A role for the transcription factor Helios in human CD4(+)CD25(+) regulatory T cells

Abstract

Relative upregulation of the Ikaros family transcription factor Helios in natural regulatory T cells (Tregs) has been reported by several groups. However, a role for Helios in regulatory T cells has not yet been described. Here, we show that Helios is upregulated in CD4(+)CD25(+) regulatory T cells. Chromatin-immunoprecipitation (ChIP) experiments indicated that Helios binds to the FoxP3 promoter. These data were further corroborated by experiments showing that knocking-down Helios with siRNA oligonucleotides results in down-regulation of FoxP3. Functionally, we found that suppression of Helios message in CD4(+)CD25(+) T cells significantly attenuates their suppressive function. Taken together, these data suggest that Helios may play an important role in regulatory T cell function and support the concept that Helios may be a novel target to manipulate Treg activity in a clinical setting.

Fig. 1

Helios expression correlates with FoxP3 expression in CD4 T cells. CD4 T cells were enriched using magnetic beads and CD25⁺ and CD25⁻ populaitons FACS sorted. RT-PCR was used to assess message levels of Helios and FoxP3. (A) Relative Helios and Foxp3 message levels in CD25⁻ and CD25⁺ subsets of human and mouse CD4⁺ T cells. (B) Helios and FoxP3 message comparison in FoxP3GFP⁻ CD4⁺CD25⁻ T cells skewed towards Regulatory T cells using TGFb and IL2. (C) Relative expression of Helios and FoxP3 message in naïve T cells post activation (left panel) or post Treg induction (right panel).

Fig. 2

Ectopic expression of Helios induces Apoptosis. Jurkat T cells were transfected with the indicated constructs and GFP positive cells were analyzed for apoptosis via Annexin V and 7AAD staining. (A) Representative FACS plot of GFP positive cells (B) graphical representation of Annexin V positive GFP positive Jurkat T cells of 2 independent experiments.

Fig. 3

Helios binds the FoxP3 promoter. EL4 cells were transfected with Myc tagged Helios or insert control and rested overnight. Chip assay performed using an anti-Myc antibody after stimulation with anti-CD3e, anti-CD28, and TGFb. One iteration of three independent experiments is shown.

Fig. 4

Helios knockdown results in decreased FoxP3 message and diminished suppressive capacity of human Tregs in vitro. Primary CD4⁺CD25⁺ T cells were nucleofected with Helios siRNA and control siRNA and incubated for 60hrs; repeated x3. (A) Relative message levels of Helios (B) relative message levels FoxP3. Responder CD4⁺CD25⁻ T cells admixed with untransfected or siRNA transfected suppressor CD4⁺CD25⁺ T cells in a 1:1 ratio. Proliferation of responders was assayed by H³-thymidine incorporation (C) or CFSE dilution (D) after 5 days of activation. Suppression assay repeated x3.

Fig. 4

Helios knockdown results in decreased FoxP3 message and diminished suppressive capacity of human Tregs in vitro. Primary CD4⁺CD25⁺ T cells were nucleofected with Helios siRNA and control siRNA and incubated for 60hrs; repeated x3. (A) Relative message levels of Helios (B) relative message levels FoxP3. Responder CD4⁺CD25⁻ T cells admixed with untransfected or siRNA transfected suppressor CD4⁺CD25⁺ T cells in a 1:1 ratio. Proliferation of responders was assayed by H³-thymidine incorporation (C) or CFSE dilution (D) after 5 days of activation. Suppression assay repeated x3.