舒尼替尼抑制树突状细胞表面共刺激分子配体的表达

目的研究舒尼替尼(sunitinib)对肾癌患者外周血单核细胞来源的树突状细胞(DC)表面共刺激分子程序性死亡蛋白配体1(PD-L1)、PD-L2、CD80、CD86、B7-H4及疱疹病毒侵入介体(HVEM)表达的影响。方法体外诱导肾癌患者外周血单核细胞来源的DC,随机分为sunitinib联合LPS组、LPS组及DMSO组。Sunitinib联合LPS组用200 ng/m L sunitinib预处理DC 12 h,再用1μg/m L脂多糖(LPS)刺激24 h;LPS组用1μL/m L DMSO预处理12 h,再用1μg/m L LPS刺激24 h;DMSO组用1μL/m L DMSO作用36 h。倒置显微镜观察各组形态变化;流式细胞术检测DC表面PD-L1、PD-L2、CD80、CD86、B7-H4及HVEM表达变化。结果 Sunitinib联合LPS组和LPS组的DC均呈典型的树枝状突起,DMSO组细胞树突不明显;与LPS组相比,sunitinib联合LPS组表达的CD80、PD-L1及B7-H4显著下降;DMSO组的PD-L1、PD-L2、CD80、CD86、B7-H4及HVEM均呈低水平表达。结论 Sunitinib抑制LPS诱导DC表达CD80、PD-L1、B7-H4。     

重症肌无力患者外周血B7：CD28／CTLA4共刺激分子表达的动态变化

目的：研究重症肌无力（MG）患者B7：CD28／CTLA4共刺激通路相关分子表达动态变化及其与MG发病的关系。方法：用流式细胞仪检测18例MG患者和16例健康对照者外周血单个核细胞（PBMC）在经PMA（佛波酯）＋ionomycin(钙离子导入剂）刺激的0h,6h,24h,48h时B7－1、B7－2、CD28、CTLA4分子在CD4^ T细胞和CD8^ T细胞表面的表达。结果：（1）0h,MG患者B7－1、B7－2分子总体表达增加，CD4^ 、CD8^ T细胞表面B7－1、B7－2的表达未见明显增加，PMA＋ionomycin刺激后B7－1、B7－2在CD4^ 、CD8^ T细胞表面也无增加；（2）0h,CD28、CTLA4的表达增强，其中CD28^ 细胞的增多主要表现在CD4^ T细胞亚群，CTLA4的表达增强主要在CD8^ T细胞，在PMA＋ionomycin激活后，CD28表达明显增强，持续到48h都处于较高水平（与对照组相比P＜0．01），CTLA4表达出现短暂增强，6h即达高峰，以后下降，与对照组相比无显著增强（P＞0．05）。结论：MG患者外周血中B7：CD28／CTLA4通路共刺激相关分子表达增高，持续时间延长，检测共刺激分子的表达可反映机体的免疫激活状态，B7：CD28／CTLA4通路在MG发病中可能起重要作用。     

p38MAPK通路调控树突状细胞表达PD-L1的实验研究

目的探讨脂多糖刺激单核细胞源树突状细胞表达程序性死亡因子配体1(PD-L1)与p38MAPK信号通路的关系。方法体外培养树突状细胞,用p38抑制剂SB203580阻断p38MAPK通路后再用LPS刺激树突状细胞。光学显微镜观察各组细胞的形态变化;流式细胞术测定CD86和PD-L1表达的平均荧光强度;Western blot测定PD-L1蛋白表达。结果光镜下观察LPS刺激组细胞先经历梭型贴壁后逐渐恢复圆形,树突较多;SB203580和LPS共同刺激组细胞树突退化,未刺激组细胞成团悬浮,树突较多。SB203580和LPS共同刺激组细胞CD86、PD-L1较LPS刺激组的明显降低(P<0.05或P<0.01),与未刺激组的相比CD86差异无统计学意义,但PD-L1降低(P<0.05)。SB203580和LPS共同刺激组细胞PD-L1的蛋白表达量较其它两组的均明显减少(P<0.01或P<0.01)。结论 LPS通过p38MAPK信号通路调控树突状细胞表达PD-L1。     

PD-1/PD-L1在骨肉瘤中的研究进展

PD-1是一种主要表达于活化的CD4+T细胞、CD8+T细胞、B细胞、NK细胞、单核细胞和DC等免疫细胞上的跨膜蛋白.PD-1及其配体PD-L1是一对共刺激分子,在正常情况下,这对分子活化后的信号通路可调节外周组织中T细胞的分化,进而调控机体对外来或自身抗原的免疫应答反应,以防免疫过激情况的发生.但在肿瘤发生时,肿瘤细...     

重症肌无力患者外周血B7∶CD28/CTLA4共刺激分子表达的动态变化

目的研究重症肌无力(MG)患者B7∶CD28/CTLA4共刺激通路相关分子表达动态变化及其与MG发病的关系. 方法用流式细胞仪检测18例MG患者和16例健康对照者外周血单个核细胞(PBMC)在经PMA(佛波酯)+ionomycin(钙离子导入剂)刺激的0 h、6 h、24 h、48 h时B7-1、B7-2、CD28、CTLA4分子在CD4+ T细胞和CD8+ T细胞表面的表达. 结果 (1) 0 h,MG患者B7-1、B7-2分子总体表达增加,CD4+、CD8+ T细胞表面B7-1、B7-2的表达未见明显增加,PMA+ionomycin刺激后B7-1、B7-2在CD4+、CD8+ T细胞表面也无增加;(2) 0 h,CD28、CTLA4的表达增强,其中CD28+细胞的增多主要表现在CD4+ T细胞亚群,CTLA4的表达增强主要在CD8+ T细胞,在PMA+ionomycin激活后,CD28表达明显增强,持续到48 h都处于较高水平(与对照组相比P＜0.01),CTLA4表达出现短暂增强,6 h即达高峰,以后下降,与对照组相比无显著增强(P＞0.05). 结论 MG患者外周血中B7∶CD28/CTLA4通路共刺激相关分子表达增高,持续时间延长,检测共刺激分子的表达可反映机体的免疫激活状态,B7∶CD28/CTLA4通路在MG发病中可能起重要作用.     

B7家族新成员PD-L1、PD-L2的研究进展

“协同刺激信号”是1970年由Bretscher和Cohn在T细胞活化双信号模型的基础上提出的，即除了通过APC递呈MHC处理过的抗原给抗原特异性T细胞提供第1信号外，还需协同刺激分子作为辅助信号的协同作用，达到生理活化闽值后才能使T细胞产生正常的免疫应答，从而在理论上更好地解释了免疫系统对自身和非自身抗原不同识别的凋节机制。如果缺少共刺激分子的第2信号，将会导致调节性T细胞的无反应性或特异性免疫耐受。     

B7家族的新成员—B7-H1、B7-H2、B7-H3、B7-DC

T细胞的活化需要TCR与抗原肽-MHC的第一信号，以及共刺激因子的第二信号系统。B7家族是重要的共刺激分子，属于免疫球蛋白家族。除B7-1和B7-2外，近几年又发现了B7家族的新成员——B7-H1、B7-H2、B7-H3、B7-DC等，与T、B细胞的活化及免疫应答等有关。深入研究B7家族成员的结构和作用机制，有助于T、B细胞活化机制以及免疫耐受、肿瘤免疫研究的深入和发展。     

PD-1／PD-L信号通路研究进展

PD-1是近年新发现的一个负性共刺激信号分子，其配体为PD-L1和PD-L2，共属于CD28／B7家族。PD-1／PD-L通路削弱、限制和／或终止T细胞、B细胞和骨髓细胞的活化及外周炎症的效应器功能；二者相互作用导致T细胞增殖抑制和细胞因子IL-2、IL-10和IFN-γ的分泌下降，在移植免疫、自身免疫及肿瘤免疫等体系中发挥着重要作用。     

外周血CD19+B细胞PD-L1的表达与系统性红斑狼疮发病的相关性分析

目的:探讨程序性死亡配体1(Programmed death ligand-1,PD-L1)在系统性红斑狼疮(Systemic lupus erythema-tosus,SLE)患者外周血B细胞上的表达及临床意义。方法:应用流式细胞仪检测51例SLE患者和38例健康对照者外周血CD19+B细胞表面PD-L1的表达水平,比较SLE稳定组、活动组和健康对照组以及狼疮肾炎组和无狼疮肾炎组之间CD19+B细胞表面PD-L1表达阳性细胞的百分比,并分析其与临床表现及实验室检查数据的相关性。结果:SLE活动组和稳定组CD19+PD-L1+B细胞百分率均低于健康对照组,活动组又低于稳定组,差异均有统计学意义(均P<0.05)。狼疮肾炎患者CD19+PD-L1+B细胞百分率低于无狼疮肾炎患者(P<0.05)。SLE患者CD19+PD-L1+B细胞百分率与SLEDAI评分、尿蛋白定量、呈负相关,与C3呈正相关。SLE患者中抗dsDNA抗体、抗Sm抗体、抗U1snRNP抗体、抗核小体抗体阳性组外周血B细胞PD-L1表达水平均低于对应阴性组,且均有统计学意义(均P<0.05)。结论:SLE患者外周血CD19+B细胞表达PD-L1下降,与病情活动性和抗体产生有很好的相关性。     

LPS诱导肺微血管内皮细胞ICAM-1的表达及 NF-κ B作用的实验研究

目的研究 LPS诱导的肺微血管内皮细胞( PWVEC) ICAM- 1的表达及调控机制.方法 100ng/ml LPS刺激 PMVEC 0h、2h、4h、6h、8h或 10ng/ml、50ng/ml、100ng/ml LPS刺激 6h,免疫细胞化学检测 PMVEC ICAM- 1的表达.凝胶电泳迁移率变化分析检测 NF- κ B的活化.并通过加入活化阻断剂 PDTC观察对 PMVEC ICAM- 1表达的影响.结果 PMVEC ICAM- 1的表达与 LPS的刺激呈时相 - 剂量依赖方式.LPS的刺激迅速活化 NF- κ B,60min达到高峰,后逐渐下降.PDTC能显著降低 ICAM- 1的表达( P<0. 01).结论 LPS刺激诱导 NF- κ B的活化,启动 ICAM- 1的合成表达.     

Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues

PD-1 is a member of the immunoglobulin superfamily expressed on immune cells, including T and B cells, and is involved in the delivery of inhibitory signal upon engagement of its ligands, PD-L1 and PD-L2. While the expression profile of PD-1 has been well documented, the analysis of PD-L1 and PD-L2 distributions on a protein basis has not been carried out because of the lack of available monoclonal antibodies specific for the molecules. In this study, we established two monoclonal antibodies, 1-111A and 122, specific for murine PD-L1 and PD-L2, respectively, and examined their expression profiles. Based on flow cytometric analyses, the expression of PD-L1 was detected in a variety of lymphohematopoietic cell types, including a minor proportion of T and B cells in the spleen, majority of pre-B cells and myeloid cells in bone marrow and subsets of thymocytes, while the expression of PD-L2 was not observed in the lymphohematopoietic cells at all. Notably, a significant proportion of the most immature lineage-marker-negative and c-Kit-positive bone marrow cells containing stem cells did express PD-L1. Following mitogenic stimulation, essentially all lymphocytes expressed PD-L1. Furthermore, a variety of leukemic lines also expressed PD-L1, while none of them did PD-L2. Thus, present results demonstrate the distinct expression patterns of PD-L1 and PD-L2 with the cells of lymphohematopoietic tissues exclusively expressing the former.     

Reciprocal expression of co-stimulatory molecules,B7-1 and B7-2, on murine T cells following activation

The co-stimulatory B7 molecules (B7-1 and B7-2) are expressed on professional antigen-presenting cells in mice. In this study, we demonstrate that B7-1 (CD80) and B7-2 (CD86) are also expressed on murine T cells in the absence of major histocompatibility complex class II molecules. The temporal expression of these two molecules on T cells varies with the state of activation where resting T cells express B7-2 but show little or no expression of B7-1. Following activation, B7-2 expression is down-regulated and there is a concomitant increase in the expression of B7-1 on the cell surface which peaks at about 72 h. Thus these two co-stimulatory molecules are reciprocally expressed on the T cell surface. This pattern of expression of B7-1 and B7-2 on T cells suggests that these two molecules may have different roles in the generation and regulation of immune responses.     

In situ expression of B7 and CD40 costimulatory molecules by normal human lung macrophages and epithelioid cells in tuberculoid granulomas.

Normal alveolar macrophages (AM) are not efficient in inducing the proliferation of resting T lymphocytes, and, rather, tend to inhibit pulmonary immune responses. In contrast, epithelioid cells (EC), activated macrophages that play an essential role in the course of granulomatous responses, appear to stimulate T cell proliferation efficiently. The inability of macrophages to deliver potent costimulatory signals through the B7/CD28 and CD40/CD40L pathways could explain their weak accessory cell activity. Using MoAbs and immunohistochemical techniques, however, we found that essentially all AM in normal human lung tissue expressed B7-1, B7-2 and CD40 molecules, and most of these cells were strongly positive. Pulmonary macrophages in other compartments also expressed these costimulatory molecules; no differences in expression were observed comparing macrophages from smokers and non-smokers. Most AM recovered by bronchoalveolar lavage from normal lung segments also strongly expressed B7-1, B7-2 and CD40 molecules. In comparison, resting blood monocytes were B7-1- and only moderately positive for B7-2. Activation of monocytes with lipopolysaccharide (LPS) induced expression of these costimulatory molecules to levels similar to that of AM from the control subjects. EC in granulomatous lesions also expressed easily detectable levels of B7-1, B7-2 and CD40. T lymphocytes within and surrounding the granulomas expressed CD28, the counter-receptor for B7, and many of these T cells also expressed B7-1 and B7-2. These findings suggest that both AM and EC can deliver costimulatory signals through B7-1, B7-2 and CD40 molecules, and indicate that the impairment in accessory cell activity observed for normal AM cannot be attributed to the absence of expression of these costimulatory molecules.     

B7-H3： Another Molecule Marker for Mo-DCs？

Using a newly generated monoclonal antibody （2E6） against human B7-H3, we explored the expression of the molecule on dendritic cells derived from monocytes （Mo-DCs）. Its expression was examined by means of immunostaining and flow cytometric （FCM） analysis. The results showed that B7-H3 was expressed in the course of Mo-DC maturation induced with interleukin 4 （IL-4） and granulocyte/macrophage colony-stimulating factor （GM-CSF）. The expression could be detected at all the stages of Mo-DC differentiation, and remained at a quite stable level. Interestingly, B7-H3 was not expressed by T cells and B cells, even these cells were activated respectively by PHA or PWM. A weak expression could be detected on resting monocytes. These data showed that constitutive expression of B7-H3 at a high level was found on imDCs and mDCs derived from monocytes. Due to no expression on T cells and B cells, we speculate that B7-H3 might be another valuable molecule marker for Mo-DCs.     

Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis

Interactions between CD8+ T cells and endothelial cells are important in both protective and pathologic immune responses. Endothelial cells regulate the recruitment of CD8+ T cells into tissues, and the activation of CD8+ T cells by antigen presentation and costimulatory signals. PD-L1 and PD-L2 are recently described B7-family molecules which bind to PD-1 on activated lymphocytes and down-regulate T cell activation. We found that PD-L1 is expressed on interferon-gamma stimulated cultured human and mouse endothelial cells, while PD-L2 was found on stimulated human but not mouse endothelial cells. Expression was further up-regulated by TNF-alpha. Antibody blockade of endothelial cell PD-L1 and PD-L2 enhanced endothelial cell costimulation of PHA-activated human CD8+ T cells. Antibody blockade of mouse endothelial cell PD-L1 enhanced both IFN-gamma secretion and cytolytic activity of CD8+ T cells in response to endothelial cell antigen presentation. These results show that IFN-gamma activated endothelial cells can inhibit T cell activation via expression of the immunoinhibitory PD-L1 and PD-L2 molecules. Endothelial expression of PD-ligands would allow activation and extravasation of T cells without excessive vessel damage. Our findings highlight a potentially important pathway by which endothelial cells down-regulate CD8+ T cell-mediated immune responses.     

PD-L2 expression extends beyond dendritic cells/macrophages to B1 cells enriched for V(H)11/V(H)12 and phosphatidylcholine binding

B1 B cells are the major source of natural antibody that is essential for innate immunity. The B1 repertoire is skewed toward production of phosphatidylcholine (PtC)-binding V(H)11 and V(H)12 immunoglobulin that plays a key role in immune defense against bacterial infection. Programmed death-ligand 2 (PD-L2) is a ligand for the immunosuppressive receptor programmed death-1 (PD-1). It has been reported that expression of PD-L2 is restricted to dendritic cells and macrophages in mice. Here we show that 50-70% of resting peritoneal B1 cells express PD-L2, which is not present or inducible on conventional B2 B cells or PD-L2(-) B1 cells. Although PD-L2(+) and PD-L2(-) B1 cells are similar in proliferative responses and spontaneous immunoglobulin secretion, PD-L2(+) B1 cells are highly enriched for expression of V(H)11 and V(H)12 genes and encompass the bulk of PtC-binding B1 cells. These findings extend the range of known PD-L2 expression to B cells and show that B1 cells identified by this marker express a specific repertoire associated with anti-bacterial immunity.     

PD-L2 is expressed on activated human T cells and regulates their function

T-cell activation and proliferation are regulated by cosignaling adhesion molecules involved in positive or negative signals. Programmed death (PD)-1 is one of immune inhibitory molecules that is expressed in activated T cells and is a promising target for immunotherapy. Both PD-1 ligands, PD-L1 and PD-L2 are expressed on antigen presenting cells (APCs) involved in the dialogue between a T cell and an APC. Here, we analysed the expression of these ligands, especially for PD-L2, on T cells. PD-L2 appears to be expressed on activated CD4 and CD8T cell subsets. Moreover, as PD-1 molecule, PD-L2 engagement at the surface of T cells is able to down-modulate cytokine production and cell proliferation. These observations indicate that PD-L2 is expressed following activation and is involved in the regulation of T cell function, highlighting the level of complexity in the T cell cosignaling network.     

PD-L2 is expressed on activated human T cells and regulates their function

T-cell activation and proliferation are regulated by cosignaling adhesion molecules involved in positive or negative signals. Programmed death (PD)-1 is one of immune inhibitory molecules that is expressed in activated T cells and is a promising target for immunotherapy. Both PD-1 ligands, PD-L1 and PD-L2 are expressed on antigen presenting cells (APCs) involved in the dialogue between a T cell and an APC. Here, we analysed the expression of these ligands, especially for PD-L2, on T cells. PD-L2 appears to be expressed on activated CD4 and CD8T cell subsets. Moreover, as PD-1 molecule, PD-L2 engagement at the surface of T cells is able to down-modulate cytokine production and cell proliferation. These observations indicate that PD-L2 is expressed following activation and is involved in the regulation of T cell function, highlighting the level of complexity in the T cell cosignaling network.     

Surface and intracellular interleukin-2 receptor expression on various resting and activated populations involved in cell-mediated immunity in human peripheral blood

The kinetics of assembly of the high-affinity interleukin-2 receptor (IL-2R)alpha/beta/gamma were investigated by studying intracellular and surface expression of IL-2Ralpha, beta and gamma by T cells, monocytes and natural killer (NK) cells. IL-2Ralpha and IL-2Rgamma were expressed by small numbers of resting T cells. These numbers increased following stimulation, to maximal expression at 48 h and 72 h, respectively. This observation was consistent with de novo synthesis of the receptor protein in response to the stimulus. The proportion of T cells producing IL-2Rbeta was smaller and up-regulated later than the proportion of cells producing IL-2Ralpha or IL-2Rgamma. IL-2Rbeta may therefore slow the assembly of the high-affinity IL-2R on T cells. A small number of resting NK cells expressed IL-2Ralpha, both on the cell surface and intracellularly, but this increased over 72 h on stimulated NK cells. IL-2Rbeta was constitutively expressed, both on the cell surface and intracellularly, by monocytes and NK cells. An increased proportion of NK cells and monocytes produced IL-2Rbeta, 24 h and 4 h post-stimulation, respectively. Maximal or plateau expression occurred at 72 h and 24 h post-stimulation, for NK cells and monocytes, respectively. The early up-regulation of intracellular IL-2Rbeta for monocytes may facilitate the up-regulation of surface IL-2Rbeta, and early assembly of the high-affinity IL-2R, accelerating monocyte activation and function. High constitutive intracellular IL-2Rgamma expression (> 80%) in all types of leucocyte investigated, decreased over the 72 h following stimulation with a concurrent increase in surface expression. IL-2Rgamma was expressed by increased proportions of T cells, monocytes and NK cells, 4 h following stimulation. The intracellular storage of IL-2Rgamma may accelerate translocation to the cell surface after stimulation. The early translocation of IL-2Rgamma may reflect its usage as a signal transduction molecule by other cytokine receptors - IL-4, IL-7, IL-9 and IL-15. This study delineated the potential expression of the high-affinity IL-2Ralpha/beta/gamma on various stimulated leucocytes. The differential kinetics of assembly of the high-affinity IL-2Ralpha/beta/gamma on different leucocyte subsets suggests that IL-2 may regulate the inflammatory cellular responses in a sequential manner, paralleling the timed expression of IL-2Ralpha/beta/gamma on the monocytes, NK cells and T cells.     

Induction of B7-H1 receptor by bacterial cells fractions of Porphyromonas gingivalis on human oral epithelial cells: B7-H1 induction by Porphyromonas gingivalis fractions

The immune-regulatory B7-H1 receptor, also known as programmed death-ligand 1 (PD-L1), plays an important role in cell-mediated immune response. It is a co-signaling molecule that mediates regulation of T cell activation and tolerance and is able to negatively regulate activated T cell functions and survival. High expression of B7-H1 in host cells may contribute to the chronicity of inflammatory disorders and represents a possible mechanism of immune evasion. Porphyromonas gingivalis is regarded as a keystone pathogen in periodontitis and is able to invade host cells and disposes a variety of virulence factors including lipopolysaccharide (LPS), fimbriae and proteases such as gingipains. Based on previous studies that demonstrated the capability of P. gingivalis to induce up-regulation of PD-L1 in malignant and non-malignant oral epithelial cells, the aim of the present work was to analyse the potential of various cellular components of P. gingivalis to induce the PD-L1 receptor. Human squamous carcinoma cells and primary gingival keratinocytes were stimulated with total, inner and outer membrane fractions, cytosolic proteins, as well as LPS and peptidoglycans. PD-L1 protein expression was investigated by Western blot analysis and RT-PCR. It was demonstrated that the total membrane fraction induced the highest up-regulation in B7-H1 expression, followed by the outer and inner membrane, whereas cytosolic proteins and LPS did not. In conclusion, we provide evidence that the membrane fraction of P. gingivalis is responsible for up-regulation of the immune-regulatory receptor PD-L1 in squamous carcinoma cells and gingival keratinocytes, and thus may support immune evasion of oral carcinomas.