The role of extracellular vesicles and PD-L1 in glioblastoma-mediated immunosuppressive monocyte induction.
细胞外囊泡和 PD-L1 在胶质母细胞瘤介导的免疫抑制单核细胞诱导中的作用。
- 作者列表："Himes BT","Peterson TE","de Mooij T","Garcia MLMC","Jung MY","Uhm S","Yan D","Tyson J","Li HJL","Parney D","Abukhadra M","Gustafson MP","Dietz AB","Johnson AJ","Dong H","Maus RL","Markovic S","Lucien F","Parney IF
BACKGROUND:Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed death ligand-1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role for EV-associated PD-L1 in the formation of Immunosuppressive monocytes. METHODS:Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T cell proliferation. PD-L1 constitutitive overexpression or shRNA-mediated knockdown was used to determined the role of altered PD-L1 expression. RESULTS:GBM EVs interact with both T cells and monocytes but do not directly inhibit T cell activation. However, GBM EVs induce immunosuppressive monocytes including myeloid-derived suppressor cells (MDSCs) and non-classical monocytes (NCMs). MDSCs and NCMs inhibit T cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes' T cell inhibition. CONCLUSION:These findings indicate GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.
背景: 胶质母细胞瘤 (GBM) 的免疫抑制是有效免疫治疗的障碍。GBM 来源的免疫抑制单核细胞对此至关重要。程序性死亡 ligand-1 (PD-L1) 是一种免疫检查点分子，由 GBM 细胞和 GBM 细胞外囊泡 (EVs) 表达。我们试图确定 EV 相关 PD-L1 在免疫抑制单核细胞形成中的作用。 方法: 收集健康供者的单核细胞，用 GBM 来源的 EVs 条件诱导免疫抑制单核细胞的形成，通过流式细胞仪进行定量。供体匹配的 T 细胞随后与 EV 条件单核细胞共培养，以评估对 T 细胞增殖的影响。PD-L1 组成型过表达或 shRNA 介导的敲除被用来确定改变 PD-L1 表达的作用。 结果: GBM EVs 与 T 细胞和单核细胞均相互作用，但不直接抑制 T 细胞活化。然而，GBM EVs 诱导免疫抑制单核细胞，包括髓源性抑制细胞 (MDSCs) 和非经典单核细胞 (NCMs)。MDSCs 和 NCMs 在体外抑制 T 细胞增殖，原位发现在 GBM 内。EV PD-L1 表达诱导 NCMs 而不是 MDSCs，并且不影响 EV 条件单核细胞的 T 细胞抑制。 结论: 这些发现表明 GBM EV 介导的免疫抑制是通过诱导免疫抑制单核细胞而不是直接 T 细胞抑制发生的，而 PD-L1 表达对诱导特异性免疫抑制单核细胞群体很重要。通过 EVs 的免疫抑制信号机制是复杂的，不限于 PD-L1。
METHODS::Macrophages (MΦ) play a critical role in tumor growth, immunosuppression and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that a macrophage Syk-PI3K axis drives polarization of immunosuppressive MΦs which establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacological inhibition of Syk and/or PI3Kγ in MΦs promotes a pro-inflammatory MΦphenotype, restores CD8+ T cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for Transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow derived macrophages (BMDMs) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first in class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.
METHODS::Dysfunction of invariant natural killer T (iNKT) cells in tumor microenvironment hinders their anti-tumor efficacy, and the underlying mechanisms remain unclear. Here we report that iNKT cells increase lipid biosynthesis after activation, and that is promoted by PPARγ and PLZF synergically through enhancing transcription of Srebf1. Among those lipids, cholesterol is required for the optimal IFN-γ production from iNKT cells. Lactic acid in tumor microenvironment reduces expression of PPARγ in intratumoral iNKT cells and consequently diminishes their cholesterol synthesis and IFN-γ production. Importantly, PPARγ agonist pioglitazone, a thiazolidinedione drug for type 2 diabetes, successfully restores IFN-γ production in tumor-infiltrating iNKT cells from both human patients and mouse models. Combination of pioglitazone and alpha-galactosylceramide treatments significantly enhances iNKT cell-mediated anti-tumor immune responses and prolongs survival of tumor-bearing mice. Our studies provide a strategy to augment the anti-tumor efficacy of iNKT cell-based immunotherapies via promoting their lipid biosynthesis.
METHODS::Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a monoclonal antibody conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, anti-CD25-IR700-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Tregs), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44/CD25 NIR-PIT also resulted in some complete remissions, whereas this was not achieved with either type of NIR-PIT alone. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.