Fig. 6. ISR-dependent translation of PD-L1 requires the alternative translation initiation factor eIF5B.

a,b,c, Western blots of H1944 cells expressing the indicated sgRNAs and transfected with siRNAs targeting EIF2D (a), EIF2A (b), or EIF5B (c). d, Quantification of western blots shown in a-c normalized to protein levels in control siRNA-treated cells. e, Western blots of LLC cells expressing the indicated shRNAs targeting Urod, or Eif5b. All experiments (a-c and e) were performed two independent times with similar results, data shown are from a representative experiment. f, Quantification of tumor volumes of LLC cells expressing the indicated shRNAs in syngeneic C57BL/6 mice. Tumor volumes of individual mice are shown (n=12 Scrambled shRNA, n=7 Eif5b shRNA, n=11 Urod shRNA and n=6 Eif5b + Urod shRNA mice). p value (Scrambled shRNA vs Eif5b shRNA = 0.0251, p value (Scrambled shRNA vs Urod shRNA) = 4.075e-10, p value (Urod shRNA vs Urod + Eif5b shRNA) = 8.666e-12. A linear mixed effect model was used to determine statistical significance, as described in Statistics and Reproducibility. g, Western blot analysis in H1944 cells expressing eGFP or EIF5B cDNA. h, qRT-PCR analysis of EIF5B and PD-L1 in H1944 cells shown in g. Bar graph represents mean relative expression of EIF5B and PD-L1 from 3 technical replicates, with individual data points plotted. Experiments in g and h were performed two independent times with similar results, data shown are from a representative experiment. i, Dual luciferase reporter analysis of the PD-L1 5′ UTR in H358 cells with transient overexpression of a control eGFP or EIF5B cDNA. Error bars represent SDs from the mean relative luciferase activity (Firefly/Renilla) across n=3 biological replicates/group. A student’s two-tailed t-test was performed to assess statistical significance with **p = 0.005. j, Dual luciferase reporter analysis of the PD-L1 5′ UTR in MEFs expressing a control eGFP or EIF5B cDNA. Error bars represent SDs from the mean relative luciferase activity (Firefly/Renilla) across n=3 biological replicates/group. A student’s two-tailed t-test was performed to assess statistical significance with ****p = 0.000083. Experiments in i and j were performed two independent times with similar results. Data from representative experiments are shown. k, Model of translational control of PD-L1 in response to ISR activation. Under normal conditions, a functional ternary complex consisting of eIF2α bound to GTP and Met-tRNA initiates translation of PD-L1 at the canonical AUG. Inhibitory upstream open reading frames (uORFs) suppress translation at the canonical AUG, limiting PD-L1 translation. Phosphorylation of eIF2α and ISR activation induce bypass of inhibitory uORFs in the human PD-L1 5′ UTR. In the absence of a functional ternary complex, eIF5B can substitute for eIF2α, culminating in enhanced PD-L1 translation and suppression of anti-tumor immunity.