Phenotypic and functional dysregulations of CD8 + T Cells in myasthenia gravis

Abstract

Myasthenia Gravis (MG) is a heterogeneous autoimmune disorder characterized by fluctuating muscle weakness caused by autoantibodies targeting neuromuscular junction components. While the role of CD4 + T cells in MG is well established, the contribution of CD8 + T cells remains poorly understood. In this study, we analyze CD8 + T cells in 36 MG patients and 38 age- and gender-matched controls using flow cytometry to evaluate subset distribution, granzyme expression, and cytokine production. MG patients exhibit an altered CD4 + /CD8 + T cell ratio and significant changes in CD8 + T cell subsets, including increased central memory CD8 + T cell (Tcm) proportions and decreased effector memory CD8 + T cell (Tem) proportions. Granzyme B expression in Tcm cells is significantly elevated in MG patients, whereas no significant changes are observed in other subsets or GZMK expression. Cytokine analysis reveals increased IL-21, GM-CSF, and IL-17A production by CD8 + T cells in MG patients. These phenotypic and functional alterations of CD8 + T cells persist during the acute phase of the disease, regardless of immunotherapy usage, and vary between ocular and generalized MG. Subgroup and correlation analyses further identify age-dependent and age-independent dysregulations of CD8 + T cells, indicating complex and subtype-specific roles of CD8 + T cells in the immunopathological processes underlying MG. Our findings provide novel insights into the involvement of CD8 + T cells in MG pathogenesis, laying a foundation for future research and potential therapeutic strategies targeting CD8 + T cells.

Keywords: CD8 + T cells; GM-CSF; Granzyme B; IL-21; Myasthenia gravis; Pathogenesis.

Fig. 1

Altered frequencies and subset distribution of CD8 + T cells in MG patients. (A) Representative flow cytometry plots showing the gating strategy for identifying CD4 + and CD8 + T cells among total T cells in MG and control group. (B) Quantification of CD4 + and CD8 + T cell frequencies among total T cells and the CD4/CD8 ratio in MG patients (n = 36) and controls (n = 38). (C) Representative flow cytometry plots demonstrating the classification of CD8 + T cell subsets based on CCR7 and CD45RA expression in MG and control group. (D) Quantification of naïve, central memory (Tcm), effector memory (Tem), and terminally differentiated effector memory (Temra) CD8 + T cell subsets in MG patients and controls. Data are presented as means ± SEM. Statistical significance was determined using Student’s t-test for normally distributed data and Mann–Whitney test for not normally distributed data (ns, not significant; *P < 0.05, **P < 0.01)

Fig. 2

Expression of GZMB in CD8 + T cells from MG patients and controls. (A) Representative flow cytometry plots showing GZMB expression in CD8 + T cells and their subsets from controls and MG patients. (B) Quantification of GZMB expression in total CD8 + T cells and across Tcm, Tem, and Temra subsets from MG patients (n = 36) and controls (n = 38). (C) Proportions of GZMB + Tcm, Tem, and Temra cells within total CD8 + T cells in MG patients and controls. Data are presented as means ± SEM. Statistical significance was determined using Student’s t-test for normally distributed data and Mann–Whitney test for not normally distributed data (ns, not significant; ***P < 0.001, ****P < 0.0001)

Fig. 3

Increased production of IL-21, GM-CSF, and IL-17A by CD8 + T cells from MG patients. (A) Representative flow cytometry plots showing the expression of IFN-γ and IL-21 in CD8 + T cells from controls and MG patients. (B) Quantification of IFN-γ, IL-21, and dual IFN-γ/IL-21-producing CD8 + T cells in MG patients (n = 36) and controls (n = 38). (C) Representative flow cytometry plots showing the expression of GM-CSF and IL-17A in CD8 + T cells from controls and MG patients. (D) Quantification of GM-CSF, IL-17A, and dual GM-CSF/IL-17A-producing CD8 + T cells in MG patients and controls. Data are presented as means ± SEM. Statistical significance was determined using Student’s t-test for normally distributed data and Mann–Whitney test for not normally distributed data (ns, not significant; *P < 0.05, **P < 0.01, ****P < 0.0001)

Fig. 4

Subgroup analysis of CD8 + T cells based on immunotherapy status in MG patients. (A) Quantification of the proportion of CD8 + T cells, the CD4/CD8 ratio, and the distribution of CD8 + T cell subsets (naïve T, Tcm, Tem, and Temra) in immunotherapy-naïve (n = 19) and immunotherapy-treated (n = 17) MG patients. (B) Comparison of GZMB expression within CD8 + T cell subsets between immunotherapy-naïve and immunotherapy-treated MG patients. (C) Comparison of GZMK expression within CD8 + T cell subsets between the two patient groups. (D) Quantification of IFN-γ and IL-21 production by CD8 + T cells in immunotherapy-naïve and immunotherapy-treated MG patients. (E) Quantification of GM-CSF and IL-17A production by CD8 + T cells in the two patient groups. Data are presented as means ± SEM. Statistical significance was determined using Student’s t-test for normally distributed data and Mann–Whitney test for not normally distributed data (ns, not significant)

Fig. 5

Comparison of CD8 + T cells between OMG and GMG patients. (A) Quantification of the proportion of CD8 + T cells, the CD4/CD8 ratio, and the distribution of CD8 + T cell subsets (naïve T, Tcm, Tem, and Temra) in OMG (n = 13) and GMG (n = 23) patients. (B) Comparison of GZMB expression within CD8 + T cell subsets between OMG and GMG patients. (C) Comparison of GZMK expression within CD8 + T cell subsets in the two patient groups. (D) Quantification of IFN-γ and IL-21 production by CD8 + T cells in OMG and GMG patients. (E) Quantification of GM-CSF and IL-17A production by CD8 + T cells in OMG and GMG patients. Data are presented as means ± SEM. Statistical significance was determined using Student’s t-test for normally distributed data and Mann–Whitney test for not normally distributed data (ns, not significant; *P < 0.05, **P < 0.01)

Fig. 6

Different phenotypic and functional characteristics of CD8 + T Cells in EOMG and LOMG. (A) Quantification of CD8 + T cell subsets (naïve T, Tcm, Tem, and Temra) in EOMG (n = 9) and LOMG (n = 27) patients compared to their respective age-matched controls (n = 9 for controls under 50 years and n = 29 for controls over 50 years). (B) Comparison of GZMB expression in Tcm, Tem, and Temra subsets between MG patients and age-matched controls. (C) Comparison of GZMK expression across CD8 + T cell subsets from MG patients and age-matched controls. (D) Quantification of IFN-γ and IL-21 production by CD8 + T cells from MG patients and age-matched controls. (E) Quantification of GM-CSF and IL-17A production by CD8 + T cells from MG patients and age-matched controls. (F) Correlation analysis between age and the proportion of naïve T cells as well as Tcm cells in MG patients and controls. (G) Correlation analysis between age and cytokine production (IFN-γ and IL-21) by CD8 + T cells in MG patients and controls. Data are presented as means ± SEM. Statistical significance in multiple comparsions (A-E) was determined by Šidák test for equal variances and Dunnett’s T3 test for unequal variances, or by Dunn’s test for not normally distributed data. (ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001). Correlation analysis (F-G) was performed using Spearman’s rank correlation coefficient