Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: NOX2 Activation of Natural Killer T Cells Is Blocked by the Adenosine A 2A Receptor to Inhibit Lung Ischemia–Reperfusion Injury

doi: 10.1164/rccm.201506-1253OC

Figure Lengend Snippet: Activation of invariant natural killer T (iNKT) cells by hypoxia–reoxygenation (HR) is mediated by NADPH oxidase and attenuated by A2A receptor agonist. (A) Exposure of wild-type (WT) iNKT cells to HR significantly increased IL-17 production compared with normoxia (Norm), which was significantly attenuated by ATL313 (ATL) or apocynin (Apo) pretreatment. KT 5720 (KT) pretreatment of iNKT cells significantly counteracted the ATL313-mediated mitigation of IL-17 production after HR. IL-17 production by iNKT cells from p47phox−/− mice (p47−/−) was significantly attenuated after HR. *P < 0.05 versus Norm WT; #P < 0.05 versus HR WT; **P < 0.05 versus HR WT + ATL; δP < 0.05 versus Norm p47−/−; n = 5–8/condition. (B) IL-17 production by primary human CD3+CD56+ NKT cells was significantly increased after HR, which was attenuated by ATL313 treatment. *P < 0.001 versus Norm; #P < 0.001 versus HR; n = 8/condition. (C) ATL313 treatment significantly attenuated superoxide anion production by HR-exposed human CD3+CD56+ NKT cells compared with HR alone. *P < 0.05 versus Norm; #P < 0.05 versus HR; n = 6/condition. Data are presented as mean ± SEM. RLU = relative light units.

Article Snippet: Furthermore, pretreatment of HR-exposed iNKT cells with a PKA inhibitor KT 5720 (0.1 μM; Tocris Bioscience, Bristol, UK) abolished the ATL313-mediated attenuation of IL-17 production (93.19 ± 6.41 pg/ml vs. 33.29 ± 4.38 pg/ml).

Techniques: Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Extracellular adenosine regulates naive T cell development and peripheral maintenance

doi: 10.1084/jem.20130249

Figure Lengend Snippet: PKA stimulation by A 2A R activation prevents TCR-induced down-regulation of IL-7Rα. (A) Wild-type T cells were stimulated with varying concentrations of plate-bound anti-CD3 or recombinant mouse IL-7 in the presence or absence of 1 µM CGS 21680 (CGS) or vehicle control (<0.1% DMSO). CD127 (IL-7Rα) staining was performed after incubation at 37°C (5% CO 2 ) for 24 h ( n = 4, from three independent experiments). (B) Isolated wild-type T cells were stimulated by transfer to tissue culture plates pretreated with 5 µg/ml anti–mouse CD3 and 2 µg/ml anti–mouse CD28 antibodies in the absence or presence of 1 µM CGS 21680 or CGS 21680 + equimolar SCH 58621. Serine 473 and threonine 308 phosphorylation of AKT was detected by immunoblotting for the indicated times (experiments were repeated three times for serine 473 and twice for threonine 308 detection). (C) Basal serine 473 phosphorylation of Akt in Adora2a +/+ versus Adora2a −/− cells was measured by flow cytometry. (D) 100 nM of the selective PKA inhibitor KT 5720 was added to cultures of T cells stimulated with anti-CD3 in the presence or absence of 1 µM CGS 21680 or vehicle control (<0.1% DMSO). CD127 staining was performed after incubation at 37°C (5% CO 2 ) for 24 h. (E) 2 µM of the selective PI3K inhibitor LY 294002 was added to cultures of stimulated T cells in the presence or absence of 1 µM CGS 21680 or vehicle control (<0.1% DMSO). CD127 staining was performed after incubation at 37°C (5% CO 2 ) for 24 h. Data in C–E are representative of two independent experiments with similar results ( n = 3). (F) 2 µM of the selective PI3K inhibitor LY 294002 was added to cultures of naive T cells stimulated with the indicated concentrations of rmIL-7. CD127 staining was performed after incubation at 37°C (5% CO 2 ) for 24 h ( n = 5, from two independent experiments with similar results). (G) Enriched CD44 lo and CD44 hi cells were stimulated by 1 µg/ml plate-bound anti-CD3 antibody in the presence or absence of 1 µM CGS 21680. CD127 (IL-7Rα) staining was performed after incubation at 37°C (5% CO 2 ) for 24 h ( n = 4, from two independent experiments with similar results). *, P < 0.05; ***, P < 0.001 by two-way ANOVA and Bonferroni post-hoc analysis. Error bars are SEM.

Article Snippet: To evaluate the effects of A 2A R signaling on TCR-induced IL-7Rα down-regulation, we stimulated isolated T cells with plate-bound anti-CD3 antibody in the presence or absence of 1 µM of the selective A 2A R agonist CGS 21680, 100 nM of the PKA inhibitor KT 5720, or 2 µM of the selective PI3K inhibitor LY 294002 (Invitrogen).

Techniques: Activation Assay, Recombinant, Staining, Incubation, Isolation, Western Blot, Flow Cytometry