocytes (CTL) provides a promising therapeutic approach for treating a variety of diseases, including cancer. We have demonstrated that CTLs encoding a chimeric T-cell receptor (IL13-zetakine), consisting of an extracellular IL-13 domain and a cytoplasmic CD3 domain, can be re-directed to target malignant glioma both in vitro and in animal models. This chimera re-targets the antigen-specific effector functions of modified CTLs to recognize glioblastomas due to the high expression of IL13R in these tumors. However, practical application of this approach is limited by the fact that patients undergoing surgical resection of the tumor often require treatment with glucocorticoids to control the resulting inflammation. Such treatment blocks the activity of the re-directed CTL clones and thus inhibits their therapeutic action. To overcome this limitation and render these tumor-specific CTLs resistant to glucocorticoids we have chosen to employ engineered ZFNs to specifically disrupt the endogenous glucocorticoid receptor (GR) gene. Heterodimeric ZFNs, consisting of the cleavage domain of the restriction enzyme FokI linked to engineered zinc finger DNA-binding domains, can be designed to specifically cleave a predetermined site in the genome. We have shown that these ZFN-induced double strand breaks can promote homologous recombination with high efficiency. In the present study we have investigated the use of ZFNs to simultaneously effect functional inactivation of human GR via specifically targeting the integration of the IL13-zetakine expression cassette into the GR locus itself. We can show that GR-specific ZFNs cleave their intended target sequences with high specificity and efficiency - resulting in the disruption of GR and the creation of glucocorticoid resistant cells. Moreover, we can demonstrate that these ZFNs coupled with an appropriate IL13-zetakine containing donor-DNA molecule can stimulate the integration of this chimeric T-cell receptor directly into the GR locus. Thus, this procedure results in the simultaneous knockout of GR and addition of the IL13-zetakine in a genetically defined manner. These data support the notion that ZFN-modified cells can be engineered to express chimeric antigen receptors from a predetermined genomic locus and may provide a general approach to generating effective cellular immunotherapy strategies.

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Increased p300 Expression Inhibits Glucocorticoid Receptor-T-Cell Receptor Antagonism but Does Not Affect Thymocyte Positive Selection

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4556-4566.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4556-4566 ◽

Cited By ~ 7

Author(s):

Cheng-Tai Yu ◽

Ming-Hsien Lin Feng ◽

Hsiu-ming Shih ◽

Ming-Zong Lai

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

Glucocorticoid Receptor ◽

Positive Selection ◽

T Cell Receptor ◽

Critical Role ◽

Interleukin 2 ◽

Cell Receptor ◽

Selection Of

ABSTRACT Positive selection of T cells is postulated to be dependent on the counterinteraction between glucocorticoid receptor (GR)- and T-cell-receptor (TCR)-induced death signals. In this study we used T-cell-specific expression of p300 to investigate whether GR-TCR cross talk between thymocytes was affected. Activation of the p300-transgenic T cells led to enhanced thymocyte proliferation and increased interleukin 2 production. Thymocyte death, induced by TCR engagement, was no longer prevented by dexamethasone in p300-transgenic mice, indicating an absence of GR-TCR cross-inhibition. This was accompanied by a 50% reduction in the number of thymocytes in p300-transgenic mice. However, the CD4/CD8 profile of thymocytes remained unchanged in p300-transgenic mice. There was no effect on positive selection of the bulk thymocytes or thymocytes with transgenic TCR in p300-transgenic mice. In addition, there was no apparent TCR repertoire “hole” in the selected antigens examined. Our results illustrate a critical role of CBP/p300 in thymic GR-TCR counterinteraction yet do not support the involvement of GR-TCR antagonism in thymocyte positive selection.

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