Fig 1: HpBARI suppresses ST2 detection, and suppresses IL-33 responses in vitro.(A) Naive murine lung cells were cultured at 37°C overnight with HES or recombinant HpBARI, and ST2 expression measured by flow cytometry. (B–D) Naive murine bone marrow cells cultured for 5 days with IL-2, IL-7 and IL-33 (all at 10 ng/ml) followed by ELISA of cell-free supernatants for IL-5 (B), IL-6 (C) and IL-13 (D). Dotted line indicates levels with IL-2, IL-7 and IL-33 alone. All data are representative of >3 repeat experiments, with three technical replicates per measurement. Error bars show SEM.
Fig 2: 6PGD blockade induce mitochondrial ROS and expression of antioxidant enzymes(A) Naive CD8+ T cells from 6PGD-/- and 6PGDfl/fl mice were stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL), and expression of CD62L and CD44 was examined by flow cytometry.(B–L) Naive CD8+ stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL), mitochondrial ROS was assessed using MitoSOX Red (B), and lipid peroxidation capacity was assessed using BODIPY 581/591 C11 (C) and flow cytometry. (D–L) The indicated enzymes of ROS metabolism pathway were assessed by real-time PCR (D–K), and their quantitative changes in 6PGDfl/fl and 6PGD-/- T cells during activation were also depicted by the heatmap (L). Results are representative of three independent experiments with n = 3 per experiment. Gsr, glutathione reductase; Gpx4, glutathione peroxidase 4; Nrf2, nuclear factor erythroid 2-related factor 2; Prdx2, peroxiredoxin 2; SOD2: superoxide dismutase 2; Txn1, thioredoxin 1; Txnrd1, thioredoxin reductase 1. Error bars represent ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 3: 6PGD-/- CD8+ T cells have enhanced cytolytic function in vitro and potent pathogen-specific and tumor-specific activity in vivo(A and B) Freshly isolated CD8+ T cells from OTI/6PGDfl/fl and OTI/6PGD-/- mice were adoptively transferred to wild-type mice followed by inoculation of Lm-Ova 24 h later. On day 3 after inoculation, bacterial burden was assessed in the spleen (A) and IFN-? production (B) was evaluated by flow cytometry on tetramer-positive cells.(C) CD8+ T cells from OTI/6PGDfl/fl and OTI/6PGD-/- mice were cultured with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL) for 4 days. CTL activity was assessed as described in the STAR methods. Results are representative of three independent experiments with n = 7 mice per group.(D and E) CD8+ T cells from OTI/6PGDfl/fl and OTI/6PGD-/- mice were adoptively transferred to mice bearing EG7 lymphoma as indicated in the schema (D). Tumor size was measured every 48 h, and tumor volume was calculated (E). Results are representative of two independent experiments with n = 10 mice per group.(F–I) On day 10 after adoptive transfer, fractions of tumor-specific, tetramer-positive T cells in tumor-infiltrating lymphocytes (TILs) were assessed by tetramer staining (F), and expression of granzyme B (G) and mitochondrial mass (H and I) in tetramer-positive, tumor-specific T cells was assessed by flow cytometry. Results are representative of two independent experiments with n = 7–8 mice per group. Error bars represent ± SEM. *p < 0.05; **p < 0.01.
Fig 4: Proposed mechanism: Cbl-b KO CD4+ T cells promote Treg resistance through enhanced IL-2 production.(a). Interaction between WT Tregs and WT Teff. When WT CD4+ T cells are stimulated in the presence of Tregs, IL-2 produced by the CD4+ T cells are immediately acquired by regulatory T cells which express a higher level of IL-2 receptors. This scenario favors T cell suppression. (b). Interaction between WT Treg and Cbl-b KO Teff. Cbl-b KO CD4+ T cells hyper-secrete IL-2 which provides a sufficient amount of IL-2 for T cell activation and survival as well as Treg expansion. Cbl-b KO CD4+ T cells also upregulate IL-2 receptors which increase the potential for T cells to acquire IL-2. Together, Cbl-b KO CD4+ T cells override the ability of Tregs to restrict T cell activation.
Fig 5: Cbl-b deficient CD4+ T cells upregulate CD25 levels and IL-2 production.IL-2 secretion by Cbl-b KO CD4+FoxP3− T cells was measured without (a) or with Treg cells (b). Supernatants were collected from a Treg suppression assay on day 1 and 3 post-stimulation, and ELISA was performed to quantitate IL-2 levels. (c). CD25 expression on stimulated WT and Cbl-b KO CD4+FoxP3− T cells. CD4+CD25− T cells were stimulated with anti-CD3 and irradiated APCs, and CD25 expression was evaluated on day 0, 1 and 3 post-stimulation by flow cytometry. (d). CD25 expression on WT and Cbl-b KO CD4+FoxP3− T cells in response to different concentrations of anti-CD3. (e). Following CD4+ Teff cell stimulation, proliferation and CD25 expression was measured using flow cytometry every 12 hours for 3 days. (f). CD25, CD122 and CD132 expression on WT or Cbl-b KO CD4+ effector T cells, or IL-2 supplemented (20 ng/ml) CD4+FoxP3− T cells were measured on day 3 post-stimulation.
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