PD-L1免疫负调节作用与延长移植物存活及其机制
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摘要
【目的】
I型糖尿病是一种主要由T细胞介导的，CD4~+、CD8~+T细胞和巨噬细胞浸润胰岛导致分泌胰岛素的β细胞受损，使胰岛素分泌减少，导致胰岛素绝对缺乏。目前已证实该疾病和自身免疫耐受缺失有关，自身反应性T细胞在I型糖尿病的免疫发病机理中起主导作用。PD-L1已被证实为免疫负性调节受体PD-1的配体，文献证明PD-L1在免疫负性调节及外周耐受中发挥重要作用。本实验构建PD-L1表达载体，转染NIT细胞，通过体内外实验探讨PD-L1免疫调节作用，研究其在链脲佐菌素(STZ)诱导的糖尿病小鼠模型中延长胰岛移植存活时间及其作用机制。
【方法】
1．稳定表达pPD-L1的NIT-1细胞株的建立
用Lipofectamine~(TM)2000将pPD-L1-EGFP n1和pEGFPn1两个质粒分别转染NIT-1细胞系获得稳定转染细胞株，分别命名为NIT-PD-L1和NIT-EGFP。
2．致敏淋巴细胞的制备
分别用三组刺激细胞NIT-1、NIT-PD-L1和NIT-EGFP体内腹腔注射Balb／c小鼠，间隔1周再腹腔注射一次，细胞免疫后第14天分离Balb／c小鼠脾脏单个核细胞作为致敏淋巴细胞备用。
3．混合细胞培养
将经过丝裂霉素C处理的NIT-1、NIT-PD-L1和NIT-EGFP三组细胞作为刺激细胞分别与新鲜分离的脾脏淋巴细胞和(或)上述致敏淋巴细胞进行混合培养7天。
4．细胞增殖实验
用CFSE预染上述小鼠淋巴细胞，待细胞混合培养7天后，FCM检测淋巴细胞的增殖。
5．细胞凋亡检测
收集上述细胞混合培养7天后的淋巴细胞，Annexin V-cy5和PI染色，FCM检测淋巴细胞的凋亡。
6．细胞毒实验
分别用三组刺激细胞NIT-1、NIT-PD-L1和NIT-EGFP体内腹腔注射Balb／c小鼠，间隔1周再腹腔注射一次，细胞免疫后第14天分离小鼠脾淋巴细胞，将刺激细胞与上述获得的脾淋巴细胞以1：10的比例混合培养，3天后收集淋巴细胞作为效应细胞。将NIT-1细胞用CFSE染色，作为靶细胞，并以1：20的比例与效应细胞混合培养，4小时后用PI染色，FCM检测坏死细胞数。
7．细胞因子检测
收集上述混合细胞培养上清，ELISA检测细胞因子的分泌水平；收集上述混合培养后的淋巴细胞，FCM检测淋巴细胞内细胞因子的表达。
8．PD-L1诱导同种胰岛移植耐受实验
(1)STZ诱导糖尿病模型
用小剂量多次腹腔注射STZ的方法诱导糖尿病模型。
(2)细胞腹腔移植
将NIT-1、NIT-EGFP或NIT-PD-L1细胞(1.5×10~7)注射到Balb／c糖尿病模型小鼠腹腔。
(3)血糖、体重、胰岛素释放实验以及生存时间观察
移植后每隔一天监测一次血糖、体重及生存时间；在移植后第7天和24天，用放免法检测葡萄糖刺激的胰岛素释放量实验。
(4)细胞增殖、细胞凋亡、培养上清细胞因子及胞内细胞因子的测定方法同上
(5)小鼠血清细胞因子测定
移植后第7天，收集小鼠血清，ELISA检测细胞因子的表达。
(6)腹腔淋巴细胞凋亡以及腹腔冲洗细胞免疫荧光染色
移植后第7天，收集小鼠腹腔冲洗细胞，FCM检测腹腔淋巴细胞凋亡；免疫荧光染色检测移植细胞的排斥情况。
【结果】
1．PD-L1对同种淋巴细胞的调节作用
(1)PD-L1对同种淋巴细胞的增殖与凋亡的影响：新鲜分离的小鼠脾脏淋巴细胞作为反应细胞，与未修饰的NIT-1或PD-L1修饰的NIT-1(NIT-PD-L1)作为刺激细胞混合培养，FCM检测结果表明未修饰的NIT-1刺激的淋巴细胞增殖率显著高于NIT-PD-L1刺激的增殖率；
未修饰的NIT-1致敏淋巴细胞作为反应细胞，与未修饰的NIT-1或NIT-PD-L1作为刺激细胞混合培养，FCM检测结果表明未修饰的NIT-1刺激的淋巴细胞增殖率显著高于NIT-PD-L1刺激的增殖率，而NIT-1诱导的淋巴细胞凋亡率低于NIT-PD-L1诱导的淋巴细胞凋亡率；
NIT-PD-L1致敏的淋巴细胞作为反应细胞，与未修饰的NIT-1或NIT-PD-L1作为刺激细胞混合培养，NIT-PD-L1刺激的淋巴细胞增殖率显著低于未修饰的NIT刺激的淋巴细胞增殖，且可诱导反应细胞凋亡。结果表明PD-L1可抑制新鲜分离的初次反应性淋巴细胞和致敏淋巴细胞的增殖，并促进其凋亡。
(2)PD-L1对细胞因子的表达与分泌的影响：FCM检测淋巴细胞内细胞因子以及ELISA检测分泌性细胞因子结果表明，与NIT-1或NIT-EGFP诱导的淋巴细胞相比，NIT-PD-L1细胞诱导的淋巴细胞表达与分泌IL-4、IL-10水平显著增高，而IFN-γ水平明显降低。
(3)PD-L1对CTL活性的影响：通过NIT-1、NIT-PD-L1或NIT-EGFP三组细胞分别体内外联合刺激获得的脾淋巴细胞，分别与NIT-1细胞体外共培养后，CFSE与PI双染色，FCM检测结果表明，NIT-1或NIT-EGFP细胞诱导的脾淋巴细胞介导NIT-1靶细胞中度坏死，而NIT-PD-L1细胞诱导的脾淋巴细胞介导NIT-1靶细胞轻度坏死，结果表明PD-L1可体内外抑制CTL的激活与效应。
2．PD-L1延长STZ诱导的糖尿病小鼠胰岛移植物的存活
将NIT-1、NIT-PD-L1和NIT-EGFP三组细胞分别移植入STZ诱导的Balb／c糖尿病小鼠腹腔，观察移植前后的血糖、体重、糖刺激的胰岛素释放及生存时间，以及脾脏细胞的增殖与凋亡、细胞因子的表达与分泌，实验结果表明：
(1)PD-L1对糖尿病小鼠体重、血糖及存活时间的影响：移植NIT-1、NIT-PD-L1或NIT-EGFP细胞后第3天，各组糖尿病小鼠血糖均降至正常范围，其中移植NIT-1或NIT-EGFP细胞的对照组糖尿病小鼠血糖，在第7天左右升高并超过正常血糖水平；而移植NIT-PD-L1细胞的糖尿病小鼠可维持正常血糖水平约21天，随后逐渐升高，于移植后第24天血糖升高且超过正常上限(＞11.1mmol／L)；
在移植后第7天，移植NIT-1或NIT-EGFP细胞的对照组糖尿病小鼠体重逐渐下降，而移植NIT-PD-L1的糖尿病小鼠体重逐渐增加；移植NIT-PD-L1细胞糖尿病小鼠的生存时间比移植NIT-1或NIT-EGFP细胞的糖尿病小鼠明显延长。
(2)葡萄糖刺激的胰岛素释放：进一步检测葡萄糖刺激的胰岛素释放，由于移植NIT-1或NIT-EGFP细胞的糖尿病小鼠在移植后24天内均死亡，所以24天组仅包括移植NIT-PD-L1细胞的小鼠。检测结果表明，在移植后第7天，移植NIT-1或NIT-EGFP细胞的小鼠胰岛素释放量明显低于正常小鼠，而移植NIT-PD-L1细胞的小鼠胰岛素释放量与正常小鼠对照组相比无显著性差异，表明NIT-PD-L1细胞维持正常功能；但在移植后24天，移植NIT-PD-L1细胞小鼠的胰岛素释放量低于正常对照组小鼠，提示可能发生排斥反应。
(3)PD-L1延长移植物存活的免疫学机制：为了研究PD-L1延长移植物存活的机制，在细胞移植后第7天，分别检测了脾淋巴细胞和腹腔淋巴细胞的增殖与凋亡。FCM检测结果表明，移植NIT-1或NIT-EGFP细胞的小鼠脾淋巴细胞增殖反应显著高于NIT-PD-L1细胞移植小鼠，而细胞凋亡率低于NIT-PD-L1细胞移植的小鼠；
在移植后第7天，FCM检测脾淋巴细胞内细胞因子以及ELISA检测细胞培养上清和血清细胞因子结果表明，移植NIT-1或NIT-EGFP细胞的小鼠脾淋巴细胞内IFN-γ表达明显高于NIT-PD-L1细胞移植小鼠，而IL-4和IL-10显著低于移植NIT-PD-L1细胞的小鼠；脾淋巴细胞培养上清与小鼠血清中细胞因子检测结果与胞内细胞因子的表达类似。上述结果表明PD-L1抑制淋巴细胞增殖同时促进其凋亡；胞内细胞因子和ELISA检测结果显示，PD-L1抑制IFN-γ的表达，而增加IL-4和IL-10的产生，从而促进Th1细胞向Th2细胞漂移。
(4)腹腔冲洗细胞活性：腹腔冲洗细胞免疫荧光染色结果显示，移植NIT-PD-L1细胞的小鼠腹腔中胰岛素阳性细胞数量明显高于移植NIT-1或NIT-EGFP细胞的小鼠，而移植NIT-PD-L1细胞小鼠腹腔中淋巴细胞凋亡数增高，且浸润程度显著低于移植NIT-1或NIT-EGFP细胞的小鼠组。
【结论】PD-L1基因修饰的NIT可明显抑制同种淋巴细胞的增殖反应，同时诱导其凋亡；PD-L1可抑制CTL的活性，且可诱导Th2细胞因子的分泌，抑制Th1细胞因子的分泌；从而移植延长糖尿病小鼠移植物存活时间，并维持较长的正常血糖水平，研究结果为进一步通过诱导免疫耐受重建IDDM胰岛细胞功能研究奠定了基础。
【Objective】Type 1 diabetes (TID) is a T-cell-mediated autoimmune disease thatresults in the destruction of the insulin-secretingβ-cells in the pancreas by CD4~+ and CD8~+T cells, as well as macrophages infiltrating the isletsre and destruction of the insulinsecretingβ-cells in the pancreas.There is compelling evidence that the disease is associatedwith loss of immunological tolerance to self.Autoreactive T-cells have been identified andbeen considered to play a direct role in type 1 diabetes immunopathogenesis.Pancreaticislettransplantation has long been considered as a safer alternative and a potentiallypreferable treatment other than conventional exogenous-insulin therapy for T1D.Programmed Death-1 Ligand (PD-L1) has been identified as the ligand for theimmunoinhibitory receptor PD-1, and has been demonstrated to play a role in the negativeregulation of immune responses and peripheral tolerance.In the present study, weinvestigated the negative regulation function of PD-L1 and improvement of allorejection inβ-cells transplantation.
【Methods】
1.Establishment of PD-L1-GFP stable transfectants in NIT-1 cells
pPD-L1-EGFP or pEGFPn1 were transfected in NIT-1 cells, for establishment of pPD-L1-EGFP or pEGFPn1 stable transfectants, namely NIT-PD-L1 and NIT-EGFP.
2.Preparation of primary reactive and primed splenic lymphocytes
The splenoeytes from Balb/C (H-2d) mice of 8-12 weeks were collected and used asprimary reactive lymphocytes.Balb/C from above were immunized twice at days 1 and 8with NIT-1, NIT-EGFP or NIT-PD-L1 cells by i.p.injection, respectively.On day 14,splenocytes from these immunized mice were collected and used as primed spleniclymphocytes.
3.Mixed cells reaction
Primary reactive and primed splenic lymphocytes were stimulated in vitro with NIT-1,NIT-EGFP or NIT-PD-L1 cells pretreated with mitomycin C for 7 days, respectivly.
4.Lymphocyte proliferation assay
Proliferation of splenic lymphocytes labeled with CFSE was analyzed by FCM.
5.Lymphocyte apoptosis assay
Apoptosis of CFSE-unlabeled splenic lymphocytes were detected by labeling AnnexinV-Cy5 and PI using FCM.
6.Combined induction in vivo and in vitro extended CTL assay
Balb/C mice were immunized twice at days 1 and 8 with NIT-1, NIT-EGFP or NIT-PD-L1cells by i.p.injection, respectively.On day 14, splenocytes were collected andrestimulated with NIT-1, NIT-EGFP or NIT-PD-L1 cells pretreated with mitomycinCrespectively, and used as effector cells.NIT-1 cells were used as target cells after labledwith CFSE.Target cells were cultured with effector cells and stained with PI beforeanalyzed by FCM.Determination of cytolysis is based on the number of dead target cells,which are characterized by CFSE and PI double positivity.
7.Cytokine detection
The supematants from above co-culture of primary reactive or primed lymphocyteswere harvested for determination of Cytokine by ELISA.Intracellular cytokine weredetected by flow cytometry.
8.The function of PD-L1 in induction of islet transplantation tolerance
The diabetes model was established by a low dose of streptozotocin (STZ) in Balb/Cmice.NIT-1, NIT-EGFP or NIT-PD-L1 cells were transplanted into diabetic mice by i.p.injection, respectively.After transplantation, body weight, survival time, BG level weremonitored every other day.At day 7 and 24 of post-transplantation, in vivo-sfimulatedinsulin secretion assay was performed on the recipients.At 7th-day of post-transplantation,proliferation and apoptosis of splenic lymphocytes from these transplanted diabetic mice were detected by FCM, the production of cytokine was detected by FCM and ELISA.On7~(th)-day of post-transplantation, peritoneal cells from these transplanted diabetic mice were collected anddetected the insulin-secreting NITs.
【Results】
1.PD-L1 leaded to negative regulation of allogeneie lymphocyte activation
PD-L1 inhibited proliferation of primary reactive lymphocytes and primarylymphocytes, and enhance apoptosis of allogeneic lymphocytes.In addition, proliferativeresponse of NIT-1 or NIT-EGFP-primed lymphocytes stimulated with NIT-PD-L1 cellswas significantly lower than these lymphocytes resfimulated with NIT-1 cells or NIT-EGFPcells.Furthermore, proliferative response of NIT-PD-L1-primed lymphocytes restimulatedwith NIT-PD-L1 cells was the lowest among all groups.The apoptosis rate of NIT-1 orNIT- EGFP- primed lymphocytes cocultured with NIT-PD-L1 cells was much higher thanthose lymphocytes cocultured with NIT-1 cells or NIT-EGFP cells.Furthermore, theapoptosis rate of NIT-PD-L1- primed lymphocytes cocultured with NIT-PD-L1 cells wasthe highest among all, groups.ELISA and intracellular cytokine assay results indicated thatPD-L1 downregulated IFN-γbut upregulated IL-4 and IL-10 production by the primedlymphocytes.The NIT-1 had a severe necrosis cultured with NIT-1 or NIT-EGFP-primedlymphocytes, however, the NIT-1 cells had moderate necrosis cultured with NIT-PD-L1-primed lymphocytes.
2.PD-L1 can prolong allograft survival
Streptozotocin-induced diabetic mice received NIT-1, NIT-EGFP or NIT-PD-L1 cellsshowed decreased BG levels after 3 days of transplantation.However, BG of diabetic micethat transplanted with NIT-1 or NIT-EGFP cells gradually rebounded by day 7 posttransplantation.On the contrary, diabetic mice that received NIT-PD-L1 cells reachednormoglycemia after 3 days of transplantation, and the lowest BG was observed at day 13of post-transplantation.These mice had maintained normoglycemic for about 21 days, then their BG increased gradually and became hyperglycemic (>11.1mmol/L) again by day 24of post-transplantation.
We examined body weight for all recipients further.Diabetic mice receiving NIT-1 orNIT-EGFP cells showed a continual decrease in body weight.However, mice transplantedwith NIT-PD-L1 cells gained weight on 7th-day after transplantation.
To evaluate the glucose-induced insulin releasing capability on 7~(th)-day and 24~(th)-day ofpost-transplantation, insulin radioimmunoassay was performed.Because all the micetransplanted with NIT-1 or NIT-EGFP cells died before day 24 post-transplantation, onlythose mice transplanted with NIT-PD-L1 cells were included at this time-point.The insulinrelease amounts of mice were assayed at 15 min, after feeding the mice with glucose bolus.At day 7 post-transplantation, insulin contents for mice transplanted with NIT-1 or NIT-EGFPcells were significantly lower than those of non-diabetic control mice, whereas, formice transplanted with NIT-PD-L1 cells, insulin content remained similar to normal mice.More importantly, their life span was significantly longer than that of mice transplantedwith NIT-1 or NIT-EGFP cells.
In order to explore the mechanisms underlying the suppressive effect of PD-L1, theproliferation and apoptosis of lymphocytes from recipients were further examined.Theseresults displayed that PD-L1 inhibited proliferation of lymphocytes and induced apoptosisof lymphocytes.The results of ELISA and intracellular cytokine assay indicated that PD-L1altered the cytokines profiles of activated lymphocytes, with increased seen in the Th2cytokines, IL-10 and IL-4, and a decreased in Th1 cytokines, IFN-γ.Immunofluorescencestaining showed that a larger member of insulin-secreting cells from NIT-PD-L1 cellstransplanted mice could be seen in peritoneal.
【Conclusions】Over-expression PD-L1 on NIT-1 cells inhibted proliferation oflymphocytes and induced apoptosis of lymphocytes.In vitro extended CTL assay showedthat PD-L1 can protect NIT-1 cells from CTL-mediated lysis.PD-L1 altered the cytokineprofiles of activated lymphocytes, with increases seen in the Th2 cytokines, IL-10 and IL-4, and a decrease in Th1 cytokine, IFN-γ.Expression of PD-L1 on pancreaticβ-cells hassignificantly prolonged allograft survival, and the understanding of this will guide rationaltherapeutic strategies for the improvement of allorejection inβ-cells transplantation.
I型糖尿病是一种主要由T细胞介导的，CD4~+、CD8~+T细胞和巨噬细胞浸润胰岛导致分泌胰岛素的β细胞受损，使胰岛素分泌减少，导致胰岛素绝对缺乏。目前已证实该疾病和自身免疫耐受缺失有关，自身反应性T细胞在I型糖尿病的免疫发病机理中起主导作用。PD-L1已被证实为免疫负性调节受体PD-1的配体，文献证明PD-L1在免疫负性调节及外周耐受中发挥重要作用。本实验构建PD-L1表达载体，转染NIT细胞，通过体内外实验探讨PD-L1免疫调节作用，研究其在链脲佐菌素(STZ)诱导的糖尿病小鼠模型中延长胰岛移植存活时间及其作用机制。
【方法】
1．稳定表达pPD-L1的NIT-1细胞株的建立
用Lipofectamine~(TM)2000将pPD-L1-EGFP n1和pEGFPn1两个质粒分别转染NIT-1细胞系获得稳定转染细胞株，分别命名为NIT-PD-L1和NIT-EGFP。
2．致敏淋巴细胞的制备
分别用三组刺激细胞NIT-1、NIT-PD-L1和NIT-EGFP体内腹腔注射Balb／c小鼠，间隔1周再腹腔注射一次，细胞免疫后第14天分离Balb／c小鼠脾脏单个核细胞作为致敏淋巴细胞备用。
3．混合细胞培养
将经过丝裂霉素C处理的NIT-1、NIT-PD-L1和NIT-EGFP三组细胞作为刺激细胞分别与新鲜分离的脾脏淋巴细胞和(或)上述致敏淋巴细胞进行混合培养7天。
4．细胞增殖实验
用CFSE预染上述小鼠淋巴细胞，待细胞混合培养7天后，FCM检测淋巴细胞的增殖。
5．细胞凋亡检测
收集上述细胞混合培养7天后的淋巴细胞，Annexin V-cy5和PI染色，FCM检测淋巴细胞的凋亡。
6．细胞毒实验
分别用三组刺激细胞NIT-1、NIT-PD-L1和NIT-EGFP体内腹腔注射Balb／c小鼠，间隔1周再腹腔注射一次，细胞免疫后第14天分离小鼠脾淋巴细胞，将刺激细胞与上述获得的脾淋巴细胞以1：10的比例混合培养，3天后收集淋巴细胞作为效应细胞。将NIT-1细胞用CFSE染色，作为靶细胞，并以1：20的比例与效应细胞混合培养，4小时后用PI染色，FCM检测坏死细胞数。
7．细胞因子检测
收集上述混合细胞培养上清，ELISA检测细胞因子的分泌水平；收集上述混合培养后的淋巴细胞，FCM检测淋巴细胞内细胞因子的表达。
8．PD-L1诱导同种胰岛移植耐受实验
(1)STZ诱导糖尿病模型
用小剂量多次腹腔注射STZ的方法诱导糖尿病模型。
(2)细胞腹腔移植
将NIT-1、NIT-EGFP或NIT-PD-L1细胞(1.5×10~7)注射到Balb／c糖尿病模型小鼠腹腔。
(3)血糖、体重、胰岛素释放实验以及生存时间观察
移植后每隔一天监测一次血糖、体重及生存时间；在移植后第7天和24天，用放免法检测葡萄糖刺激的胰岛素释放量实验。
(4)细胞增殖、细胞凋亡、培养上清细胞因子及胞内细胞因子的测定方法同上
(5)小鼠血清细胞因子测定
移植后第7天，收集小鼠血清，ELISA检测细胞因子的表达。
(6)腹腔淋巴细胞凋亡以及腹腔冲洗细胞免疫荧光染色
移植后第7天，收集小鼠腹腔冲洗细胞，FCM检测腹腔淋巴细胞凋亡；免疫荧光染色检测移植细胞的排斥情况。
【结果】
1．PD-L1对同种淋巴细胞的调节作用
(1)PD-L1对同种淋巴细胞的增殖与凋亡的影响：新鲜分离的小鼠脾脏淋巴细胞作为反应细胞，与未修饰的NIT-1或PD-L1修饰的NIT-1(NIT-PD-L1)作为刺激细胞混合培养，FCM检测结果表明未修饰的NIT-1刺激的淋巴细胞增殖率显著高于NIT-PD-L1刺激的增殖率；
未修饰的NIT-1致敏淋巴细胞作为反应细胞，与未修饰的NIT-1或NIT-PD-L1作为刺激细胞混合培养，FCM检测结果表明未修饰的NIT-1刺激的淋巴细胞增殖率显著高于NIT-PD-L1刺激的增殖率，而NIT-1诱导的淋巴细胞凋亡率低于NIT-PD-L1诱导的淋巴细胞凋亡率；
NIT-PD-L1致敏的淋巴细胞作为反应细胞，与未修饰的NIT-1或NIT-PD-L1作为刺激细胞混合培养，NIT-PD-L1刺激的淋巴细胞增殖率显著低于未修饰的NIT刺激的淋巴细胞增殖，且可诱导反应细胞凋亡。结果表明PD-L1可抑制新鲜分离的初次反应性淋巴细胞和致敏淋巴细胞的增殖，并促进其凋亡。
(2)PD-L1对细胞因子的表达与分泌的影响：FCM检测淋巴细胞内细胞因子以及ELISA检测分泌性细胞因子结果表明，与NIT-1或NIT-EGFP诱导的淋巴细胞相比，NIT-PD-L1细胞诱导的淋巴细胞表达与分泌IL-4、IL-10水平显著增高，而IFN-γ水平明显降低。
(3)PD-L1对CTL活性的影响：通过NIT-1、NIT-PD-L1或NIT-EGFP三组细胞分别体内外联合刺激获得的脾淋巴细胞，分别与NIT-1细胞体外共培养后，CFSE与PI双染色，FCM检测结果表明，NIT-1或NIT-EGFP细胞诱导的脾淋巴细胞介导NIT-1靶细胞中度坏死，而NIT-PD-L1细胞诱导的脾淋巴细胞介导NIT-1靶细胞轻度坏死，结果表明PD-L1可体内外抑制CTL的激活与效应。
2．PD-L1延长STZ诱导的糖尿病小鼠胰岛移植物的存活
将NIT-1、NIT-PD-L1和NIT-EGFP三组细胞分别移植入STZ诱导的Balb／c糖尿病小鼠腹腔，观察移植前后的血糖、体重、糖刺激的胰岛素释放及生存时间，以及脾脏细胞的增殖与凋亡、细胞因子的表达与分泌，实验结果表明：
(1)PD-L1对糖尿病小鼠体重、血糖及存活时间的影响：移植NIT-1、NIT-PD-L1或NIT-EGFP细胞后第3天，各组糖尿病小鼠血糖均降至正常范围，其中移植NIT-1或NIT-EGFP细胞的对照组糖尿病小鼠血糖，在第7天左右升高并超过正常血糖水平；而移植NIT-PD-L1细胞的糖尿病小鼠可维持正常血糖水平约21天，随后逐渐升高，于移植后第24天血糖升高且超过正常上限(＞11.1mmol／L)；
在移植后第7天，移植NIT-1或NIT-EGFP细胞的对照组糖尿病小鼠体重逐渐下降，而移植NIT-PD-L1的糖尿病小鼠体重逐渐增加；移植NIT-PD-L1细胞糖尿病小鼠的生存时间比移植NIT-1或NIT-EGFP细胞的糖尿病小鼠明显延长。
(2)葡萄糖刺激的胰岛素释放：进一步检测葡萄糖刺激的胰岛素释放，由于移植NIT-1或NIT-EGFP细胞的糖尿病小鼠在移植后24天内均死亡，所以24天组仅包括移植NIT-PD-L1细胞的小鼠。检测结果表明，在移植后第7天，移植NIT-1或NIT-EGFP细胞的小鼠胰岛素释放量明显低于正常小鼠，而移植NIT-PD-L1细胞的小鼠胰岛素释放量与正常小鼠对照组相比无显著性差异，表明NIT-PD-L1细胞维持正常功能；但在移植后24天，移植NIT-PD-L1细胞小鼠的胰岛素释放量低于正常对照组小鼠，提示可能发生排斥反应。
(3)PD-L1延长移植物存活的免疫学机制：为了研究PD-L1延长移植物存活的机制，在细胞移植后第7天，分别检测了脾淋巴细胞和腹腔淋巴细胞的增殖与凋亡。FCM检测结果表明，移植NIT-1或NIT-EGFP细胞的小鼠脾淋巴细胞增殖反应显著高于NIT-PD-L1细胞移植小鼠，而细胞凋亡率低于NIT-PD-L1细胞移植的小鼠；
在移植后第7天，FCM检测脾淋巴细胞内细胞因子以及ELISA检测细胞培养上清和血清细胞因子结果表明，移植NIT-1或NIT-EGFP细胞的小鼠脾淋巴细胞内IFN-γ表达明显高于NIT-PD-L1细胞移植小鼠，而IL-4和IL-10显著低于移植NIT-PD-L1细胞的小鼠；脾淋巴细胞培养上清与小鼠血清中细胞因子检测结果与胞内细胞因子的表达类似。上述结果表明PD-L1抑制淋巴细胞增殖同时促进其凋亡；胞内细胞因子和ELISA检测结果显示，PD-L1抑制IFN-γ的表达，而增加IL-4和IL-10的产生，从而促进Th1细胞向Th2细胞漂移。
(4)腹腔冲洗细胞活性：腹腔冲洗细胞免疫荧光染色结果显示，移植NIT-PD-L1细胞的小鼠腹腔中胰岛素阳性细胞数量明显高于移植NIT-1或NIT-EGFP细胞的小鼠，而移植NIT-PD-L1细胞小鼠腹腔中淋巴细胞凋亡数增高，且浸润程度显著低于移植NIT-1或NIT-EGFP细胞的小鼠组。
【结论】PD-L1基因修饰的NIT可明显抑制同种淋巴细胞的增殖反应，同时诱导其凋亡；PD-L1可抑制CTL的活性，且可诱导Th2细胞因子的分泌，抑制Th1细胞因子的分泌；从而移植延长糖尿病小鼠移植物存活时间，并维持较长的正常血糖水平，研究结果为进一步通过诱导免疫耐受重建IDDM胰岛细胞功能研究奠定了基础。
【Objective】Type 1 diabetes (TID) is a T-cell-mediated autoimmune disease thatresults in the destruction of the insulin-secretingβ-cells in the pancreas by CD4~+ and CD8~+T cells, as well as macrophages infiltrating the isletsre and destruction of the insulinsecretingβ-cells in the pancreas.There is compelling evidence that the disease is associatedwith loss of immunological tolerance to self.Autoreactive T-cells have been identified andbeen considered to play a direct role in type 1 diabetes immunopathogenesis.Pancreaticislettransplantation has long been considered as a safer alternative and a potentiallypreferable treatment other than conventional exogenous-insulin therapy for T1D.Programmed Death-1 Ligand (PD-L1) has been identified as the ligand for theimmunoinhibitory receptor PD-1, and has been demonstrated to play a role in the negativeregulation of immune responses and peripheral tolerance.In the present study, weinvestigated the negative regulation function of PD-L1 and improvement of allorejection inβ-cells transplantation.
【Methods】
1.Establishment of PD-L1-GFP stable transfectants in NIT-1 cells
pPD-L1-EGFP or pEGFPn1 were transfected in NIT-1 cells, for establishment of pPD-L1-EGFP or pEGFPn1 stable transfectants, namely NIT-PD-L1 and NIT-EGFP.
2.Preparation of primary reactive and primed splenic lymphocytes
The splenoeytes from Balb/C (H-2d) mice of 8-12 weeks were collected and used asprimary reactive lymphocytes.Balb/C from above were immunized twice at days 1 and 8with NIT-1, NIT-EGFP or NIT-PD-L1 cells by i.p.injection, respectively.On day 14,splenocytes from these immunized mice were collected and used as primed spleniclymphocytes.
3.Mixed cells reaction
Primary reactive and primed splenic lymphocytes were stimulated in vitro with NIT-1,NIT-EGFP or NIT-PD-L1 cells pretreated with mitomycin C for 7 days, respectivly.
4.Lymphocyte proliferation assay
Proliferation of splenic lymphocytes labeled with CFSE was analyzed by FCM.
5.Lymphocyte apoptosis assay
Apoptosis of CFSE-unlabeled splenic lymphocytes were detected by labeling AnnexinV-Cy5 and PI using FCM.
6.Combined induction in vivo and in vitro extended CTL assay
Balb/C mice were immunized twice at days 1 and 8 with NIT-1, NIT-EGFP or NIT-PD-L1cells by i.p.injection, respectively.On day 14, splenocytes were collected andrestimulated with NIT-1, NIT-EGFP or NIT-PD-L1 cells pretreated with mitomycinCrespectively, and used as effector cells.NIT-1 cells were used as target cells after labledwith CFSE.Target cells were cultured with effector cells and stained with PI beforeanalyzed by FCM.Determination of cytolysis is based on the number of dead target cells,which are characterized by CFSE and PI double positivity.
7.Cytokine detection
The supematants from above co-culture of primary reactive or primed lymphocyteswere harvested for determination of Cytokine by ELISA.Intracellular cytokine weredetected by flow cytometry.
8.The function of PD-L1 in induction of islet transplantation tolerance
The diabetes model was established by a low dose of streptozotocin (STZ) in Balb/Cmice.NIT-1, NIT-EGFP or NIT-PD-L1 cells were transplanted into diabetic mice by i.p.injection, respectively.After transplantation, body weight, survival time, BG level weremonitored every other day.At day 7 and 24 of post-transplantation, in vivo-sfimulatedinsulin secretion assay was performed on the recipients.At 7th-day of post-transplantation,proliferation and apoptosis of splenic lymphocytes from these transplanted diabetic mice were detected by FCM, the production of cytokine was detected by FCM and ELISA.On7~(th)-day of post-transplantation, peritoneal cells from these transplanted diabetic mice were collected anddetected the insulin-secreting NITs.
【Results】
1.PD-L1 leaded to negative regulation of allogeneie lymphocyte activation
PD-L1 inhibited proliferation of primary reactive lymphocytes and primarylymphocytes, and enhance apoptosis of allogeneic lymphocytes.In addition, proliferativeresponse of NIT-1 or NIT-EGFP-primed lymphocytes stimulated with NIT-PD-L1 cellswas significantly lower than these lymphocytes resfimulated with NIT-1 cells or NIT-EGFPcells.Furthermore, proliferative response of NIT-PD-L1-primed lymphocytes restimulatedwith NIT-PD-L1 cells was the lowest among all groups.The apoptosis rate of NIT-1 orNIT- EGFP- primed lymphocytes cocultured with NIT-PD-L1 cells was much higher thanthose lymphocytes cocultured with NIT-1 cells or NIT-EGFP cells.Furthermore, theapoptosis rate of NIT-PD-L1- primed lymphocytes cocultured with NIT-PD-L1 cells wasthe highest among all, groups.ELISA and intracellular cytokine assay results indicated thatPD-L1 downregulated IFN-γbut upregulated IL-4 and IL-10 production by the primedlymphocytes.The NIT-1 had a severe necrosis cultured with NIT-1 or NIT-EGFP-primedlymphocytes, however, the NIT-1 cells had moderate necrosis cultured with NIT-PD-L1-primed lymphocytes.
2.PD-L1 can prolong allograft survival
Streptozotocin-induced diabetic mice received NIT-1, NIT-EGFP or NIT-PD-L1 cellsshowed decreased BG levels after 3 days of transplantation.However, BG of diabetic micethat transplanted with NIT-1 or NIT-EGFP cells gradually rebounded by day 7 posttransplantation.On the contrary, diabetic mice that received NIT-PD-L1 cells reachednormoglycemia after 3 days of transplantation, and the lowest BG was observed at day 13of post-transplantation.These mice had maintained normoglycemic for about 21 days, then their BG increased gradually and became hyperglycemic (>11.1mmol/L) again by day 24of post-transplantation.
We examined body weight for all recipients further.Diabetic mice receiving NIT-1 orNIT-EGFP cells showed a continual decrease in body weight.However, mice transplantedwith NIT-PD-L1 cells gained weight on 7th-day after transplantation.
To evaluate the glucose-induced insulin releasing capability on 7~(th)-day and 24~(th)-day ofpost-transplantation, insulin radioimmunoassay was performed.Because all the micetransplanted with NIT-1 or NIT-EGFP cells died before day 24 post-transplantation, onlythose mice transplanted with NIT-PD-L1 cells were included at this time-point.The insulinrelease amounts of mice were assayed at 15 min, after feeding the mice with glucose bolus.At day 7 post-transplantation, insulin contents for mice transplanted with NIT-1 or NIT-EGFPcells were significantly lower than those of non-diabetic control mice, whereas, formice transplanted with NIT-PD-L1 cells, insulin content remained similar to normal mice.More importantly, their life span was significantly longer than that of mice transplantedwith NIT-1 or NIT-EGFP cells.
In order to explore the mechanisms underlying the suppressive effect of PD-L1, theproliferation and apoptosis of lymphocytes from recipients were further examined.Theseresults displayed that PD-L1 inhibited proliferation of lymphocytes and induced apoptosisof lymphocytes.The results of ELISA and intracellular cytokine assay indicated that PD-L1altered the cytokines profiles of activated lymphocytes, with increased seen in the Th2cytokines, IL-10 and IL-4, and a decreased in Th1 cytokines, IFN-γ.Immunofluorescencestaining showed that a larger member of insulin-secreting cells from NIT-PD-L1 cellstransplanted mice could be seen in peritoneal.
【Conclusions】Over-expression PD-L1 on NIT-1 cells inhibted proliferation oflymphocytes and induced apoptosis of lymphocytes.In vitro extended CTL assay showedthat PD-L1 can protect NIT-1 cells from CTL-mediated lysis.PD-L1 altered the cytokineprofiles of activated lymphocytes, with increases seen in the Th2 cytokines, IL-10 and IL-4, and a decrease in Th1 cytokine, IFN-γ.Expression of PD-L1 on pancreaticβ-cells hassignificantly prolonged allograft survival, and the understanding of this will guide rationaltherapeutic strategies for the improvement of allorejection inβ-cells transplantation.
引文
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14. Yamazaki T, Akiba H, Iwai H, et al. Expression of programmed death 1 ligands by murine T cells and APC. J. Immunol. 2002; 169: 5538.
15. Eppihimer MJ, Gunn J, Freeman GJ, et al. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation 2002; 9: 133.
16. Schoop R, Wahl P, Le Hir M, Heemann U, Wang M, W(u|¨)thrich RP. Wuthrich. Suppressed T-cell activation by IFN-gamma-induced expression of PD-L1 on renal tubular epithelial cells. Nephrol. Dial. Transplant. 2004; 19: 2713.
17. Nakazawa A, Dotan I, Brimnes J, et al. The expression and function of costimulatory molecules B7H and B7-H1 on colonic epithelial ceils. Gastroenterolog. 2004; 126: 1347.
18. Ishida M, Iwai Y, Tanaka Y, et al. Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues. Immunol. Lett. 2002; 84: 57.
19. Sandner SE, Clarkson MR, Salama AD, et al. Role of the programmed death-1 pathway in regulation of alloimmune responses in vivo. J. Immunol. 2005; 174: 3408.
20. Keir ME, Liang SC, Guleria I, et al. Tissue expression of PD-L1 mediates peripheral T cell tolerance. Jem. 2006; 203: 883.
21. Hamaguchi K, Gaskins HR, Leiter EH. NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse. Diabetes. 1991; 40: 842.
22. Holstad M, Sandier S. Prolactin protects against diabetes induced by multiple low doses of streptozotocin in mice. Endocrinology. 1999; 163: 163.
23. Wang M, Wang P, Liu YQ, et al. The immunosuppressive and protective ability of glucose-regulated protein 78 for improvement of alloimmunity in beta cell transplantation. Clin Exp Immunol. 2007; 150: 546.
24. Holstad M, Sandier S. Prolactin protects against diabetes induced by multiple low doses of streptozotocin in mice. J. Endocrinol. 1999; 163: 229.
25. Meier-Kriesche HU, Schold JD, Kaplan B. Long-term renal allograft survival: have we made significant progress or is it time to rethink our analytic and therapeutic strategies? Am. J. Transplant. 2004; 4: 1289.
26. Ito T, Ueno T, Clarkson MR, et al. Analysis of the role of negative T cell costimulatory pathways in CD4 and CD8 T cell-mediated alloimmune responses in vivo. J. Immunol. 2005; 174: 6648.
27. Subudhi SK, Zhou P, Yerian LM, et al. Local expression of B7-H1 promotes organspecific autoimmunity and transplant rejection. J. Clin. Invest. 2004; 113: 694.
28. Yadav D, Sarvetnick N. Costimulafion and Pancreatic Autoimmunity: The PD-1/PD-L Conundrum. Diabetic Studies. 2006; 3: 6.
1. GenomicsTivol, E. A. et al. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3, 541-7 (1995).
2. Waterhouse, P. et al. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270, 985-8 (1995).
3. Iwai, Y. et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 99, 12293-7 (2002).
4. Konishi, J. et al. B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res 10, 5094-100 (2004).
5. Webster, W. S. et al. Targeting molecular and cellular inhibitory mechanisms for improvement of antitumor memory responses reactivated by tumor cell vaccine. J Immunol 179, 2860-9 (2007).
6. Rodig, N. et al. Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and eytolysis. Eur J Immunol 33, 3117-26 (2003).
7. Keir, M. E. et al. Tissue expression of PD-L1 mediates peripheral T cell tolerance. J Exp Med 203, 883-95 (2006).
8. Tseng, S. Y. et al. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J Exp Med 193, 839-46 (2001).
9. Wu, C. et al. Immunohistochemical localization of programmed death-1 ligand-1 (PD-L1) in gastric carcinoma and its clinical significance. Aeta Histochem 108, 19-24 (2006).
10. Kitazawa, Y. et al. Involvement of the programmed death-1/programmed death-1 ligand pathway in CD4+CD25+ regulatory T-cell activity to suppress alloimmune responses. Transplantation 83, 774-82 (2007).
11. Chemnitz, J. M., Parry, R. V., Nichols, K. E., June, C. H. & Riley, J. L. SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J Immunol 173, 945-54 (2004).
12. Brown, J. A. et al. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol 170, 1257-66 (2003).
13. Raimondi, G., Shufesky, W. J., Tokita, D., Morelli, A. E. & Thomson, A. W. Regulated compartmentalization of programmed cell death-1 discriminates CD4+CD25+ resting regulatory T cells from activated T ceils. J Immunol 176, 2808-16 (2006).
14. Petrovas, C. et al. PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J Exp Med 203, 2281-92 (2006).
15. Ansari, M. J. et al. The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 198, 63-9 (2003).
16. Eppihimer, M. J. et al. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation 9, 133-45 (2002).
17. Schreiner, B. et al. Interferon-beta enhances monocyte and dendritic cell expression of B7-H1 (PD-L1), a strong inhibitor of autologous T-cell activation: relevance for the immune modulatory effect in multiple sclerosis. J Neuroimmunol 155, 172-82 (2004).
18. Sheppard, K. A. et al. PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta. FEBS Lett 574, 37-41 (2004).
19. Liu, J. et al. Plasma cells from multiple myeloma patients express B7-H1 (PD-L1) and increase expression after stimulation with IFN-{gamma} and TLR ligands via a MyD88-, TRAF6-, and MEK-dependent pathway. Blood 110, 296-304 (2007).
20. Parsa, A. T. et al. Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nat Med 13, 84-8 (2007).
21. Zhong, X., Tumang, J. R., Gao, W., Bai, C. & Rothstein, T. L. PD-L2 expression extends beyond dendritic cells/macrophages to B1 cells enriched for V(H)11/V(H)12 and phosphatidylcholine binding. Eur J Immunol 37, 2405-10 (2007).
22. Loke, P. & Allison, J. P. PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells. Proc Natl Acad Sci U S A 100, 5336-41 (2003).
23. Blazar, B. R. et al. Blockade of programmed death-1 engagement accelerates graft-versus-host disease lethality by an IFN-gamma-dependent mechanism. J Immunol 171, 1272-7 (2003).
24. Bennett, F. et al. Program death-1 engagement upon TCR activation has distinct effects on costimulation and cytokine-driven proliferation: attenuation of ICOS, IL-4, and IL-21, but not CD28, IL-7, and IL-15 responses. J Immunol 170, 711-8 (2003).
25. Nguyen, L. T. et al. Cross-linking the B7 family molecule B7-DC directly activates immune functions of dendritic cells. J Exp Med 196, 1393-8 (2002).
26. Radhakrishnan, S., Iijima, K., Kobayashi, T., Kita, H. & Pease, L. R. Dendritic cells activated by cross-linking B7-DC (PD-L2) block inflammatory airway disease. J Allergy Clin Immunol 116, 668-74 (2005).
27. Grabie, N. et al. Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8+ T-cell mediated injury in the heart. Circulation 116, 2062-71 (2007).
28. Keir, M. E., Freeman, G. J. & Sharpe, A. H. PD-1 regulates self-reactive CD8+ T cell responses to antigen in lymph nodes and tissues. J Immunol 179, 5064-70 (2007).
29. Salama, A. D. et al. Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis. J Exp Med 198, 71-8 (2003).
30. Baecher-Allan, C., Brown, J. A., Freeman, G. J. & Hailer, D. A. CD4+CD25+regulatory cells from human peripheral blood express very high levels of CD25 ex vivo. Novartis Found Symp 252, 67-88; discussion 88-91,106-14 (2003).
31. Krupnick, A. S. et al. Murine vascular endothelium activates and induces the generation of allogeneic CD4+25+Foxp3+ regulatory T cells. J Immunol 175, 6265-70 (2005).
32. Totsuka, T. et al. Regulation of murine chronic colitis by CD4+CD25- programmed death-1+ T cells. Eur J Immunol 35, 1773-85 (2005).
33. Sandner, S. E. et al. Role of the programmed death-1 pathway in regulation of alloimmune responses in vivo. J Immunol 174, 3408-15 (2005).
34. Vadivel, N., Trikudanathan, S. & Chandraker, A. Transplant tolerance through costimulation blockade--are we there yet? Front Biosci 12, 2935-46 (2007).
35. Kean, L. S., Gangappa, S., Pearson, T. C. & Larsen, C. P. Transplant tolerance in non-human primates: progress, current challenges and unmet needs. Am J Transplant 6, 884-93 (2006).
36. Tao, R. et al. Differential effects of B and T lymphocyte attenuator and programmed death-1 on acceptance of partially versus fully MHC-mismatched cardiac allografts. J Immunol 175, 5774-82 (2005).
37. Ozkaynak, E. et al. Programmed death-1 targeting can promote allograft survival. J Immunol 169, 6546-53 (2002).
38. Nomi, T. et al. Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer. Clin Cancer Res 13, 2151-7 (2007).
39. Jun, H. et al. B7-H1 (CD274) inhibits the development of herpetic stromal keratitis (HSK). FEBS Lett 579, 6259-64 (2005).
40. Barber, D. L. et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439, 682-7 (2006).
41. D'Souza, M. et al. Programmed death 1 expression on HIV-specific CD4+ T cells is driven by viral replication and associated with T cell dysfunction. J Immunol 179, 1979-87 (2007).
42. Beswick, E. J., Pinchuk, I. V., Das, S., Powell, D. W. & Reyes, V. E. Expression of the programmed death ligand 1, B7-H1, on gastric epithelial cells after Helicobacter pylori exposure promotes development of CD4+ CD25+ FoxP3+ regulatory T cells. Infect Immun 75, 4334-41 (2007).
43. Terrazas, L. I., Montero, D., Terrazas, C. A., Reyes, J. L. & Rodriguez-Sosa, M. Role of the programmed Death-1 pathway in the suppressive activity of alternatively activated macrophages in experimental cysticercosis. Int J Parasitol 35, 1349-58 (2005).
44. Hamanishi, J. et al. Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer. Proc Nat Acad Sci U S A 104, 3360-5 (2007).
45. Dong, H. et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 8, 793-800 (2002).
46. Matsumoto, K. et al. B7-DC regulates asthmatic response by an IFN-gamma-dependent mechanism. J Immunol 172, 2530-41 (2004).
47. Hirano, F. et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity. Cancer Res 65, 1089-96 (2005).
48. Wong, R. M. et al. Programmed death-1 blockade enhances expansion and functional capacity of human melanoma antigen-specific CTLs. Int Immunol 19, 1223-34 (2007).
49. Fukushima, A., Yamaguchi, T., Azuma, M., Yagita, H. & Ueno, H. Involvement of programmed death-ligand 2 (PD-L2) in the development of experimental allergic conjunctivitis in mice. Br J Ophthalmol 90, 1040-5 (2006).
50. Fife, B. T. et al. Insulin-induced remission in new-onset NOD mice is maintained by the PD-1-PD-L1 pathway. J Exp Med 203, 2737-47 (2006).
51. Gotsman, I. et al. Proatherogenic immune responses are regulated by the PD-1/PD-L pathway in mice. J Clin Invest 117, 2974-82 (2007).
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[5] Nishimura H and Honjo T. PD-1: an inhibitory immunoreceptor involved in peripheral tolerance. Trends Immunol 2001; 22: 265-268.
[6] Keir ME, Freeman GJ and Sharpe AH. PD-1 regulates self-reactive CD8+ T cell responses to antigen in lymph nodes and tissues. J Immunol 2007; 179: 5064-5070.
[7] Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 2000; 192: 1027.
[8] Yamazaki T, Akiba H, Iwai H, et al. Expression of programmed death 1 ligands by murine T cells and APC. J. Immunol. 2002; 169: 5538.
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1. Greenwald R J, Freeman GJ, Sharpe AH (2005) The B7 family revisited. Annu Rev Immunol 23: 515-548.
2. Chen L (2004) Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev Immunol 4: 336-347.
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6. Latchman YE, Liang SC, Wu Y et al. PD-L1-deficient mice show that PD-L1 on T cells, antigenpresenting cells, and host tissues negatively regulates T cells. Proc Natl Acad Sci USA 2004; 101: 10691-6.
7. Ansari MJ, Salama AD, Chitnis T et al. The programmed death- 1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 2003; 7:63-83.
8. Tsushima F, Iwai H, Otsuki N et al. Preferential contribution of B7-H1 to programmed death-1-mediated regulation of haptenspecific allergic inflammatory responses. Eur J Immunol 2003; 33:2773-82.
9. Smith P, Walsh CM, Mangan NE et al. Schistosoma mansoni worms induce anergy of T cells via selective up-regulation of programmed death ligand 1 on macrophages. J Immunol 2004; 173:1240-8.
10. Reynoso, E. D., Elpek, K. G., Francisco, L., Branson, R., Bellemare-Pelletier, A., Sharpe, A. H., Freeman, G. J., Turley, S. J., Intestinal tolerance is converted to autoimmune enteritis upon PD-1 ligand blockade. J Immunol 2009. 182:2102-2112.
11. Blank, C, Brown, I., Peterson, A. C, Spiotto, M., Iwai, Y., Honjo, T. and Gajewski, T. F., PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8+ T cells. Cancer Res 2004. 64: 1140-1145.
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1. Castano L, Eisenbarth GS. Type-Ⅰ diabetes: a chronic autoimmune disease of human, mouse, and rat. Annu Rev Immunol 1990; 8: 647.
2. Atkinson MA, Kaufman DL, Campbell L, et al. Response of peripheral-blood mononuclear cells to glutamate decarboxylase in insulin-dependent diabetes. Lancet 1992; 339: 458.
3. Naquet P, Ellis J, Tibensky D, et al. T-cell autoreactivity to insulin in diabetic and related non-diabetic individuals. J Immunol 1988; 140: 2569.
4. Hawkes CJ, Schloot NC, Marks J, et al. T-cell lines reactive to an immunodominant epitope of the tyrosine phosphatase-like autoantigen IA-2 in type 1 diabetes. Diabetes 2000; 49: 356.
5. Roep BO. The role of T-cells in the pathogenesis of type 1 diabetes: from cause to cure. Diabetologia 2003; 46: 305.
6. Giraud S, Claire B, Eugene M, Debre P, Richard F, Barrou B. A new preservation solution increases islet yield and reduces graft immunogenicity in pancreatic islet transplantation. Transplantation 2007; 83: 397.
7. Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 1992; 11: 3887.
8. Nishimura H, Honjo T. PD-1: an inhibitory immunoreceptor involved in peripheral tolerance. Trends Immunol. 2001; 22: 265
9. Carreno BM, Collins M. The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu. Rev. Immunol. 2002; 20: 29.
10. Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 2000; 192: 1027.
11. Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, costimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 1999; 5: 1365.
12. Latchman Y, Wood CR, Chernova T, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat. Immunol. 2001. 2: 261.
13. Tseng SY, Otsuji M, Gorski K, et al. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J. Exp. Med. 2001; 193: 839.
14. Yamazaki T, Akiba H, Iwai H, et al. Expression of programmed death 1 ligands by murine T cells and APC. J. Immunol. 2002; 169: 5538.
15. Eppihimer MJ, Gunn J, Freeman GJ, et al. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation 2002; 9: 133.
16. Schoop R, Wahl P, Le Hir M, Heemann U, Wang M, W(u|¨)thrich RP. Wuthrich. Suppressed T-cell activation by IFN-gamma-induced expression of PD-L1 on renal tubular epithelial cells. Nephrol. Dial. Transplant. 2004; 19: 2713.
17. Nakazawa A, Dotan I, Brimnes J, et al. The expression and function of costimulatory molecules B7H and B7-H1 on colonic epithelial ceils. Gastroenterolog. 2004; 126: 1347.
18. Ishida M, Iwai Y, Tanaka Y, et al. Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues. Immunol. Lett. 2002; 84: 57.
19. Sandner SE, Clarkson MR, Salama AD, et al. Role of the programmed death-1 pathway in regulation of alloimmune responses in vivo. J. Immunol. 2005; 174: 3408.
20. Keir ME, Liang SC, Guleria I, et al. Tissue expression of PD-L1 mediates peripheral T cell tolerance. Jem. 2006; 203: 883.
21. Hamaguchi K, Gaskins HR, Leiter EH. NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse. Diabetes. 1991; 40: 842.
22. Holstad M, Sandier S. Prolactin protects against diabetes induced by multiple low doses of streptozotocin in mice. Endocrinology. 1999; 163: 163.
23. Wang M, Wang P, Liu YQ, et al. The immunosuppressive and protective ability of glucose-regulated protein 78 for improvement of alloimmunity in beta cell transplantation. Clin Exp Immunol. 2007; 150: 546.
24. Holstad M, Sandier S. Prolactin protects against diabetes induced by multiple low doses of streptozotocin in mice. J. Endocrinol. 1999; 163: 229.
25. Meier-Kriesche HU, Schold JD, Kaplan B. Long-term renal allograft survival: have we made significant progress or is it time to rethink our analytic and therapeutic strategies? Am. J. Transplant. 2004; 4: 1289.
26. Ito T, Ueno T, Clarkson MR, et al. Analysis of the role of negative T cell costimulatory pathways in CD4 and CD8 T cell-mediated alloimmune responses in vivo. J. Immunol. 2005; 174: 6648.
27. Subudhi SK, Zhou P, Yerian LM, et al. Local expression of B7-H1 promotes organspecific autoimmunity and transplant rejection. J. Clin. Invest. 2004; 113: 694.
28. Yadav D, Sarvetnick N. Costimulafion and Pancreatic Autoimmunity: The PD-1/PD-L Conundrum. Diabetic Studies. 2006; 3: 6.
1. GenomicsTivol, E. A. et al. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3, 541-7 (1995).
2. Waterhouse, P. et al. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270, 985-8 (1995).
3. Iwai, Y. et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 99, 12293-7 (2002).
4. Konishi, J. et al. B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res 10, 5094-100 (2004).
5. Webster, W. S. et al. Targeting molecular and cellular inhibitory mechanisms for improvement of antitumor memory responses reactivated by tumor cell vaccine. J Immunol 179, 2860-9 (2007).
6. Rodig, N. et al. Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and eytolysis. Eur J Immunol 33, 3117-26 (2003).
7. Keir, M. E. et al. Tissue expression of PD-L1 mediates peripheral T cell tolerance. J Exp Med 203, 883-95 (2006).
8. Tseng, S. Y. et al. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J Exp Med 193, 839-46 (2001).
9. Wu, C. et al. Immunohistochemical localization of programmed death-1 ligand-1 (PD-L1) in gastric carcinoma and its clinical significance. Aeta Histochem 108, 19-24 (2006).
10. Kitazawa, Y. et al. Involvement of the programmed death-1/programmed death-1 ligand pathway in CD4+CD25+ regulatory T-cell activity to suppress alloimmune responses. Transplantation 83, 774-82 (2007).
11. Chemnitz, J. M., Parry, R. V., Nichols, K. E., June, C. H. & Riley, J. L. SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J Immunol 173, 945-54 (2004).
12. Brown, J. A. et al. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol 170, 1257-66 (2003).
13. Raimondi, G., Shufesky, W. J., Tokita, D., Morelli, A. E. & Thomson, A. W. Regulated compartmentalization of programmed cell death-1 discriminates CD4+CD25+ resting regulatory T cells from activated T ceils. J Immunol 176, 2808-16 (2006).
14. Petrovas, C. et al. PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J Exp Med 203, 2281-92 (2006).
15. Ansari, M. J. et al. The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 198, 63-9 (2003).
16. Eppihimer, M. J. et al. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation 9, 133-45 (2002).
17. Schreiner, B. et al. Interferon-beta enhances monocyte and dendritic cell expression of B7-H1 (PD-L1), a strong inhibitor of autologous T-cell activation: relevance for the immune modulatory effect in multiple sclerosis. J Neuroimmunol 155, 172-82 (2004).
18. Sheppard, K. A. et al. PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta. FEBS Lett 574, 37-41 (2004).
19. Liu, J. et al. Plasma cells from multiple myeloma patients express B7-H1 (PD-L1) and increase expression after stimulation with IFN-{gamma} and TLR ligands via a MyD88-, TRAF6-, and MEK-dependent pathway. Blood 110, 296-304 (2007).
20. Parsa, A. T. et al. Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nat Med 13, 84-8 (2007).
21. Zhong, X., Tumang, J. R., Gao, W., Bai, C. & Rothstein, T. L. PD-L2 expression extends beyond dendritic cells/macrophages to B1 cells enriched for V(H)11/V(H)12 and phosphatidylcholine binding. Eur J Immunol 37, 2405-10 (2007).
22. Loke, P. & Allison, J. P. PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells. Proc Natl Acad Sci U S A 100, 5336-41 (2003).
23. Blazar, B. R. et al. Blockade of programmed death-1 engagement accelerates graft-versus-host disease lethality by an IFN-gamma-dependent mechanism. J Immunol 171, 1272-7 (2003).
24. Bennett, F. et al. Program death-1 engagement upon TCR activation has distinct effects on costimulation and cytokine-driven proliferation: attenuation of ICOS, IL-4, and IL-21, but not CD28, IL-7, and IL-15 responses. J Immunol 170, 711-8 (2003).
25. Nguyen, L. T. et al. Cross-linking the B7 family molecule B7-DC directly activates immune functions of dendritic cells. J Exp Med 196, 1393-8 (2002).
26. Radhakrishnan, S., Iijima, K., Kobayashi, T., Kita, H. & Pease, L. R. Dendritic cells activated by cross-linking B7-DC (PD-L2) block inflammatory airway disease. J Allergy Clin Immunol 116, 668-74 (2005).
27. Grabie, N. et al. Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8+ T-cell mediated injury in the heart. Circulation 116, 2062-71 (2007).
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