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. 2020 Aug 15:345:577279.
doi: 10.1016/j.jneuroim.2020.577279. Epub 2020 May 27.

Imbalance in T follicular helper cells producing IL-17 promotes pro-inflammatory responses in MuSK antibody positive myasthenia gravis

Yingkai Li  1 , Jeffrey T Guptill  2 , Melissa A Russo  2 , James F Howard Jr  3 , Janice M Massey  2 , Vern C Juel  2 , Lisa D Hobson-Webb  2 , Doug Emmett  2 , Manisha Chopra  3 , Shruti Raja  2 , Weibin Liu  4 , John S Yi  5
Affiliations

Imbalance in T follicular helper cells producing IL-17 promotes pro-inflammatory responses in MuSK antibody positive myasthenia gravis

Yingkai Li et al. J Neuroimmunol. .

Abstract

A detailed understanding of the role of Tfh cells in MuSK-antibody positive myasthenia gravis (MuSK-MG) is lacking. We characterized phenotype and function of Tfh cells in MuSK-MG patients and controls. We found similar overall Tfh and follicular regulatory (Tfr) T cell frequencies in MuSK-MG and healthy controls, but MuSK-MG patients exhibited higher frequencies of Tfh17 cells and a higher ratio of Tfh:Tfr cells. These results suggest imbalanced Tfh cell regulation, further supported by increased frequencies of CD4 T cells co-producing IL-21/IL-17 and IL-17/IFN-γ, and increased Tfh-supported IgG production. These results support a role for Tfh cell dysregulation in MuSK-MG immunopathology.

Keywords: Autoimmunity; MuSK antibody; Myasthenia gravis; T follicular helper cells; Tfh17 cells.

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Figures

Figure 1
Figure 1
Comparable frequencies of circulating Tfh cells in healthy controls and MuSK-MG. (1A) Tfh cells were identified by gating CXCR5 on CD4 T cells. Percentages of Tfh cells in CD4 T cells in lymphocytes in MG patients were not different than healthy controls. The overall frequencies of Tfh cells in MuSK-MG patients according to MGFA classification were similar. Frequencies of PD-1+/ICOS+ Tfh cells (1B), CD38+/ICOS+ Tfh cells (1C), Ki-67+/ICOS+ Tfh cells (1D) and Ki-67+/CD38+ Tfh cells (1E) were not different in MuSK-MG patients compared with controls.
Figure 2
Figure 2
Tfh subsets in MuSK-MG patients. (2A) Gating strategy of Tfh subsets in CD4 T cells using CXCR3 and CCR6 in MuSK-MG and healthy controls. Tfh1 was defined as CXCR3+CCR6-. Tfh2 was defined as CXCR3-CCR6-. Tfh17 was defined as CXCR3-CCR6+. (2B, 2C) Tfh1 and Tfh2 frequencies were not different between MuSK-MG and healthy controls. (2D) Tfh17 frequencies were significantly increased in MuSK-MG patients. (2E) The ratio of Tfh17/Tfh1 subsets is increased in MuSK-MG patients compared with healthy controls. *, significant difference, p<0.05; **, significant difference, p<0.01.
Figure 3
Figure 3
Comparison of Tfr subsets between MuSK-MG and healthy control groups. (3A) Gating strategy Tfr cells in controls and MuSK-MG patients. (3B) Overall Tfr frequencies and (3C) Tfr frequencies among MuSK-MG patients with different MGFA classifications were not significantly different compared to controls. (3D) The overall Tfh:Tfr ratio is increased in MuSK-MG patients compared to controls. (3E) The Tfh17/Tfr ratio is also increased in MuSK-MG patients. **, significant difference, p<0.01; ***, significant difference, p<0.001.
Figure 4
Figure 4
CD4 T cell function assessed by intracellular cytokine production in MuSK-MG and healthy controls. (4A) IL-21, (4B) IFN-γ and (4C) IL-17 cytokine production is increased in CD4 T cells in MuSK-MG compared with healthy controls. (4D) Demonstration of IL-17+/IFN-γ+ gating strategy among CD4 T cells (left). IL-17+/IFN-γ+ CD4+ pathogenic Th17 cells are increased in MuSK-MG compared with controls (right). (4E) IL-17+/IL-21+ gating strategy in CD4 T cells between controls and patients with MuSK-MG (left). IL-17+/IL-21+ CD4 T cells are significantly increased in MuSK-MG patients (right). *, significant difference, p<0.05.
Figure 5
Figure 5
Tfh cells promote B cell IgG production in vitro. Sorted peripheral CD19+CD127-IgD- naïve B cells (2*105/well) were co-cultured with Tfh cells (2*105/well) from MuSK-MG patients (N=4) or healthy controls (N=4) in the presence of CD3/CD28. Naïve B cells only served as a control condition. Production of IgG Abs in cultured supernatants was detected by ELISA. In the B cell only condition IgG production was not different between MuSK-MG patients or healthy controls. As expected, adding Tfh cells to naïve B cells resulted in increased IgG production in both controls and MuSK-MG patients. However, IgG production was significantly increased in MuSK-MG patients compared with controls. *, significant difference, p<0.05; **, significant difference, p<0.01.
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