Journal: Cell reports

Article Title: NKT cells adopt a glutamine-addicted phenotype to regulate their homeostasis and function

doi: 10.1016/j.celrep.2022.111516

Figure Lengend Snippet: (A–E) Sorted splenic NKT cells from C57BL/6 mice were labeled with 5 μM CellTrace Violet (CTV) and stimulated for 3 days in the indicated culture conditions. (A) The data show percentages of cell survival as measured by live/dead marker staining (left panel) and cell proliferation (right panel) in media containing the indicated amounts of glutamine (n = 3). (B) The schematic depicts key branches of glutamine metabolism producing αKG and GSH, as well as utilization of glutamine in the HBP to synthesize O-GlcNAc. Pathway-specific inhibitors are shown in bold, and the names of target enzymes are italicized. (C) The graph shows glutamate levels in NKT cells activated in the presence or absence of glutamine (n = 3). (D) Cell survival and proliferation of NKT cells activated in the presence or absence of CB839 (n = 4). (E) Total live cell numbers (left panel), the percentages of live cells (middle panel), and cell proliferation from WT and GLS1 KO mice are shown after 3 days of activation (n = 3). (F) Sorted splenic NKT cells were activated in the presence or absence of glutamine. GSH levels on day 3 of activation are shown (n = 3). (G and H) NKT cells were activated for 3 days in the presence or absence of BSO (G) or EGCG (H). Cell survival and proliferation are shown (n = 3). (I) The levels of O-GlcNAc in NKT cells with and without activation were compared (n = 3). (J) Sorted NKT cells were stimulated for 3 days in the presence or absence of either glutamine or glucose as indicated. The graph shows the relative mean fluorescent intensity (MFI) of O-GlcNAc on day 3 of activation (n = 3). (K and L) NKT cells were activated for 3 days in the presence or absence of DON (K) or OSMI (L). Cell survival and proliferation are shown (n = 3). All relative levels were calculated using the average of the control values as a reference point. All data are representative of or combined from at least three independent experiments. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01. ns, not significant.

Article Snippet: Mouse: GLS1 fl/fl ( Gls tm2.1Sray /j ) , The Jackson Laboratory , Stock No 017894.

Techniques: Labeling, Marker, Staining, Activation Assay, Control

Journal: Cell reports

Article Title: NKT cells adopt a glutamine-addicted phenotype to regulate their homeostasis and function

doi: 10.1016/j.celrep.2022.111516

Figure Lengend Snippet: (A) The graph shows hexokinase 2 (HK2) expression in NKT and CD4 T cells with and without stimulation (n = 3). (B) Heatmap shows relative levels of the indicated PPP metabolites in resting NKT cells compared with resting CD4 T cells analyzed after LC-MS/MS analysis (n = 3). (C) Heatmap shows PPP metabolites in NKT cells with and without stimulation as analyzed by LC-MS/MS (n = 3). (D and E) Cell survival and proliferation of sorted NKT cells stimulated in the presence or absence of EGCG (20 μM) with or without sodium pyruvate (1 mM) or DMαKG (1.5 mM) (n = 3). (F and G) WT and GLS1 KO mice were injected with αGalCer (5 μg/mouse), and splenic NKT cells were sorted on day 3 of activation. The representative graphs show ECAR using the glycolytic stress test (F) and OCR using the Mito Stress test (G) in Seahorse assay (n = 6 replicates per group pooled from two mice). The graph in (G) shows basal respiration (BR), maximum respiration capacity (MRC), and reserve capacity (RC) (n = 3). (H and I) NKT from WT and GLS1 KO mice were stimulated in the presence or absence of DMαKG (1.5 mM). (H) Cell survival and proliferation of NKT cells are shown (n = 3). (I) Representative histograms and summary graphs show mitochondrial mass and membrane potential (n = 3). All relative levels were calculated using the average control value as a reference point. All data are representative of or combined from at least two or three different experiments. Seahorse data are shown as mean ± SD. All other data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Mouse: GLS1 fl/fl ( Gls tm2.1Sray /j ) , The Jackson Laboratory , Stock No 017894.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Injection, Activation Assay, Membrane, Control

Journal: Cell reports

Article Title: NKT cells adopt a glutamine-addicted phenotype to regulate their homeostasis and function

doi: 10.1016/j.celrep.2022.111516

Figure Lengend Snippet: (A) Sorted splenic NKT cells from C57BL/6 mice were stimulated for 3 days with or without glutamine (Gn), and cytokine expression was compared after PMA/Ionomycin re-stimulation. (B) Representative dot plots show cytokine expression in sorted NKT cells from WT and GLS1 KO mice stimulated for 3 days. Graphs show cumulative data from three independent experiments. (C and D) Sorted splenic NKT cells from C57BL/6 mice were stimulated for 3 days in the presence or absence of DON (6 μM). Intracellular cytokine expression (C) and the levels of cytokine secreted into the media by ELISA (D) are shown (n = 3). (E) The graph shows cytokine expression in NKT cells stimulated in the presence or absence of BSO (n = 3). (F) NKT cells were stimulated for 3 days in the presence or absence of glutamine (2 mM) in combination with DMαKG (1.5 mM). Graphs show relative percentages of cytokine-positive NKT cells (n = 3). Data are shown as mean ± SEM. All data are representative of or combined from at least three independent experiments. *p < 0.05, **p < 0.01.

Article Snippet: Mouse: GLS1 fl/fl ( Gls tm2.1Sray /j ) , The Jackson Laboratory , Stock No 017894.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: NKT cells adopt a glutamine-addicted phenotype to regulate their homeostasis and function

doi: 10.1016/j.celrep.2022.111516

Figure Lengend Snippet: WT and GLS1 KO mice were injected with either 10 5 CFUs/mouse of LM-Ova (Lm) or PBS intraperitoneally. Two days after infection, spleens and livers were harvested and analyzed for bacterial load, NKT cell proliferation, and IFNγ expression. (A and B) Graphs show levels of CD98 expression and GSH in NKT cells from the spleen (left panel) and the liver (right panel) of PBS- and Lm-injected WT mice (n = 6). (C) Graphs show bacterial loads in the spleens and livers of infected WT and GLS1 KO mice (n = 6). (D) Graphs show CD69 expression in splenic and hepatic NKT cells from WT and GLS1 KO mice (n = 6). (E) Representative dot plots and graphs show cell proliferation as measured by Ki-67 expression in NKT cells from the spleens and livers of WT and GLS1 KO mice (n = 6). (F) To assess IFNγ expression, we incubated total splenocytes in the presence of Monensin for 2 h to prevent cytokine secretion followed by comparing intracellular expression of IFNγ in splenic (top panel) and hepatic (bottom panel) NKT cells (n = 6). The data are pooled from two independent experiments. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: Mouse: GLS1 fl/fl ( Gls tm2.1Sray /j ) , The Jackson Laboratory , Stock No 017894.

Techniques: Injection, Infection, Expressing, Incubation

Journal: Cell reports

Article Title: NKT cells adopt a glutamine-addicted phenotype to regulate their homeostasis and function

doi: 10.1016/j.celrep.2022.111516

Figure Lengend Snippet:

Article Snippet: Mouse: GLS1 fl/fl ( Gls tm2.1Sray /j ) , The Jackson Laboratory , Stock No 017894.

Techniques: Recombinant, Virus, Functional Assay, Adhesive, SYBR Green Assay, Staining, Software