Journal: Nature immunology

Article Title: NF-κB inducing kinase maintains T cell metabolic fitness in antitumor immunity

doi: 10.1038/s41590-020-00829-6

Figure Lengend Snippet: a-c , Flow cytometric analysis of cellular ROS levels (Cell ROX) in naive or in vitro activated (anti-CD3 plus anti-CD28 24 h) OT-I CD8 + T cells from wildtype (WT) and Map3k14 tKO (tKO) mice ( a ) or in OT-I CD8 + T cells freshly isolated from the spleen of day 7 L. monocytogenes (L.M.) -infected ( b ) or in tumor infiltrating CD8 + T cells from day 16 tumor of B16F10-implanted ( c ) wildtype and Map3k14 tKO mice ( b , c , n=4 per genotype). d , e , Flow cytometric analysis of cellular ROS levels in activated (anti-CD3 plus anti-CD28 48h) total T cells from wildtype and NIK iTg mice ( d ) or in tumor infiltrating CD8 + T cells from day-32 MC38-implanted wildtype and NIK iTg mice ( e ) ( e , n=6 per genotype). f , g , Immunoblot analysis of HK2 expression in wildtype or Map3k14 tKO (KO) OT-I CD8 + T cells activated with anti-CD3 plus anti-CD28 for the indicated time periods in the presence of the ROS inhibitor NAM ( f ), GSH and Vit C ( g ) or solvent control DMSO. The right panel of f is a summary graph of densitometric quantification data based on three independent experiments. h , Whole-cell NADPH concentration measured in 1 x 10 6 in vitro activated (anti-CD3 plus anti-CD28 24 h) wildtype or Map3k14 tKO OT-I CD8 + T cells (left), or OT-I CD8 + T cells isolated from the spleen of day 7 L. monocytogenes (L.M.)-infected wildtype and Map3k14 tKO mice (right) (n=4 per genotype). i , Whole-cell NADPH concentration measured with in vitro activated (anti-CD3 plus anti-CD28 48h) CD8 + T cells prepared from wildtype or NIK iTg mice (n=4 per getotype). j , Immunoblot (upper) and qRT-PCR (lower) analysis of G6PD expression in wildtype or Map3k14 tKO OT-I CD8 + T cells activated with anti-CD3 plus anti-CD28 for the indicated time points. k , G6PD activity in wildtype and Map3k14 tKO OT-I CD8 + T cells activated in vitro for 24 h with anti-CD3 plus anti-CD28 (left, n=3 per genotype) or isolated from L. monocytogenes (L.M.)-infected mice (right, n=4 per genotype). l , G6PD activity in total T cells isolated from wildtype or NIK iTg mice injected with tamoxifen for 5 days and activated in vitro with anti-CD3 plus anti-CD28 for 24 h (n=4 per genotype). m , Immunoblot analysis of HK2 and G6PD (using both G6PD and Flag antibodies) in NIK-deficient CD8 + T cells transduced with (+) either an empty vector or Flag-tagged G6PD vector. Data are representative of three independent experiments. Summary data are shown as mean ± s.e.m. with P values determined by two-tailed Student’s t test.

Article Snippet: In brief, the assay was carried out in 20 μl of reaction mixture containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 5 mM MgCl 2 , 0.1uM OA, 500 ng recombinant human G6PD (Novus Biologicals) as the substrate, and different doses (200 ng and 400 ng) of recombinant human NIK protein (ThermoFisher Scientific).

Techniques: In Vitro, Isolation, Infection, Western Blot, Expressing, Solvent, Control, Concentration Assay, Quantitative RT-PCR, Activity Assay, Injection, Transduction, Plasmid Preparation, Two Tailed Test

Journal: Nature immunology

Article Title: NF-κB inducing kinase maintains T cell metabolic fitness in antitumor immunity

doi: 10.1038/s41590-020-00829-6

Figure Lengend Snippet: a-c , Whole-cell NADPH concentration ( a ), immunoblot ( b ), right panel in b is a summary graph of densitometric quantification of three independent experiments, and qRT-PCR ( c ) assays using total T cells isolated from wildtype ( G6pd WT ) or G6PD mut mice, in vitro activated with anti-CD3 plus anti-CD28 for 72 h ( a , b ) or as indicated ( c ) ( a , n=2 per genotype; c , n=4 per genotype). d , Immunoblot analysis using lysates of G6PD WT or G6PD mut T cells, stimulated with anti-CD3 plus anti-CD28 for 48 h in the presence (+) or absence (−) of NAM. Right panel is a summary graph of densitometric quantification of two independent experiments (n=4 per genotype). e , Immunoblot analysis using lysates of G6PD mut CD8 + T cells transduced with either a control vector or Flag-G6PD. f,g , Immunoblot ( f ) and flow cytometric analysis of ROS level ( g ) using total T cells isolated from chimeric mice transferred with G6PD WT or G6PD mut bone marrows, in vitro activated with anti-CD3 plus anti-CD28 for 48 h. h , Flow cytometric analysis of CD4 + and CD8 + T cells in the spleen of G6PD WT and G6PD mut mice. i , ELISA of IFN-γ and IL-2 in the supernatant of G6PD WT and G6PD mut CD8 + T cell cultures stimulated with anti-CD3 plus anti-CD28 for 66 h. j , CoIP analysis of endogenous NIK-G6PD interaction using whole-cell lysates of CD8 + T cells isolated from NIK iTg mice and activated for 48 h with anti-CD3 plus anti-CD28 in the presence of 4OH-tamoxifen. MG132 and BV6 were added during the last 4 h to block NIK degradation. An immunoprecipitation with IgG was included as a negative control. k , l , Summary graph of G6PD activity and NADPH concentration ( k ) and flow cytometric analysis of ROS level ( l ) in splenic CD8 + T cells of chimeric mice adoptively transferred with G6PD mut bone marrow cells transduced with an empty vector (vector) or expression vectors encoding G6PD wildtype (WT) or mutants. m-p , Tumor growth curves ( m , o ) and flow cytometric analysis of tumor-infiltrating CD8 + T cells producing IFN-γ ( n , p ) in chimeric mice adoptively transferred with G6PDmut bone marrow cells transduced with the indicated expression vectors ( m , n , vector: n=7, 1 survived; WT: n=4, 3 survived; S40D: n=5, 5 survived); o , p , WT: n=4, 3 survived; S40A: n=5, 2 survived). q , Immunoblot using T cells of chimeric mice adoptively transferred with G6PD mut bone marrow cells that had been transduced with an empty vector or HK2. r , Flow cytometric analysis of IFN-γ-producing CD8 + T cells derived from chimeric mice of vector- or HK2-transduced G6PD mut bone marrow cells (described in q ), in vitro stimulated for 5 h with PMA plus Ionomycin in the presence of monensin (n=3 per genotype). Data are representative of one ( k-r ), two ( a ,d, e-i ), or three ( b,c, , j ) independent experiments. Summary data are shown as mean ± s.e.m. with P values determined by two-tailed Student’s t test.

Article Snippet: In brief, the assay was carried out in 20 μl of reaction mixture containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 5 mM MgCl 2 , 0.1uM OA, 500 ng recombinant human G6PD (Novus Biologicals) as the substrate, and different doses (200 ng and 400 ng) of recombinant human NIK protein (ThermoFisher Scientific).

Techniques: Concentration Assay, Western Blot, Quantitative RT-PCR, Isolation, In Vitro, Transduction, Control, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Immunoprecipitation, Negative Control, Activity Assay, Expressing, Derivative Assay, Two Tailed Test

Journal: Nature immunology

Article Title: NF-κB inducing kinase maintains T cell metabolic fitness in antitumor immunity

doi: 10.1038/s41590-020-00829-6

Figure Lengend Snippet: a , Seahorse analysis of basal ECAR (measured after glucose injection, Glc) and maximum ECAR (measured after oligomycin injection, Oligo) and Seahorse analysis of baseline OCR (no treatment) and maximum OCR (FCCP injection) in G6PD mut or wildtype (WT) control T cells activated by anti-CD3 plus anti-CD28 for 48 h. b,c, Immunoblot analysis of the indicated proteins ( b ) and ROS detection ( c ) in wildtype or Nfkb2 lym1/+ (Lym1/+) CD8 T cells, which were either not treated (NT) or stimulated with anti-CD3 plus anti-CD28 for the indicated time periods. d , CoIP analysis of NIK-G6PD interaction (upper) and immunoblot analysis of HA-NIK and Flag-G6PD expression in whole-cell lysates (WCL) of 293 cells transfected with (+) or without (−) Flag-G6PD and HA-NIK. e , Phos-tag SDS PAGE and Immunoblot analysis of phosphorylated (p-) and total G6PD as well as GST-NIK in an in vitro kinase assay mix containing 500 ng recombinant His-G6PD and the indicated amounts of recombinant GST-NIK. f , NIK-induced G6PD phosphorylation sites identified by mass spectrometry analysis of G6PD phosphorylated in vitro by NIK. g , G6PD activity in 293T cells transiently transfected with Flag-tagged wildtype G6PD or the indicated G6PD mutants along with either an empty vector or HA-NIK. h , i , Schematic of experimental design ( h ) and immunoblot analysis of G6PD expression in T cells isolated from bone marrow chimeric mice constructed using G6PDmut bone marrow cells reconstituted with either an empty vector (Vector) or the indicated G6PD expression vectors ( i ). For a and g, data are shown as representative plots, each circle represents a well. Data are representative of one ( f ) or three ( a-e,g-i ) independent experiments. Summary data are shown as mean ± s.e.m. with P values determined by two-tailed Student’s t test.

Article Snippet: In brief, the assay was carried out in 20 μl of reaction mixture containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 5 mM MgCl 2 , 0.1uM OA, 500 ng recombinant human G6PD (Novus Biologicals) as the substrate, and different doses (200 ng and 400 ng) of recombinant human NIK protein (ThermoFisher Scientific).

Techniques: Injection, Control, Western Blot, Expressing, Transfection, SDS Page, In Vitro, Kinase Assay, Recombinant, Phospho-proteomics, Mass Spectrometry, Activity Assay, Plasmid Preparation, Isolation, Construct, Two Tailed Test