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miR-155 inhibition sensitizes CD4+ Th cells for TREG mediated suppression.

Stahl HF, Fauti T, Ullrich N, Bopp T, Kubach J, Rust W, Labhart P, Alexiadis V, Becker C, Hafner M, Weith A, Lenter MC, Jonuleit H, Schmitt E, Mennerich D - PLoS ONE (2009)

Bottom Line: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations.Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3.Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

View Article: PubMed Central - PubMed

Affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Respiratory Diseases Research, Genomics Group, Biberach an der Riss, Germany.

ABSTRACT

Background: In humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.

Principal findings: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.

Conclusion: Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

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Human Exon Array Genechip expression profiling showed BIC as one of the highest up-regulated genes after T cell activation.(A) Schematic overview of different T cell populations out of 10 human donors. Expression profiles were analyzed of freshly isolated resting CD4+ Th cells and of nTregs. In addition, both populations were profiled upon 4 h and 16 h anti-CD3/anti-CD28 stimulation. (B) The expression profiling revealed the BIC transcript specifically up-regulated upon activation in both populations: the CD4+ Th cells and nTregs. The median relative expression level of BIC in logarithmic scale and the standard deviation is shown (n = 10).
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pone-0007158-g001: Human Exon Array Genechip expression profiling showed BIC as one of the highest up-regulated genes after T cell activation.(A) Schematic overview of different T cell populations out of 10 human donors. Expression profiles were analyzed of freshly isolated resting CD4+ Th cells and of nTregs. In addition, both populations were profiled upon 4 h and 16 h anti-CD3/anti-CD28 stimulation. (B) The expression profiling revealed the BIC transcript specifically up-regulated upon activation in both populations: the CD4+ Th cells and nTregs. The median relative expression level of BIC in logarithmic scale and the standard deviation is shown (n = 10).

Mentions: Autoimmune diseases are characterized by hypersensitive immune responses to self-antigens. One proposed explanation for autoimmune pathogenesis is that naturally occurring CD4+CD25+ regulatory T cells (nTregs) lose their suppressive potential. Another possibility is that CD4+ Th cells have become insensitive to nTreg cell-mediated suppression. To identify the molecular mechanisms underlying the activation of T cells respective Treg cell-mediated suppression, we performed gene expression profiling of primary human T cells freshly-isolated by leukapheresis [14]. Using this method, we were able to isolate a sufficient number of T cells such that in vitro expansion was not necessary. Per donor, more than 5.0×107 pure nTregs and more than 2.5×108 pure CD4+ Th cells were obtained (Figure S1: FACS analysis of freshly isolated, not expanded, human CD4+ Th cell and nTreg cell populations). We compared, in a so-called “paired” analysis, resting versus 4 h and 16 h activated nTregs, to similarly activated CD4+ Th cells from the same healthy volunteer. We distinguished between early and late activated genes (Fig. 1A). To gain statistically relevant data, the study was performed with T cells from 10 different healthy human volunteers (n = 10).


miR-155 inhibition sensitizes CD4+ Th cells for TREG mediated suppression.

Stahl HF, Fauti T, Ullrich N, Bopp T, Kubach J, Rust W, Labhart P, Alexiadis V, Becker C, Hafner M, Weith A, Lenter MC, Jonuleit H, Schmitt E, Mennerich D - PLoS ONE (2009)

Human Exon Array Genechip expression profiling showed BIC as one of the highest up-regulated genes after T cell activation.(A) Schematic overview of different T cell populations out of 10 human donors. Expression profiles were analyzed of freshly isolated resting CD4+ Th cells and of nTregs. In addition, both populations were profiled upon 4 h and 16 h anti-CD3/anti-CD28 stimulation. (B) The expression profiling revealed the BIC transcript specifically up-regulated upon activation in both populations: the CD4+ Th cells and nTregs. The median relative expression level of BIC in logarithmic scale and the standard deviation is shown (n = 10).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2743997&req=5

pone-0007158-g001: Human Exon Array Genechip expression profiling showed BIC as one of the highest up-regulated genes after T cell activation.(A) Schematic overview of different T cell populations out of 10 human donors. Expression profiles were analyzed of freshly isolated resting CD4+ Th cells and of nTregs. In addition, both populations were profiled upon 4 h and 16 h anti-CD3/anti-CD28 stimulation. (B) The expression profiling revealed the BIC transcript specifically up-regulated upon activation in both populations: the CD4+ Th cells and nTregs. The median relative expression level of BIC in logarithmic scale and the standard deviation is shown (n = 10).
Mentions: Autoimmune diseases are characterized by hypersensitive immune responses to self-antigens. One proposed explanation for autoimmune pathogenesis is that naturally occurring CD4+CD25+ regulatory T cells (nTregs) lose their suppressive potential. Another possibility is that CD4+ Th cells have become insensitive to nTreg cell-mediated suppression. To identify the molecular mechanisms underlying the activation of T cells respective Treg cell-mediated suppression, we performed gene expression profiling of primary human T cells freshly-isolated by leukapheresis [14]. Using this method, we were able to isolate a sufficient number of T cells such that in vitro expansion was not necessary. Per donor, more than 5.0×107 pure nTregs and more than 2.5×108 pure CD4+ Th cells were obtained (Figure S1: FACS analysis of freshly isolated, not expanded, human CD4+ Th cell and nTreg cell populations). We compared, in a so-called “paired” analysis, resting versus 4 h and 16 h activated nTregs, to similarly activated CD4+ Th cells from the same healthy volunteer. We distinguished between early and late activated genes (Fig. 1A). To gain statistically relevant data, the study was performed with T cells from 10 different healthy human volunteers (n = 10).

Bottom Line: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations.Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3.Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

View Article: PubMed Central - PubMed

Affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Respiratory Diseases Research, Genomics Group, Biberach an der Riss, Germany.

ABSTRACT

Background: In humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.

Principal findings: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.

Conclusion: Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

Show MeSH
Related in: MedlinePlus