Fig 2: IL-6 and IL-21 induced the exhaustion of NK cells in vitro. (A–C) Representative flow histograms and summary dot plots showing the ΔMFI of NKG2D and PD1 on total NK cells, CT NK cells between control, IL-6, IL-21, and IL-6/IL-21 group. (D–F) Summary dot plots showing the frequency of perforin, granzyme B, CD107a, IFN-γ, and IL-10 production by NK-cell subsets between the four groups. (G) Quantification of the percentage of the killing of K562 cells by NK cells between the four groups. (H, I) Representative flow cytometry plots and summary dot plots representing the percentage of viable (Annexin V−/7-ADD−), early apoptotic (Annexin V+/7-ADD−), and late apoptotic (Annexin V+/7-ADD+)/necrotic (Annexin V-/7-ADD+) NK cells. ΔMFI value was calculated by subtracting the MFI value of the FMO control. MFI, median fluorescence intensity; CT NK, cytotoxic CD56dimCD16+ NK; FMO, fluorescence minus one control. Experiments were measured for eight blood NK-cell samples. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: IL-6/IL-21 regulates SOCS2/STAT5 signaling in NK cells. (A) Volcano plot showing the DEGs between MGFA I (n = 3) and MGFA II-IV (n = 4) group. (B–D) GO enrichment analysis of the DEGs in cellular components, biological process, and molecular function. (E) KEGG enrichment pathway analysis of the DEGs. (F) Protein-protein interaction network for DEGs. (G) The mRNA expression of S100A8/9, SOCS2, JCHAIN, FHL3, and FCMR in NK cells between control, IL-6, IL-21, and IL-6/IL-21 group. (H–K) Protein bands and levels of p-STAT5, p-STAT5/STAT5, and SOCS2 between the four groups. MGFA, Myasthenia Gravis Foundation of America. RT-qPCR was measured for eight blood NK-cell samples and western blot was measured for five samples. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Monocytes and Tfh cells greatly expand in myasthenia gravis, leading to the large secretion of IL-6 and IL-21. IL-6 and IL-21 work cooperatively to mediate NK-cell exhaustion by up-regulating SOCS2 and impairing the phosphorylation of STAT5. Subsequently, NK cells exhibit exhausted signature, characterized by low cytokine production (IFN-γ), decreased cytotoxicity (perforin, granzyme B, and CD107a), downregulated activating receptor NKG2D, and upregulated inhibitory receptor PD1.

Fig 5: Changes of plasma cytokines in myasthenia gravis patients. (A–F) Levels of plasma IL-2, IL-6, IL-12 p70, IL-15, IL-18, IL-21 between HC (n = 20), MGFA I (n = 18), and MGFA II-IV (n = 35) group. (G) Correlation analysis between plasma IL-6, IL-21, and expression of NKG2D, PD1, perforin, granzyme B, CD107a, IFN-γ, and IL-10 in total NK cells for all subjects (n = 73). (H) Correlation analysis between plasma IL-6, IL-21 and expression of NKG2D, PD1, perforin, granzyme B, CD107a, IFN-γ, and IL-10 in CT NK cells and CS NK cells for all subjects. The numbers in the lower left part of the heatmap show the values of the correlation coefficient. (I–K) Representative flow cytometry plots and summary violin plots showing the frequency of IL-6 and IL-21 secretion by major immune cells for all subjects. (L, M) Summary dot plots showing the frequency of IL-6+ monocytes and IL-21+ Tfh cells between HC, MGFA I, and MGFA II-IV group. HC, healthy control; MGFA, Myasthenia Gravis Foundation of America; CT NK, cytotoxic CD56dimCD16+ NK; CS NK, cytokine-secreting CD56brightCD16− NK. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.