Session Type: Abstract Submissions (ACR)
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” FoxP3hi 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–/FoxP3hi) 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.
S. A. Hasni,
G. G. Illei,
E. M. Shevach,
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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/