Natural and inducible Tregs in swine: Helios expression and functional properties

• Porcine iTregs can be generated in vitro via CD3 + IL-2 + TGF-β stimulation.
• Porcine iTregs suppress the proliferation but not the IFN-γ production of PBMC.
• The transcription factor Helios might be an activation marker for porcine Tregs.

Within the population of regulatory T cells (Tregs) natural Tregs (nTregs) and inducible Tregs (iTregs) can be distinguished. Although information about Tregs in swine exists, porcine iTregs were not under investigation yet. In this study, Foxp3+ iTregs were generated from CD4+Foxp3− T cells by in vitro stimulation in the presence of IL-2 and TGF-β. In comparison to ex vivo Tregs these iTregs had a similar suppressive capacity on the proliferation of CD3-stimulated PBMC, caused higher levels of IL-10 in PBMC/Treg co-cultures, but did not suppress IFN-γ levels. The Ikaros family member Helios is currently discussed to distinguish iTregs and nTregs or to serve as an activation marker of Tregs. In this study, we demonstrate the cross-reactivity of an anti-mouse/human Helios mAb with porcine Helios. Flow cytometric analyses with this antibody showed that porcine iTregs do not express Helios after in vitro iTreg induction. Nevertheless, thymic Foxp3+ T cells, which arise at the CD4/CD8α single-positive stage of T-cell development and are defined as nTregs, entirely expressed Helios. Although this might suggest the suitability of Helios as an nTreg–iTreg differentiation marker we also found that Helios− Tregs displayed a phenotype of naive CD4+ T cells in vivo. Since iTregs are by definition activated/differentiated Tregs, this finding precludes that all Helios− Tregs are iTregs and thus also the use of Helios as a selection marker for porcine nTregs. Furthermore, Helios+ Tregs displayed a more differentiated phenotype indicating that Helios might rather serve as a Treg activation/differentiation marker.