LAUSR

Dear Editor, PD-L1 is a well-known transmembrane protein, which is highly expressed on many types of cancer cells. By binding to its receptor PD-1 on T cells, PD-L1 significantly inhibits T cells activation and activity, and thus plays a pivotal role in driving the escape of tumor cells from immune surveillance. Antibody blockade of PD-L1/PD-1 interaction has revolutionized cancer therapy with promising clinical outcomes in many cancer types, including melanoma, lung cancer, bladder cancer, colorectal cancer, and renal-cell cancer. However, in some others such as prostate cancer, ovarian cancer, and breast cancer, the response rate of PD-L1/PD-1 antibody therapy is less satisfactory. Recent studies have shown that PD-L1 can be regulated by post-translational regulations such as ubiquitination, phosphorylation and glycosylation, providing opportunity for marker-guided effective combinational therapy with immune checkpoint therapy. Palmitoylation is one of the coand post-translational modifications of proteins in which a palmitate is covalently linked to a cysteine residue as vast majority via a thioester linkage (also known as S-palmitoylation). By affecting protein membrane anchoring, trafficking, interaction and degradation, palmitoylation plays important roles in human physiological and pathological processes, including cancers. For instance, several cancerrelated proteins, such as EZH2, TEAD, and c-Met, are palmitoylated for stabilization, and knockdown of ZDHHC5, a palmitoyltransferase of EZH2, significantly inhibits glioma tumor growth. Thus, palmitoylation or palmitoyltransferases could be novel targets for cancer therapy. In the current study, we searched for other possible metabolic-related modifications of PD-L1 such as lipid modification, and unexpectedly discovered that palmitoylation occurred on PD-L1 and played an important role in PD-L1 stability. Targeting PD-L1 palmitoylation sensitized tumor cells to T-cell killing and inhibited tumor growth. Previously, we reported that saccharide regulates PD-L1 stability via glycosylation. Recently, lipid modification has also been shown to play an important role in regulation of cell membrane proteins, we were curious whether PD-L1 might also be regulated by lipid modification. Since palmitoylation is an important and broadly studied post-translational lipid modification of proteins, we first explored the possibility whether PD-L1 is regulated by palmitoylation. We treated breast cancer cell lines MDA-MB231 and BT549 with a general palmitoylation inhibitor, 2-bromopalmitate (2-BP). As shown in Fig. 1a, b, PD-L1 protein level significantly decreased upon 2-BP treatment in a doseand time-dependent manner, suggesting that PD-L1 expression is regulated by protein palmitoylation. On the basis of prior studies, which indicated that palmitoylation could regulate protein stability, we speculated that PD-L1 itself undergoes palmitoylation to maintain stability. To validate this hypothesis, we subjected PD-L1-expressing MDA-MB-231 (MB231-PD-L1) and BT549 (BT549-PD-L1) breast cancer cells to acyl-biotin exchange (ABE) assays in which free cysteine thiol groups of the proteins are irreversibly blocked by N-ethylmaleimide (NEM), whereas palmitoylated cysteines are later cleaved by hydroxylamine (HAM) and biotinylated. Palmitoylation of PD-L1 was detected using streptavidin-HRP, following immunoprecipitation with α-Flag beads (Fig. 1c). Consistently, by using ABE assay, the palmitoylation of PD-L1 was confirmed in primary human tumor samples (Fig. 1d). These results unveiled a novel post-translational modification of PD-L1. To further investigate the role of palmitoylation on PD-L1, we used an online software CSS-Palm (csspalm.biocuckoo.org) to predict the palmitoylation site(s) on PD-L1. The result revealed a single palmitoylation site at Cys272 located in cytosolic domain of PD-L1 that is highly conversed among different species (Supplementary information, Fig. S1a). On the basis of the above results, endogenous PD-L1 in MDA-MB231 and BT549 cells was knocked out and substituted with PD-L1 or PD-L1 (termed as MB231-PD-L1, MB231-PD-L1, BT549-PD-L1, and BT549-PD-L1). Results of ABE assay revealed that mutation of Cys272 to Ala substantially abolished PD-L1 palmitoylation (Fig. 1e), suggesting that this cysteine residue is the major palmitoylation site of PD-L1. Furthermore, in line with 2-BP treatment, which blocks palmitoylation, PD-L1 exhibited a faster turnover rate as well as less cell surface distribution than PD-L1 in both MB231 and BT549 cells treated with protein synthesis inhibitor cycloheximide (CHX) (Fig. 1f; Supplementary information, Fig. S1b). Given that PD-L1 induces T-cell exhaustion through binding to its receptor PD-1, we next investigated whether palmitoylation of PD-L1 affects T-cell killing activity. In order to adequately mimic the in vivo circumstance, we first constructed 4T1-mPD-L1 and 4T1-mPD-L1 Flag mouse cell lines with endogenous mPD-L1 knocked out and substituted with mPD-L1 or mPD-L1. Consistent with the results in MB231 and BT549 cells (Fig. 1e, f; Supplementary information, Fig. S1b), mutation of Cys272 to Ala dramatically abolished mPD-L1 palmitoylation (Fig. 1g), resulting in much less protein level of mPD-L1 (Fig. 1h) as well as its cell surface distribution (Supplementary information, Fig. S1c). It is worth noting that compared with that observed in MB231 and BT549 cells, mPD-L1 was substantially less stable in 4T1 cells (Fig. 1f vs. 1h; Supplementary information, Fig. S1b vs. S1c), which could be attributed to faster degradation of PD-L1 in 4T1 cells (Supplementary information, Fig. S1d). Next, we isolated CD8 T cells from 4T1 tumor infiltrating lymphocytes (TILs) and performed T-cell killing assay with 4T1-mPD-L1 and 4T1mPD-L1 cells. As shown in Fig. 1i, 4T1-mPD-L1 cells were more sensitive to T-cell killing than 4T1-mPD-L1 cells, with results that were similar to mPD-L1 antibody blockade. Together, our data suggested that palmitoylation at Cys272