The percentage of FoxP3+Helios+ Treg cells correlates positively with disease activity in systemic lupus erythematosus - PubMed (original) (raw)

The percentage of FoxP3+Helios+ Treg cells correlates positively with disease activity in systemic lupus erythematosus

Amit Golding et al. Arthritis Rheum. 2013 Nov.

Erratum in

Abstract

Objective: To assess the use of Helios in combination with FoxP3 as a superior method for identifying non-cytokine-producing human Treg cells in patients with systemic lupus erythematosus (SLE) and to determine if FoxP3+Helios+ Treg cells are maintained at normal levels in patients with clinically active disease.

Methods: Peripheral blood mononuclear cells (PBMCs) were purified from the blood of healthy volunteer donors and from 52 consecutive patients with SLE of varying clinical activity (Systemic Lupus Erythematosus Disease Activity Index scores of 0, 2-4, and ≥ 5). PBMCs (either fresh or after 4 hours of stimulation for cytokine production) were then analyzed by flow cytometry for the expression of cell surface markers (CD4, CD25, CD127, and CD45RA) and transcription factors (FoxP3 and Helios), as well as for the production of cytokines (interleukin-2 and interferon-γ).

Results: FoxP3+Helios+ Treg cells were found to be non-cytokine producing in both SLE patients and healthy controls. Patients with clinically active SLE had higher percentages of FoxP3+Helios+ Treg cells than did patients with inactive SLE or healthy controls. When corrected for the total CD4 cell count, the absolute numbers of FoxP3+Helios+ Treg cells in patients with moderately-to-highly active SLE were normal.

Conclusion: Previous reports of a deficiency in Treg cell number or function in SLE are limited by their use of CD25, either alone or in combination with other markers, to identify human Treg cells. Helios in combination with FoxP3 is a superior method for detecting all non-cytokine-producing Treg cells, irrespective of CD25 or CD45RA expression. Using this method, we showed that FoxP3+Helios+ Treg cell numbers are not reduced in patients with clinically active SLE.

Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

PubMed Disclaimer

Figures

Figure 1

Figure 1

The non−cytokine-producing FoxP3+Helios+ Treg cell subset of CD4+FoxP3+ cells from patients with systemic lupus erythematosus (SLE). A, Peripheral blood mononuclear cells (PBMCs) from 18 patients with SLE (9 with a Systemic Lupus Erythematosus Disease Activity Index score of 0, 4 with a score of 2, and 5 with a score of ≥4) were stimulated with 12-_O_-tetradecanoylphorbol-13-acetate (PMA)/ionomycin/GolgiStop to induce cytokine production and then stained for CD4, intracellular Helios, intercellular FoxP3, and either interleukin-2 (IL-2) or interferon-γ (IFNγ). * = P = 0.015; ** = P = 0.00001 by Student’s unpaired _t_-test. B, PBMCs from 6 healthy donors (HD) were also stimulated and stained for intracellular cytokines as for those from the SLE patients. * = P = 0.05; ** = P = 0.001 by Student’s unpaired _t_-test. Values are the mean ± SD.

Figure 2

Figure 2

CD25, a poor marker of Treg cells in systemic lupus erythematosus (SLE). Representative histograms and fluorescence-activated cell sorter (FACS) plots for cells from 12 healthy donors (HD) and 19 SLE patients are shown. A, CD4+ T cells gated on FoxP3+Helios+ (Treg cells) or Helios−FoxP3− (conventional T cells), showing relative CD25 expression. Vertical lines indicate CD25 expression levels that overlap between FoxP3+Helios+ and FoxP3−Helios− cells in SLE patients but not healthy donors. B, FACS analysis of total peripheral blood mononuclear cells, showing gating strategy for the CD4+CD25high subset based on the CD25 histograms shown in A. C, Helios and FoxP3 expression in the gated CD4+CD25high subset shown in B. D, Ratio of the CD25 mean fluorescence intensity (MFI) in FoxP3+ versus FoxP3− CD4+ T cells in 8 patients with inactive SLE (Systemic Lupus Erythematosus Disease Activity Index [SLEDAI] score of 0), 11 patients with active SLE (SLEDAI score of >0), and 12 healthy donors. Values are the mean ± SD. * = P > 0.05; ** = P < 0.05 by Student’s unpaired _t_-test.

Figure 3

Figure 3

Presence of non-cytokine-producing FoxP3+Helios+ cells irrespective of the expression of CD45RA or the level of FoxP3 in healthy donors and patients with systemic lupus erythematosus (SLE). All panels are gated on CD4+ cells, and subtypes of FoxP3+ cells are based on the previously published system (18). A, Cell samples from 20 SLE patients and 17 healthy donors were stained for CD45RA in addition to CD4, Helios, and FoxP3, and the Treg cell subsets were determined as a percentage of the total CD4+FoxP3+ cells. rTreg = resting Treg cells; aTreg = activated Treg cells. B, Percentage of Helios expression in Treg cell subsets was determined in the samples shown in A. Patients in A and B were categorized according to Systemic Lupus Erythematosus Disease Activity Index [SLEDAI] scores. Values in A and B are the mean ± SD. C, Peripheral blood mononuclear cells from an SLE patient were directly stimulated ex vivo with 12-_O_-tetradecanoylphorbol-13-acetate (PMA)/ionomycin/GolgiStop, as described in Patients and Methods, prior to surface and intracellular staining. Costaining for the cytokines interferon-γ (IFNγ) and interleukin-2 (IL-2) was performed. Results are representative of cells from 6 different patients. Numbers in each compartment are the percentages of positive cells. Cell fractions I, II, and III are indicated. D, IFNγ production in 6 SLE samples was determined as described in C. Values are the mean ± SD. P values were determined by Student’s unpaired _t_-test.

Figure 4

Figure 4

Hypomethylation of the FoxP3 locus Treg-specific demeth-ylation region (TSDR) in Helios+ versus Helios− cells, even within the previously described (18) “non–Treg cell” FoxP3lowCD45RA− cell subtype. A, Nine sequentially CpG methylated or unmethylated sites within the FoxP3 locus TSDR, as described by Floess et al (19) (ideal results of methylation sequencing), are shown. Site 6 was unreliable in our experiments and was therefore excluded. Due to X-inactivation in females, at most only 50% of the potential TSDR CpG sites in sorted Treg cells are demethylated, as compared with 100% demethylation of TSDR CpG sites in sorted Treg cells from males, where both X chromosome FoxP3 genes are expressed. B, Sorted CD4+CD45RA− FoxP3+ mature Treg cells from a healthy male donor show almost complete demethylation of CpG sites. C, Sorted CD45RA− subsets from a female patient with systemic lupus erythematosus (SLE) show FoxP3 locus TSDR methylation. Maximum (50%) TSDR demethylation is observed in sorted FoxP3highCD45RA− cells as well as sorted FoxP3lowCD45RA–Helios+ cells as compared to sorted FoxP3– CD45RA– cells and FoxP3lowCD45RA−Helios− cells.

Figure 5

Figure 5

Lack of association between increasing clinical activity of systemic lupus erythematosus (SLE) and a drop in the absolute numbers of FoxP3+Helios+ Treg cells, but positive correlation with an increasing percentage of FoxP3+Helios+ Treg cells among total CD4+ T cells. A and B, Percentages of FoxP3+Helios+ cells (A) and FoxP3+Helios− cells (B) in healthy donors (HD) and in SLE patients with different levels of clinical disease activity. C, Numbers of CD4+ T cells in SLE patients with different levels of clinical disease activity. D, Absolute numbers of FoxP3+Helios+ Treg cells in SLE patients with different levels of clinical disease activity. Each data point represents a single subject; bars show the mean ± SD. P values were determined by Student’s unpaired _t_-test.

Figure 6

Figure 6

Lack of direct correlation between prednisone use and FoxP3+Helios + Treg cells in patients with systemic lupus erythematosus (SLE). A, Spearman’s rank correlation between the mean daily prednisone dose and clinical disease activity, as determined by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores. B, Spearman’s rank correlation between the percentage of FoxP3+Helios+ Treg cells and the daily prednisone dosage in SLE patients of all clinical disease activity groups.

Comment in

References

    1. Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol. 2010;10:490–500. - PubMed
    1. Kinnunen T, Chamberlain N, Morbach H, Choi J, Kim S, Craft J, et al. Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells. Blood. 2013;121:1595–1603. - PMC - PubMed
    1. Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet. 2001;27:20–21. - PubMed
    1. Michels-van Amelsfort JM, Walter GJ, Taams LS. CD4+CD25+ regulatory T cells in systemic sclerosis and other rheumatic diseases. Expert Rev Clin Immunol. 2011;7:499–514. - PubMed
    1. Scheinecker C, Bonelli M, Smolen JS. Pathogenetic aspects of systemic lupus erythematosus with an emphasis on regulatory T cells. J Autoimmun. 2010;35:269–275. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources