重组人HLA-G蛋白纠正ITP患者异常细胞免疫功能机制的研究

Altered human leukocyte antigen (HLA)-G and its receptor immunoglobulin-like transcripts (ILTs) is associated with kinds of autoimmune diseases. Our previous study demonstrated that impaired function of mesenchymal stem cell in inducing tolerogenic DCs and suppression self-reactive T cell proliferation as well as elevated profile of Th1 in patients with immune thrombocytopenia (ITP). HLA-G secreted by human mesenchymal stem cells (MSCs), which is probably related with its disorder in cellular immunological modulation, but the precise mechanisms remain to be elucidated. In addition, our recently research revealed that the down-regulated soluble HLA-G was found and which is positively related to the platelet count in ITP. Besides, the ratio of CD19+HLA-G+ILT2+ cells and CD14+HLA-G+ILT4+ cells from peripheral blood mononuclear cells significantly decreased. Based on our above-mentioned study, we speculate that the dysfunction of HLA-G and its receptor molecules played pivotal roles in pathogenesis of ITP. To test the hypothesis, we intend to use a variety of molecular biological methods, such as blocking antibodies, engineering technologies, to neutralize, down-regulate or up-regulate the expression of HLA-G and next evaluate the effect of these interventions on the HLA-G-specific tolerogenicity to cell-mediated immunity. Moreover, the feasibility of exogenous recombinant human HLA-G protein for disrupting the pathophysiology process of ITP will be evaluated, thus providing new clues for the management of ITP.

HLA-G及其受体分子异常在多种自身免疫性疾病中起关键作用。课题组前期研究发现ITP患者MSCs体外诱导调节性DCs、抑制自身反应性T细胞增殖功能障碍，并且患者T细胞亚群向Th1方向趋化过度。MSCs可表达HLA-G，并且MSCs的免疫抑制功能失调很可能与HLA-G的异常有关，但具体机制不详。课题申请者最新研究发现：ITP患者外周血可溶性HLA-G表达下调，并与血小板计数正相关。另外ITP患者外周血单个核细胞中CD19+HLA-G+ILT2+以及CD14+HLA-G+ILT4+细胞比例显著降低。本研究首先提出HLA-G及其受体分子异常是ITP发病的重要机制假说。为验证假说，本研究拟应用基因工程等技术中和、阻断、上调HLA-G表达，观察其对患者免疫细胞功能的影响，双向验证其调节患者细胞免疫功能的机制；同时探讨外源性重组人HLA-G对临床干预ITP发生发展的可能性，为ITP治疗提供新思路。

人白细胞抗原G（HLA-G）是非经典的主要组织相容性复合体I类抗原，具有强大的免疫抑制功能。HLA-G通过与受体、免疫球蛋白样转录因子（ILTs）相互作用，对人类同种异体移植和自身免疫性疾病有益。然而，目前对HLA-G在免疫血小板减少症（ITP）中的作用知之甚少。我们的研究发现，与自身抗体阴性的患者或健康对照组相比，抗自身抗体阳性的ITP患者血浆中HLA-G明显减少。在患者和健康对照组中，HLA-G与血小板数量呈正相关。在患者的CD4+细胞和CD14+细胞上膜结合的HLA-G和ILTs表达率低。另外，应用重组人HLA-G（rhHLA-G）可分别上调CD4+和CD14+细胞膜表面ILT2和ILT4的表达。同时, rhHLA-G可上调患者外周血单核细胞分泌IL-4和IL-10，以及下调TNF-α, IL-12和IL-17的表达。提示rhHLA-G可促进Th2细胞方向分化和抑制Th1、Th17细胞方向分化。应用rhHLA-G可下调ITP患者树突状细胞CD80和CD86表达比例，抑制CD4+T细胞增殖。同时rhHLA-G干预可减少血小板凋亡。综上，我们的数据表明，HLA-G和ILTs参与了ITP的发病机制，rhHLA-G可通过上调ILTs纠正这一异常，提示HLA-G可作为ITP的诊断标志物和治疗选择。