【作者】 王伟刚；

【导师】 傅耀文； 李巍；

【作者基本信息】 吉林大学 ， 外科学， 2011， 博士

【摘要】 器官移植是治疗多种终末期疾病的有效手段。FK506等免疫抑制剂的问世,对预防移植物发生排斥反应、延长移植物存活起了重要的作用。但是,现有免疫抑制剂的毒副作用仍困扰着病人,导致许多病人死于全身免疫抑制剂的毒副作用,如严重的感染以及肿瘤等。移植物的慢性排斥反应仍未得到有效地控制,长期存活率并未能得到明显提高。于是,如何诱导移植物特异的免疫耐受成为目前移植免疫研究的热点。随着研究的不断深入,人们已揭示出影响同种异体免疫反应过程的一些信号传导机制。如果能够发现启动同种异体免疫反应的关键分子通路,并对其及相关配体进行有效地调控将会更有效地调控移植免疫反应的强度,并且有可能诱导移植物免疫耐受。PD-L1是PD-1的配体。目前已知它在激活T细胞及造成T细胞无反应性的过程中起关键性作用。该分子可能是我们要寻找并研究的靶点。PD-L1,与B7同源。它可表达于多种组织及细胞上。这些细胞包括T细胞,如CD4、CD8、NKT细胞、B细胞、APC。PD-L1可负向调节T细胞功能,阻止T细胞在胸腺内阳性选择和发育,在调节外周耐受中起关键性作用。目前已知,用PD-L1免疫球蛋白合成蛋白刺激能诱导PD-1分子活性增强,继而可促进胰岛移植物长期存活。应用PD-L1单克隆抗体阻断PD-L1,可促进皮肤移植物发生排斥反应。同样,该方法也可引起心脏移植物产生动脉血管性疾病。但是,不同细胞亚型上的PD-L1分子在调节同种异体免疫反应中的确切作用目前尚不得而知。实验目的：验证PD-L1/PD-1信号通路在同种异体移植免疫反应中的作用及其作用机制。实验方法：应用PD-L1基因敲除小鼠和同种异体心脏移植模型,(1)通过流式细胞技术比较了PD-L1-/-小鼠及野生型B6小鼠脾脏T细胞及其各亚型的表型特征；(2)通过MLR研究PD-L1-/-小鼠外周T淋巴细胞及APC的功能特点；(3)通过ELISPOT实验研究T细胞及APC上的PD-L1分子在同种异体免疫反应中诱导辅助性T细胞产生细胞因子的特点：(4)在体内实验中,在MHC完全错配的小鼠品系间进行心脏移植,检查PD-L1/PD-1信号通路对同种异体心脏移植物存活的影响。采用ELISPOT、MLR、CTL和流式细胞技术来评价移植受体的免疫功能状态。实验结果：(1)与野生型对照组相比,PD-L1-/-小鼠脾脏中,CD11c+DC明显升高(5.44±2.2vs3.54±1.1,P<0.05),CDllb+巨噬细胞也明显升高(12.7-4.7vs5.44±0.4,P<0.05),但是,其Foxp3+CD4+T调节细胞显著降低(5.7±1.9vs 7.3±1.5,P<0.05)所有CD4+CD25+T细胞均表达Treg的特异转录因子Foxp3。在野生型小鼠中这些Treg表达较高的PD-L1。PD-L1在PD-L1敲除小鼠中并不表达。CD11c+DC在野生型小鼠表达较高的PD-L1,但PD-L2表达并不升高。PD-L1-/-小鼠CD11c+DC中CD80、CD95L. OX40L、PD-L2和MHCⅡ(IAb)表达轻度升高。(2)与野生型对照组相比,体外实验中PD-L1-/-小鼠T细胞同种异体免疫反应较强。并且,PD-L1-/-APC对同种异基因T细胞的激活能力亦显著增强。(3)PD-L1-/-小鼠T细胞产生Th1(IFN-γ)及Th2(IL-4,IL-10)型细胞因子明显增加(与野生型对照组相比,IFN-γ,P<0.05；IL-4,P<0.01；IL-10,P<0.05)。由同种异基因PD-L1-/-小鼠脾细胞刺激的T细胞可造成IFN-γ水平升高,IL-4水平降低(与野生型对照组相比,P<0.05)。IL-10及IL-2水平两组间无显著差异。(4)与野生型小鼠对比,PD-L1-/-小鼠无论作为供体还是受体均发生了加速的急性排斥反应。C3H受体PD-L1-/-移植物平均生存时间(mean survival time,MST)为9.3±1.1天,而对照组为36±37天(P<0.01)；PD-L1-/-受体的C3H移植物MST为8.54±0.6天,而对照组为13.3±1.3天(P<0.01)。相似地,在B6与BALB/c小鼠间进行心脏移植实验中,BALB/c受体PD-L1-/-移植物在术后第7天出现排斥反应。这早于对照组的134±1.9天(P<0.01)。同样,PD-L1-/-受体BALB/c移植物也发生了加速的排斥反应,其MST为7.0±0.8天。对照组为13±1.6天(P<0.001)。(5)于移植术后第7天MLR实验结果显示：与野生型对照组相比,PD-L1-/-受体及PD-L1-/-小鼠作供体的受体中T细胞增殖反应均明显增高(P<0.05)。(6)细胞毒T细胞及NK细胞活性实验表明：两组受体脾细胞的抗供体CTL活性(抗EL-4[H2b]及R1.1[H2k])均显著增强(前者P<0.01,后者P<0.05)。但是,NK细胞活性增加仅体现在PD-L1-/-小鼠作受体时,并未发生在PD-L1-/-小鼠作供体的受体小鼠中。(7)于移植术后第7天ELISPOT实验结果显示：与野生B6小鼠对比,PD-L1-/-小鼠无论作供体或受体,细胞因子分泌均产生了变化。PD-L1-/-小鼠受体T细胞中Thl(IL-2,P<0.05；IFN-γ,P<0.05)及Th2(IL-4,P<0.05)的细胞因子水平显著增加。但是,PD-L1-/-小鼠作供体的受体,其T细胞中仅Thl的细胞因子(IFN-γ,P<0.05)水平显著升高。而且,在PD-L1-/-小鼠心脏移植物及其受体脾脏的冰冻切片免疫组化染色中显示：IFN-γ分泌水平显著升高。但是,与野生型对照组相比,PD-L1-/-小鼠作受体的心脏移植物及脾脏中,IL-2产生明显升高。实验结论：(1)PD-L1基因缺陷改变了外周淋巴细胞各亚系之间的比例平衡,PD-L1-/小鼠外周Foxp3 +CD4+T调节细胞下降,APC增多并表达较强的共刺激分子。(2)PD-L1-/T细胞对同种异基因刺激产生的特异性增殖免疫反应明显增强。(3)PD-L1-/- APC激活同种异基因T细胞反应的能力显著增强。(4)PD-L1-/-APC促进T细胞产生Th1细胞因子,同时抑制产生Th2细胞因子。(5)PD-L1-/-小鼠作为心脏移植的供体或受体,均可加速心脏移植物的急性排斥反应,移植物存活时间明显缩短。(6)PD-L1-/-介导的心脏移植物加速排斥反应与诱导受体T细胞增强抗供体增殖反应和Thl细胞因子的产生有关。(7)PD-L1信号参与了细胞毒T细胞的反应,但可能未直接参与到NK细胞的功能。(8)PD-L1-/-小鼠作为供体或受体中,PD-L1信号所介导的免疫反应的机制是不同的。其原因可能是由于PD-L1可表达于多种类型的细胞上。实验意义：PD-L1不仅传递了负向共刺激信号给活性T细胞,调节对自体抗原的外周耐受,而且在诱导同种异体耐受方面也起到关键性作用。如果我们能对引起同种异体免疫反应的启动中心进行调控,我们就可能有机会抑制该反应。PD-L1,这一PD-1的配体,可能就是我们要寻找的靶点。它可能调控T细胞的激活及APC反应的过程。本课题为全面理解PD-L1/PD-1信号通路在体内同种异基因免疫反应中的作用机制提供了新的理论基础,丰富了人们对于PD-L1/PD-1信号通路的认识,并为临床诱导、维持免疫耐受,提高移植物存活时间提供了新的思路,具有临床应用前景。更多还原

【Abstract】 Organ transplantation is an effective treatment for several end-stage diseases. Tremendous advances have been made during the last decade in therapeutic strategies to prevent allograft rejection. However, toxicities and complications of current immuno-suppressive drugs make apparent the need for new targets involved in allograft tolerance induction. Manipulation of a ligand central to the initiation of alloimmune responses may previde an opportunity to control the host response to an allograft with enhanced precision.PD-L1, a homolog to B7, has recently been identified as a ligand for PD-1 and is expressed on a variety of tissues and cells including T cells (CD4, CD8, natural killer [NK] T cells), B cells, and antigen-presenting cells (APCs). PD-L1 negatively regulates T-cell function, inhibits T-cell positive selection and development in the thymus, and plays a critical role in the regulation of peripheral tolerance. It has also been shown that PD-1 engagement by PD-L1 immunoglobulin (Ig) fusion protein administration induces long-term islet allograft survival. PD-L1 blockade using anti-PD-L1 monoclonal antibodies accelerates skin allograft rejection and cardiac graft arterial disease. The exact role of the regulation of alloimmune responses by PD-L1 on each cell type remains unclear.Aims:We used PD-L1-/- mice to evaluate the role of the PD-1/PD-L1 signal on allogeneic immune responses in vivo and the underlying mechanisms.Methods:(1) We compared the characteristics of APCs and T cells from the spleens of PD-L1-/- and WT B6 mice by four-color flow cytometry. (2) To determine the functional abilities of peripheral T lymphocytes and APCs from PD-L1-/- mice, a standard mixed lymphocytes reaction assay was performed. (3) To determined the cytokine profiles of PD-L1-/- T cells under allostimulation, or the T cells stimulated by allogeneic SCs from PD-L1-/- mice, ELISPOT assays for cytokine were performed. (4) Heart transplantation was performed from PD-L1-/- donors or recipients in major histocompatibility complex fully mismatched mouse combinations. The immunologic function of allograft recipients was evaluated ex vivo by enzyme-linked immunospot, mixed lymphocytes reaction, cytotoxic T lymphocyte, and flow cytometry.Results:(1) The data revealed a significant increases in CD11c+ DCs (5.4±2.2 Vs 3.5±1.1, P<0.05) and CDllb+ macrophages (12.7±4.7Vs 5.4±0.4, P<0.05) in the spleens of PD-L1-/- mice compared with WT controls, but a significant decrease in Foxp3+CD4+ Treg from PD-L1-/- mice compared with WT B6 mice (5.7±1.9Vs 7.3±1.5, P<0.05). All of the CD4+CD25+ T cells expresses Foxp3, phenotype Tregs. As expected, these Treg expressed higher PD-L1 on the surface in WT, but not knockout mice. The CD11c+ DCs demonstrated higher levels of PD-L1, but not PD-L2 expression on their surface in the WT B6 mice, whereas CD11c+ DCs from PD-L1-/- mice expressed slightly higher levels of CD80, CD95L, OX40L, PD-L2, and MHC II (IAb). (2) PD-L1-/- T cells demonstrated enhanced proliferation when stimulated with allogeneic SCs. When PD-L1-/- SCs served as stimulators, they demonstrated enhanced allostimulatory ability to C3H spleen T cells compared with their response to WT controls. (3) We performed ELIPOT assays for cytokine IL-2, IFN-y, IL-10, IL-4 production. T cells from PD-L1-/- mice demonstrated enhanced cytokine production in both Thl and Th2 types (vs. WT controls, P<0.05 for IFN-y; P<0.01 for IL-4; P<0.05 for IL-10) compared with the WT B6 mice. However, the T cells stimulated by allogeneic PD-L1-/- SCs produced an increased level of IFN-y and a decreased level of IL-4 (vs. WT controls, P<0.05) compared with the T cells stimulated by WT SCs. IL-10及IL-2 productions were equivocal between groups. (4) We performed a series of allogeneic HTx between two MHC fully mismatched strain combinations, B6 and C3H, and B6 and BALB/c, respectively. PD-L1-/- mice were tested as donors or recipients in both strain combinations. The heart allograft survival in all strain combinations demonstrated accelerated acute rejection in PD-L1-/- donors or recipients when compared with their comparable WT strain crosses. The mean survival time (MST) of PD-L1-/- allografts in C3H recipients was 9.3±1.1 days compared with 36±37 days in WT controls (P<0.01); the MST of C3H allografts in PD-L1-/ recipients was 8.5±0.6 days compared with 13.3±1.3 days in WT B6 recipients (P<0.01). Similarly, in B6 and BALB/c strain combinations, PD-L1-/- grafts in the BALB/c recipients were rejected at an accelerated rate at day 7 posttransplantation compared with 13±1.9 days in WT grafts (P<0.01). Although BALB/c grafts in the PD-L1-/- recipients were also rejected at an accelerated rate, MSTs were 7.0±0.8 days vs.13±1.6 days in WT recipients (P<0.001). (5) Standard MLR assays were performed on purified spleen T cells of heart allograft recipients at day 7 posttransplantation. The results demonstrated a significant increase in T-cell proliferation in both PD-L1-/- recipients and the recipients of PD-L1-/- grafts compared with the WT controls (P<0.05). (6) We examined the cytotoxic T cell and NK cell activities of recipient splenocytes both in recipients of PD-L1-/- allografts and PD-L1-/- recipients of WT allografts at day 7 posttransplantation. Antidonor CTL activities (against EL-4[H2b] and R1.1[H2k]) of recipients SCs were markedly increased both in recipients of PD-L1-/- grafts (P<0.01) and PD-L1-/- recipients (P<0.05) compared with WT controls. However, the NK cell activity increased moderately only in the PD-L1-/- recipients and not in the recipients of PD-L1-/- grafts compared with the WT controls. (7) The cytokine production of spleen T cells of heart allograft recipients was evaluated by ELISPOT assay in the combination of C3H to PD-L1-/- and PD-L1-/- to C3H mice at day 7 posttransplantation. WT B6 mice were used as controls. PD-L1-/- donors or recipients demonstrated a different pattern of cytokine production. Both Th1-(IL-2, P<0.05 and IFN-γ, P<0.05) and Th2 (IL-4, P<0.05)-cytokine production were increased significantly in the T cells of PD-L1-/- recipients. However, the T cells from the recipients of PD-L1-/-donors recealed a significant increase only in Thl cytokine IFN-y(P<0.05). Moreover, immunohistochemistry straining of frozen sections of the heart allografts and recipient spleens revealed significant elevations of IFN-γsecretion in the PD-L1-/- heart allografts and recipient spleens. However, increased IL-2 production was detected in the heart allografts and spleens of PD-L1-/- recipients compared with WT controls.Conclusions:(1) PD-L1 deficiency altered the balance of peripheral lymphocytes, resulting in a decrease in the number of Foxp3 +CD4+ Treg and an increase in APCs and their co-stimulatory molecule expression in the PD-L1-/- mice. (2) In vitro PD-L1-/-T cells respond more vigorously to allogeneic stimulation than those from WT mice. (3) PD-L1-/- APCs significantly enhance the response of allogeneic T cells compared with those of WT mice. (4) Absence of PD-L1 signal on APCs promotes Thl-cytokine production and suppresses Th2 cytokine production of allogeneic T cells. PD-L1/PD-1 signal induces different immune responses depending on the cell types. (5) Our in vivo data of heart allograft survival clearly support the role of PD-L1 signaling in modulating allogeneic immune responses after HTx. (6) The communication through PD-L1 is bidirectional, because both PD-L1-/- recipients and recipients of PD-L1 allografts demonstrated an augmented antidonor response. (7) The PD-L1 signaling is involved in the cytotoxic T cells, but may not be directly in involved in NK cell funciton. (8) The mechanisms of the PD-L1 signal-mediated immune response are different in recipients of PD-L1-/- allografts and PD-L1-/- recipients of WT allografts. These may be due to PD-L1 expressed on multiple types of cells.Significance:Our data suggest that PD-L1 not only delivers a negative costimulatory signal to activated T cells, regulating peripheral tolerance to self-antigen, but is also critical to the development of allogeneic tolerance. Manipulation of a ligand central to the initiation of alloimmune responses may provide an opportunity to control with enhanced precision the host response to an allograft. PD-L1, the ligand for PD-1, seems to be integral to the modulation of T-cell activation and the APC responses, suggesting that it is such a target. This subject can support a new theoretical foundation by fully understand the mechanisms of PD-1/PD-L1 signal in transplantation immunology; provide a new idea on clinical tolerance induction and maintenance; and have a good clinical application for allografts long-term survival.更多还原