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The inherent abilities of natural killer (NK) cells to recognize and kill target cells place them among the first cells with the ability to recognize and destroy infected or transformed cells. Cancer cells, however, have mechanisms by which they can inhibit the surveillance and cytotoxic abilities of NK cells with one believed mechanism for this: their ability to release exosomes. Exosomes are vesicles that are found in abundance in the tumor microenvironment that can modulate intercellular communication and thus enhance tumor malignancy. Recently, our lab has found cancer cell exosomes to contain the inhibitor of apoptosis (IAP) protein survivin to be associated with decreased immune response in lymphocytes and cellular death. The purpose of this study was to explore the effect of survivin and lymphoma-derived survivin-containing exosomes on the immune functions of NK cells. NK cells were obtained from the peripheral blood of healthy donors and treated with pure survivin protein or exosomes from two lymphoma cell lines, DLCL2 and FSCCL. RNA was isolated from NK cell samples for measurement by PCR, and intracellular flow cytometry was used to determine protein expression. Degranulation capacity, cytotoxicity, and natural killer group 2D receptor (NKG2D) levels were also assessed. Lymphoma exosomes were examined for size and protein content. This study established that these lymphoma exosomes contained survivin and FasL but were negative for MHC class I-related chains (MIC)/B (MICA/B) and TGF-β. Treatment with exosomes did not significantly alter NK cell functionality, but extracellular survivin was seen to decrease natural killer group 2D receptor (NKG2D) levels and the intracellular protein levels of perforin, granzyme B, TNF-α, and IFN-γ.

Heather R Ferguson Bennit,1,2 Amber Gonda,1,3 Laura J Oppegard,2 David P Chi,2 Salma Khan,1,2 Nathan R Wall1,2 1Center for Health Disparities & Molecular Medicine, 2Division of Biochemistry, Department of Basic Sciences, 3Department of Anatomy, Loma Linda University School of Medicine, Loma Linda, CA, USA Abstract: Exosomes are nanosized lipid vesicles secreted into blood and other body fluids and serve as vehicles for intercellular communication. Despite being an important component of the tumor microenvironment (TME), exosomal targeting and uptake into recipient cells are still not fully understood. Few studies have looked at lymphoma exosomes and their interactions with circulating blood cells. In this study, we examine the exosomal uptake distribution among peripheral blood leukocytes (PBLs) using vesicles derived from a diffuse large B cell lymphoma cell line, WSU-DLCL2. Lymphoma cells survive, proliferate, and are protected from the cytotoxic effects of chemotherapeutic agents by soluble factors or by direct contact with inflammatory and stromal cells within the TME. In an attempt to close the gap in knowledge concerning lymphoma TME immunosuppression, we have treated normal human PBLs with PKH67-labeled lymphoma exosomes and monitored the uptake by measuring fluorescence at different time points using flow cytometry and fluorescent microscopy. Our results show that of the four populations examined, B cells and monocytes demonstrated uptake of PKH67-labeled exosomes, while T cells and NK cells displayed significantly less uptake. Keywords: exosome, non-Hodgkin’s lymphoma, B cell

Tumors alter their microenvironment to promote survival using methods such as angiogenesis promotion, growth signals, and immune suppression. The immune system, whose regular function is to recognize and eradicate infected or abnormal cells, often seems unresponsive to transformed neoplastic cells. Many of the methods employed by tumors to reduce immune function are unclear, but there is evidence for T cell suppression, increased myeloid-derived suppressor cells (MDSCs), and reduced natural killer (NK) cell activity. NK cells are among the first cells to recognize and destroy abnormal cells due to their inherent killing capabilities. However, many types of cancers inhibit the surveillance and cytotoxic abilities of NK cells by releasing exosomes, vesicles that can modulate tumor microenvironment and intercellular communication for the purpose of enhancing tumor malignancy. Exosomes as a means for immunomodulation of tumor microenvironment have been the focus of recent research. These 50-150 nm sized lipid bound vesicles are secreted by many cell types, including immune cells and tumor cells, and the unique protein, lipid, mRNA and miRNA contents contribute to the complex intercellular communication occurring between malignant and normal cells. Cancer patients often have increased numbers of exosomes circulating through their body, including patients with hematological malignancies, such as lymphoma. Recently, cancer cell exosomes have been found to contain Survivin, an Inhibitor of Apoptosis (IAP) protein that prevents cell death and induces Th2 polarity shift and decreases proliferation and cytotoxicity of CD8+ T lymphocytes. The purpose of this study is to explore the effect of Survivin and cancer cell-derived exosomes, which contain Survivin, on the immune functions of NK cells. The hypothesis is that Survivin and exosomes will decrease NK cell functions by suppressing NK cell production or release of cytokines and cytotoxic granules. NK cells were obtained from peripheral blood using magnetic NK cell isolation kit from Miltenyi Biotec and exosomes were obtained from conditioned media from two lymphoma cell lines (WSU-DLCL2 and WSU-FSCCL) using ExoQuickTM (System Biosciences). Flow cytometry methods were used to evaluate degranulation capacity by measuring levels of CD107a, as well as expression of activating receptor NKG2D, cytotoxic granules (perforin, Granzyme B) and cytokines (TNF-α, IFN-γ). RNA message expression was investigated using both block RT-PCR and qPCR. Results showed little significant difference in ability to degranulate when exposed to stimuli, and NKG2D levels did not change after exposure to Survivin or lymphoma exosomes. Although ImageJ analysis of block PCR showed some instances of decreased band densities in some donors treated with Survivin protein, RNA levels in general did not show a trend between NK donors in response to treatment. However, protein expression of Granzyme B, perforin, TNF-α, and IFN-γ did appear to decrease after Survivin treatment. This work is still in the early stages and results are inconsistent due to donor variability. More samples are required, as well as further studies on NK cell functional cytotoxicity. Citation Format: Heather R. Ferguson Bennit, Amber Gonda, Jenniffer Licero Campbell, Laura Oppegard, David Chi, Nathan R. Wall. Effects of survivin and lymphoma cell-derived exosomes on natural killer cell function. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A03.