半相合造血干细胞移植（haplo-HSCT）目前是治疗恶性血液病的有效方法，但缓慢的免疫重建触发的免疫缺陷和严重感染是高死亡率和移植失败的主要原因。其中之一的突出问题是胸腺损伤，与移植反应性移植物抗宿主病（GVHD）和预处理（包括辐射和化疗）有关。因此，加速损伤的胸腺的修复和移植后的胸腺来源供体T细胞的快速增殖是解决问题的关键。本研究旨在加速移植后胸腺来源T细胞的恢复。在haplo-HSCT小鼠模型中，采用具有正常免疫力的野生型小鼠作为受体，模拟临床haplo-HSCT。建立了一种改进的细胞培养系统，使用Notch配体Delta4和IL-7，能够在体外诱导和扩增造血干细胞分化和增殖为前体T（preT）细胞。haplo-HSCT方案包括preT和G-CSF移动的供体脾单个细胞核（MNC）共同输注或仅输注MNC。通过多色免疫荧光追踪、流式细胞术和T细胞受体Vβ检测，比较了两组中的胸腺GVHD、胸腺修复和胸腺来源T细胞发育情况。观察了异基因preT细胞的胸腺寄生和T细胞再生。证明了preT细胞在加速免疫重建、恢复胸腺结构、减弱GVH效应和增强免疫耐受性等方面的功能。进一步研究发现，由含有IL7和Delta4的培养系统诱导的异基因preT细胞高度表达ccr9和RANKL。有趣的是，在preT细胞的胸腺寄生后促进了RANK表达。这些结果表明RANK/RANKL途径可能在胸腺寄生中发挥重要作用。我们的结果为优化haplo-HSCT提供了潜在的治疗选择。它进一步开辟了一种新的T细胞治疗领域，用于在体外人工诱导异基因前体T细胞，修复因辐射和化疗而受损的胸腺，并补偿由多种原因引起的免疫缺陷患者的免疫功能恢复。 版权所有 © 2023 Elsevier Inc.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is currently an effective treatment for malignant hematological disease, but the immune deficiency and severe infection triggered by slow immune reconstitution are the main causes of high mortality and transplant failure. One of these outstanding problems is thymus damage, which is associated with graft-versus-host disease (GVHD), and preconditioning including irradiation and chemotherapy. Therefore, rapid repair of damaged thymus and rapid proliferation of thymus-derived donor T cells after transplantation are key to solving the problem. This study is designed to accelerate the recovery of thymus-derived T cells after transplantation. Wild-type mice with normal immunity were used as recipients in a haplo-HSCT mouse model to mimic clinical haplo-HSCT. A modified cell culture system using Notch ligand Delta4 and IL-7 was established that is capable of inducing and amplifying the differentiation and proliferation of hematopoietic stem cells into precursor T (preT) cells in vitro. Haplo-HSCT protocol included the preT and G-CSF mobilized donor splenic mononuclear cells (MNC) co-infusion or MNC alone. Thymic GVHD, thymic repair, and thymus-derived T cell development were compared in two groups by polychromatic immunofluorescence tracking, flow cytometry and detection of T cell receptor Vβ. The thymus homing and T-cell regeneration of allogenic preT cells were observed. The functions of preT cells in accelerating immune reconstitution, restoring thymic architecture, weakening GVH effects, and enhancing immuno-tolerance after transplantation were demonstrated. Further studies revealed that allogeneic preT cells induced by a culture system containing IL7 and Delta4 highly express ccr9 and RANKL. Interestingly, the RANK expression was promoted after preT cells' thymus homing. These results suggested that the RANK/RANKL pathway may play an important role in thymus homing. Our results provide a potential therapeutic option to optimize haplo-HSCT. It further opened up a new field of T cell therapy for artificial induction of allogeneic precursor T cells in vitro to repair the damaged thymus from irradiation and chemotherapy, and to compensate for the recovery of immune function in patients with immune deficiency caused by multiple reasons.Copyright © 2023. Published by Elsevier Inc.