Journal: Nature Biomedical Engineering

Article Title: Ablation of prostaglandin E 2 signalling through dual receptor knockout in CAR T cells enhances therapeutic efficacy in solid tumours

doi: 10.1038/s41551-025-01610-6

Figure Lengend Snippet: ( a ) 5 ×10 6 CD45.1 x CD4 Cre Ptger2 − / − Ptger4 fl/fl OT-I T cells and 5 ×10 6 CD90.1 OT-I T cells were co-injected into D4M.3A-SIINFEKL (10 6 cells s. c.) bearing mice. On days 2, 5, 9 and 14 the abundance of T cell populations was analyzed using flow cytometry; gating strategy depicted. T cell populations in spleen ( b ) and blood ( c ) were analyzed (data shown as mean ± SEM of n = 5 mice per group). Statistical analysis was done with a two-way ANOVA. ( d ) 10 ×10 6 CD4 Cre Ptger2 − / − Ptger4 fl/fl anti-EpCAM-CAR T cells and 10 ×10 6 anti-EpCAM-CAR T cells were intravenously injected into Panc02-OVA-EpCAM (2 ×10 6 cells s. c.) bearing mice. Tumor growth and survival are depicted for n = 5 mice per group. ( e ) Schematic representation of Ep2 −/− Ep4 −/− OT-I T cells production workflow. OT-I T cells were submitted to genetic editing by inserting the RNP complex through electroporation. ( f ) OT-I T cells with Ep2 and/or Ep4 knockout were treated with 2000 ng/ml PGE 2 . CREB phosphorylation was measured by flow cytometry. Depicted is the gating strategy for flow cytometry analysis. ( g ) 10 7 OT-I splenocytes with Ep2 and/or Ep4 knockout were adoptively transferred (i. v.) into recipient mice bearing D4M.3A-SIINFEKL tumors (10 6 cells s. c.). Single tumor growth curves of n = 3 independent repetitions with n = 5 mice per group are shown. Illustrations in e created with BioRender.com .

Article Snippet: The following antibodies and staining reagents were used for flow cytometry or cell sorting: Fixable Viability Dye eFluor 780 (1:1,000, Invitrogen), APC anti-human CD3 (1:100, clone OKT3, BioLegend), Pacific Blue anti-mouse CD3 (1:100, clone 17A2, BioLegend), Pacific Blue anti-mouse CD4 (1:100, clone GK1.5, BioLegend), FITC anti-mouse CD4 (1:100, clone GK1.5, BioLegend), PE-Cy7 anti-human CD4 (1:100, clone OKT4, BioLegend), PerCP anti-human CD8 (1:100, clone HIT8a, BioLegend), Pacific Blue anti-mouse CD8a (1:100, clone 53-6.7, BioLegend), FITC anti-mouse CD8 (1:100, clone 53-6.7, BioLegend), BV605 anti-human CD8 (1:100, clone SK1, BioLegend), BV785 anti-mouse CD8 (1:100, clone 53-6.7, BioLegend), Alexa Fluor 647 mouse anti-CREB (pS133)/ATF-1 (pS63) (1:20, clone J151-21, BD Biosciences), BV650 anti-human CD69 (1:100, clone FN50, BioLegend), PE/Cy7 anti-human CD62L (1:100, clone DREG-56, BioLegend), BV510 anti-human CD44 (1:100, clone IM7, BioLegend), Alexa Fluor 700 anti-human PD-1 (1:100, clone EH12.2H7, BioLegend), BV510 anti-human TIM-3 (1:100, clone F38-2E2, BioLegend), APC anti-human LAG-3 (1:100, clone 7G2C65, BioLegend), PE-Cy7 anti-mouse IFNγ (1:100, clone XMG1.2, eBioscience), BV421 anti-human CD45 (1:100, clone 2D1, BioLegend), BV605 anti-human CD4 (1:100, clone UCHL1, BioLegend), Alexa Fluor 700 anti-mouse CD4 (1:100, clone GK1.5, BioLegend), BV711 anti-mouse CD45.1 (1:100, clone A20, BioLegend), APC anti-rat CD90/mouse CD90.1 (Thy-1.1) (1:100, clone OX-7, BioLegend), BV421 anti-human CD69 (1:100, clone F50, BioLegend), PerCP-Cy5.5 anti-human CD25 (1:100, clone BC96, BioLegend) and FITC anti-human/mouse/rat c-myc (1:100, clone SH1-26E7.1.3, Miltenyi).

Techniques: Injection, Flow Cytometry, Electroporation, Knock-Out, Phospho-proteomics

Journal: Nature Biomedical Engineering

Article Title: Ablation of prostaglandin E 2 signalling through dual receptor knockout in CAR T cells enhances therapeutic efficacy in solid tumours

doi: 10.1038/s41551-025-01610-6

Figure Lengend Snippet: a , 5 × 10 6 CD45.1 × CD4 Cre Ptger2 − / − Ptger4 fl/fl OT-I T cells and 5 × 10 6 CD90.1 OT-I T cells were co-injected into D4M.3A-SIINFEKL (10 6 cells s.c.) bearing mice. b , c , On days 2, 5, 9 and 14, the abundance of T cell populations in tumours ( b ) and lymph nodes ( c ) was determined by flow cytometry (data shown as mean ± s.e.m. of n = 5 mice per group). Statistical analysis was done with a two-way ANOVA. d , OT-I T cells with Ep2 and/or Ep4 knockout were treated with 2,000 ng ml −1 PGE 2 and cAMP levels were determined in a luciferase-based readout (data shown as mean ± s.d. of n = 3 independent experiments with 2 technical replicates each; statistical analysis was done with an ordinary one-way ANOVA). e , OT-I T cells with Ep2 and/or Ep4 knockout were treated with 1,600 ng ml −1 PGE 2 and CREB phosphorylation was measured by flow cytometry. Pooled data of n = 3 representative experiments (mean ± s.d.) with 2 technical replicates each and a representative change of the pCREB MFI are shown. Statistical analysis was done with a two-way ANOVA. Mice bearing D4M.3A-SIINFEKL tumours (10 6 cells s.c.) were treated with 10 7 adoptively transferred (i.v.) OT-I T cells with Ep2 and/or Ep4 knockout. f , g , Tumour growth ( f ) was monitored over time and survival ( g ) was determined. Pooled results of n = 3 independent repetitions with n = 5 mice per group are shown as mean ± s.e.m. Statistical analysis was done with a repeated measurements mixed-effects analysis with Dunnett’s multiple comparison correction ( f ) and log-rank (Mantel–Cox) test ( g ). Panel a created with BioRender.com .

Article Snippet: The following antibodies and staining reagents were used for flow cytometry or cell sorting: Fixable Viability Dye eFluor 780 (1:1,000, Invitrogen), APC anti-human CD3 (1:100, clone OKT3, BioLegend), Pacific Blue anti-mouse CD3 (1:100, clone 17A2, BioLegend), Pacific Blue anti-mouse CD4 (1:100, clone GK1.5, BioLegend), FITC anti-mouse CD4 (1:100, clone GK1.5, BioLegend), PE-Cy7 anti-human CD4 (1:100, clone OKT4, BioLegend), PerCP anti-human CD8 (1:100, clone HIT8a, BioLegend), Pacific Blue anti-mouse CD8a (1:100, clone 53-6.7, BioLegend), FITC anti-mouse CD8 (1:100, clone 53-6.7, BioLegend), BV605 anti-human CD8 (1:100, clone SK1, BioLegend), BV785 anti-mouse CD8 (1:100, clone 53-6.7, BioLegend), Alexa Fluor 647 mouse anti-CREB (pS133)/ATF-1 (pS63) (1:20, clone J151-21, BD Biosciences), BV650 anti-human CD69 (1:100, clone FN50, BioLegend), PE/Cy7 anti-human CD62L (1:100, clone DREG-56, BioLegend), BV510 anti-human CD44 (1:100, clone IM7, BioLegend), Alexa Fluor 700 anti-human PD-1 (1:100, clone EH12.2H7, BioLegend), BV510 anti-human TIM-3 (1:100, clone F38-2E2, BioLegend), APC anti-human LAG-3 (1:100, clone 7G2C65, BioLegend), PE-Cy7 anti-mouse IFNγ (1:100, clone XMG1.2, eBioscience), BV421 anti-human CD45 (1:100, clone 2D1, BioLegend), BV605 anti-human CD4 (1:100, clone UCHL1, BioLegend), Alexa Fluor 700 anti-mouse CD4 (1:100, clone GK1.5, BioLegend), BV711 anti-mouse CD45.1 (1:100, clone A20, BioLegend), APC anti-rat CD90/mouse CD90.1 (Thy-1.1) (1:100, clone OX-7, BioLegend), BV421 anti-human CD69 (1:100, clone F50, BioLegend), PerCP-Cy5.5 anti-human CD25 (1:100, clone BC96, BioLegend) and FITC anti-human/mouse/rat c-myc (1:100, clone SH1-26E7.1.3, Miltenyi).

Techniques: Injection, Flow Cytometry, Knock-Out, Luciferase, Phospho-proteomics, Comparison