不变自然杀伤 T (iNKT) 细胞是 T 淋巴细胞的一小部分，具有很强的细胞毒性和免疫调节特性。我们之前表明，人类培养扩增的 iNKT 细胞可防止同种异体反应并裂解原发性白血病母细胞。在这里，基于免疫调节能力，iNKT 细胞比 T 细胞有几个优势。由于嵌合抗原受体 (CAR) 增加了免疫效应细胞的益处，因此它们在提高 iNKT 细胞等新型细胞疗法的细胞毒能力方面发挥着至关重要的作用。在本研究中，我们研究了 NK 细胞的反式激活以及通过 CD19 定向 CAR 转导的 iNKT 细胞预防同种异体反应。 iNKT 细胞通过磁性细胞分离技术从外周血单核细胞中分离出来，并用 CD19-CAR 逆转录病毒转导。通过流式细胞术测量转导效率、纯度和细胞亚群。已经建立了反式激活和细胞毒性测定法来研究 CD19-CAR-iNKT 细胞反式激活原代 NK 细胞的能力。进行混合淋巴细胞反应（MLR）以探讨 CD19-CAR-iNKT 细胞对同种异体反应性 CD3 T 细胞的抑制作用。 CD19-CAR-iNKT 细胞能够不依赖于细胞接触而反式激活 NK 细胞：用 CD19-CAR-iNKT 细胞预处理的 NK 细胞中，激活标记物 CD69 的表达显着增加，促炎细胞因子干扰素-γ 的产生也更高。因此，此类 NK 细胞的细胞毒活性显着增加，能够比没有事先反式激活更有效地裂解白血病细胞。将 CD19-CAR-iNKT 细胞添加到 MLR 中，会导致用 HLA 不匹配的树突状细胞刺激的同种异体反应性 CD3 T 淋巴细胞上 T 细胞激活标记物 CD25 的表达降低。此外，在这种情况下，同种异体反应性 CD3 T 淋巴细胞的增殖显着减少。我们证明，尽管用 CAR 转导，CD19-CAR-iNKT 细胞仍保持其免疫调节特性，使其成为同种异体造血细胞移植后应用的有吸引力的效应细胞群。通过反式激活 NK 细胞、增加其细胞毒性活性并抑制同种反应性 T 细胞，它们可能会通过预防复发和移植物抗宿主病进一步改善治疗结果。版权所有 © 2024 国际细胞学会

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.