Abstract
BackgroundThe 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).MethodsMembrane 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.ResultsUsing fractionated conditioned growth media, factors >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.ConclusionsFor 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.
Highlights
The suppressive nature of immune cells in the tumor microenvironment plays a major role in regulating anti-tumor immune responses
Upon demonstrating uptake of tumor-derived exosomes (TDEs) by Suppressor T cells (Ts) cells, we further show that these dysfunction-causing exosomes are secreted by a number of different head and neck cancer cell lines, and contain unique proteins, including galectin-1(Gal-1), which is known to play a role in immune regulation
We lastly demonstrate that RNA purified from exosomes is able to induce the suppressor phenotype when transfected into normal donor CD8+ T cells indicating that there is more than one factor responsible for the dysfunctional processes induced by TDEs
Summary
The suppressive nature of immune cells in the tumor microenvironment plays a major role in regulating anti-tumor immune responses. Recent studies have demonstrated that a large fraction of tumor-resident CD8+ T cells have phenotypic changes associated with exhaustion and dysfunction typified by the loss of expression of the immune co-receptors CD27 and CD28, as well as the expression of immune checkpoint inhibitor proteins such as programed death receptor 1 (PD-1), T cell immunoglobulin and mucin protein 3 (Tim-3), among many others [3,4,5]. The mechanism by which the cytotoxicity of tumorresident CD8+ T cells becomes blunted in the tumor microenvironment is incompletely understood, though it has been associated with the presence of significant populations of immune regulators including CD4+ regulatory T cells (Treg), and suppressive myeloid cells such as tumor-associated macrophages and myeloid-derived suppressor cells (MDSC) [9, 10]
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