Regulatory T Cell Responses to High-Dose Methylprednisolone in Active Systemic Lupus Erythematosus

Abstract

Background/purpose: A slight increase in the proportion of circulating regulatory T (Treg) cells has been reported in systemic lupus erythematosus (SLE) patients taking oral prednisone. The effects of intravenous (IV) high dose methylprednisolone (MP) on Tregs have not yet been described, especially in active SLE.

Methods: We prospectively analyzed the proportion of circulating CD4+ Treg cell subsets defined as follows: (1) naïve Treg (nTreg) FoxP3lowCD45RA+ cells; (2) effector Treg (eTreg) FoxP3highCD45RA- cells; and (3) non-suppressive FoxP3lowCD45RA- cells (non-regulatory Foxp3low T cells). Peripheral blood mononuclear cells of patients with active SLE were analyzed before the first infusion of IV high dose MP (day 0) and the following days (day 1, day 2, ±day 3 and ±day 8). The activity of SLE was assessed by the SLEDAI score.

Results: Seventeen patients were included. Following MP infusions, the median (range) percentage of eTregs significantly increased from 1.62% (0.53-8.43) at day 0 to 2.80% (0.83-14.60) at day 1 (p = 0.003 versus day 0), 4.64% (0.50-12.40) at day 2 (p = 0.06 versus day 1) and 7.50% (1.02-20.70) at day 3 (p = 0.008 versus day 2), and declined to baseline values at day 8. Expanding eTreg cells were actively proliferating, as they expressed Ki-67. The frequency of non-regulatory FoxP3low T cells decreased from 6.39% (3.20-17.70) at day 0 to 4.74% (1.03-9.72) at day 2 (p = 0.005); nTreg frequency did not change. All patients clinically improved immediately after MP pulses. The absence of flare after one year of follow up was associated with a higher frequency of eTregs at day 2.

Conclusion: IV high dose MP induces a rapid, dramatic and transient increase in circulating regulatory T cells. This increase may participate in the preventive effect of MP on subsequent flares in SLE.

Fig 1. Cytofluorometric analysis of CD4⁺FoxP3⁺ T cell subsets following IV methylprednisolone pulses in SLE patients.

Fresh PBMCs from SLE patients were analyzed by flow cytofluorometry, gated on CD4⁺ T lymphocytes, for the expression of FoxP3 and CD45RA (top rows) and Ki-67 (bottom rows). FoxP3⁺CD4⁺ T cells can be divided into CD4⁺CD45RA⁻FoxP3^bright effector Tregs (eTregs) and CD4⁺CD45RA⁺FoxP3⁺ naïve Tregs (nTregs), while the remaining CD4⁺CD45RA⁻FoxP3^low include a notable amount of non-regulatory, cytokine-secreting, activated T cells (non-regulatory FoxP3⁺ T cells) [8]. FoxP3^highKi-67⁺ (right gate), which correspond to the eTregs, and FoxP3⁻Ki-67⁺ (left gate), which correspond to non-regulatory T-cells, are shown on the lower FACS panel. Percentages of the different subsets are shown. Representative analyses from one SLE patient are shown (pt #3).

Fig 2. Frequencies of CD4⁺FoxP3⁺ T cell subsets following IV methylprednisolone pulses in SLE patients.

Fresh PBMCs from SLE patients were analyzed by flow cytofluorometry as described in Fig 1. Kinetics of (A) effector Tregs, (B) naïve Tregs and (C) non-regulatory FoxP3⁺ T cells were performed in patients with active SLE undergoing IV high dose MP pulse treatment at baseline. Seventeen patients at baseline and day 2, 8 patients at day 3 and 6 patients at day 8 were assessed. (A) Right panel, eTreg cells frequencies at day 3 are displayed (except for two patients for whom these data are missing; eTreg cells frequencies at day 2 are shown instead). Each dot represents an individual assessed in an independent experiment, and the grey bar shows median values. Statistical analyses were performed using the Wilcoxon matched pairs signed ranks test.

Fig 3. Frequencies of eTreg cells in SLE patients according to their clinical response following IV methylprednisolone pulses.

(A) Evolution of the SELENA-SLEDAI score at baseline, month 3, 6 and 12 following IV MP pulses according to the responder status of the patients defined after 12 months of follow-up. (B) Effector Treg frequencies at day 0 (baseline) and day 2 and the ratio of effector Tregs between day 2 and day 0 in poor and good responders after 12 months of follow-up. (A-B) Each dot represents an individual and lines show median values. Statistical analyses were performed using the Wilcoxon matched pairs signed ranks test (A) and the Mann-Whitney U test (B).