Limits...
A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

Jebbawi F, Fayyad-Kazan H, Merimi M, Lewalle P, Verougstraete JC, Leo O, Romero P, Burny A, Badran B, Martiat P, Rouas R - J Transl Med (2014)

Bottom Line: We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes.We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes.These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

View Article: PubMed Central - PubMed

Affiliation: Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles, 121, Boulevard de Waterloo, 1000, Bruxelles, Belgium. Fadi.Jebbawi@ulb.ac.be.

ABSTRACT

Background: Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation.

Methods: We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes.

Results: The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo.

Conclusions: We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

No MeSH data available.


Related in: MedlinePlus

Purified human CD8+CD25+natural Tregs express FOXP3 and CTLA-4. Separated cell fractions, CD8+CD25+ and CD8+CD25− T cells, were analyzed by multicolor FACS using the following antibodies: anti-CD3 PerCP, anti-CD8-APC (BD biosciences) and anti-CD25-PE (Miltenyi Biotec) to assess purity for each isolation. Intracellular FOXP3 and cell-surface CTLA-4 were assessed using human anti-FOXP3-PE detection kit and anti-CTLA-4-PE (BD biosciences). Flow cytometry was performed using a FACSCalibur applying CellQuest software (BD Biosciences). CD3 and CD8 purity was above 97% among isolated CD8+ T cells. CD8+CD25+ nTregs express FOXP3 and CTLA-4 when compared to CD8+CD25− T cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4440568&req=5

Fig1: Purified human CD8+CD25+natural Tregs express FOXP3 and CTLA-4. Separated cell fractions, CD8+CD25+ and CD8+CD25− T cells, were analyzed by multicolor FACS using the following antibodies: anti-CD3 PerCP, anti-CD8-APC (BD biosciences) and anti-CD25-PE (Miltenyi Biotec) to assess purity for each isolation. Intracellular FOXP3 and cell-surface CTLA-4 were assessed using human anti-FOXP3-PE detection kit and anti-CTLA-4-PE (BD biosciences). Flow cytometry was performed using a FACSCalibur applying CellQuest software (BD Biosciences). CD3 and CD8 purity was above 97% among isolated CD8+ T cells. CD8+CD25+ nTregs express FOXP3 and CTLA-4 when compared to CD8+CD25− T cells.

Mentions: CD8+CD25+ and CD8+CD25− T cells were immunomagnetically purified from fresh human cord blood by negative selection of CD8+ T cells, followed by CD25-based positive selection (Miltenyi Biotec, Bergisch Gladbach, Germany). To confirm the purity of the separated cell fractions, CD8+CD25+ and CD8+CD25− T cells were analyzed by flow cytometry. Purity was always >96%. CD8+ natural Treg cells expressed more intracellular FOXP3 and cell-surface CTLA-4 proteins compared with CD8+ CD25− T cells (Figure 1).Figure 1


A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

Jebbawi F, Fayyad-Kazan H, Merimi M, Lewalle P, Verougstraete JC, Leo O, Romero P, Burny A, Badran B, Martiat P, Rouas R - J Transl Med (2014)

Purified human CD8+CD25+natural Tregs express FOXP3 and CTLA-4. Separated cell fractions, CD8+CD25+ and CD8+CD25− T cells, were analyzed by multicolor FACS using the following antibodies: anti-CD3 PerCP, anti-CD8-APC (BD biosciences) and anti-CD25-PE (Miltenyi Biotec) to assess purity for each isolation. Intracellular FOXP3 and cell-surface CTLA-4 were assessed using human anti-FOXP3-PE detection kit and anti-CTLA-4-PE (BD biosciences). Flow cytometry was performed using a FACSCalibur applying CellQuest software (BD Biosciences). CD3 and CD8 purity was above 97% among isolated CD8+ T cells. CD8+CD25+ nTregs express FOXP3 and CTLA-4 when compared to CD8+CD25− T cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4440568&req=5

Fig1: Purified human CD8+CD25+natural Tregs express FOXP3 and CTLA-4. Separated cell fractions, CD8+CD25+ and CD8+CD25− T cells, were analyzed by multicolor FACS using the following antibodies: anti-CD3 PerCP, anti-CD8-APC (BD biosciences) and anti-CD25-PE (Miltenyi Biotec) to assess purity for each isolation. Intracellular FOXP3 and cell-surface CTLA-4 were assessed using human anti-FOXP3-PE detection kit and anti-CTLA-4-PE (BD biosciences). Flow cytometry was performed using a FACSCalibur applying CellQuest software (BD Biosciences). CD3 and CD8 purity was above 97% among isolated CD8+ T cells. CD8+CD25+ nTregs express FOXP3 and CTLA-4 when compared to CD8+CD25− T cells.
Mentions: CD8+CD25+ and CD8+CD25− T cells were immunomagnetically purified from fresh human cord blood by negative selection of CD8+ T cells, followed by CD25-based positive selection (Miltenyi Biotec, Bergisch Gladbach, Germany). To confirm the purity of the separated cell fractions, CD8+CD25+ and CD8+CD25− T cells were analyzed by flow cytometry. Purity was always >96%. CD8+ natural Treg cells expressed more intracellular FOXP3 and cell-surface CTLA-4 proteins compared with CD8+ CD25− T cells (Figure 1).Figure 1

Bottom Line: We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes.We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes.These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

View Article: PubMed Central - PubMed

Affiliation: Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles, 121, Boulevard de Waterloo, 1000, Bruxelles, Belgium. Fadi.Jebbawi@ulb.ac.be.

ABSTRACT

Background: Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation.

Methods: We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes.

Results: The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo.

Conclusions: We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

No MeSH data available.


Related in: MedlinePlus