PD-1/CD80+ small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity.


Ethics oversight
The study was conducted strictly based on the guidelines setting forth by the Medical Ethics Committee of Hospital of Stomatology Wuhan University.All patients had signed the informed consents and blood samples from primary head and neck squamous cell carcinoma (HNSCC) patients used in this expriment were collected at the Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology Wuhan University.All participants received a compensation of transportation expanses.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

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All studies must disclose on these points even when the disclosure is negative.Data exclusions No sample was excluded in our analysis.

Replication
All replicates reported in the manuscript are biological replicates.All the statistics reported in the manuscript are based on at least 3 biologically independent replicates.All attempts to replicate the experiments were successful.
Randomization All samples used in this study were randomly allocated into different experimental groups.

Blinding
The assessment of clinical responses for patients was performed independently in a double-blind fashion.For mice studies, the experiments were performed in a blinded fashion when possible.The investigators were not blinded to sample allocation during experiments because the information of the value of tumor size and different treatments among groups was essential and correctly to conduct the studies.Downstream analyses of mouse samples (immunofluorescence staining, flow cytometry and ELISA) were performed in a blinded fashion, which means that people performing the assays were not aware of the treatment groups until the data analyses were completed.

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Antibodies
Antibodies used All primary antibodies used in this study were shown in Supplementary Table 2.
The following primary antibodies were used for western blotting (Immunoblotting).They are listed as antigen first, followed by dilution, host, supplier, catalog number and clone/lot number as applicable.