Soluble glucocorticoid-induced tumor necrosis factor receptor regulates Helios expression in myasthenia gravis

Abstract

Background: Helios is important for functional and phenotype stability of regulatory T cells (Tregs). However, the role of Helios in autoimmune diseases and its regulation remains unclear. This study aimed to investigate the role of Helios⁺ Tregs in myasthenia gravis (MG) and glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) in the modulation of Helios.

Method: Multicolor flow cytometry was performed to analyze Helios⁺ Tregs in peripheral blood from MG patients and healthy donors (HDs). Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of soluble GITRL/GITR in plasma. Tregs were isolated via magnetic separation and treated with recombinant GITRL and GITR-Fc. Membrane GITRL on Tregs and expression of Helios and other markers (FOXP3, CD25, CD39, CTLA-4, PD-L1 and IL-10) involved in immunosuppressive activity were determined by flow cytometry.

Result: Both Helios⁺ Tregs and soluble GITR were decreased in generalized MG (GMG) patients (n = 14), compared with HDs (n = 14) and ocular MG (OMG) patients (n = 16). Helios⁺ Tregs possessed greater immunosuppressive capacity compared to Helios^- Tregs. Further analysis indicates soluble GITR was negatively correlated with quantitative MG score and promoted Helios expression and enhanced function of Tregs independently of membrane GITRL.

Conclusion: This work demonstrates abnormal changes in Helios⁺ Tregs and soluble GITR in MG, as well as direct regulation of Helios by GITR in the context of Tregs. This work provides new insight into the role of GITR in the regulatory pathway of Helios and pathogenesis of MG.

Keywords: GITR; GITRL; Helios; Myasthenia gravis; Regulatory T cells.

Fig. 1

Frequencies of Helios⁺ Tregs in MG patients compared to HDs. a The FMO and isotype controls are shown. b Analysis of CD25, FOXP3, and Helios expression among CD4⁺ T cells in representative peripheral blood samples from HDs and patients with MG. c Frequencies of Helios⁺ Tregs among CD4⁺ T cells in HDs (n = 14), and patients with OMG (n = 14), and GMG (n = 16) (median/interquartile range values). d Correlation between frequencies of Helios⁺ Tregs among CD4⁺ T cells in MG patients with their QMG scores. e, f Frequency of Tregs in GMG patients decreased compared to HDs (p = 0.0059) and there was no different of CD4⁺ T frequency in each groups (p = 0.1435) (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001)

Fig. 2

Change of QMG scores and frequency of Helios⁺ Tregs after glucocorticoid therapy in MG patients (n = 10). Although the QMG scores significantly decreased after therapy (p = 0.0039), frequency of Helios⁺ Tregs showed no difference (p = 0.3125). (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001)

Fig. 3

Analysis of suppressive function-related molecules in Helios⁺ Tregs and Helios⁻ Tregs. a Histogram showing the representative comparison of FOXP3, CD39, CTLA-4, PD-L1, CD25 and IL-10 expression in Helios⁺ Tregs and Helios⁻ Tregs. b Helios⁺ Tregs expressed higher levels of FOXP3 and CD39 (p = 0.033, p = 0.0415, respectively) in MG patients and HDs. (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001)

Fig. 4

Level of soluble GITR and soluble GITRL in peripheral plasma. Soluble GITR in GMG patients was significantly lower than in HDs (p = 0.0377) and OMG patients (p = 0.0002). For soluble GITRL, there was no significant difference among HDs, OMG patients, and GMG patients (p = 0.8250). There was a positive correlation between soluble GITR and frequency of Helios⁺ regulatory T cells (r = 0.4001, p = 0.0285). (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001)

Fig. 5

GITR and GITRL directly promoted Helios expression and function in Tregs in vitro. a, b Recombinant GITRL had a modest effect on promoting Helios expression, although there is no statistical significance. c, d GITR-Fc promoted Helios expression in Tregs (ANOVA, p < 0.0001) in a dose-dependent manner (post linear trend test, p < 0.0001). e Purity of Tregs after isolation by magnetic separation. f Expression of FOXP3 and CTLA-4 increased (p = 0.0219, p = 0.002, respectively) after treatment with GITR-Fc (10 µg/mL). (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001)

Fig. 6

sGITR regulated Helios-independent membrane GITRL in Tregs. a Tregs were gated from CD4⁺ cells. b FMO and isotype controls and gating strategy are shown. c Analysis of GITRL expression shows that GITRL was not expressed on the surface of Tregs, even when activated by pre-coating with anti-CD3 mAbs in both HDs (n = 4) and MG patients (n = 3)