Fig 2: Patients with Parkinson’s disease (PD) and mice with experimental PD exhibit decreased regulatory T (Treg) and increased T helper 1 (Th1) cell numbers in the blood. Treg cells were defined as CD3+CD4+CD25+FoxP3+ cells, whereas Th1 cells were identified as CD3+CD4+T-bet+ cells. (a) Representative plots of Treg and Th1 cells in patients with PD and healthy volunteers (HVs). (b) Representative plots of Treg and Th1 cells in control mice (Saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. (c) Patients with PD exhibited lower Treg cell levels and greater Th1 cell levels in the blood than HVs. n = 20. ****, p < 0.0001 by a two-tailed Student’s t tests. (d) MPTP-induced experimental PD mice had fewer Treg and more Th1 cells in the circulation than saline-treated mice. n = 9/group in each experiment performed in triplicate. ***, p < 0.001; ****, p < 0.0001, according to Student’s t test.

Fig 3: α4β7 expression on lymphocyte subsets in inflammatory bowel disease (IBD) patients. (A) Representative flow cytometric stainings of α4β7 expression on Th1, Th2, and Th17 polarized CD4+ T cells (previously gated on CD3 and CD4) before and after induction therapy with vedolizumab (VDZ) (week 14). Gray line: isotype control. (B,C) Quantification of α4β7 expression in different subsets of peripheral T cells (previously gated on CD3, CD4, and T-bet, IFNγ, IL-4, GATA-3, or IL-17, respectively) from patients with Crohn’s disease (CD) [(B), n = 12] and ulcerative colitis (UC) [(C), n = 10] before initiation of anti-integrin therapy with VDZ (indicated by “0”) (week 0) and directly before the second (week 2), third (week 6), and fourth (week 14) administration of 300 mg VDZ (indicated by “1,” “2,” and “3,” respectively). (D,E) Quantitative comparison of α4β7 expression in different subsets of lamina propria T cells in patients with UC [(D), n = 11] and CD [(E), n = 12]. α4β7 expression was compared between remitters and non-remitters for each disease after completed induction therapy with VDZ (UC: n = 5 remitters, n = 6 non-remitters; CD: n = 6 remitters, n = 6 non-remitters). (F) Cryosections of colonic biopsies from IBD patients were stained with fluorescein isothiocyanate (FITC)-VDZ or FITC-IgG1. After counterstaining with DAPI (Thermo Fischer Scientific, Cat. D1306), cryosections were analyzed with confocal microscopy. The arrows indicate α4β7-expressing mucosal cells. (G) The amount of α4β7-expressing cells within the lamina propria was assessed in 15 IBD patients (8 CD and 7 UC) patients prior to the commencement of VDZ therapy. For each patient a mean of 5 high power field (HPF) were analyzed by fluorescence microscopy and the amount of α4β7-expressing cells prior to VDZ therapy was quantified. UC and CD patients with clinical remission toward subsequent VDZ exhibited significantly higher levels of α4β7-expressing cells within the lamina propria compared with non-remitters. (H) Histopathological scoring from the same remitters and non-remitters [as shown in (G)] prior to VDZ therapy. Results are presented as mean ± SEM. Differences between samples are compared with the Mann–Whitney U test (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig 4: Homozygous RhoA deletion in Treg cells dampens Treg cell homeostasis and induces Treg cell plasticity. (A) Left, representative flow cytogram of Foxp3 staining in CD4+ cells from the spleen of RhoA+/+Foxp3YFP-Cre and RhoAFlox/FloxFoxp3YFP-Cre mice. The numbers indicate percentages of CD4+Foxp3+ Treg cells. Middle, average percentages of CD4+Foxp3+ Treg cells. Right, average numbers of CD4+Foxp3+ Treg cells. (B) Treg cell proliferation. Percentages of CD4+Foxp3+ Treg cells incorporated with BrdU are shown. (C) Treg cell apoptosis. The expression levels (MFI: Mean fluorescence intensity) of active caspase 3 in CD4+Foxp3+ Treg cells are shown. (D) The expression levels of Foxp3 in Treg cells. (E) Left, representative flow cytogram of IL-17, IFN-γ, and IL-4 staining in CD4+Foxp3+ Treg cells. The numbers indicate percentages of IL-17+, IFN-γ+, and IL-4+ Treg cells. Right, average percentages of IL-17+, IFN-γ+, and IL-4+ Treg cells. (F) Left, representative flow cytogram of RORγT, T-bet and GATA3 staining in CD4+Foxp3+ Treg cells. The numbers indicate percentages of RORγT +, T-bet+, and GATA3+ Treg cells. Right, average percentages of RORγT +, T-bet+, and GATA3+ Treg cells. (G) Left, representative histogram of the expression levels of CTLA-4, GITR and PD-1 in CD4+Foxp3+ Treg cells. The numbers above the graphs indicate MFI. Right, average MFI of CTLA-4, GITR and PD-1 in CD4+Foxp3+ Treg cells. n = 3 mice. Data are representative of two independent experiments. Error bars indicate SD. **p < 0.01.

Fig 5: Heterozygous RhoA deletion in Treg cells induces Treg cell plasticity and increases CD4+ effector T cells but does not result in autoimmunity. (A) Body weight of RhoA+/+Foxp3YFP-Cre and RhoAFlox/+Foxp3YFP-Cre mice. (B) Images of H&E staining of the indicated organs. (C) Representative flow cytogram of Foxp3 staining in CD4+ cells from the spleen of RhoA+/+Foxp3YFP-Cre and RhoAFlox/+Foxp3YFP-Cre mice. The numbers indicate percentages of CD4+Foxp3+ Treg cells. (D) Left, average percentages of CD4+Foxp3+ Treg cells. Right, cell numbers of CD4+Foxp3+ Treg cells. (E) Proliferation of Foxp3+YFP+ Treg cells from RhoA+/+Foxp3YFP-Cre/+ and RhoAFlox/+Foxp3YFP-Cre/+ female mice. Percentages of Foxp3+YFP+ Treg cells incorporated with BrdU are shown. (F) Apoptosis of Foxp3+YFP+ Treg cells from RhoA+/+Foxp3YFP-Cre/+ and RhoAFlox/+Foxp3YFP-Cre/+ female mice. The expression levels (MFI: mean fluorescence intensity) of active caspase 3 in Foxp3+YFP+ Treg cells are shown. (G) The expression levels of Foxp3 in Treg cells from RhoA+/+Foxp3YFP-Cre and RhoAFlox/+Foxp3YFP-Cre mice. (H) Representative flow cytogram of IFN-?, IL-17 and IL-4 staining in CD4+Foxp3+ Treg cells. The numbers indicate percentages of IFN-?+, IL-17+ and IL-4+ Treg cells. (I) Average percentages of IFN-?+, IL-17+ and IL-4+ Treg cells. (J) Representative flow cytogram of ROR?T, T-bet and GATA3 staining in CD4+Foxp3+ Treg cells. The numbers indicate percentages of ROR?T +, T-bet+, and GATA3+ Treg cells. (K) Average percentages of ROR?T +, T-bet+, and GATA3+ Treg cells. (L) Representative flow cytogram of IFN-?, IL-17 and IL-4 staining in CD4+Foxp3- cells. The numbers indicate percentages of IFN-?+CD4+, IL-17+CD4+ and IL-4+CD4+ effector T cells. (M) Average percentages of IFN-?+CD4+, IL-17+CD4+ and IL-4+CD4+ effector T cells. (N, O) In vitro Treg suppressive activity. (N), representative flow cytogram of CFSE dilution in T cells (responder). The numbers above the gating are percentages of cells low for CFSE, representing proliferating T cells. (O), average percentages of cells low for CFSE. Foxp3+YFP+ Treg cells were obtained by flow cytometry sorting of CD4+YFP+ cells from the spleen of RhoA+/+Foxp3YFP-Cre/+ and RhoAFlox/+Foxp3YFP-Cre/+ female mice. (A, C–O) Error bars indicate SD of 5-6 mice (A, C–M) or triplicates (N, O). Data are representative of two independent experiments. Error bars indicate SD. **p < 0.01. (B) Data are representative of 3 mice.