Fig 2: Molecular actions of LipC6, aCTLA4 Ab, or their combination on T cells. Pan T cells purified from splenocytes in wild-type mice with negative selection beads. These cells respectively received the treatments of LipC6, aCTLA4 Ab or both in the presence of anti-CD3 & anti-CD28 Abs. Ghost (LipC6 vehicle control) and isotype Ab (aCTLA4 Ab control) were used in control group (W/O). 24 h later, the cells were harvested to extract total RNAs for detecting the expression of the indicated genes by qPCR. The expression level of the indicated genes was normalized to housekeeping genes. The fold changes of each gene were shown for Klf2 (A), Ctla4 (B), Pdcd1 (C), Foxp3 (D), Gzmb (E), Tnf (F), and Ifng (G). *p < .05, **p < .01, ***p < .001. n = 3, Error bars represent means ± SD. (G) Schematic diagram depicting the signal pathways mediating combinatorial function of LipC6 and aCTLA4 Ab

Fig 3: 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-?, and IL-4 staining in CD4+Foxp3+ Treg cells. The numbers indicate percentages of IL-17+, IFN-?+, and IL-4+ Treg cells. Right, average percentages of IL-17+, IFN-?+, and IL-4+ Treg cells. (F) Left, 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. Right, average percentages of ROR?T +, 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 4: Combination therapy-mediated MB49 tumour rejection is dependent on CD4+ and CD8+ T cells.Isolated TILs from 13–15 days tumours, 1–3 days after the last treatment with a-CTLA-4, a-PD-1, combination of both (Combo) were analyzed by flow cytometry for (a) Total CD4+ T cells and CD8+ T cells. Representative flow panels are shown on the left and pooled results from five mice are depicted in bar graphs on the right. (b) Foxp3+ Treg in CD4+ T cells. Pooled results from five mice are shown in the right bar graph. (c) Ratios of Foxp3- to Foxp3+ in CD4+ T cells. (d) Ratios of CD8+ T cells to Foxp3+ CD4+ T cells. Mice depleted of CD4+ (GK1.5), CD8+ (2.43) or both (GK1.5+2.43) were treated with the combo and mouse survivals are presented (e). Data in the bar graphs are means±s.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by one-way ANOVA with Bonferroni's post hoc test. Data are representative of three independent experiments.

Fig 5: a-CTLA-4 upregulates the PD-1/PD-L1 inhibitory pathway in human and murine bladder tumours.(a,b) Surface expression of CTLA-4 and PD-1 in Foxp3-CD4+ TILs isolated from patients with bladder cancer (a) and from murine MB49 bladder tumours (b). (c,d) PD-L1 expression on human (c) and mouse (d) bladder tumour cells analyzed by flow cytometry. (e) PD-1 expression on immune cells from pre- and post-a-CTLA-4-treated human bladder tumour by IHC. Representative IHC photos (arrows indicate positively-stained cells) and pooled results from all the patients are shown. (f) PD-1 expression on Foxp3-CD4+ TILs isolated from MB49 tumour-bearing mice treated with (a-CTLA-4) or without a-CTLA-4 (UnTx) were analyzed by flow cytometry. Histograms of PD-1 (left) and pooled results (right) from five mice in one representative experiment are shown. (g,h) PD-L1 expression on tumour cells (g) and immune cells (H) of pre- and post-a-CTLA-4-treated human bladder samples by IHC. Representative IHC photos (arrows indicate positively-stained cells) and pooled results from all the matched cases are shown. Data in scatter plots and the bar graph are means±s.e.m. Representative results (b, d, and f) from three independent experiments are shown. *P<0.05; **P<0.01; ***P<0.001 by two-tailed unpaired Student's t-test.