Tumor-associated macrophages mediate immune suppression by secreting PD-L1+ exosomes in epithelial ovarian cancer

Purpose: To study the role of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) signaling pathway in patients with epithelial ovarian cancer (EOC). Methods: A total of 10 EOC specimens and 10 benign ovarian tumor were obtained from surgery and the pathological type. We used the methods of immunofluorescence confocal microscopy, western blot, MTT assay, apoptosis detection and co-culture to verify the aim of the research. Results: In the present study, it was validated that the number of PD-L1+ tumor-associated macrophages (TAMs) per field was significantly increased in EOC tissues compared with benign ovarian tumor tissues. Furthermore, it was demonstrated that PD-L1 was expressed on the membrane of TAM-derived exosomes, which may inhibit the proliferation and induce the apoptosis of T cells by activating the caspase 3 signaling pathway. The analysis of the supernatant of T cells co-cultured with TAM drived exosome revealed that the levels of pro-inflammatory cytokines and tumor necrosis factor α decreased compared with those T cells co-cultured with monocyte drived exosome. However, the expression of the immuno-suppressive cytokine, interleukin 10 and markers of T cell exhaustion (the inhibitory molecule lymphocyte activated gene-3, T-cell immunoglobulin and mucin domain-containing protein-3 and PD-1) increased. Conclusions: The present study demonstrated that the M2-derived exosomes regulate immune suppression in the EOC microenvironment. The findings of the present study provide a theoretical basis for future target therapy on exosomes from immune cells to treat EOC. Total Protein Lysis buffer (as manufacturer's instruction). The detection of PD-L1 was performed using an anti-PD-L1 antibody (ab58810, Abcam, Cambridge, UK) by western blot analysis. CD63 was used as a control reference.

PD-1 is expressed by activated T cells and triggers an inhibitory signal via the Src homology 2 domain-containing phosphatase-1 (5,6). PD-1 has two known ligands, PD-L1 (7,8) and PD-L2 (9,10). A number of studies have demonstrated that PD-L1 is selectively expressed on a number of tumor cells and also other cells within the tumor microenvironment in response to inflammatory stimuli (11,12).
In addition, PD-L1 is upregulated in solid tumors, therefore it is able to inhibit cytokine production and the cytolytic activity of PD-1 + (CD)4 + and CD8 + T cells in the tumor microenvironment (11,13,14). In 2012, in a study involving an antibody that specifically binds to PD-L1, Brahmer et al (9) demonstrated that 12-41% of 207 patients with advanced cancer and who received antibody treatment resulted in a prolonged stabilization of disease in non-small-cell lung cancer (NSCLC), melanoma and renal-cell cancer. However, the efficacy of the antibody was not sufficient for ovarian cancer (9).
An interesting question is the difference between the efficacy of the antibody between EOC and other tumors. A previous study supported the existence of metastasis-supportive microenvironments, termed pre-metastatic niches, which are potential metastatic sites that allow the colonization and growth of disseminated tumor cells (15). Furthermore, our previous study revealed that the peritoneum of patients with EOC contained a large number of immune cells, 70% of which were macrophages (16). These macrophages, termed tumor associated-macrophages (TAMs), serve an important function in the regulation of T cell differentiation and function in the tumor microenvironment by delivering microRNAs (miRNAs) through exosomes (unpublished data).
Exosomes are vesicles of endocytic origin, which range between 30 and 100 nm in size (17)(18)(19), and may mediate the intercellular communication by horizontal transfer of information via their cargo. The cargo includes proteins on the membrane, DNA, mRNA and miRNAs (20)(21)(22)(23). Exosomes may arise from numerous cell types, but the biological functions of exosomes remain unknown, as they serve complex and diverse functions in immunobiology. Previous studies demonstrated that exosomes secreted by dendritic cells may stimulate the immune system by antigen presentation, while the tumor-associated exosomes may either promote or inhibit tumor immunity, depending on the pathophysiological context (24)(25)(26). Herbst et al (12) indicated that PD-L1-positive tumor-infiltrating 4 immune cells (macrophages, dendritic cells and T cells) were more common compared with PD-L1positive tumor cells. Since the exosomes secreted by TAMs exhibit specific markers from the TAMs, it was hypothesized in the present study that the PD-1 + TAM-exosomes may regulate T cell dysfunction and apoptosis in the tumor micro-environment and participate in the formation of pre-metastatic niches, therefore contributing to immune evasion and metastasis of EOC cells. In the present study, it was demonstrated that exosomes that were released from TAMs express increased levels of PD-L1 which inhibit the proliferation and induce the apoptosis of T cells by activating the caspase 3 signaling pathway, thus regulating T cell dysfunction in the EOC microenvironment.  Subsequently, M0/M2-exosomes (50 ng/ml) were added and co-cultured for 1,2 and 3 days. The control group was treated with PBS alone. Subsequently, 20 μl MTS Solution Reagent (Promega Biosciences, CA, USA) to each well and incubated for 2 hours at 37°C, 5% CO2 atmosphere. The absorbance was recorded at 490 nm using a 96-well plate reader. and calculated using the standard curve performed on the first day.

Methods
Detection of apoptosis of Jurkat T cells following treatment with M0/M2-exosomes. Jurkat T cells were cultured in 6-well plates (1x10 6 cells/well) with RPMI-1640, supplemented with 10% FBS at 37°C, 5% CO2 atmosphere. Subsequently, M0/M2-exosomes (50 ng/ml) were added and cultured for 3 days. The cells were collected and stained using the annexin V Apoptosis detection kit fluorescein isothiocyanate (eBioscience; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Flow cytometric analysis was performed using a fluorescence activated cell sorter (FACS) Calibur cytometer and data was analyzed with CellQuest software.
PD-L1 expression is significantly increased in M2-exosomes. The expression of PD-L1 was increased in the exosomes that were released by M2 macrophages. A previous study by the authors revealed that the exosomes released by M2 macrophages might regulate the differentiation of T cells via miRNAs (data in submission). Since exosomes carry specific proteins from the host cell, exosomes from M2 macrophages were purified and examined using an electron microscope ( Fig. 2A). Subsequently, PD- vs M0: P<0.001. ANOVA) (Fig. 3).

Exosome released from M2 macrophages regulate the apoptosis of Jurkat T cells. Mediation of T cell
apoptosis by PD-L1 is a novel mechanism for tumor immune escape (4,5). In the present study, the apoptosis of Jurkat T cells was determined following co-culture with M2-exosome/M0 (Thp-1)-exosome  (Fig. 6 B). It was also validated that M2 macrophages were able to induce apoptosis and exhaustion of Jurkat T cells by activating caspase 3 (P=0.011, ANOVA) ( Fig. 6C and D)
Multiple monoclonal antibodies to PD-L1 are under development in clinical trials. It has been demonstrated that BMS-936559 (anti-PD-L1) exhibited therapeutic efficacy in a phase I clinical trial (ClinicalTrials.gov identifier, NCT00729664) (9). In addition, a study of metastatic urothelial bladder cancer identified MPDL3280A (an engineered anti-PD-L1 Immunoglobulin G1 monoclonal antibody) to have marker activity in controlling tumor growth; the objective response rates (ORRs) were 43% for PD-L1 + tumor and 11% for PD-L1tumors. (ClinicalTrials.gov identifier, NCT01375842) (34). Furthermore, in a phase I trial, patients with different types of cancer (melanoma, NSCLC and renal cell carcinoma) responded to MPDL3280A with an ORR of 23% total patients with NSCLC and an ORR of 85% in patients with high PD-L1 expression (NCT01375842) (12). However, ovarian cancer has not been demonstrated to respond to MPDL3280A (9). Therefore, in the present study, it was hypothesized that other types of cells, in addition to tumor cells, may express PD-L1 and contribute to tumor proliferation and metastasis. According to a previous study by the authors, the peritoneum of patients with EOC contained a high number of immune cells, 70% of which were TAM cells. In the present study, it was validated that TAM cells in the EOC tumor microenvironment expressed PD-L1, which was consistent with the results of Herbst et al (12), and that PD-L1 may be expressed on the membrane of exosomes (small vesicles ranging between 30 and 100 nm in size) that are released from TAM cells.
Exosomes are one of the major means of communication employed by immune cells. Exosomes contain a combination of ligands and receptors that may concurrently interact with a number of cellsurface receptors, therefore mediating the exchange of membrane and cytosolic components without cell-cell contact. For example, antigen-presenting cells release exosomes exhibit major histocompatibility complex (MHC) class II/antigen complexes, resulting in the activation of T cells via specific T cell receptor-peptide interactions (35,36). Additionally, mast cell-derived exosomes may stimulate T cells via the MHC class II molecules (37). A previous study in 2012 revealed that the exosomes released from the first trimester and term placenta regulates immune tolerance (6).
Furthermore, it was demonstrated that dendritic cells that were exposed to tumor peptides generated exosomes that were able to induce a T-cell-mediated anti-tumor immune response (26). In the present study, it was observed that the PD-L1 + exosomes released from M2 macrophages, similar to TAM cells in the EOC microenvironment, may inhibit the proliferation of T cells and induce the apoptosis of T cells by activating the caspase 3 signaling pathway. According to Duraiswamy et al (7), it was demonstrated that the expansion of ovarian antigen-specific CD8 + tumor infiltrating lymphocytes was dependent on the amount of PD-L1 signaling by tumor cells, tumor-derived myeloid cells and T regulatory cells. The present results were partly consistent with these findings. Furthermore, the present results offer a better explanation for the low efficacy of molecular targeted therapeutic drugs specific to PD-L1. As exosomes are small and are released in a huge amount, if the PD-L1 on tumor or TAM cells is inhibited, the nomadic and diffused TAM-derived PD-L1 + exosomes may serve their function in immune suppression. The small size of the exosomes enables a diffuse spread of these vesicles, therefore forming pre-metastatic niches on the peritoneum for EOC tumor cells.
In the present study, it was demonstrated that the M2-derived exosomes were able to regulate the The results of the present study identified that TAMs mediated the dysfunction of T cells by releasing PD-L1 + exosomes in the EOC tumor microenvironment, therefore promoting the proliferation of tumor cells and the formation of pre-metastatic niches on the peritoneum. Further investigation on the release of exosomes from immune cells may enable the identification of a novel treatment for EOC.

Funding
The present study was supported by the National Science Foundation of China (grant no. 81372787)

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Dr. Zhou X have done the experiments, Dr. Zhu Q and Dr. Liu L helped to collected the clinical tissues, Dr. Zhang Y analysised the data and Pro. Wang designed and wrote the article.

Ethics approval and consent to participate
The study protocol was approved by the Institutional Review Board of Shanghai First Maternity and Infant Hospital (Shanghai, China).

Consent for publication
All authors have reviewed the final version of the manuscript and approve it for submission.