Tumor tissue sample

Human ENKTCL and HVLPD tissues were obtained from patients with ENKTCL and HVLPD who had undergone surgical resection in the Department of Dermatology, West China Hospital, Sichuan University, Chengdu, Sichuan, China. And we utilized 5 mycosis fungoides (MF) and 9 inflammatory dermatoses (include 3 pityriasis lichenoides et varioliformis actua, 3 lichen planus and 3 psoriasis) as controls. The study was approved by the Ethics Committee of West China Hospital, Sichuan University, and all aspects of the study complied with the Declaration of Helsinki.

Immumohistochemical staining

Tissue specimens were fixed in neutral buffered formalin and embedded in paraffin for processing. Tissue sections that were stained with hematoxylin–eosin for PD-L1, IL-10, and p-STAT3 were chosen for phenotypic identification of the infiltrating cells by immunohistochemistry according to the routine immunostaining protocols of the Department of Pathology of the West China Hospital. Antibody against PD-L1 was purchased from Cell Signaling Technology (CST, USA); p-STAT3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); IL-10 was purchased from HuaAn Biotechnology Co., Ltd (Huabio, Hangzhou, CN).

Cell culture

CTCL cell lines, Hut-78 (ATCC TIB-161), H9 (ATCC HTB-176), and lymphoblastoid cell lines (LCLs) cells, BL2009 (EBV-positive, ATCC CRL-5961), Human Burkitt lymphoma cells, CA46 (EBV-negative, ATCC CRL-1648) were kindly provided by Procell Life Science & Technology Co., Ltd. Cells were maintained at 37 °C in RPMI 1640 medium, supplemented with 10% heat-inactivated FBS and 2 mM l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, in a humidified atmosphere with 5% CO2. Before cell treatment, cells were seeded (5 × 105 cells/ml) in 6-well plates and grown 24 h at 37 °C with 5% CO2.

Isolation and identification of LCL/Burkitt lymphoma cell-derived exosomes

Exosomes were isolated from the BL2009 and CA46 supernatant by using an Hieff@ Quick exosome isolation kit (for Cell Culture Media, Yeasen, CN) in accordance with the manufacturer’s instructions. These exosomes were then characterized by Western blotting analysis of exosome surface markers, such as CD9, CD63, and CD81.

Transmission electron microscopy on exosomes

Ten milliliters of exosome suspension in 1xPBS was dried onto freshly glow discharged 200-mesh formvar-carbon-coated copper grids (Ted Pella, Redding, CA), negatively stained with 2% aqueous uranyl acetate and observed with a JEM-1400PLUS transmission electron microscope (TEM) (JEM-1400PLUS, Japan Electronics Co. LTD).

Real-time RT-PCR

Total RNA was isolated by using the TRIcom Reagent kit (Tianmo Biotech, CN). The iScript one-step RT-PCR kit with SYBR Green (BioRad, Hercules, CA) was used as a supermix, and 20 ng RNA was used as template on a Bio-Rad MyIQ Single Color Real-Time PCR Detection System (BioRad). The primers used for quantification of EBER, IL-10, PD-L1, and GAPDH mRNA were purchased from Tsingke Biological Technology Co., Ltd.

ELISA

IL-10 cytokine release was measured in cell culture supernatants by the Elikine™ Human IL-10 ELISA Kit (Abbkine, lnc., CN) in accordance with the manufacturer’s instructions.

Western blot analysis

Whole-cell or nuclear extracts were solubilized with 1% sodium dodecyl sulfate buffer and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 4 to 15% gel (Bio-Rad, Hercules, CA). We transferred proteins onto polyvinylidene difluoride membranes and probed them with specific primary antibodies and horseradish peroxidase–conjugated secondary antibodies. Proteins were visualized using an ECL system (Amersham, Little Chalfont, UK). Antibodies against PD-L1 were purchased from Cell Signaling Technology (CST, USA); STAT3, pSTAT3, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); CD9, CD81, CD63 were purchased from HuaAn Biotechnology Co., Ltd (Huabio, Hangzhou, CN).

Flow cytometry analysis of exosomes-treated CTCLs

PD-L1 expressions on the cell surface were detected by flow cytometry. Pacific anti-human PD-L1 antibody (CST, USA) was used.

Treatment of C188-9

The STAT3 inhibitor C188-9 was purchased from Selleck (China). Different concentrations of C188-9 (0, 40, 100, and 200 μM) were added into the culture medium. The cultures were incubated for 2 h followed by the treatment with exosome suspension.

Statistics

Statistical significance was determined using Student t-test or Mann–Whitney U-test. p < 0.05 was considered statistically significant. Curves were analyzed with GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA).