促T细胞CCR7表达诱导异基因造血干细胞移植后免疫耐受的机制研究

Clinical studies have demonstrated that mesenchymal stem cells(MSCs) infusion and mobilization of hematopoietic stem cells with recombinant granulocyte colony-stimulating factor (G-CSF) can increase the expression of CCR7 chemokine receptor on some T lymphocyte subsets which may lead to immune tolerance after allo-HSCT. Because CCR7 is the unique receptor guiding migration of immune cells into secondary lymphoid organs(SLOs), which depends on the expression of its cognate ligands,CCL19 and CCL21, in high endothelial venules (HEV) and fibroblastic reticular cells(FRC) in lymph nodes. But the mechanisms underlying immune tolerance induction in LNs by up-regulating CCR7 have not been investigated after allo-HSCT. In this study, we will probe the possible mechanisms for immune tolerance induced after using G-CSF or MSCs. In vitro studies, we will explored whether FRC could induce T cell tolerance , either directly or via CCL19/CCL21expression, by inhibiting CCR7+ T cell expansion ,differentiation and transform to effector T cells. By establishing murine acute graft versus host disease (aGVHD) model after allo-HSCT, we will explore whether FRC would be able to regulate more CCR7 positive immune cells entry into LNs via higher expression of CCL19 and CCL21 after conditioning regimen. After CCR7 positive cells entry into Lymph nodes (LNs), they could bind CCL19/CCL21-expressed FRC, which lead to a delayed or inhibition of CCR7-expressed T cell exit from LNs. Within LNs，FRC may inhibit CCR7+T cell expansion ,differentiation and transform to effector cells by expression of CCL19/CCL21, resulting in reduced T lymphocyte trafficking to target organs at early stage after transplantation and induction of immune tolerance. In clinical studies, the ratio and composition of T cell subsets would be changed in the grafts after hematopietic stem cell mobilization with G-CSF in healthy donors. We will study the occurrence of GVHD and survival, which may be associated with the changes in the ratio of CCR7+T cell and its subsets or dose infused in the grafts. These studies may reveal that increased expression of CCR7 chemokine receptor on T cell or its subsets could be a new immunology mechanisms to induce immune tolerance and attenuate the severity of GVHD.

临床研究证实， MSCs和G-CSF动员诱导allo-HSCT免疫耐受与上调T细胞表达趋化因子受体CCR7有关,该受体是免疫细胞向次级淋巴器官归巢的唯一分子,其作用依赖于淋巴结内高内皮微静脉和基质纤维网状细胞(FRC)表达CCR7配体CCL19/CCL21,但促CCR7表达如何在淋巴结内诱导免疫耐受的机制尚不清楚。本研究拟通过应用G-CSF和MSC诱导T细胞表达CCR7，应用小鼠移植模型和体外研究等方法，证明FRC通过直接或上调CCL19/CCL21的表达滞留并延迟CCR7+T细胞迁出淋巴结，并抑制CCR7+T细胞增殖分化及向效应T细胞转化，在移植早期减少T细胞向GVHD靶器官浸润而诱导免疫耐受。临床方面，研究G-CSF动员后移植物CCR7+T细胞各亚群的比例和输注数量对GVHD发生和生存的影响，揭示促CCR7表达诱导免疫耐受而减轻GVHD发生的免疫学新机制。

在诱导移植后免疫耐受的过程中，CCR7促进了淋巴细胞向次级淋巴器官的归巢。归巢后淋巴细胞的命运受到SLO内部环境中基质细胞、巨噬细胞、树突状细胞等的调节。本课题组围绕CCR7开展一系列体内外研究，探讨了这些细胞的来源和功能，及其如何参与CCR7+细胞的调节：1.体外应用表观遗传学药物可上调活化T细胞上CCR7表达，通过CCR7参与的途径抑制T细胞活化及向TH1/TC1的分化。2.分离自小鼠淋巴结的FRC可诱导骨髓细胞向特殊类型巨噬细胞（FM）分化，偏向抗炎的M2型，在体外可有效抑制T细胞的活化及向Th1的极化，回输至GVHD模型可明显抑制受鼠脾脏中供者T细胞活化及Th1/Tc1极化，降低炎性细胞因子水平，减轻GVHD靶器官的损害，延长小鼠生存。3.直接回输FRC同样可以有效减轻移植小鼠GVHD严重程度，延长生存，机制在于回输FRC可以降低淋巴细胞向靶器官的浸润，减少靶器官的损害，抑制脾脏中供者来源T细胞活化及向Th1/Tc1的分化。4.脾脏中DC也能通过上调PD-L1/L2的表达发挥诱导移植后免疫耐受的作用。5.临床研究发现G-CSF动员可上调移植物中T细胞CCR7、抑制CCR5的表达，多因素分析表明动员后供者CD4+T细胞下调CCR7表达是aGVHD的独立危险因素。这些研究结果从细胞水平、动物模型和临床分析等多个方面阐明aGVHD发病机制，为预防GVHD、诱导异基因造血干细胞移植后免疫耐受提供了新的思路和方法。