潜在新细胞因子LYG1在急性移植物抗宿主病中的作用及机制研究

LYG1 (Lysozyme G-like 1) is a novel potential cytokine first identified using the strategy of Immunogenomics in my PhD laboratory. Our previous study found that LYG1 could promote the activation, proliferation and IFN-γ production of CD4+T cells. Some studies have demonstrated that donor derived T cells and their producing cytokines (like IFN-γ) play a central role in aGVHD (acute graft-versus-host disease), which is the main complication of allo-HSCT (allogeneic hematopoietic stem cell transplantation). Therefore, based on our previous study, we hypothesized LYG1 might participate in initiation and development of GVHD by regulating the activation, proliferation and cytokine production of donor derived CD4+T cells. In this study, in order to verify the hypothesis, we will use human LYG1 recombiant protein and Lyg1-/- mice (we have obtatined the homozygous knock out mice) to explore the function and mechanism of the novel potential cytokine LYG1 in aGVHD. First, in vitro study, the effect of LYG1 on the function of human or mice allogenic T cell will be investigated in the mixed lymphocyte reaction (MLR). Second, in vivo study, we will explore the role and mechanism of LYG1 in the MHC-mismatched aGVHD murine models by evaluating aGVHD development, histopathology analysis and activation, expansion, differentiation, apoptosis and cytokine secretion of donor-derived T cells. At last, we will examine the clinical correlation of aGVHD and LYG1 expression in the serum of recipients before and after allo-HSCT. This study will provide a new mechanism of the immunopathophysiology of GVHD, which may potentially help to develop a new strategy for preventing GVHD.

LYG1（Lysozyme G-like 1）是通过免疫组学策略发现的一个潜在新细胞因子。我们前期研究结果发现，LYG1能够促进CD4+T细胞的活化、增殖和细胞因子IFN-γ的分泌。而供者来源的CD4+T细胞及其分泌的细胞因子（如IFN-γ）在急性移植物抗宿主病（aGVHD）的发生发展中发挥关键核心作用。基于此，我们推测LYG1可能作为aGVHD发病过程中新的调控分子，通过调节受者体内供者来源的CD4+T细胞的活化、扩增和细胞因子的分泌，参与aGVHD的发生发展。为了验证假说，本研究拟通过体外混合淋巴细胞反应和体内aGVHD小鼠模型，利用LYG1重组蛋白和Lyg1基因敲除小鼠，研究LYG1在aGVHD的中的作用及其机制，并对LYG1与aGVHD严重程度的临床相关性进行分析。本研究结果将揭示GVHD的免疫学调控新机制，为GVHD的防治提供新的靶点和方向。

LYG1（Lysozyme G-like 1）是通过免疫组学策略发现的一个潜在新细胞因子。前期研究结果发现，LYG1能够促进CD4+T细胞的活化、增殖和细胞因子IFN-γ的分泌。而供者来源的CD4+T细胞及其分泌的细胞因子在急性移植物抗宿主病（Acute graft-versus-host disease，aGVHD）的发生发展中发挥关键作用。基于此，我们推测LYG1可能是aGVHD发病过程中新的调控分子。本项目通过体外混合淋巴细胞反应和体内aGVHD小鼠模型，利用Lyg1敲除小鼠和LYG1重组蛋白，系统研究了LYG1在aGVHD中的作用及其机制。此外，本项目分析了人体内LYG1表达水平与aGVHD严重程度的相关性。在体外，Lyg1敲除（Lyg1-/-）小鼠和野生型（Lyg1+/+）小鼠（均为C57BL/6背景小鼠）的脾细胞分别作为反应细胞，BALB/c小鼠脾细胞作为刺激细胞，我们发现LYG1缺失抑制CD4+T细胞的活化，降低Th1细胞比例，增加Treg细胞比例。在体内，将Lyg1-/-或Lyg1+/+小鼠脾细胞和Lyg1+/+小鼠骨髓细胞移植到750cGy剂量照射的BALB/c小鼠构建MHC不相和异基因移植aGVHD模型。与Lyg1+/+脾细胞移植组相比，Lyg1-/-脾细胞移植组小鼠aGVHD发病显著改善，体重下降、临床评分、脏器病理评分均减轻，生存延缓。受鼠脾脏和肝脏中Lyg1-/-来源的CD4+T细胞活化降低，Th1比例降低，Treg比例增加。Lyg1-/-来源的CD4+T细胞在肝脏中浸润减少，CXCL9、CXCL10表达降低。在上述模型中静脉注射P815肿瘤细胞，发现LYG1缺失不影响移植物抗肿瘤作用。此外，在aGVHD小鼠模型中，注射LYG1重组蛋白加重aGVHD，IFN-γ分泌增多，验证了LYG1敲除小鼠在aGVHD中的结果。最后，利用LYG1的单克隆及多克隆抗体，我们建立了LYG1的微球检测方法，与行异基因造血干细胞移植后发生0-1度aGVHD相比，发生2-4度aGVHD患者血清中LYG1表达明显升高，提示LYG1与aGVHD严重程度具有临床相关性。综上，本研究证实LYG1是aGVHD新的调控分子，其可通过调节异基因T细胞反应和Th1/Treg分化平衡，参与aGVHD的发生发展。LYG1作为潜在靶点为防治aGVHD提供了新的策略和方向。