TY - JOUR T1 - Tumor-derived exosomes induce CD8<sup>+</sup> T cell suppressors JF - Journal for ImmunoTherapy of Cancer JO - J Immunother Cancer DO - 10.1186/s40425-017-0269-7 VL - 5 IS - 1 SP - 65 AU - Brian T. Maybruck AU - Lukas W. Pfannenstiel AU - Marcela Diaz-Montero AU - Brian R. Gastman Y1 - 2017/12/01 UR - http://jitc.bmj.com/content/5/1/65.abstract N2 - Background The suppressive nature of immune cells in the tumor microenvironment plays a major role in regulating anti-tumor immune responses. Our previous work demonstrated that a soluble factor from tumor cells is able to induce a suppressor phenotype (SP) in human CD8+ T cells typified by loss of CD27/CD28 expression and acquisition of a potent suppressor function. The present study hypothesized that the soluble mechanism that is inducing the SP in CD8+ T cells are tumor-derived exosomes (TDEs).Methods Membrane vesicles and TDEs from multiple head and neck cancer cell line’s conditioned growth media were isolated by ultracentrifugation and precipitation, respectively. Human purified CD3+CD8+ T cells were assessed for their induction of the T cell SP by flow cytometry identifying loss of CD27/CD28 expression and in vitro suppression assays. Furthermore, the T cell SP was characterized for the attenuation of IFN-γ production. To delineate exosomal proteins contributing to T cell SP, mass spectrometry was used to identify unique proteins that were present in TDEs. CRISPR/Cas9 knockout constructs were used to examine the role of one of these proteins, galectin-1. To assess the role of exosomal RNA, RNA purified from TDEs was nucleofected into CD8+ T cells followed by suppression analysis.Results Using fractionated conditioned growth media, factors &gt;200 kDa induced CD8+ T cell SP, which was determined to be an exosome by mass spectrometry analysis. Multiple head and neck cancer-derived cell lines were found to secrete T cell SP-inducing exosomes. Mass spectrometry analysis revealed that an immunoregulatory protein, galectin-1 (Gal-1), was expressed in those exosomes, but not in TDEs unable to induce T cell SP. Galectin-1 knockout cells were found to be less able to induce T cell SP. Furthermore, RNA purified from the T cell SP-inducing exosomes were found to partially induce the SP when transfected into normal CD8+ T cells.Conclusions For the first-time, TDEs have been identified to induce a SP in CD8+ T cells and their mode of action may be synergistic effects from exosomal proteins and RNA. One protein in particular, galectin-1, appears to play a significant role in inducing T cell SP. Therefore, tumor-derived immunosuppressive exosomes are a potential therapeutic target to prevent T cell dysfunction and enhance anti-tumor immune responses.Abbreviations:BrdUBromodeoxyuridineCaco-2Human colon carcinoma cell lineCGMConditioned growth mediaECLEnhanced chemiluminescenceFBSFetal-bovine serumGal-1Galectin-1MDSCMyeloid-derived suppressor cellsPBSPhosphate-buffered salinePD-1Programed death receptor 1RIPARadioimmunoprecipitation assay bufferSPSuppressor phenotypeTDEsTumor-derived exosomesTim-3T cell immunoglobulin and mucin protein 3TregCD4+ regulatory T cellsTsSuppressor T cellsTu167, SCC0209, and HN60Head and neck cancer cell lines ER -