LAUSR

Dear Editor, The tumor-microenvironment interactions play important roles in tumor progression, metastasis, and therapeutic resistance, and increasing evidence indicates that tumor cellderived exosomes can systematically modulate or reprogram the tumor microenvironment by transferring molecules, such as microRNAs, mRNAs, and proteins from donor cells to recipient cells. PD-L1 is a classical immune surface protein, which inhibits anti-tumor function of T cells by binding to its receptor programmed cell death-1 (PD-1) and effectively protects tumor from immune surveillance. Exosomes have been reported to contain certain types of proteins, including membrane proteins, e.g., EGFR and MET, that promote cancer metastasis. As a membrane-bound protein, whether PD-L1 exists in cancer cellderived exosomes and whether it plays a role in tumor progress are largely unknown. We isolated exosomes from the supernatant of cell cultures of MDA-MB-231 (231) human breast cancer cells and 4T1 mouse mammary tumor cells with PD-L1 expression or PD-L1 knockout (PD-L1) by sequential centrifugation. Transmission electron microscopy images showed that these exosomes are typically spherical and membrane encapsulated with a size of 30–100 nm (Supplementary information, Figure S1a). PD-L1 was detected in exosomes isolated from the culture media of PD-L1-expressing human breast cancer cells (231-PD-L1) and mouse mammary tumor cells (4T1-PD-L1), but not 231-PD-L1 or 4T1-PD-L1 cells with similar levels of exosome makers, CD63 and CD81, whose expression indicates exosome production (Fig. 1a; Supplementary information, Figure S1b and c). Notably, treatment with exosome secretion inhibitor, GW4869, reduced exosome production (as indicated by the reduction of exosome markers CD63 and CD81 or the total amount of exosomal protein) in 231 cells as well as the levels of PD-L1 in exosomes, but had no effect on PD-L1 expression in the cell lysates (Fig. 1b; Supplementary information, Figure S1d). In addition, in vitro binding assay showed that PD-1Fc protein simultaneously pulled down PD-L1 and CD81 in 231PD-L1-derived exosomes (term as exosome-PD-L1) (Fig. 1c). Immunofluorescence (IF) staining of 231 cells (Fig. 1d) and immunohistochemistry (IHC) double staining of human breast cancer tissues (Supplementary information, Figure S1e) demonstrated co-localization of PD-L1 and CD63 in the multivesicular bodies (MVBs), which are the precursor form of exosomes inside cells before released. These data further supported the presence of PD-L1 in exosomes in vitro and in vivo. To evaluate the biological functions of exosome-PD-L1, we first asked whether it could transfer PD-L1 to other cells with low (MCF7) or no PD-L1 expression (BT549-PD-L1). We detected the transfer of PD-L1 from exosome-PD-L1 to MCF7 or BT549-PD-L1 cells but not from exosomes derived from 231-PD-L1 cells (exosome-PD-L1; Fig. 1e). The acquisition of PD-L1 protein was not a result of PD-L1 gene expression as indicated by the lack of PD-L1mRNA in these cells (Supplementary information, Figure S1f). We also established 231-PD-L1 cells and visually demonstrated the transfer of PD-L1 from 231-PD-L1-derived exosomes to BT549 cells (Supplementary information, Figure S1g). To examine whether this also occurs in vivo, we co-injected mouse 4T1-PDL1 cells with exosomes derived from 4T1-PD-L1 (EX-PDL1), 4T1-PD-L1 (EX-PD-L1), or PBS into the mammary fat pad of BALB/c mice. Tumors were harvested after 5 days. IF staining of tumor tissue sections showed that EX-PD-L1 but not EX-PD-L1 rendered 4T1-PD-L1 cells PD-L1 positive (Supplementary information, Figure S1h). Importantly, results from flow cytometric analysis further revealed that the PD-L1 transported by exosomes was located on the surface of target cells and able to bind to PD-1 (Supplementary information, Figure S1i). These results indicated that exosomes are capable of transferring functional PD-L1 to other cells. Given that PD-L1 of exosomes can directly bind to PD-1 (Fig. 1c), we next examined whether exosomal PD-L1 affects T cell functions. As shown in Fig. 1f, exosome-PD-L1, but not exosome-PD-L1 or PBS, significantly inhibited the T cell killing effect on BT549-PD-L1 cells. Next, to explore whether exosomal PD-L1 inhibits CD3/CD28-triggered T cell activation signaling pathway, we generated T cell blasts by treating peripheral blood mononuclear cells (PBMCs) with phytohemagglutinin (PHA) to induce PD-1 expression. The results showed that exosome-PD-L1 but not exosome-PD-L1 markedly inhibited CD3/CD28-induced ERK phosphorylation and NF-κB activation of T cells in a dosedependent manner (Supplementary information, Figure S2a and b) as well as PHA-induced interleukin-2 (IL-2) secretion (Supplementary information, Figure S2c), all of which are indicators of T cell activation. Furthermore, exosomal PD-L1 from other cancer cell lines such as colon (RKO) and lung (HCC827) cancer cells has similar function in blocking T cell activation (IL-2 production; Supplementary information, Figure S2d and e). Together, these data supported that exosomal PD-L1 inhibits T-cell activation. Next, to evaluate the role of exosomal PD-L1 in tumor microenvironment and tumor progression in vivo, we measured tumor growth of 4T1-PD-L1 cells co-injected with EX-PD-L1, EX-PD-L1, or PBS. Consistent with the previous report, PD-L1 deficiency in 4T1-PD-L1 cells led to substantial tumor regression; however, EX-PD-L1 but not EX-PD-L1 remarkably restored tumor growth of 4T1-PD-L1 cells (Fig. 1g). We then exposed 4T1PD-L1 cells to increasing amounts of EX-PD-L1 and showed that EX-PD-L1 promoted tumor growth in a dose-dependent manner (Supplementary information, Figure S2f). Moreover, 4T1PD-L1 tumors with EX-PD-L1 co-injection exhibited much less granzyme B expression, indicating reduced cytotoxic T cell activity, in tumor area compared with those with EX-PD-L1 or PBS co-