Fig 2: Deletion of 6-phosphogluconate dehydrogenase (6PGD) in regulatory T cells (Tregs) induces a fatal autoimmune phenotype.(A–B) YFP+ cells were sorted from Pgd+/+Foxp3Cre (wild-type [WT]) and Pgdfl/flFoxp3Cre mice and deletion of Pgd was confirmed by western blot (A) and real-time PCR (B). (C) Representative image of 21-day-old WT and Pgdfl/flFoxp3Cre mice. (D) Representative image of lymphadenopathy in Pgdfl/flFoxp3Cre compared to WT mice. (E) T cells absolute number per spleen and peripheral lymph nodes (pLNs) of WT and Pgdfl/flFoxp3Cre mice are shown (N = 12 mice per group). (F) Survival curve of WT and Pgdfl/flFoxp3Cre mice. Representation of 15 mice per group. (G) Splenocytes from 20-day-old WT and Pgdfl/flFoxp3Cre mice were harvested and distribution of CD4+ versus CD8+ T cells were evaluated. (H) Deletion of 6PGD induces enhanced effector phenotype (CD44high CD62Llow) both in CD4+ T cells (top panel) and in CD8+ T cells (bottom panel) in 20-day-old Pgdfl/flFoxp3Cre compared to WT mice. (I) Both CD4+ and CD8+ T cells express higher levels of CD69 activation marker in Pgdfl/flFoxp3Cre compared to WT mice. (J) Splenocytes from 20-day-old WT and Pgdfl/flFoxp3Cre mice were stimulated with PMA (50 ng/ml)/ionomycin (1 µg/ml) plus GolgiPlug (1 µl/ml) for 4 hr and expression of IFN-? was evaluated by flow cytometry. (K) Granzyme B (top panel) and CD107a degranulation activation marker (bottom panel) expression was assessed on splenocytes of 20-day-old WT and Pgdfl/flFoxp3Cre mice by flow cytometry. (L–M) Serum was collected from 20-day-old WT and Pgdfl/flFoxp3Cre mice and levels of serum antibodies (L) and IFN-?, IL-17A, IL-4, and IL-5 (M) were detected as described in supplemental information. Results are representative of 12 mice per group. (N) Hematoxylin and eosin staining of lung: original magnification (X10), pancreas (X10), skin (X10), and intestine (X10) of WT and Pgdfl/flFoxp3Cre mice. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: Conjugating peptide antigens to CPPs increases antigen-specific T cell responses in vitro and in vivo. (A) Schematic structure of antigen-CPP conjugates prepared using azide/alkyne click chemistry and sequences of eight tested CPPs. (B and C) Splenocytes from C57BL/6 mice were pulsed for 1 h with the indicated gp100 peptide or CPP conjugate then cocultured with naïve CFSE-labeled pmel-1 CD8+ T cells. Pmel-1 T cell activation was assessed by flow cytometry analysis of CD69 up-regulation at 24 h (B) and proliferation (CFSE dilution) at 72 h (C). Significance relative to the gp100 long peptide was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test: ****P < 0.0001. (D–H) C57BL/6 mice (n = 4 to 5 animals per group) were immunized twice with 5 nmol of the indicated antigen and 25 µg cyclic-di-GMP, followed by restimulation of peripheral blood mononuclear cells (PBMCs) at day 21 with peptide to detect cytokine-producing antigen-specific T cells by flow cytometry. (D) Timeline of immunization experiments. (E) Representative flow cytometry plots (Left) and quantification (Right) of gp100-specific IFN-γ+/TNF-α+ CD8+ T cells. (F) Representative flow cytometry plots (Left) and quantification (Right) of Adpgk-specific IFN-γ+/TNF-α+ CD8+ T cells. (G) Linker placement for the antigen gp100 to pAntp and MPG. The original, a click linkage between the antigen C terminus and the CPP N terminus, is denoted “c-n”; “c-c” denotes a click linker between the antigen C terminus and the CPP C terminus; “–“ denotes a variant with a peptide bond linking the antigen and the CPP (i.e., synthesizing the antigen-CPP as a single long peptide). (H) Percentage of IFN-γ+ of CD8+ T cells after a prime and boost with the CPP conjugates shown in G. Significance relative to the gp100 long peptide (or Adpgk) was determined by two-way ANOVA followed by Dunnett’s multiple comparisons test: ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig 4: Production of ROS and inflammatory factors in the ischemic brain at day 14 after onset in mice. C57BL/6 (B6) mice were subjected to sham operation, photothrombosis, or 60 mins MCAO for 14 days. a At day 14 after surgery, bioluminescence images show ROS generation in photothrombosis and MCAO mice. b Quantification analysis showed ROS generation in photothrombosis and MCAO mice 14 days after ischemia. n = 6 mice per group. c At 14 days after ischemia, brain tissues were obtained from photothrombosis and MCAO mice. Brain tissues from sham-operated mice were used as control. Brain homogenates were prepared, and cytokine concentrations were measured by a customized Mouse Cytokines ELISA kit, including IL-1α, IL-1β, IFN-γ, MCP-1, IL-6, TNF-α, and CD69. Results shown are from three independent experiments with a pool of 4 mice per group. Error bars represent s.e.m.; *p < 0.05; **p < 0.01, sham vs. stroke group (photothrombosis/MCAO) by one-way ANOVA

Fig 5: Brain-infiltrating lymphocytes express CD69 and IFN-γ at day 14 after brain ischemia in mice. a Flow cytometry dot plots show IFN-γ and CD69 expression in CD4+ T, CD8+ T, and NK cells in single cell suspension from brains of photothrombosis- or MCAO-operated mice at 14 days after stroke. b, c Bar graphs summarize the cumulative data for quantifying IFN-γ (b) and CD69 (c) expression in CD4+ T, CD8+ T, and NK cells from brains of photothrombosis- or MCAO-operated mice at 14 days after stroke. n = 8 mice per group. Error bars represent s.e.m.; **p < 0.01, sham vs. stroke group (photothrombosis/MCAO) by one-way ANOVA