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

Abstract

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.

Keywords: FoxP3; GARP/LAP; Helios; Immune response; Immunity; Immunology and Microbiology Section; regulatory T cells.

Figure 1. Expression of GARP and LAP on different FoxP3^+/−Helios^+/− non-activated T-cell subsets

Thawed PBMCs isolated from healthy donors were stained for CD3, CD4, GARP and LAP surface markers followed by FoxP3 and Helios intracellular staining. A. Representative flow cytometric plots showing FoxP3 expression against LAP or GARP, as gated on CD3⁺CD4⁺ T cells. B. Representative flow cytometric plots showing FoxP3⁻Helios⁻, FoxP3⁻Helios⁺, FoxP3⁺Helios⁺ and FoxP3⁺Helios⁻ T-cell subsets and the expression of GARP/LAP within these subsets in non-activated cells. C. Scatter plots show the mean percentage ± SEM of GARP⁺LAP⁺ within FoxP3⁻Helios⁻, FoxP3⁻Helios⁺, FoxP3⁺Helios⁺ and FoxP3⁺Helios⁻ T-cell subsets in non-activated PBMCs isolated from 14 healthy donors.

Figure 2. Expression of GARP and LAP on different FoxP3^+/−Helios^+/− T-cell subsets in the activated setting

PBMCs from healthy donors were activated by plate-bound anti-CD3/28 followed by surface staining for CD3, CD4, GARP and LAP and intracellular staining for FoxP3 and Helios. A. Representative flow cytometric plots showing FoxP3⁻Helios⁻, FoxP3⁻Helios⁺, FoxP3⁺Helios⁺ and FoxP3⁺Helios⁻ T-cell subsets and the expression of GARP/LAP within these subsets in activated PBMCs. B. Representative overlaid histogram plots show the MFIs of GARP and LAP within the different FoxP3/Helios subsets. C. Scatter plots show the mean percentage ± SEM of GARP⁺LAP⁺ within FoxP3⁻Helios⁻, FoxP3⁻Helios⁺, FoxP3⁺Helios⁺ and FoxP3⁺Helios⁻ T-cell subsets in activated PBMCs isolated from 19 healthy donors.

Figure 3. Intracellular cytokine secretion from different GARP^+/−LAP^+/− CD4⁺ T-cell subsets

A. Representative flow cytometric plots showing GARP^+/−LAP^+/− T-cell subsets and cytokine release (IFNγ and IL-10) from these subsets following PBMCs activation. B. Mean percentage ± SEM of IL-10-secreting cells within GARP^+/−LAP^+/− CD4⁺ T-cell subsets in activated PBMCs isolated from 10 healthy donors. C. Flow cytometric plots showing that some CD4⁺ T cells express higher levels of GARP/LAP, and all GARP^high/LAP^high cells secreted IL-10, compared to GARP^inter/LAP^inter. The overlaid histogram plot shows that MFI of IL-10 expression is higher within GARP^high/LAP^high cells (red) than GARP^inter/LAP^inter cells (blue). D. Representative flow cytometric plots show the secretion of IL-10 and IFN-γ from CD4⁺ T cells; IL-10-secreting CD4⁺ T cells express GARP/LAP, while IFN-γ-secreting CD4⁺T cells lack the expression of GARP/LAP.

Figure 4. Intracellular cytokine secretion from different FoxP3^+/−Helios^+/− T-cell subsets

Representative flow cytometric plots showing FoxP3^+/−Helios^+/− T-cell subsets and intracellular cytokine secretion of IFNγ and IL-10 (A) and IL-2 (B) from these different subsets following PBMCs activation. Mean percentage ± SEM of IFNγ (C. n = 9), IL-2- (D. n = 6) and IL-10-secreting cells (E. n = 8) within the different FoxP3^+/−Helios^+/− CD4⁺T-cell subsets.

Figure 5. CFSE-based proliferation assays and CD25 expression within different FoxP3^+/−Helios^+/− T-cell subsets

A. Representative flow cytometric plots showing FoxP3⁻Helios⁻, FoxP3⁻Helios⁺, FoxP3⁺Helios⁺ and FoxP3⁺Helios⁻ T-cell subsets (first plot) and their proliferation (second plot) as measured by CFSE loss. B. Mean percentage ± SEM of cell proliferation of these different subsets in PBMCs isolated from 9 healthy donors. Flow cytometric plots and mean percentage ± SEM of CD25 expression in non-activated (C) and activated (D) FoxP3^+/−Helios^+/− T-cell subsets in PBMCs isolated from 5 healthy donors.