Fig. 4. ACLY is a critical effector molecule downstream of PD-1.
a, Schematic of glucose flux to histone acetyl groups. b, Flow cytometry for H3K27ac in ITK–SYK+ cells after 4 h of in vitro culture with the ACLY inhibitor BMS-303141 (‘iACLY’, 15 μM) or DMSO. ITK-SYK+ cells were sorted by flow cytometry from ITK-SYKCD4-CreERT2 mice on day 5 after tamoxifen injection and 12 h of anti-PD-L1 or isotype control antibody treatment (intraperitoneal, 200 μg). P, paired two-sided Student’s t-test. Data are presented as the mean ± s.d. c, Western blot analysis in lysates from ITK–SYK+PD-1+ cells. Spleen-derived single-cell suspensions were sorted by flow cytometry from ITK-SYKCD4-CreERT2 mice on day 5 after tamoxifen injection and cultured in vitro overnight with anti-PD-L1 or isotype control antibodies and the PI3K inhibitors (‘iPI3K’) wortmannin (20 nM), LY294002 (20 μM) or DMSO. d, Flow cytometry for CD4 and eGFP in viable lymphocytes derived from spleens of acutely induced ITK-SYKCD4-CreERT2;Pdcd1−/− mice after 3 d of in vitro culture with BMS-303141 (5 μM) or DMSO. e, Fold change in viable ITK–SYK-expressing eGFP+ cells in medium supplemented with BMS-303141 (5 μM) or DMSO. Spleen-derived lymphocytes, sorted by flow cytometry from ITK-SYKCD4-CreERT2 and ITK-SYKCD4-CreERT2;Pdcd1−/− mice on day 5 after tamoxifen injection, were incubated for 3 d in vitro. P, two-sided Student’s t-test. Data are presented as mean ± s.d. f, Western blot for ACLY in ITK–SYK+PD-1− cells electroporated with Acly-targeting (sgACLY) or non-targeting control (sgMOCK)RNP complexes. Protein lysates were generated after 48 h of in vitro culture. g, Survival of recipient mice transplanted with Acly (‘sgACLY’) or mock-edited (‘sgMOCK’) ITK–SYK+PD-1− cells. P, two-sided log-rank test. h, Gene-wise global H3K27ac scores from anti-H3K27ac ChIP–seq in a 1.5-kb window around the TSS. ITK–SYK+ cells were sorted by flow cytometry from ITK-SYKCD4-CreERT2 and ITK-SYKCD4-CreERT2;Pdcd1−/− mice on day 5 after tamoxifen injection, fixed and processed for anti-H3K27ac ChIP–seq analysis. b, Representative data from three independent experiments with three biological replicates per group. c, Representative data from two independent experiments with two biological replicates. d,e, Representative data from three independent experiments. f,g, Data from one experiment with n = 5 mice per group. h, Data from a single experiment with two biological replicates per group.