区域免疫细胞Tfr/Tfh平衡失调在慢性移植物抗宿主病中的作用机制研究

Chronic graft-versus-host disease (cGVHD) remains the most common long-term complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and it decreases the success of transplantation by increasing the risk of death and disability. Chronic GVHD is the leading cause of non-relapse mortality in transplant survivors otherwise cured of their diseases, and its adverse effects include physical, functional, and psychosocial deficits, inability to return to work, and poor quality of life (QOL). Progress in improving cGVHD prevention and therapy has been hindered by complexities in cGVHD pathogenesis, lack of uniform treatment response criteria, paucity of controlled trials and access to new therapies with an established proof-of-concept or strong pathophysiological basis in preclinical models. In our previous study, we used mass cytometry analysis to depict an immune atlas of patients with cGVHD compared with that of patients without cGVHD in 42 simultaneous dimensions. Mass cytometry analysis reveals the immune cell diversity between patients with cGVHD and patients without cGVHD. We also found that compared with patients without cGVHD, the number of T follicular help (Tfh) cells was significantly increased, however that of T follicular regulatory (Tfr) cells was decreased, which suggested that the unbalance between Tfr and Tfh cells contributes to the development of cGVHD. Based on our previous study findings, we hypothesized that in the development of cGVHD, co-inhibitory receptors including programmed death-1 (PD-1) and CTL-associated antigen 4 (CTLA-4) paly different roles in regulating the balance between Tfr and Tfh cells. Tfh cells limit Tfr cell differentiation by IL-21 production, which binds the transcription factors STAT3 and IRF4 to induce expression of Blimp1, the transcription factor shown to inhibit Tfr cell differentiation. In the present study, we attempt to elucidate the role and mechanisms of Tfr and Tfh cells, and aberrant Tfr/Tfh homeostasis in the development of cGVHD by clinical studies, ex vivo cell studies, transplantation animal model studies and gene knock-out mouse model studies. Our study may provide valuable information to elucidate pathophysiological basis of cGVHD, which may improve cGVHD prevention and the survival of patients with cGVHD post-HSCT.

慢性移植物抗宿主病（cGVHD）是引起移植患者晚期死亡最常见的原因之一。本课题组在前期研究中，首次利用单细胞质谱流式CyTOF发现滤泡调节性T淋巴细胞（Tfr）/滤泡辅助性T淋巴细胞（Tfh）平衡失调参与cGVHD的病理过程。本项目在前期研究基础上，提出以下假说：免疫检查点分子PD-1和CTLA-4在维持Tfr/Tfh平衡中具有拮抗作用，Tfh细胞通过激活IL21/STAT3/IRF4/Blimp1信号通路，抑制Tfr细胞分化，导致Tfr/Tfh平衡失调，B细胞稳态失衡，触发cGVHD的多脏器损害。本项目拟通过临床研究、体外细胞实验和基因敲除动物移植模型研究，首次明确区域免疫反应细胞Tfr、Tfh、Tfr/Tfh平衡失调在cGVHD中的作用和机制，为最终明确cGVHD的免疫耐受失衡机制，探索靶向特定免疫机制的治疗新策略提供线索。

慢性移植物抗宿主病(cGVHD)是移植后患者非复发性死亡的最主要原因。以闭塞性细支气管炎综合征(bronchiolitis obliterans syndrome，BOS)为表现的肺部cGVHD (cGVHD-BOS) 是cGVHD最严重的类型。目前，cGVHD的病理机制并不十分明确。本研究通过单细胞质谱流式(CyTOF)、单细胞转录组、单细胞TCR测序等技术围绕临床cGVHD患者循环免疫细胞、两种小鼠cGVHD模型开展研究，经过4年的具体实施，基本完成预定工作计划，并取得一些重要进展：(1)基于单细胞质谱流式技术（CyTOF）绘制cGVHD患者的循环免疫细胞图谱；(2)进一步对cGVHD-BOS相关T细胞亚群进行功能分析，发现CD8+GranzymeK (GZMK)+T细胞可能参与了cGVHD-BOS病理过程；(3)构建cGVHD-BOS小鼠模型，通过单细胞转录组测序技术对移植后小鼠肺组织免疫细胞进行检测，发现cGVHD-BOS组小鼠相较于对照组小鼠，肺组织中CD8+GZMK+T细胞浸润明显增加，单细胞TCR测序证实CD8+GZMK+T细胞在cGVHD-BOS小鼠肺部出现了明显的TCR克隆扩增；(4)通过小分子化合物库筛选，发现酪氨酸激酶抑制剂Bosutinib可以有效抑制CD8+GZMK+T细胞，显著缓解cGVHD-BOS小鼠肺部症状，减轻肺组织中血管和细支气管周围的炎症细胞浸润以及胶原沉积现象；(5)以B10.D2小鼠为供鼠，BALB/c小鼠为受鼠，通过异基因骨髓移植的方法成功构建慢性移植物抗宿主病-硬皮病(scleroderma, Scl) (cGVHD-Scl)小鼠模型。cGVHD-Scl小鼠脾脏细胞相较于control组存在不同的免疫细胞分布，且与区域免疫平衡直接相关的滤泡辅助性T细胞(T follicular help, Tfh) /滤泡调节性T细胞(T follicular regulatory, Tfr) 细胞比例明显增高，可能与cGVHD的病理机制有关；(6)发现临床上用来治疗多发性骨髓瘤的抗CD38单克隆抗体-达雷妥尤单抗(Daratumumab, Dara) 对GVHD具有潜在治疗作用，具有上调免疫抑制T细胞：Treg细胞和Tfr细胞的作用。

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