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

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.

Figure 1

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.

Figure 2

Functional assessment of CD8 ⁺ CD25 ⁺ nTreg cell suppressive capacity toward proliferation of allogeneically activated CFSE-labeled T lymphocytes. CFSE dilution analysis shows CD8⁺CD25⁺ nTreg-mediated suppression of allogeneic T cell proliferation in 5 days mixed leukocyte reaction (MLR) compared to control MLR and compared to MLR with CD8⁺CD25⁻ non-Tregs. CFSE dilution histograms are shown for 1:1 suppressor/responder cell ratio. Shown is one representative experiment out of four independent experiments performed. (A) CFSE-labeled allogeneic T cells alone at d0 before MLR. (B) Proliferation of CFSE-labeled allogeneic T cells after 5 days of allo-MLR (control MLR). (C) Allo-MLR in presence of CD8⁺CD25⁻ non-Treg cells, at day 5. (D) Allo-MLR in presence of CD8⁺CD25⁺ nTregs, at day 5.

Figure 3

Differential expression of miR-24, −335, −155, −31, −210, −449, −509, −214, −205 and −9 between CD8 ⁺ CD25 ⁺ nTregs and CD8 ⁺ CD25 ⁻ T cells. Data obtained by qRT-PCR amplification of miRs are plotted. p values for each miRNA relative expression are presented. Boxes represent SE; and Error bars SD Pooled data from five independent experiments are shown. (*p < 0.05; **p < 0.01 CD8⁺CD25⁺ nTregs versus CD8⁺CD25⁻ T cells (Student's t test).

Figure 4

MicroRNA specific activities. (A) MiR-335 negatively regulates luciferase expression in a plasmid coupling its coding sequence with FOXP3 3′UTR. Renilla luciferase reporter assays with constructs holding FOXP3 3′-UTR sequences from the indicated genes were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-335. (B) MiR-9 and miR-155 negatively regulate luciferase expression in a plasmid coupling its coding sequence with CTLA-4 3′UTR. Renilla luciferase reporter assays with constructs holding CTLA-4 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with a firefly luciferase transfection control plasmid either alone or together with miR-9, −155. MiR-24 (C) and miR-335 (D) specifically targets GARP 3′UTR and negatively regulate luciferase reporter expression. Renilla and firefly luciferase reporter assays with constructs holding GARP 3′-UTR sequences, wild type or miR-site deleted, were co-transfected into HEK293T cells along with miR-24, −335. Relative luciferase values normalized to transfections without miRNA are shown. Data represent mean ± SD (error bars) of three independent experiments, each performed in triplicate. (*p < 0.05; **, p < 0.01, Student's t test).

Figure 5

Differential expression of miR-24, −335, −155 and −9 in CD8 ⁺ CD25 ⁺ natural Treg cells after transduction by lenti-miR-24, −335, −155 and −9. Data obtained by qRT-PCR amplification of miRs are plotted for CD8⁺CD25⁺nTregs versus non-transduced CD8⁺CD25⁺ nTregs. p values for each miRNA relative expression are presented. Boxes represent SE; and Error bars SD. Pooled data from five independent experiments are shown. (*p < 0.05; **p < 0.01 Student's t test).

Figure 6

MicroRNA specific activities. (A) In primary human Treg cells, miR-335 specifically regulates FOXP3 expression. Relative miR-335 and FOXP3 expression in CD8⁺CD25⁺ Treg cells transduced by lenti-miR-335 compared with CD8⁺CD25⁺ Treg cells transduced by lenti-miR-Ctrl, as determined by relative qRT-PCR. (B) In primary human Treg cells, miR-9 and −155 specifically regulate CTLA-4 expression. Relative miR-9, miR-155 and CTLA-4 expression in CD8⁺CD25⁺ Treg cells transduced by lenti-miR-9 or lenti-miR-155 compared with CD8⁺CD25⁺ Treg cells transduced by lenti-miR-Ctrl, as determined by relative qRT-PCR. (C) In primary human Treg cells, miR-24 and −335 specifically regulate GARP expression. Relative miR-24 and GARP expression in CD8⁺CD25⁺ Treg cells transduced by lenti-miR-24 compared with CD8⁺CD25⁺ Treg cells transduced by lenti-miR-Ctrl, as determined by relative qRT-PCR. Relative miR-335 and GARP expression in CD8⁺CD25⁺ Treg cells transduced by lenti-miR-335 compared with CD8⁺CD25⁺ Treg cells transduced by lenti-miR-Ctrl. as determined by relative qRT-PCR. CD8⁺CD25⁻ T cells were considered to be non-Tregs; *p < 0.05, **p < 0.01, ***p < 0.001 (as determined by Student's t-test).