Background/Purpose: FoxP3 is not a reliable marker for distinguishing regulatory T (Treg) cells in humans due to the fact that FoxP3 may be up-regulated in activated, conventional T (Tconv) cells. Recently, the marker CD45RA has been added to distinguish RA⁺, “resting” FoxP3⁺ Tregs from RA^– “activated” FoxP3^hi Tregs (Miyara et al, 2009). This scheme has also included a third “non-Treg” group that is RA^– and expresses lower levels of FoxP3 similar to that seen in RA⁺ Tregs. Previous data demonstrated that in normal individuals as well as in patients with systemic lupus erythematosus (SLE), the largest subset of FoxP3⁺ cells is, in fact, the “non-Treg” group. The transcription factor Helios has recently been defined as a marker of thymus-derived Tregs in both mouse and man (J. Immunol. 2010). The goal of this study was to determine if expression of Helios could reliably indicate what fraction of FoxP3⁺cells are true Tregs in healthy controls and in SLE patients.

Methods: Samples included 35 healthy donors and 52 SLE patients (23 SLEDAI 0; 19 SLEDAI 2-4; 10 SLEDAI 6-20). Ficoll-purified PBMCs were surface stained followed by fixation/pemeabilization and intracellular staining prior to FACS analysis. When appropriate, cells were pre-stimulated for 4-5 hours with PMA/ionomycin/golgistop. CpG methylation analysis of sorted cells was performed using the Qiagen EpiTect platform.

Results: We found that CD4⁺ T cells in SLE patients contain both resting (RA⁺) and activated (RA^–/FoxP3^hi) Tregs and that the majority of cells in each group were Helios⁺, (3/4 and 4/5, respectively). Surprisingly, even within the sub-group defined as “non-Tregs”, the majority of the cells in both healthy controls (2/3) and SLE patients (3/4) were Helios⁺. All FoxP3⁺ Helios⁺ cells, including those within the “non-Treg” RA^– subset, were found to be demethylated at the FoxP3 locus (a gold standard epigenetic mark of the Treg lineage), as compared to Helios^– cells. In pre-stimulated cells, FoxP3⁺ Helios^– cells consistently produced significantly high amounts of cytokines (IFN-gamma and IL-2), whereas FoxP3⁺Helios⁺ cells produced essentially no cytokines, which is characteristic of Tregs. In SLE patients with mild and highly active disease, there was a significant increase in both the % Foxp3⁺ Helios⁺ and % FoxP3⁺Helios^– relative to both healthy controls and inactive patients (p<0.05, Mann-Whitney test). There was not a significant difference for absolute numbers of either Helios⁺ or Helios^– cells, likely due to the significant reduction in total CD4 counts in more active patients (p<0.05, Mann-Whitney test).

Conclusion: Expression of Helios is a highly useful tool for distinguishing true Tregs from FoxP3⁺ cells that include activated Tconvs. The use of Helios has allowed us to de-convolute the largest subset of CD45RA-based grouping of FoxP3 cells. Furthermore, we have also shown that active SLE patients do have a higher frequency of FoxP3⁺Helios⁺ Tregs, but that in active SLE patients these are counter-balanced with a higher frequency of Foxp3⁺Helios^– cells that contain cytokine-producing Tconvs. Future studies may make use of Helios to reliably monitor both true Tregs and activated Tconvs in SLE and other autoimmune diseases.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/expression-of-helios-facilitates-distinction-between-foxp3-treg-and-foxp3-activated-t-conventional-cells-in-patients-with-systemic-lupus-erythematosus/