单倍体相合造血干细胞移植模式下NK功能重建规律及调控机制研究

Rapid NK-cell function reconstitution is important for the clinical outcomes of haploidentical hematopoietic stem cells transplantation (HSCT). The recognition of self-MHC class I by inhibitory Killer cell Ig-like receptors (KIR) is involved in the calibration of NK cell effector capacities during a developmental stage. To add further complexity to the KIR system, each gene shows considerable polymorphism. KIR and HLA class I genotype polymorphism predictive of varying degrees of receptor-ligand binding affinities affect NK cells function. Our previous study has demonstrated that recipient expression of ligands for KIRs enhances NK-cell function to control leukemic relapse after haploidentical HSCT, and IL2 can enhance NK-cell function recontitution. Based on the clinic haploidentical HSCT model and mouse model, the aims of this study are to explore differences in donor KIR and recipient HLA class I binding affinities affecting NK cells function reconstitution and influencing GVHD/GVL effect post haploidentical HSCT:1) the effect of different affinities of donor KIR bind cognate recipient HLA class I ligands on NK-cell function reconstitution as well as clinical outcomes post our novel unmanipulated blood and marrow haploidentical transplantation; 2) the effect of low dose of IL2 administrated early post transplantation on receptor-ligand binding affinities and NK-cell function reconstitution; 3) building up humanized mice models with the expression of human HLA through Crispr-Cas9 technique and comparing the functional change of donor NK cells after transferred into different humanized mice with different receptor-ligand binding affinities. Through clarifying the mechanism of NK cells function reconstitution under haploidentical hematopoietic stem cells transplantation, it would be helpful for us to explore the new ways to enhance NK cells function reconstitution and select the best donor for haploidentical transplantation.

NK细胞功能重建决定移植预后。KIR基因与HLA相互作用决定NK的成熟及获得功能，KIR与HLA基因多态性可影响KIR与HLA相互作用的亲和力强弱，调控NK细胞功能。申请人前期工作发现在非体外去除T细胞的单倍型相合移植体系下，供者KIR基因与患者HLA匹配程度促进移植后NK功能重建、降低白血病复发，且IL2可促进NK功能重建。本研究旨在以NK功能重建为主线，借助临床队列及动物模型，探讨供者KIR与患者HLA亲和力强弱对单倍体相合移植后NK功能重建调控：1）研究供者KIR与受者HLA亲和力强弱对单倍体相合移植后NK功能重建及其对预后的影响；2）结合前瞻性临床试验，探讨小剂量IL-2治疗对KIR与HLA亲和力及NK功能重建的影响；3）借助人源化小鼠模型，研究KIR与HLA亲和力改变对NK功能的影响。阐明NK细胞功能重建的调控机制，为建立促NK细胞重建新方法和指导单倍体移植的供者选择奠定基础。

NK细胞功能重建决定移植预后。抑制性受体KIR与HLA相互作用决定NK细胞的成熟及获得功能。本项目首先通过研究健康人KIR-HLA亲和力和NK细胞功能的关系，发现KIR-HLA亲和力强的NK细胞具有更强的功能。接下来在单倍体移植背景下，研究重建的NK细胞表型及功能与KIR和供受者HLA基因型的相关性，发现供者和受者的HLA基因型在NK细胞的功能重建过程中均起重要作用，而表达所有KIR配体的HLA基因型的供受者组合相比其他具有更低的复发率和更长的生存期。此外，通过健康人和重建的NK表面活化性受体检测发现DNAM-1的表达与KIR-HLA亲和力具有明显的正相关，而KIR-HLA高亲和性NK细胞较强的功能是由于DNAM-1，NKP30和NKG2D的协同作用；根据供受者KIR-Bw4和供者KIR3DL1等位基因，将移植后重建的KIR3DL1可以分为高亲和力组（high），低亲和力组（low），和无亲和力组（bw6）；移植后90天和180天，供受者HLA-Bw4位点对KIR3DL1+NK细胞亲和力越高，重建的KIR3DL1+ NK细胞功能越强，且亲和力越高的KIR3DL1+NK细胞DNAM-1的表达越强，因此高亲和力组患者移植后复发率最低。结合临床试验探讨了小剂量IL-2治疗对NK细胞重建的影响。并在NCG白血病小鼠模型和临床中论证了体外扩增的NK细胞产品的抗白血病作用和安全性。据此，阐明了NK细胞重建过程中供受者KIR-HLA调控的重要作用，并通过筛选和阻断活化性受体探讨了KIR-HLA调控NK细胞功能的机制。通过这些发现，在临床上我们可通过供受者HLA基因型预测白血病移植治疗的预后，并指导单倍体移植的供者选择，在未来指导NK重建的干预和调控具有重要意义。