Immune Skew of Circulating Follicular Helper T Cells Associates With Myasthenia Gravis Severity

Abstract

Objective: To clarify functional alterations of follicular helper T cells (Tfh) in myasthenia gravis (MG) because Tfh play important roles in helping B cells generate antibody-producing cells.

Methods: A total of 24 immunotherapy-naive patients with anti-acetylcholine receptor (AchR) antibody-positive MG and 18 age-matched healthy subjects (HS) were enrolled. Samples from 6 patients were available for posttreatment analysis. Subsets of circulating Tfh (cTfh) and B cells were identified by flow cytometry analysis of surface molecules. Cytokine production by isolated cTfh subsets from 5 patients with MG and 5 HS was measured in vitro. Analysis was performed to examine the correlation between the frequency of cTfh subsets and that of plasmablasts and between cTfh subsets and the quantitative MG score.

Results: cTfh increased with elevated expression of inducible T-cell costimulator (ICOS) in patients with MG. cTfh shifted to Th2 and Th17 over Th1 in MG. ICOS^highcTfh produced significantly higher levels of interleukin (IL)-21, IL-4, and IL-17A than ICOS^low cTfh only in patients with MG. The frequency of cTfh within CD4 T cells was more closely associated with disease severity than the serum anti-AchR antibody titer and frequency of plasmablasts within B cells. Abnormalities of cTfh were improved after immunotherapy in parallel with clinical improvement.

Conclusions: Alternation of cTfh is a key feature in the development of MG and may become a biomarker for disease severity and therapeutic efficacy.

Classification of evidence: This study provides Class II evidence that the level of cTfh is associated with disease severity in patients with MG.

Figure 1. cTfh Frequency and ICOS Expression on cTfh and cTfh Subsets

(A) Representative flow cytometry dot plots of CXCR5 expression on CD4 T cells from patients with MG and HS (left) and representative flow cytometry dot plots showing expression of CXCR5 and ICOS (right). (B) Frequency of cTfh within CD4 T cells shown for comparison between patients with MG and HS. Frequency of ICOS^high cTfh within CD4 T cells (C) and within cTfh (D) for comparison between patients with MG and HS. Frequency of cTfh1 (CXCR3+ CCR6- cTfh), cTfh2 (CXCR3-CCR6-cTfh), and cTh17 (CXCR3-CCR6+ cTfh) within CD4 T cells (E) and cTfh1, cTfh2, and cTfh17 subsets within cTfh (F) was also analyzed. Data are indicated as the mean ± SEM. cTfh = circulating follicular helper T cell; HS = healthy subjects; ICOS = inducible T-cell costimulator; MG = myasthenia gravis; PBMC = peripheral blood mononuclear cell.

Figure 2. Cytokine Production by cTfh and Correlation With ICOS Expression on cTfh

cTfh from 5 patients with MG and the same number of HS were cultured under PMA and ionomycin stimulation for 48 hours. CXCR5+CD4 T cells as cTfh, ICOS^high cTfh, and ICOS^high cTfh were sorted as shown in (A). Levels of IFN-γ, IL-4, IL-17A, and IL-21 production by cTfh were measured and compared between patients with MG and HS (B). The heatmap shows cytokine production by ICOS^high and ICOS^low cTfh from patients with MG and HS (C). Each column represents the mean ± SEM. CCR6 = C-C receptor 6; cTfh = circulating follicular helper T cell; CXCR5 = C-X-C motif receptor 5; HS = healthy subjects; ICOS = inducible T-cell costimulator; IFN-γ = interferon-γ; IL = interleukin; MG = myasthenia gravis; PMA = phorbol 12-myristate 13-acetate.

Figure 3. Association of Disease Severity With Phenotype of cTfh and Tfh Phenotypic Change Before and After Treatment

The QMG score was correlated with the frequency of the following subsets within CD4 T cells: (A) cTfh, (B) ICOS^high cTfh, (C) ICOS^low cTfh. In (A), the circle indicates EOMG, and the triangle indicates LOMG. The QMG score and frequency of cTfh subsets within either CD4 T cells or cTfh were analyzed before and after immunotherapy. (D) QMG score. (E) Frequency of cTfh and (F) ICOS^highcTfh within CD4 T cells. cTfh = circulating follicular helper T cell; ICOS = inducible T-cell costimulator; MG = myasthenia gravis; post = after immunotherapy; pre = before immunotherapy; QMG score = quantitative MG score.