靶向抑制CCR5功能及表达预防急性GVHD免疫学机制的研究

Chemotaxis of T cells to GVHD target organs directed by chemokines and their cognate receptors play a key role in the pathogenesis of GVHD. Targeting chemokine receptors may be an important modality in the prophylaxis of acute GVHD without affecting the whole body immunity. Although many chemokine receptors are related to the onset and progression of GVHD, only suppression of CCR5 by gene knocking out or by targeting CCR5 to inhibit its function by maraviroc could effectively prevent acute GVHD. The mechanisms underlying this preventive effect by targeting CCR5 have not been investigated. In this study, we will probe the possible mechanisms for prophylaxis of acute GVHD by targeting CCR5 using CCR5-gene knocking out mouse model to inhibit the expression and CCR5 antagonist maraviroc (MVC) to block CCR5 to bind its ligands, and through in vitro and in vivo studies to prove the hypothesis which may be as follows.⒈By targeting CCR5, less T lymphocyte trafficking to GVHD target organs or tissues where the cognate ligands for CCR5 are higher expressed.⒉ There exist a cross-talk on expression between CCR5 and other chemokine receptors. When CCR5 expression is inhibited or its function suppressed，other chemokine receptors will be disregulated which lead to inhibit T lymphocyte trafficking to GVHD target organs.⒊ Targeting CCR5 affects T lymphocyte function which result in production of less chemokines and cytokines and reduction of T lymphocyte infiltration to GVHD target organs.⒋ Targeting CCR5 may change the differentiation ability of CD4+ cells, which may lead to production of more CD4+CD25+ Foxp3+Treg cells and induction of immunotolerance to alloantigens. ⒌ Targeting CCR5 may change the function of Th cells which causes imbalance of Th1/Th2/Th17 and skew to Th2 anti-inflammation direction.

趋化因子及其受体介导的T细胞向靶器官定向迁移是GVHD发生的关键环节，靶向抑制趋化因子受体（CR）的功能或表达可预防GVHD但对整体免疫功能无重大影响。尽管与GVHD发生相关的CR众多，单独敲除CCR5基因或用Maraviroc（MVC）抑制CCR5功能仍能有效抑制GVHD发生，其机制尚未阐明。应用CCR5基因敲除鼠或MVC抑制CCR5与配体结合，通过体内外对照研究证明靶向抑制CCR5预防GVHD的可能机制：①效应T细胞向CCR5配体表达水平较高的GVHD靶器官迁移减少；②CCR5与其他CR间具有交叉效应，抑制CCR5的同时也抑制了其它CR的表达；③使效应T细胞分泌更少的炎性细胞因子从而降低炎症反应，减轻GVHD发生；④改变了受者CD4+T细胞的分化能力，增加Treg的产生，从而诱导对同种异体的免疫耐受；⑤改变了受者体内Th细胞功能，使Th1/Th2/Th17平衡向抗炎的Th2极倾斜。

趋化因子受体CCR5在急性移植物抗宿主病（acute graft-versus-host disease，aGVHD）的发生和发展中发挥重要作用。有研究表明，单独敲除CCR5基因或应用CCR5拮抗剂Maraviroc（MVC）能有效抑制aGVHD的发生，其机制尚未阐明。本课题通过体内外实验探讨了单独靶向CCR5或联合应用免疫抑制剂、阻断其他趋化因子受体等预防aGVHD的可能机制：1. 体外实验中，CCR5拮抗剂MVC能抑制CCL5/CCR5介导的T细胞的趋化，同时高浓度的MVC能抑制T细胞产生炎症因子IFN-γ和TNF-α。2. 小鼠aGVHD模型中，单独阻断CCR5不能改善aGVHD，而靶向抑制CCR5联合免疫抑制剂环孢素A（CsA）可减少供者来源T细胞向靶器官的迁移、抑制脾脏中树突状细胞成熟、减低供者Th1及Th17的比例等机制减轻aGVHD靶器官肝脏和肠道的病理损伤。3. 除CCR5外，趋化因子CXCR3也参与了aGVHD的发生发展。小鼠aGVHD模型中，靶向CXCR3联合CsA通过抑制脾脏中T细胞活化及分化减轻aGVHD。联合阻断CCR5和CXCR3可进一步改善小鼠aGVHD，其机制与联合阻断CCR5及CXCR3可滞留供者来源T细胞至二级淋巴器官、减少其向aGVHD靶器官的浸润并抑制供者来源T细胞的功能有关。4. 通过构建异基因造血干细胞移植（allo-HSCT）后小鼠特发性肺炎综合症（IPS）模型，我们证明骨密质来源间充质干细胞移植可通过调控T细胞表面趋化因子受体的表达（下调CXCR3和CCR5的表达、上调CCR7的表达），进而改变供者来源T细胞在受鼠体内的迁移能力，从而减轻急性IPS的发生。5. 临床研究发现，rhG-CSF动员可引起供者CD4+T细胞表面CCR5的下调和CCR7的上调，从而减轻aGVHD的发生。6. 健康供者CCR5基因启动子单核苷酸多态性（尤其rs2227010AG/GG型、rs2734648GG/GT型、rs1799987AA/AG型）对受者移植后发生II-IV°aGVHD具有独立预测价值。综上所述，CCR5在aGVHD发生和发展中发挥重要作用，通过不同的手段靶向CCR5为allo-HSCT后aGVHD的预防提供了新的思路。