Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: ( a–d ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with or without B16 melanoma cells at an E:T ratio of 2:1 ( a ), with B16 melanoma cells at an E:T ratio of 4:1, 2:1 or 1:2 ( b ), with or without YAC-1, CT26 and LLC tumour cells at an E:T ratio of 1:4 ( c ), or with or without RMA/RMA-S cells at an E:T ratio of 1:4 ( d ) for 18 h before analysis of CD25 expression by flow cytometry. ( e – k ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells for 18 h at an E:T ratio of 2:1, washed and put back into culture with IL15 (7.5 ng/mL) with or without ± IL2 (20 ng/mL) for 18 h before analysis by flow cytometry. ( e ) IFNγ production by CD25 high and CD25 neg NK cells cultured in IL2 alone, with B16 cells or with B16 cells plus IL2. ( f ) IFNγ production in CD25 high NK cells cultured with B16 ± IL2. ( g , h ) Analysis of NK cells cultured with B16 cells + IL2 comparing CD25 high and CD25 neg NK cells (g, left panel) for effector functions, IFNγ production and granzyme (Gnzb) B expression. ( i , j ) Analysis of cell size (FSC), cMyc and CD71 expression, and levels of phosphorylated S6 ribosomal protein (pS6) in CD25 high and CD25 neg NK cells from B16 cells + IL2 co-cultures. ( k,l ) Rates of fluorescent transferrin uptake were measured in CD25 high and CD25 neg NK cells from B16 cells + IL2 co-cultures. Data is representative ( a , c , d , e , g , i , k ) or mean ± SEM (b,f,h,j,l) of 3–5 independent experiments. Data was analyzed using a paired students t -test. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Cultured NK cells were purified using MACS purification EasySepTM Mouse NK cell isolation kit (Stemcell technologies, Vancouver, Canada), prior to stimulation.

Techniques: Cell Culture, Purification, Expressing, Flow Cytometry

Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells and purified T cells at NK:T:B16 ratio of 1:1:2. The T cells were either naïve or αCD3/αCD28-activated T cells. The NK cells were analysed by flow cytometry for cell size and CD71 ( a , b ), NKp46 expression ( c ) IFNγ production and Gnzb expression ( a , d , e ) in CD25 low and CD25 high NK cells as indicated. Data is representative ( a – e ) or mean ± SEM ( b – e ) of 4 independent experiments. Data was analyzed using a paired students t -test. (* p < 0.05, *** p < 0.001, ns non-significant).

Article Snippet: Cultured NK cells were purified using MACS purification EasySepTM Mouse NK cell isolation kit (Stemcell technologies, Vancouver, Canada), prior to stimulation.

Techniques: Cell Culture, Purification, Flow Cytometry, Expressing

Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: Cultured NK cells (6 days in IL15 10 ng/mL) were purified and stimulated with a plate bound α-NK1.1 antibody (10 μg/mL) in media supplemented with low dose IL15 (5 ng/mL) ± IL2 (20 ng/mL) as indicated, with IL2 (20 ng/mL) plus IL12 (10 ng/mL) or left unstimulated (IL15 - 5 ng/mL). Cells were analysed for rates of glycolysis ( a,f,g ) and OXPHOS ( b,d,e ) using the seahorse extracellular flux analyser or by flow cytometry for the expression of CD25 ( c ), CD71 ( h ) or CD98 ( i ). Data is representative ( a–d,f ) or mean ± SEM (a,b,e, g–i ) of 4–5 independent experiments. Data was analyzed using a one way ANOVA and tukey post test (* p < 0.05, ** p < 0.01, *** p < 0.001, ns non-significant).

Article Snippet: Cultured NK cells were purified using MACS purification EasySepTM Mouse NK cell isolation kit (Stemcell technologies, Vancouver, Canada), prior to stimulation.

Techniques: Cell Culture, Purification, Flow Cytometry, Expressing

Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: Cultured NK cells (6 days in IL15 10 ng/mL) were stimulated with a plate bound α-NK1.1 antibody (10 μg/mL) in media supplemented with low dose IL15 (5 ng/mL) ± IL2 (20 ng/mL) as indicated, or left unstimulated (IL15; 5 ng/mL). Inhibitors of glycolysis, 2-deoxyglucose (2DG, 1 mM) or oxalate (2 mM), or OXPHOS, oligomycin (4 nM) were added as indicated. Cells were analysed by flow cytometry for the production of IFNγ ( a,b,e–g ) and the expression of granzyme B ( c-e,g ). ( h,i ) NK cells were purified directly ex vivo from murine splenocytes and stimulated with plate bound α-NK1.1 antibody (unprimed). Alternatively splenocytes were maintained for 18 h in IL15 (10 ng/mL) prior to NK cell purification and stimulation with plate bound α-NK1.1 antibody (primed). Cells were analysed for CD25 expression by flow cytometry. Data is representative ( a,c,h ) or mean ± SEM ( b,d–g,i ) of 3–5 independent experiments. Data was analyzed using a one way ANOVA and tukey post test (* p < 0.05, ** p < 0.01, *** p < 0.001, ns non-significant).

Article Snippet: Cultured NK cells were purified using MACS purification EasySepTM Mouse NK cell isolation kit (Stemcell technologies, Vancouver, Canada), prior to stimulation.

Techniques: Cell Culture, Flow Cytometry, Expressing, Purification, Ex Vivo

Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: ( a–e ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and stimulated with a plate bound α-NK1.1 antibody (10 μg/mL) in media supplemented with low dose IL15 (5 ng/mL) ± IL2 (20 ng/mL) ± rapamycin (20 nM) as indicated, or left unstimulated (IL15; 5 ng/mL). Cells were analysed by immunoblot analysis ( a ), by flow cytometry for levels of phosphorylated S6 ribosomal protein (pS6) ( b ), or the expression of CD71 and CD98 ( e ). Alternatively, rates of OXPHOS ( c ) or glycolysis (d) were measured using the seahorse extracellular flux analyser. Data is representative ( a–e ) or mean ± SEM ( b–d ) of 5–6 independent experiments. Data was analyzed using a one way ANOVA and tukey post test ( b ), a paired students t -test ( c,d ) (* p < 0.05, ** p < 0.01).

Article Snippet: Cultured NK cells were purified using MACS purification EasySepTM Mouse NK cell isolation kit (Stemcell technologies, Vancouver, Canada), prior to stimulation.

Techniques: Cell Culture, Purification, Western Blot, Flow Cytometry, Expressing

Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: ( a–h ) Cultured NK cells (6 days in IL15 10 ng/mL) were stimulated with a plate bound α-NK1.1 antibody (10 μg/mL) plus IL2 (20 ng/mL) ± rapamycin (20 nM) ( a , d ) or ±BCH (25 mM) ( e–h ) as indicated. Cells were analysed by flow cytometry for the production of IFNγ ( a,b,e,f ) or granzyme B expression ( c,d,g,h ). Data is representative ( a,c,e,g ) or mean ± SEM ( b,d,f,h ) of 3–6 independent experiments. Data was analyzed using a paired students t- test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Cultured NK cells were purified using MACS purification EasySepTM Mouse NK cell isolation kit (Stemcell technologies, Vancouver, Canada), prior to stimulation.

Techniques: Cell Culture, Flow Cytometry, Expressing

Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: (a) Cultured NK cells (3 days in IL15 10 ng/mL) were purified and stimulated with a plate bound α-NK1.1 antibody (10 μg/mL) ± IL2 (20 ng/mL) for 24–72 h and analysed by flow cytometry for cell viability. ( b–c ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells or for 18 h, washed and put back into culture with IL2 (20 ng/mL) ± rapamycin for 24 h before analysis of CD25 high NK cells by flow cytometry for phosphorylated S6 ribosomal protein (pS6), IFNγ production and granzyme B expression. ( d–g ) Cultured (6 days in IL15 10 ng/mL) from cMyc fl/fl Tamox-Cre and cMyc wt/wt Tamox-Cre were treated with tamoxifen (0.6 μM) and then co-cultured with B16 melanoma cells with IL2 (20 ng/mL) for 18 h before analysis by flow cytometry for cell size and the expression of CD25, cell size, CD71, granzyme B and granzyme B mRNA by rtPCR ( g ). ( h,i ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells or for 18 h, washed and put back into culture with IL2 (20 ng/mL) ± BCH (25 mM), ± rapamycin (20 nM) as indicated for 24 h before analysis of CD25 high NK cells by flow cytometry for phosphorylated S6 ribosomal protein (pS6), cMyc and cell viability. Data is representative ( b,e,f,g,h,i ) or mean ± SEM ( a,c,d,f,g,i ) of 3–5 independent experiments. Data was analyzed using a one way ANOVA with a tukey post test or a paired students t -test. (** p < 0.01, *** p < 0.001).

Article Snippet: Cultured NK cells were purified using MACS purification EasySepTM Mouse NK cell isolation kit (Stemcell technologies, Vancouver, Canada), prior to stimulation.

Techniques: Cell Culture, Purification, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction