Table 1.
Clinical characteristics of participants.
Fig 1.
Comparison of frequency and phenotypes of CD4+FOXP3+ Tregs among the HC, TS (–) and TS (+) groups.
(A) Both the TS (–) and TS (+) groups had lower frequencies of CD4+ T cells than the HC group as shown by the dotted boxes. The frequencies of CD4+FOXP3+ Tregs (among total lymphocytes) did not significantly differ among the three groups as shown by the lined boxes. Numbers within the plots indicate the mean (standard deviations) percentage of each subset. (B) The TS (–) and TS (+) groups exhibited higher percentages of FOXP3+ among their CD4+ T cells than did the HC group. (C) There were no significant differences among the HC, TS (–) and TS (+) groups in the expression of CTLA-4 and GITR in CD4+FOXP3+ Tregs. The percentages of CD4+FOXP3+ Tregs expressing CXCR3, or both CCR4 and CCR6 were also comparable among the three groups. Grey bars and error bars indicate means and standard deviations, respectively. Values from the three groups were compared by ANOVA, and the between-group values were compared via Bonferroni post-hoc analysis. Abbreviations: HC, healthy control subjects; TS (−), Turner syndrome patients without thyroid autoimmunity; TS (+), Turner syndrome patients with thyroid autoimmunity; CTLA4, cytotoxic T lymphocyte antigen-4; MFI, mean fluorescence intensity; GITR, glucocorticoid-induced tumor necrosis factor receptor family related.
Fig 2.
Comparison of the frequency of CD4+FOXP3+ Treg subsets according to CD45RA expression between the TS and HC groups.
(A) PBMCs were stained with Abs against CD4, CD45RA, and FOXP3, and then analyzed by flow cytometry. CD4+FOXP3+ Tregs were categorized into three distinct subsets: CD45RA+FOXP3lo resting Treg (TregI), CD45RA–FOXP3hi activated Treg (TregII), and CD45RA–FOXP3lo non-suppressive Treg (TregIII) cells. (B) Then cytokine production in the Treg subsets was measured by stimulating PBMCs with PMA and ionomycin in the presence of GolgiStop®, and by intracellular staining with Abs against IFN-γ and IL-17. Numbers on the dot plot indicate the frequency of cytokine-producing cells. (C) While the frequency of TregI cells was lower in the TS (–) than in the HC group, the frequency of TregII cells was higher in the TS (+) than in the HC group. The frequency of TregIII cells was higher in the TS (–) and TS (+) than in the HC group. Grey bars and error bars indicate means and standard deviations, respectively. Values from the three groups were compared by ANOVA, and the between-group values were compared via Bonferroni post-hoc analysis. Abbreviations: Tregs, regulatory T cells; HC, healthy control subjects; TS (−), Turner syndrome patients without thyroid autoimmunity; TS (+), Turner syndrome patients with thyroid autoimmunity.
Fig 3.
Baseline target cell proliferation and cytokine production and inhibitory index of proliferation among the HC, TS (–) and TS (+) groups.
Whereas baseline target cell proliferation (at Treg: target cell ratio of 0:1) was similar among the three groups (A), CD4+CD25– target cells from TS (–) and TS (+) patients produced higher levels of TNF-α, IFN-γ, and IL-17 compared to cells from the HC group (B). The percentage of the inhibitory index of cell proliferation was calculated as described in Methods. The inhibitory index of proliferation indicating the suppressive function of Tregs was significantly lower in the TS (–) and TS (+) than the HC group (C). Bars and error bars indicate means and standard deviations, respectively. Values (B) from the three groups were compared by ANOVA, and the between-group values were compared via Bonferroni post-hoc analysis. P-values (C) were calculated using generalized estimating equations. Abbreviations: HC, healthy control subjects; TS (−), Turner syndrome patients without thyroid autoimmunity; TS (+), Turner syndrome patients with thyroid autoimmunity.
Fig 4.
Comparison of CD4+ T cell subsets among the HC, TS (−), and TS (+) groups.
Both the TS (–) and TS (+) groups had lower frequencies of naïve cells (P = 0.001 for both) but higher frequencies of EM cells than did the HC group. Numbers within the plots indicate the mean (standard deviations) percentage of each subset. Values from the three groups [HC, TS (-), and TS (+)] were compared by ANOVA, and the between-group values were compared via Bonferroni post-hoc analysis. Abbreviations: HC, healthy control subjects; TS (−), Turner syndrome patients without thyroid autoimmunity; TS (+), Turner syndrome patients with thyroid autoimmunity.