Fig 2: Anti-IL-6R reduces the expression of IFN-? in activated Xiap-/- Treg cells. a, b Anti-IL-6R decreases IFN-? expression in re-stimulated Xiap-/- Treg cells. WT and Xiap-/- iTreg cells were stimulated with anti-CD3/CD28 and IL-2, or with additional IL-12 as indicated, in the absence or presence of anti-IL-6R (50 µg ml-1) for 4 days. iTreg cells were re-stimulated with TPA/A23187 for 24 h and IFN-? production was determined by ELISA (a), or reactivated with TPA/A23187 for 5 h and the expressions of Foxp3 and IFN-? were determined by intracellular staining (b). c, d Anti-IL-6R inhibits IFN-? expression in human Treg cells. Control and human XIAP-knockdown iTreg cells were stimulated as in (a, b), with additional IL-6 as indicated, and secretion (c) or intracellular expression (d) of IFN-? was determined. e Inability of anti-TNF or anti-IL-1R to inhibit the production of IFN-? in activated Xiap-/- Treg cells. WT and Xiap-/- iTreg cells were stimulated, as described in (a), in the presence or absence of anti-TNF or anti-IL-1R (50 µg ml-1 each) for 4 days. Treg cells were re-stimulated with TPA/A23187 for 24 h and the secreted IFN-? was determined by ELISA. f Anti-IL-6R rescues the impaired suppressive activity of Xiap-/- tTreg cells in vivo. CD45.2+ WT or Xiap-/- tTreg cells were co-transferred with CD45.1+ CD4+CD25- effector T cells into male CD45.1+ RagI-/- mice. Anti-IL-6R antibody (500 µg per mouse) was intraperitoneally administrated at day 0, followed by weekly dosing of 500 µg. The body weights of mice were monitored at the indicated time-points. ***P < 0.001 by two-way ANOVA. g, h Anti-IL-6R restores Foxp3 stability and reduces IFN-? expression in Xiap-/- tTreg cells transferred in vivo. Lymphocytes were isolated from spleen and mesenteric lymph nodes of mice in (f) and reactivated with TPA/A23187. The expressions of Foxp3 (g) and IFN-? (h) in CD45.2+ T cells were determined by intracellular staining. Values are mean ± SD of triplicate samples in an experiment. *P < 0.05, **P < 0.01, ***P < 0.001 for unpaired t-test (a, c, g, h). Experiments (a–e) were independently repeated three times with similar results

Fig 3: Combination anti-IL-6R and Xiap-/- Treg cells rescue mice from infection-induced inflammation. a, b Xiap-/- iTreg cells plus anti-IL-6R rescue Xiap-/- mice from C. albicans infection. Male Xiap-/- mice (KO) were intravenously administered with C. albicans and Xiap-/- iTreg cells (1 × 106) or anti-IL-6R antibody (500 µg) at day 2, or both, as indicated. Survival of mice is presented as a Kaplan–Meier survival curve (a). n = 11 for KO, n = 10 for KO + KO iTreg, n = 6 for KO + Ab, n = 9 for KO + KO iTreg + Ab. ***P < 0.001 for Log-rank (Mantel–Cox) Test (a). n.s., not significant. The serum level of IL-6 was determined on day 14 after infection (b). *P < 0.05 for unpaired t-test (b). c, d Anti-IL-6R prevents the conversion of the transferred Xiap-/- Treg cells. CD45.1+ Xiap-/- iTreg cells were transferred into CD45.2+ Xiap-/- mice infected with C. albicans as in (a). CD45.1+ T cells were recovered at day 10 post-infection, and the expression of Foxp3 and IFN-? was determined (c). The Foxp3+IFN-?+ fractions in the CD45.1+ Treg cells were quantitated (d), n = 5. **P < 0.01 for unpaired t-test (d). e, f Anti-IL-6R reduces kidney neutrophil infiltration and fungal load in C. albicans-infected Xiap-/- mice with KO iTreg cells transfer. Male Xiap-/- mice were infected with C. albicans, as described in (a). Kidneys were isolated 14 days post-infection and neutrophils (e) and C. albicans titres (f) were quantitated. *P < 0.05, **P < 0.01 for unpaired t-test. n.s., not significant. g Anti-IL-6R does not affect IL-17 production. Male CD45.1+ Xiap-/- mice were infected with C. albicans and treated with anti-IL-6R and anti-IL-6R plus CD45.2+ KO iTreg cells. Spleen T cells and B cells were isolated 7 days after treatments. CD45.2+ iTreg cells were depleted. T cells were incubated with B cells and heat-killed C. albicans (HKCA) for 4 days, and the production of IL-17A after stimulation with TPA/A23187 (10/100 ng ml-1) was determined by ELISA47, 48. Values are mean ± SD of triplicate samples in an experiment. Experiments were independently repeated three times with similar results

Fig 4: Targeting the CDK6-PTPs axis enhances the efficacy of T cell killing and adoptive T cell immunotherapy.(A) In vitro tumor killing assays showed that targeting the CDK6-PTPs axis increased splenic CD8+ T cell killing capacity against OVA-expressing B16F10 cells. The control groups were without OT-I CD8+ T cells. n = 3.(B) CAR T tumor killing assay was applied to examine the cytotoxicity of human T cells targeting Raji cells expressing GFP upon compound treatments. n = 3. For (A) and (B), 1 µM PTP1B-IN2, 1 µM Palbo, or 10 µM BSJ-03–123 was applied.(C) Graph showing the mean of B16F10-OVA tumor volumes in C57BL/6-Ly5.1 mice receiving CD8+ T cells from OT-I mice. n = 5.(D) Activities of tumor-infiltrating OT-I CD8+ T cells (top) and analysis of memory T cells among tumor-infiltrating OT-I T cells from C57BL/6-Ly5.1 mice (bottom). Ly5.2+ T cells were quantified in this experiment. n = 5.(E and F) Bioluminescence images (E) and overall survival graph (F) of tumor progression in Raji tumor-bearing mice with/without receiving CAR T cells under treatment with BSJ-03–123 (50 mg/kg) or PTP1B-IN2 (5 mg/kg). Shown are representative bioluminescence images (E, top) and quantification of the regions of interest (ROIs) (E, bottom). n = 11/group.(G) Memory T cell analysis of CAR T cells from Raji tumor-bearing mice receiving treatment with PTP1B-IN2 or BSJ-03–123 for 2 weeks. n = 4/group.(H) Overall survival (left) and tumor-free survival (right) of mice challenged with B16F10 melanoma cells with/without receiving pmel CD8+ T cells.(I) Schematic depicting the presented study model. Created with BioRender.(A), (B), (D), and (G) represent mean ± SEM, 1-way ANOVA (Dunnett’s test); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: Foxp3 instability in activated Xiap−/− Treg cells. a, b Reduced Foxp3+ population in activated Xiap−/− tTreg cells. WT and Xiap−/− tTreg cells were stimulated with anti-CD3/CD28 in the presence of IL-2 for 4 days. Foxp3 expression of activated tTreg cells was determined by intracellular staining and analyzed by flow cytometry. The gated section represents the Foxp3+ population and the number indicates the percentage of each population (a). The Foxp3+ fractions in WT and Xiap−/− tTreg cells were quantitated (b), n = 5. c, d Diminished Foxp3high population in adoptively transferred Xiap−/− tTreg cells. CD45.2+ tTreg cells were recovered from CD45.1+ Rag1−/− mice into which CD45.2+ tTreg cells and CD45.1+ CD4+CD25− T cells had been transferred a month earlier. The isolated CD45.2+ CD4+ T cells were reactivated with TPA/A23187 and Foxp3 expression was determined by intracellular staining (c). The Foxp3+ fractions in the transferred WT and Xiap−/− tTreg cells re-isolated from Rag1−/− mice were quantitated (d), n = 9. *P < 0.05 for unpaired t-test