Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP

Regulatory T cells (Tregs) are key players of immune regulation/dysregulation both in physiological and pathophysiological settings. Despite significant advances in understanding Treg function, there is still a pressing need to define reliable and specific markers that can distinguish different Treg subpopulations. Herein we show for the first time that markers of activated Tregs [latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP, or LRRC32)] are expressed on CD4⁺FoxP3^- T cells expressing Helios (FoxP3^-Helios⁺) in the steady state. Following TCR activation, GARP/LAP are up-regulated on CD4⁺Helios⁺ T cells regardless of FoxP3 expression (FoxP3^+/-Helios⁺). We show that CD4⁺GARP^+/-LAP⁺ Tregs make IL-10 immunosuppressive cytokine but not IFN-γ effector cytokine. Further characterization of FoxP3/Helios subpopulations showed that FoxP3⁺Helios⁺ Tregs proliferate in vitro significantly less than FoxP3⁺Helios^- Tregs upon TCR stimulation. Unlike FoxP3⁺Helios^- Tregs, FoxP3⁺Helios⁺ Tregs secrete IL-10 but not IFN-γ or IL-2, confirming they are bona fide Tregs with immunosuppressive characteristics. Taken together, Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP, and FoxP3⁺Helios⁺ Tregs have more suppressive characteristics, compared with FoxP3⁺Helios^- Tregs. Our work implies that therapeutic modalities for treating autoimmune and inflammatory diseases, allergies and graft rejection should be designed to induce and/or expand FoxP3⁺Helios⁺ Tregs, while therapies against cancers or infectious diseases should avoid such expansion/induction.