Journal: Journal of Clinical Investigation

Article Title: ATR kinase inhibitor AZD6738 potentiates CD8+ T cell–dependent antitumor activity following radiation

doi: 10.1172/jci96519

Figure Lengend Snippet: Figure 2. CD8+ T cells are required for maximal efficacy of AZD6738 plus radiation in CT26 tumors. (A–D) Response of CT26 over time to treatment with AZD6738, IR, or the combination of AZD6738 plus IR in CD8-depleted BALB/c (A and B) and athymic nude (C and D) mice. Dose and time of administra- tion of AZD6738 were the same as in Figure 1. (A and B) Response in CD-depleted BALB/c mice, with 250 μg anti-CD8 antibody (αCD8) administered on days 1–2. Data represent individual tumor volumes (A) or mean tumor volumes ± SEM (B) from 2 independent experiments. n per arm (mice) = 9 vehicle, 8 AZD6738, 8 IR, 10 AZD6738 + IR. ***P < 0.001, ANOVA with Holm-Šidák multiple-comparisons test comparing change in tumor volume from day 1 to day 15 for AZD6738 + IR vs. AZD6738 and AZD6738 + IR vs. IR. Statistical significance not shown for other comparisons/time points. (C and D) Response in athymic nude mice. Data represent individual tumor volumes (C) or mean tumor volumes ± SEM (D) from 1 experiment. n per arm (mice) = 7 vehicle, 7 AZD6738, 8 IR, 8 AZD6738 + IR. Unpaired, 2-tailed t test comparing change in tumor volume from day 1 to day 14 for AZD6738 + IR vs. IR. Statistical significance not shown for other time points.

Article Snippet: Anti-CD8 antibody (αCD8, clone YTS 169.4, BioXCell) was diluted in 1× PBS and administered i.p. (100 μl volume).

Techniques:

Journal: Journal of Clinical Investigation

Article Title: ATR kinase inhibitor AZD6738 potentiates CD8+ T cell–dependent antitumor activity following radiation

doi: 10.1172/jci96519

Figure Lengend Snippet: Figure 3. AZD6738 attenuates radiation-induced PD-L1 expression in CT26 tumors. (A) Schematic showing schedules of IR, AZD6738, and time points for tumor PD-L1 expression analyses. Dose and time of administration of AZD6738 were the same as in Figure 1. (B) Representative histograms of PD-L1 expres- sion on CT26 tumor cells at day 5 for the designated treatments and isotype control, and corresponding quantitation of the fold change in PD-L1 median flu- orescence intensity (MFI) relative to the average MFI of vehicle controls (within a given experiment). Data from 4 independent experiments (3 for AZD6738), each with 1–4 mice per arm. n = 10 vehicle, 7 AZD6738, 9 IR, 8 AZD6738 + IR. (C) Quantitation of fold change in PD-L1 MFI following treatment in vitro with AZD6738 (300 nM), 6 Gy IR, AZD6738 plus IR, or DMSO control. Data represent 6 independent biological replicates. (D) Quantitation of the percentage of tumor-infiltrating CD8+ T cells expressing IFN-γ or IFN-γ and TNF-α following stimulation with PMA/ionomycin at day 5. Data from 3 independent experi- ments (1 for AZD6738), each with 1–3 mice per arm. n = 6 vehicle, 3 AZD6738, 5 IR, 5 AZD6738 + IR. (B–D) Mean and SD bars shown. *P < 0.05, **P < 0.01, ****P < 0.0001, ANOVA with Tukey’s multiple-comparisons test. Brackets not shown for comparisons that were not statistically significant.

Article Snippet: Anti-CD8 antibody (αCD8, clone YTS 169.4, BioXCell) was diluted in 1× PBS and administered i.p. (100 μl volume).

Techniques: Expressing, Control, Quantitation Assay, In Vitro

Journal: Journal of Clinical Investigation

Article Title: ATR kinase inhibitor AZD6738 potentiates CD8+ T cell–dependent antitumor activity following radiation

doi: 10.1172/jci96519

Figure Lengend Snippet: Figure 4. AZD6738 impacts T cell infiltration in CT26 tumors following radiation. (A) Schematic showing schedules of IR and AZD6738 treatments and time points for tumor-infiltrating lymphocyte (TIL) analyses. Dose and time of administration of AZD6738 were the same as in Figure 1. (B) Quantitation of the number of TIL CD8+ cells per 104 cells stained at days 5, 9, and 12. (C) Quantitation of the number of TIL Tregs per 104 cells stained at days 5 and 9. (D) CD8+/Treg ratios at days 5, 9, and 12. (B–D) Data from 3 independent experiments per time point, each with 1–3 mice per arm. n at day 5 = 7 per arm; n at day 9 = 6 vehicle, 4 AZD6738, 7 IR, 7 AZD6738 + IR; n at day 12 = 6 per arm (7 IR). Mean and SD bars shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ANOVA with Tukey’s multiple-comparisons test. Brackets not shown for comparisons that were not statistically significant.

Article Snippet: Anti-CD8 antibody (αCD8, clone YTS 169.4, BioXCell) was diluted in 1× PBS and administered i.p. (100 μl volume).

Techniques: Quantitation Assay, Staining

Journal: Journal of Clinical Investigation

Article Title: ATR kinase inhibitor AZD6738 potentiates CD8+ T cell–dependent antitumor activity following radiation

doi: 10.1172/jci96519

Figure Lengend Snippet: Figure 5. AZD6738 impacts proliferating splenic and tumor-infiltrating T cells in CT26 tumor–bearing mice. (A) Quantitation of the percentages of pro- liferating (Ki67+) splenic and tumor-infiltrating (TIL) CD8+ T cells at days 5, 9, and 12. (B) Representative contour plots depicting Ki67+ expression on splenic and TIL CD8+ T cells for the designated treatment groups at day 12. (C) Representative contour plots depicting Ki67+ expression on splenic and TIL Tregs for the designated treatment groups at day 5. (D) Quantitation of the percentages of proliferating (Ki67+) splenic and TIL Tregs at days 5 and 9. (B and D) Data from 3 independent experiments per time point, each with 1–3 mice per arm. n at day 5 = 7 per arm; n at day 9 = 6 vehicle, 4 AZD6738, 7 IR, 7 AZD6738 + IR; n at day 12 = 6 per arm (7 IR). Mean and SD bars shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ANOVA with Tukey’s multiple-comparisons test. Brackets not shown for comparisons that were not statistically significant.

Article Snippet: Anti-CD8 antibody (αCD8, clone YTS 169.4, BioXCell) was diluted in 1× PBS and administered i.p. (100 μl volume).

Techniques: Quantitation Assay, Expressing

Journal: Journal of Clinical Investigation

Article Title: ATR kinase inhibitor AZD6738 potentiates CD8+ T cell–dependent antitumor activity following radiation

doi: 10.1172/jci96519

Figure Lengend Snippet: Figure 7. AZD6738 attenuates coexpression of CD8+ T cell exhaustion markers and promotes CD8+ T cell effector function in CT26 tumors following radiation. (A) Representative contour plots depicting PD-1 and LAG-3 expression on splenic and tumor-infiltrating (TIL) CD8+ T cells for the designated treatment groups at day 12. (B) Quantitation of the percentage of TIL CD8+ T cells that coexpress PD-1 and LAG-3 or PD-1 and Tim-3 at day 12. Data from 3 independent experiments per time point, each with 1–3 mice per arm. n at day 12 = 6 per arm (7 IR). (C) Representative contour plots depicting IFN-γ and TNF-α expression by splenic and tumor-infiltrating (TIL) CD8+ T cells for the designated treatment groups following stimulation with PMA/ionomycin at day 12. (D) Quantitation of the percentage of TIL CD8+ T cells that elicit IFN-γ or IFN-γ and TNF-α following stimulation with PMA and ionomycin at days 9 and 12. Day 9 data from 1 experiment with the IR/AZD6738 + IR arms and vehicle/AZD6738 arms staggered and harvested/stained on separate days. n at day 9 = 5 per arm (4 IR). Day 12 data from 3 independent experiments, each with 1–3 mice per arm, with harvesting/staining for all arms performed on the same day within a given experiment. n at day 12 = 5 vehicle, 6 AZD6738, 6 IR, 7 AZD6738 + IR. (B and D) Mean and SD bars shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ANOVA with Tukey’s multiple-comparisons test. Brackets not shown for comparisons that were not statistically significant.

Article Snippet: Anti-CD8 antibody (αCD8, clone YTS 169.4, BioXCell) was diluted in 1× PBS and administered i.p. (100 μl volume).

Techniques: Expressing, Quantitation Assay, Staining