前凋亡蛋白肿瘤坏死因子相关凋亡诱导配体（TRAIL）在免疫细胞中生理表达，并在感染、自身免疫疾病和癌症中发挥调节功能，在其中作为肿瘤抑制剂。来自脂肪的间充质干细胞（AD-MSC）在原发和获得性免疫反应中也可能扮演免疫调节角色。我们此前已经证明了一种基于工程化AD-MSC分泌可溶性TRAIL变体（sTRAIL）的抗癌基因治疗的疗效，用于胰腺癌。然而，AD-MSC sTRAIL对白细胞亚群的影响尚未被考虑，以预测这种基于细胞的抗癌策略的可能的免疫毒性谱。 单核细胞、多形核细胞和T淋巴细胞从健康献血者的外周血中新鲜分离。流式细胞仪检测了免疫表型和功能性（DR4和DR5）以及诱骗（DcR1和DcR2）TRAIL受体。然后通过代谢试验和流式细胞仪评估了接受基因修饰AD-MSC释放的sTRAIL或与AD-MSC sTRAIL共培养的白细胞的存活率。此外，通过多重酶联免疫吸附测定检测了共培养中的细胞因子。 单核细胞和多形核细胞分别对DR5和DcR2高度阳性，而T细胞对所有TRAIL受体的表达均极小。不考虑细胞膜上TRAIL受体的存在，白细胞对基因修饰AD-MSC分泌的sTRAIL所显示的前凋亡效应不敏感，而AD-MSC sTRAIL直接与T细胞和单核细胞接触对其存活率的影响很小。在T细胞和AD-MSC sTRAIL共培养中，突显了淋巴细胞分泌的白细胞介素10、肿瘤坏死因子α和干扰素γ以及AD-MSC释放的血管内皮生长因子A和白细胞介素6之间的细胞因子相互作用。 总之，本研究证明了基于表达前凋亡分子sTRAIL的AD-MSC的抗癌方法的免疫安全性，从而证明了其在临床上的可行性。版权所有©2023年国际细胞与基因治疗学会。由Elsevier Inc.出版。版权所有。

The proapoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is physiologically expressed by immune cells and performs regulatory functions in infections, autoimmune diseases and cancer, where it acts as a tumor suppressor. Adipose-derived mesenchymal stromal cells (AD-MSCs) also may play immunomodulatory roles in both primary and acquired immune responses. We have previously demonstrated the efficacy of an anticancer gene therapy based on AD-MSC engineered to secrete a soluble TRAIL variant (sTRAIL) against pancreatic cancer. However, the impact of AD-MSC sTRAIL on leukocyte subsets has been not yet considered also to predict a possible immunotoxicity profile in the clinical translation of this cell-based anticancer strategy.Monocytes, polymorphonuclear cells and T lymphocytes were freshly isolated from the peripheral blood of healthy donors. Immunophenotype and functional (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors were tested by flow cytometry. The viability of white blood cells treated with sTRAIL released by gene-modified AD-MSC or co-cultured with AD-MSC sTRAIL was then evaluated by both metabolic assays and flow cytometry. In addition, cytokine profile in co-cultures was analyzed by multiplex enzyme-linked immunosorbent assay.Monocytes and polymorphonuclear cells showed high positivity for DR5 and DcR2, respectively, whereas T cells revealed negligible expression of all TRAIL receptors. Irrespective of TRAIL receptors' presence on the cell membrane, white blood cells were refractory to the proapoptotic effect displayed by sTRAIL secreted by gene-modified AD-MSC, and direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on T-cell and monocyte viability. Cytokine crosstalk involving interleukin 10, tumor necrosis factor alpha, and interferon gamma secreted by T lymphocytes and vascular endothelial growth factor A and interleukin 6 released by AD-MSC was highlighted in T-cell and AD-MSC sTRAIL co-cultures.In summary, this study demonstrates the immunological safety and thus the clinical feasibility of an anticancer approach based on AD-MSC expressing the proapoptotic molecule sTRAIL.Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.