研究激活性KIR受体基因在异基因HSCT中分离aGVHD和GVL的不同作用机制

The intrinsic anti-leukemic effect of allogeneic hematopoietic cell transplantation (allo-HSCT) is dependent on genetic disparity between donor and recipient, intimately associated with graft-versus-host disease (GVHD). Natural killer (NK) cell alloreactivity in human leukocyte antigen (HLA) cell of recipients is regulated by killer immunoglobulin-like receptors (KIRs) on donor NK cells after allo-HSCT. Three decades of intense effort have not identified clinical strategies that can reliably separate the graft-versus-leukemia (GVL) effect from the alloimmune reaction that drives clinical GVHD. Recent study confirmed that the KIR genetype was associated with the development of acute GVHD and relapse after HSCT, but the mechanism was unclear. Our study findings suggested that activating KIR(aKIR) and inhibitory KIR(iKIR) may offer different effect on the lowest probability of GVHD and the highest probability of a potent GVL. So we speculated that aKIR receptor on donor NK cells which missed the HLA ligand in the recipient could promote imbedded and prevent aGVHD passage kill APC cell by specific expression of aKIR, and the high expression of aKIR could kill leukemia cell for prevention of post-transplant relapse. But the over-activation aKIR gene leading to high expression may both contributed to the ITAM phosphorylation of DAP12 and released IFN-γ、IL-15 of NK cells responses cytokine production leading to aGVHD. The result will illustrate that KIRs might be the most important fundamental impact on outcomes of HSCT. Also this project is going to establish the KIR/HLA mismated experimental model and animal model in vitro and in vivo to explore the mechanism of aKIR development in combination with clinical data analysis using flow cytometry, western blot and real-time PCR methods. It is demonstrated that aKIR gene play a crucial role in prognosis of HSCT providing experimental foundation for new protocol for dissecting GVHD and GVL by specific donor natural killer cell infusion. These findings provide new idea and significance that may be beneficial to prevention aGVHD and relapse and improve the clinical outcomes of allo-HSCT.

HSCT中如何降低aGVHD和复发一直是临床迫切需要解决的关键问题。KIR是NK细胞表面最重要的受体基因，其在aGVHD和GVL的作用机制知之甚少。我们预实验结果提示：aKIR基因在预防aGVHD和复发中发挥不同作用。我们提出假说：移植早期特异性表达aKIR基因的NK细胞，通过杀伤宿主APC促进移植物植入并抑制aGVHD发生，后期高表达aKIR的NK细胞通过杀伤宿主残留白血病细胞调控复发，但过度活化aKIR在杀伤宿主细胞的同时释放细胞因子可能诱发aGVHD。为了验证该假说，我们将通过体外分选表达供者特异性aKIR基因NK细胞，与APC和白血病细胞株建立KIR/HLA错配模型、aGVHD和白血病模型，从分子、细胞、组织、动物整体水平，结合临床资料多方面探讨aKIR基因在HSCT中的作用，明确aKIR基因表达和功能的因故调控机制，为从分离aGVHD和GVL这个新视点防治GVHD和复发提供新思路

中文摘要(对项目的背景、主要研究内容、重要结果、关键数据及其科学意义等做简单概 述，800字以内)：. 本课题围绕NK细胞表面最重要的受体KIR基因，包括aKIR（2DS1/2DS2/2DS3/2DS4/2DS5/3DS1)和iKIR(2DL1/2DL2/3DL1) 重要基因，在HSCT后发生aGVHD和复发中的调控作用进行体内外实验和临床研究，完成如下主要和关键研究结果：⑴根据中国人群KIR和HLA基因的分布特点和频率、KIR基因型、单体型、Cen端/Tel端基序分型、KIR/HLA受配体模式、供者来源特征性KIR基因等数据库，自主建立了适用于中国人群KIR和HLA基因分型遴选供者的策略和临床分层诊断模式。⑵自行建立应用荧光定量PCR方法检测aKIR和iKIR基因在mRNA转录水平表达的实验方法学，并进行特异性、灵敏度、重复性、仪器适用范围等方法学性能验证。⑶研究HSCT后供者来源特征性与非特征性aKIR基因的免疫重建规律；并且动态监测各种特异性aKIR 和iKIR 基因mRNA转录水平的表达，及aKIR 基因和iKIR基因表达不同水平的发生机制。⑷体内外研究表明在HSCT后不同时期aKIR 和iKIR基因mRNA转录水平表达存在差异性，aKIR 基因通过调控iKIR基因的表达差异，实现NK细胞杀伤白血病细胞和清除宿主细胞而发挥异源反应活性的功能，降低GVHD和复发的作用机制。. 本研究成果中自主建立中国人群KIR和HLA基因分型遴选供者分层诊断模式，已在全国实施亲缘和非亲缘移植中心进行推广应用，取得了较好的社会和经济效益。自行建立荧光定量PCR方法检测aKIR和iKIR基因在mRNA转录水平表达，有望成为早期预测GVHD发生和程度，及白血病复发的新标志物。研究证实了检测KIR基因可作为供者来源基因植入的指标，在HSCT后aKIR比iKIR基因更早呈现mRNA转录水平高表达的态势。aKIR和iKIR基因mRNA转录水平持续低表达，与HSCT后GvHD发生及程度密切相关，且aKIR的下降趋势比iKIR更为明显；而在复发时aKIR和iKIR基因呈现高表达。尤其是在aKIR中2DS1和3DS1基因mRNA转录水平，从低表达、高表达、持续高表达的变化过程，对于预示移植后GvHD、复发的发生和发展，疾病转归及生存率，将成为临床诊断和治疗中重要的新策略和新思路