Article Figures & Data
Figures
- FIGURE 1.
CD28 is required for maximal glucose uptake in T cell activation. A, Purified T cells were stimulated on plates coated with or without anti-CD28 and with indicated doses of anti-CD3. Glucose uptake was measured after 1 day. B and C, T cells were cocultured with LPS-stimulated bone marrow-derived macrophages. T cells were stimulated with anti-CD3 Abs with and without CTLA4-Ig (B) or anti-ICAM Ab (C). After 1 day of coculture, T cells were separated from macrophages and glucose uptake was analyzed (∗, p < 0.005 and ∗∗, p < 0.01).
- FIGURE 2.
Costimulation induced Glut1 protein expression and trafficking to the cell surface. A and B, Purified T cells were stimulated on plates coated with anti-CD3 and anti-CD28 Abs at the indicated concentrations for 1 day before lysis and (A) immunoblotting and (B) PNGgase F treatement to deglycosylate followed by immunblot. Numbers indicate the quantification of the deglycosylated Glut1 normalized to Actin control. C, T cells were cocultured with LPS-stimulated bone marrow-derived macrophages and stimulated with anti-CD3 Abs with and without CTLA4-Ig for 1 day before lysis and immunoblotting. D and E, T cells from mice reconstituted with hematopoietic stem cells infected with a Myc-tagged Glut1 were purified, stimulated on plates coated with the indicated dose of anti-CD3 with and without 5 μg/ml anti-CD28 for 1 day, and cells were immunoblotted for Myc-Glut1 levels (D) and stained with anti-Myc to detect surface levels or Glut1 by flow cytometry (E). Means and SDs of five samples are shown (∗, p < 0.05).
- FIGURE 3.
T cells require glucose for cell survival, IL-2 production, proliferation, and IFN-γ production. Purified T cells were stained with CFSE, stimulated with 5 μg/ml anti-CD3 with or without 1 μg/ml or 5 μg/ml anti-CD28 in glucose-free media supplemented with glucose to the indicated concentrations, and cultured for 2 days before analysis. A, Cell survival was determined by propidum iodide exclusion as analyzed by flow cytometry. B, IL-2 production was assayed through ELISA on T cell supernatants. C, Reduction in CFSE staining was used to determine cell division by flow cytometry. D, IFN-γ production was assayed through ELISA on T cell supernatants.
- FIGURE 4.
Glut1 over-expression in T cells does not alter T cell development or homeostasis. A, Thymocytes and purified resting mature T cells from Glut1 transgenic and nontransgenic littermates were lysed and immunoblotted to determine Glut1 levels. Immunoblot was uniformly contrasted and digitally rearranged for ease of viewing (indicated by white bar). B, Thymocytes and purified resting mature T cells from Glut1 transgenic animals were assayed for glucose uptake (∗, p < 0.005). C, Thymocytes and whole spleen cell suspensions were stained with fluorescently conjugated Abs against CD4 and CD8 and analyzed by flow cytometry. D, The number of cells in splenocyte suspensions was determined by particle size analyzer and multiplied by the percentage of whole spleen determined to be B cells, CD4+, or CD8+ cells as indicated by staining with B220, CD4, or CD8 fluorescently conjugated Abs and flow cytometry. E, Levels of the activation markers CD25 and CD44 were determined in resting purified T cells from Glut1 transgenic and nontransgenic littermates by staining with fluorescent Abs and flow cytometry.
- FIGURE 5.
Transgenic Glut1 over-expression increases T cell size and cytokine production. A, The size of resting T cells from Glut1 transgenic and nontransgenic animals was determined by particle size analyzer (∗, p < 0.005). B, T cells were stimulated 0.5 μg/ml anti-CD3, with or without 5 μg/ml anti-CD28, for 2 days and supernatant was collected and analyzed by ELISA for IL-2 production (∗, p < 0.05). C, T cells were stimulated with 5 μg/ml anti-CD3, with or without 5 μg/ml anti-CD28, for 3 days and supernatant was collected and analyzed by ELISA for IFN-γ production.
- FIGURE 6.
Glut1 over-expression led to accumulation of memory-phenotype T cells in aged mice. A, Resting T cells purified from Glut1 transgenic and age-matched nontransgenic animals >1 year old were stained with fluorescently labeled Abs to detect the T cell activation markers CD25, CD44, and CD69 and analyzed by flow cytometry. B and C, Resting T cells purified from Glut1 transgenic and age-matched nontransgenic animals >1 year old were stimulated with 1 μg/ml anti-CD3, with or without 5 μg/ml anti-CD28, for 1 day and supernatants were collected and analyzed for cytokine production by ELISA (∗, p < 0.05; ∗∗, p < 0.005; ND = Not Detected). D, Serum from representative aged-matched Glut1 transgenic and nontransgenic mice was analyzed by ELISA for Ig isotypes (∗, p < 0.04; ∗∗, p < 0.01). E, Kidney sections from 8-wk-old and 1-year-old aged matched Glut1 transgenic and nontransgenic mice were stained for anti-Mouse Ig and examined microscopically.
- FIGURE 7.
Constitutively active Akt increases glucose uptake in resting cells but cannot compensate for CD28 signals at low levels of CD3 stimulation. A, T cells from Akt1−/− animals and Akt1+/+ littermates were purified, stimulated with 1 μg/ml anti-CD3 with and without 5 μg/ml anti-CD28 for 18 h and glucose uptake and Glut1 protein levels were analyzed. B and C, T cells were purified from mAkt transgenic (A) and nontransgenic (N) littermates and stimulated with CD3 with and without 5 μg/ml anti-CD28 (B) for 1 h before cell lysis and analysis by immunoblotting for phospho-Akt which appeared larger in the transgenic mice than the nontransgenic due to the presence of an HA tag in the transgene and (C) for 1 day before cell lysis and analysis by immunoblotting for Glut1. D, Purified T cells were pretreated with LY294002 or vehicle control for 30 min and stimulated with anti-CD3 and anti-CD28 at the indicated concentrations for 1 day before T cell lysis and immunoblotting for Glut1. E, Purified resting T cells from mAkt and nontransgenic animals were analyzed for glucose uptake (∗, p < 0.02). F, T cells purified from mAkt and nontransgenic animals were stimulated with the indicated dose of anti-CD3 with and without 5 μg/ml anti-CD28 for 1 day before analysis of glucose uptake (∗, p < 0.005).
- FIGURE 8.
T cell glucose uptake and activation are increased in Glut1 and mAkt double transgenic animals. A, Glucose uptake was analyzed on purified resting T cells from littermates (∗, p < 0.01; ∗∗, p < 0.05; ∗∗∗, p < 0.005). B, Cell size was determined by particle size analyzer analysis on purified resting T cells from nontransgenic, Glut1, mAkt, and Glut1/mAkt double transgenic cells (∗, p < 0.01 and ∗∗, p < 0.005). C, Levels of T cell activation markers CD25, CD44, and CD69 were determined with fluorescently labeled Abs and flow cytometric analyses of resting purified T cells from the indicated genotypes. D, Purified T cells were stained with CFSE, stimulated with 5 μg/ml anti-CD3, with and without 5 μg/ml anti-CD28, for 3 days, and levels of CFSE depletion to indicate cell proliferation were determined by flow cytometry.
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