Phenotypic and functional properties of in vitro-induced Helios+ regulatory T cells

Objective To investigate the phenotypic and functional properties of in vitro-induced Helios+ regulatory T cells (Helios+ iTreg) and to analyze the differences between Helios+ iTreg and Helios+ thymic-derived natural Treg (Helios+ nTreg). Methods CD4+ CD25- effector T cells (CD4+ CD25- Teff) that were separated from healthy subjects were cultured with anti-CD3 and anti-CD28 antibodies and stimulated with IL-2 and TGF-β to induce the generation of Helios+ iTreg. Flow cytometry and real-time PCR were respectively used to analyze the differences in phenotype and CpG methylation in Treg-specific demethylated region (TSDR) between Helios+ iTreg and Helios+ nTreg derived from the same donor. Results CD45RA was highly expressed on Helios+ iTreg, but not on Helios+ nTreg. Additionally, Helios+ iTreg expressed significantly higher levels of CD127, ICOS and PD-1, but lower levels of CXCR3, CCR4 and CD25 than Helios+ nTreg did. IFN-γ and IL-2 that expressed by Helios+ iTreg were more than those by Helios+ nTreg. It was also found that the level of CpG methylation in TSDR was much higher in Helios+ iTreg than in Helios+ nTreg. Conclusion Both nTreg and iTreg expressed Helios, but the phenotypic and functional properties of the two Treg subsets were different. It was suggested that Helios+ iTreg and Helios+ nTreg might play different roles in the immune system. Key words: Regulatory T cell; Helios; Interleukin-2; Immunophenotype; Methylation