Qa-1介导的NK细胞对供体来源DC细胞杀伤作用的抑制在移植免疫中的作用及其机制研究

The development of high-efficient immune suppression agents has greatly propelled clinical allogeneic organ transplantation, and significantly improved the outcomes of patients with severe organ dysfunctional diseases. However, immune suppression agents also raise the risk of infection and tumor. The induction of long-term donor specific tolerance to the grafts is still a huge challenge for the immunologists.In allogeneic organ transplantation, donor's dendritic cells(DCs) in the grafts could migrate into the peripheral lymph nodes of the host, where these cells encounter and activate host's T lymphocytes as antigen-presenting cells. Besides presenting alloantigen to host's APCs donor DCs could also priming host's na?ve T cells directly, which induce a potent polyclonal T-cell response activating up to 10% of the entire peripheral T-cell repertoire. Thus donor DCs in the grafts play an important role in the process of graft rejection. And DCs were considered to be contribute to the strong immunogeneicity of skin graft for numerous DCs reside in dermis. Natural killer cells are potent cytolytic cells, and mainy act as act as effector cells in immune reaction. However, recent studies showed that host's NK cells could directly kill these graft-derived donor DCs, leading to inhibition of the direct priming of alloreactive T cells. and have important clinical implications in tolerance induction. Qa-1 is a nonclassical class I MHC molecule with roles in the regulation of both innate and adaptive immunity. The ligand of Qa-1 is NKG2, which forms an additional and distinct class I receptor, adjacent to CD94 and forms a heterodimer. CD94/NKG2A could transduce negative signal through ITIM motif and the SHP-1/2 signaling pathway, and suppress the cell lysis activity of NK cells. Qa-1 could also downregulate the expression and secretion of INF-γ and IL-12. In our pilot study, the survival time of allogeneic skin graft derived from Qa-1 knockout transgeneic mice was significantly prolonged when compared with which from the wild-type mouse. As Qa-1 expressing cells in the skin grafts are mainly DCs, this result implied that Qa-1 may affect the antigen-presenting process of DCs. Based on these results, we hypothesize that blocking the interaction between Qa-1 on donor derived DCs and CD94/NKG2A on host NK cells may release the expression of INF-γ and IL-6 by NK cells, which could enhance the cell lysis of alloDCs and partially block the antigen-presenting process by these cells, and finally promote the graft tolerance.

异基因移植中，供体DCs可通过直接抗原递呈途径多克隆激活宿主T细胞，对免疫排斥反应的发生具有重要作用。研究表明，宿主NK细胞可通过杀伤供体DCs从而抑制上述反应。Qa-1属非典型MHC分子，其通过与其配体CD94/NKG2A的结合，对NK细胞的细胞毒性具有显著的抑制作用。根据前期研究结果，我们猜测供体DCs通过其表达的Qa-1与宿主NK表面NKG2A相互作用，影响下游SHP磷酸化；同时还抑制其表达分泌IFN-γ、IL-12等细胞因子，最终抑制了NK细胞毒性作用，使部分供体DCs免受宿主NK的杀伤。而阻断Qa-1可能抑制上述反应，并减弱移植免疫排斥反应。本项目拟在异基因移植模型中，采用流式细胞、免疫磁珠分选、免疫印迹等技术，通过体外及在体研究，探讨Qa-1对NK杀伤供体DCs的抑制作用，以及其可能的调控机制，为进一步理解Qa-1的免疫机理，以及应用Qa-1作为移植免疫调控靶点提供理论依据。

异基因器官/组织移植是现代医学的重大成就之一，然而如何诱导供体特异性的长期免疫耐受仍是免疫学的重大挑战。在异基因移植中，供体DC细胞可通过直接抗原递呈途径多克隆激活宿主T细胞，对免疫排斥反应的发生具有重要作用。而宿主的NK细胞可通过杀伤移植物中供体来源的DC细胞，阻断其介导的直接抗原递呈作用，从而显著抑制宿主T细胞对供体抗原的增殖反应。在NK细胞与DC细胞的相互作用中，Qa-1/NKG2A信号通路具有重要的意义。Qa-1属非典型MHC分子，其通过与其配体CD94/NKG2A的结合，对NK细胞的细胞毒性具有显著的抑制作用。因此，供体DC细胞可能通过其表达的Qa-1与宿主NK表面NKG2A相互作用，影响下游SHP磷酸化；同时还抑制其表达分泌IFN-γ、IL-12等细胞因子，最终抑制了NK细胞毒性作用。. 在本项目中，通过小鼠异基因皮肤移植模型证实，阻断Qa-1信号通路可延长异基因移植物的存活时间（21.57±0.65天 v.s. 14.50±0.43天，p=0.001），而该延长效果可因清除宿主NK细胞所逆转（14.00±0.52天v.s. 15.00±0.37天，p=0.223）。同时，还通过流式细胞技术分析宿主引流淋巴结中的细胞表型发现，阻断Qa-1信号后，宿主引流淋巴结中的CD11+CD86+MHC+成熟DC细胞明显减少（1.26±0.34% v.s. 3.32±0.57%）。而体外实验显示，与Qa-1缺陷的DC细胞共培养的NK细胞的INF-γ及STAT1表达明显上调，表明其细胞杀伤活性升高。此外，在本研究中还发现，清除宿主Vγ4+T细胞后，Qa-1 KO小鼠来源的皮片存活时间进一步延长36.52±13.31天（p<0.001），且引流淋巴结中的CD11c+MHC+CD86+成熟DC细胞含量亦下降。. 上述研究结果表明，阻断Qa-1信号通路，可影响NK细胞的NKG2A下游信号通路，上调INF-γ及STAT1表达，从而增强其对供体来源异基因DC细胞杀伤的活性，因而抑制供体来源异基因DC细胞的抗原递呈作用，并最终抑制免疫排斥反应。此研究结果首次证实了Qa-1/NKG2A信号通路介导的NK细胞与DC细胞的相互作用在移植免疫反应中的意义，进一步丰富了对Qa-1的免疫学机理的认识，并为临床应用Qa-1作为免疫调控靶点，诱导移植物免疫耐受提供新的理论依据。