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.