调节性γδT细胞在急性移植物抗宿主病中的作用及机理研究

Experimental and clinical studies have demonstrated that regulatory T cells (Treg) could be utilized to prevent and treat graft-versus-host disease (GVHD). However, there have been increasing reports that challenge this view by demonstrating that Treg might lead to autoimmune diseases. Treg inducing autoimmune diseases might be associated with the plasticity of Treg, and the plasticity was related to the heterogeneity of Treg population. Therefore, a further subdivision of Treg population is quite necessary. Lately, investigators found a more homogeneous cell population- regulatory γδ T cells (γδ Treg), which belonged to γδ T cells, expressed Foxp3 and CD25, and had immunosuppressive function. Some studies reported that γδ Treg could inhibit the occurrence and development of autoimmune diseases. Our previous study found that the incidence of GVHD in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) was negatively correlated with the number of γδ Treg subsets in transplant donors, suggesting that γδ Treg might have inhibitory effect to GVHD. In this study, the optimal ex vivo expansion system for γδ Treg would be established. To explore the biological characteristics of γδ Treg，as well as the effects and mechanisms of γδ Treg on GVHD, graft-versus-leukemia (GVL) and hematopoietic reconstitution, the expressions of each lymphocyte subset and related cytokines in peripheral blood and target organs of GVHD would be detected in vitro,animal model and recipients of allo-HSCT, using fluorescence activated cell sorter (FACS), polymerase chain reaction (PCR), enzyme-linked immuno sorbent assay (ELISA) and other methods. These studies will supply theoretical basis for γδ Treg in preventing and treating GVHD.

Treg致自身免疫性疾病使其用于防治GVHD受到挑战。Treg致自身免疫性疾病可能与Treg可塑性相关，而可塑性又与Treg不均一性有关，故进一步细分Treg亚群十分必要。新近发现一种比Treg更均一的细胞-γδTreg，它属于γδT细胞，表达Foxp3和CD25，有免疫抑制功能。有研究认为γδTreg可抑制自身免疫性疾病的发生与发展。我们前期研究发现allo-HSCT患者GVHD发生率与供者γδTreg数量负相关，提示γδTreg可能对GVHD有抑制作用。本研究拟①建立γδTreg体外最佳扩增体系；②在体外、动物模型和allo-HSCT患者中，采用流式、PCR和ELISA等方法，检测外周血和GVHD累及组织器官中各淋巴细胞亚群和相关细胞因子的表达等，探讨γδTreg生物学特性，揭示γδTreg对GVHD、GVL和造血重建的影响及机理，为临床开展γδTreg防治GVHD提供理论依据。

异基因造血干细胞移植(allo-HSCT)是治疗血液系统恶性肿瘤和非血液系统恶性肿瘤的主要方法。急性移植物抗宿主病(aGVHD)是移植后常见的并发症，具有较高的发病率和死亡率。近年在正常小鼠和肿瘤患者体内发现了具有免疫抑制功能的调节性γδ T细胞 (γδ Treg)，该群细胞属于γδ+ T细胞，Foxp3表达阳性且具有免疫抑制的作用。新近研究发现γδ Treg数量减少与SLE的疾病发展相关，因此提出γδTreg可能成为自身免疫性疾病新的治疗靶点。然而，关于 γδ Treg在aGVHD发生和发展中的作用目前尚不清楚。. 本课题主要研究内容：建立体外γδTreg最佳诱导扩增体系，探索体外γδTreg生物学特性与功能，探索G-CSF动员对供者γδTreg的影响以及输注不同数量供者γδTreg对患者GVHD的影响，在allo-HSCT患者和动物模型中探索γδTreg对aGVHD发生与发展以及移植物抗白血病(GVL)的影响。. 我们首先研究发现G-CSF动员后外周血Foxp3+Vδ1和CD27+Foxp3+Vδ1亚群比例均显著高于动员前，且动员后供者外周血CD27+Foxp3+Vδ1亚群表达比例与患者aGVHD发生率呈负相关。G-CSF动员后分选γδ T细胞中Foxp3+γδ T、Foxp3+Vδ1 T和Foxp3+ Vδ2 T细胞表达比例显著高于动员前分选γδ T细胞。G-CSF动员后分选γδ T细胞对自身CD4+T细胞的增殖抑制作用显著强于动员前分选γδ T细胞。接着，我们发现：配对未发生aGVHD患者Foxp3+γδ T, Foxp3+Vδ1 T, Foxp3+Vδ2 T细胞和Treg细胞表达比例显著高于aGVHD发病患者。Foxp3+γδ T, Foxp3+Vδ1 T 和 Foxp3+Vδ2 T 细胞比例与aGVHD的发生呈负相关。aGVHD患者经有效治疗后，Foxp3+γδ T, Foxp3+Vδ1 T 和Foxp3+Vδ2 T细胞表达比例显著增高；aGVHD治疗2周无效时，Foxp3+γδ T细胞表达水平呈下降的趋势。最后，我们初步研究发现预防性输注供鼠γδ Treg可减轻allo-BMT后小鼠aGVHD的发生率和aGVHD程度。本课题进一步揭示了γδTreg的生物学特性及功能，为临床应用γδTreg预防和治疗aGVHD提供理论依据。