简述影响肿瘤PD-L1表达的各种因素

程序性细胞死亡配体1(PD-L1)及其受体PD-1属于CD28/B7协同刺激分子超家族成员,具有负性协同刺激信号的作用。PD-L1/PD-1通路传导抑制性信号,抑制淋巴结CD8+T细胞的杀伤功能,对人体免疫应答起到负调节作用,降低其对肿瘤的杀伤作用。近年来,已有多种PD-L1/PD-1单克隆抗体进入肿瘤免疫治疗的临床研究阶段。本文就细胞因子、癌基因、MicroRNAs三个方面简述了调控PD-L1表达的分子机制,以期对PDL1单克隆抗体的临床研究提供参考。     

程序性死亡蛋白配体-1/程序性死亡蛋白-1在老年胃癌患者外周血CD8+T淋巴细胞中的表达及临床意义

目的 通过分析程序性死亡蛋白配体-1/程序性死亡蛋白-1(programmed death ligand-1/programmed death-1,PD-L1/PD-1)在老年胃癌患者外周血CD8+T淋巴细胞中表达情况,探讨其临床意义.方法 选择同一时期90例老年胃癌患者(具有不同临床分期)及90例老年健康体检者,分别采取新鲜外周血后经流式细胞仪检测血中CD8+T淋巴细胞表面PD-L1/PD-1表达情况,结合老年胃癌患者的临床分期,分析PD-L1/PD-1在不同胃癌分期中的表达意义.结果 PD-L1/PD-1在老年胃癌患者外周血CD8+T淋巴细胞呈高表达,相较于老年健康者差异具有统计学意义(t=7.043,P<0.05);外周血CD8+T淋巴细胞的PD-L1/PD-1阳性表达均随着临床分期的增加而增加,呈明显的正相关性(r=0.883,P<0.05);外周血CD8+T淋巴细胞的PD-1阳性表达随着临床分期的增加而增加,两者之间呈正相关性(r=0.811,P<0.05);临床分期较晚的老年胃癌患者外周血CD8+T淋巴细胞PD-L1/PD-1表达比其他相对较早的临床分期老年胃癌患者显著上升(t=4.377,P<0.05).结论 PD-L1/PD-1信号通路异常在老年胃癌患者病变的发生发展过程中发挥了重要作用,外周血CD8+T淋巴细胞PD-L1/PD-1的表达对评判患者的预后具有指导作用.     

Tim-3对人Jurkat T细胞NFAT信号通路的调控

目的利用基因过表达技术来研究关键免疫检查点Tim-3对人T淋巴细胞NFAT信号通路的调控作用。方法将NFAT-RE-Nluc、Tim-3基因编码序列制备成相应的慢病毒质粒,通过Fugene HD转染或者慢病毒介导的基因过表达技术,将NFAT-RE-Nluc和Tim-3基因编码序列导入到Jurkat T细胞中,通过萤光素酶报告基因检测系统检测细胞中的荧光信号强度。结果Tim-3和NFAT-RE-Nluc成功在人T淋巴细胞中表达,并且Tim-3成功定位到细胞膜上。Tim-3能够直接激活人T淋巴细胞的NFAT信号通路,诱导NFAT-RE-Nluc荧光素酶的表达。同时在Jurkat T细胞中,Tim-3也能够增强CD3抗体激活NFAT信号通路,诱导CD69的表达以及IL-2分泌的能力。结论成功构建了稳定表达Tim-3及NFAT-RE-Nluc报告基因的NFAT-RENluc Tim-3细胞株,为深入研究Tim-3与其配体Galectin-9或CEACAM1之间的相互作用,及筛选阻断Tim-3-Galectin-9-CEACAM1的更有效的新型免疫检查点药物提供了重要的基础。     

致敏小鼠树突状细胞和T细胞PD-1配体/受体的表达

目的 探讨致敏小鼠PD-1信号通路是否有改变.方法 RT-PCR法分别检测致敏及正常小鼠树突状细胞PD-1配体PD-L1、PD-L2及T细胞PD-1受体的mRNA表达情况,流式细胞检测PD-1和配体PD-LI、PD-12的表达.结果 [1]致敏小鼠树突状细胞PD-L1和PD-L2的mRNA表达均显著低于正常小鼠,致敏小鼠T细胞PD-1的mRNA表达也显著低于正常小鼠(P＜0.05).[2]流式细胞检测致敏小鼠树突状细胞PD-L1、PD-L2表达和T细胞PD-1的表达也显著低于正常小鼠.结论 致敏小鼠PD-1信号表达缺陷.     

HIV-1感染过程中CD8~+T细胞PD-1水平增高与其活化状态正相关

目的分析人类免疫缺陷病毒1型(HIV-1)感染者CD8+T细胞程序性死亡蛋白1(PD-1)、CD38、人白细胞抗原DR(HLA-DR)及KI-67抗原(ki67)的表达水平,探讨HIV-1感染过程中CD8~+T细胞PD-1表达水平与免疫活化和免疫耗竭的关系及意义。方法收集HIV-1感染者87例,健康志愿者22例,密度梯度法分离外周血单个核细胞;采用流式细胞术分析其CD8+T细胞PD-1、CD38、HLA-DR、ki67的表达水平;观察用抗PD-L1 m Ab阻断PD-1/PD-L1通路后CD8~+T细胞分泌肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)的水平变化。结果 HIV-1感染者CD8~+T细胞PD-1表达水平显著高于健康对照组;CD8~+T细胞PD-1水平与病毒载量呈正相关,与CD4~+T细胞数呈负相关;CD8~+T细胞PD-1与CD38、HLA-DR表达呈正相关,与ki67表达无相关性;体外阻断PD-1/PD-L1通路后TNF-α、IFN-γ的表达量增加。结论 HIV-1感染者外周血CD8~+T细胞PD-1表达增加;PD-1过表达与HIV感染过程中CD8~+T细胞功能受抑、免疫耗竭及疾病进程相关;体外阻断PD-1/PD-L1通路可恢复CD8~+T细胞功能。     

人PD-1Δex3基因的克隆、表达及其生物学活性的鉴定

目的:构建表达人PD-1Δex3(ΔPD-1)基因的真核表达载体,检测其表达和生物学活性。方法:通过RT-PCR的方法获得人PD-1全长(PD-1)基因,设计特异性引物,PCR方法获得PD-1Δex3基因的2个cDNA片段,通过重组PCR的方法获得ΔPD-1基因,将目的片段双酶切后与pIRES2-EGFP真核表达载体连接,构建重组真核表达载体pIRES2-EGFP/PD-1和pIRES2-EGFP/ΔPD-1并进行鉴定;脂质体转染法将重组载体导入293T细胞;流式细胞术(FCM)检测转染细胞膜表面PD-1的表达;Western blot法检测培养上清中可溶性PD-1(sPD-1)的表达;间接免疫荧光实验分析ΔPD-1蛋白与PD-1两个配体PD-L1和PD-L2的结合。结果:酶切和测序结果均证实插入的基因序列正确,成功构建两个真核表达载体;FCM分析和Western blot检测结果表明,PD-1转染细胞膜表面高表达PD-1蛋白,细胞培养上清中无sPD-1蛋白,而ΔPD-1转染细胞膜表面不表达PD-1蛋白,细胞培养上清中则有大量sPD-1;间接免疫荧光实验结果表明,ΔPD-1蛋白能够与PD-1两种配体产生特异性结合。结论:成功进行PD-1Δex3基因的真核表达,证实PD-1Δex3基因编码sPD-1蛋白,该蛋白具有良好的生物学活性,为进一步研究PD-1Δex3在PD-1/PD-L信号通路中的生物学作用提供了有价值的物质基础。     

肝癌患者外周血CD3~+T细胞和CD19~+B细胞中程序性死亡蛋白1(PD-1)及其配体表达水平增加

目的 观察肝细胞肝癌(HCC)患者外周血单个核细胞(PBMC)中程序性死亡蛋白1(PD-1)和配体以及γ干扰素(IFN-γ)水平的变化。方法 采集15例早期HCC患者、13例进展期HCC患者和12例健康人(对照组)的外周血并分离PBMC。流式细胞术检测PBMC中PD-1、PD-L1和PD-L2的表达,ELISA检测血清中IFN-γ的水平,利用Pearson相关系数法和One-way ANOVA分析PD-1与IFN-γ的相关性。结果 进展期HCC患者外周血CD3+T细胞和CD19+B细胞中PD-1、PD-L1和PD-L2的表达水平和血清IFN-γ水平显著高于健康人群和早期HCC患者,HCC患者外周血CD3+T细胞和CD19+B细胞PD-1、PD-L1表达水平与IFN-γ呈正相关。结论 肝癌患者PBMC中PD-1、PD-L1和PD-L2分子表达增加,且PD-1、PD-L1与IFN-γ水平有关联。     

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...     

PD-1/PD-L1信号通路与肿瘤免疫逃逸

PD-1及其配体PD-L1、PD-L2广泛表达于多种细胞表面,是一组与免疫负调控有关的共刺激分子。PD-1/PD-L1信号通路的激活有利于肿瘤逃脱机体免疫监视,但具体的肿瘤免疫逃逸机制尚未清楚。就抗PD-1和抗PD-L1抗体疗效而言,仍然需要对肿瘤免疫逃逸机制做更深入的研究。文章就现有研究成果作一综述如下。     

原发CD5阳性弥漫大B细胞淋巴瘤18例临床病理特征

目的 探讨原发CD5阳性的弥漫大B细胞淋巴瘤(diffuse large B cell lymphoma, DLBCL)的临床病理及分子学特征。方法 收集18例原发CD5⁺ DLBCL的临床资料，采用免疫组化EnVision两步法检测CD3、CD5、CD7、CD20、PAX-5、CD10、BCL-6、BCL-2、C-MYC、MUM1等表达；应用FISH法检测C-MYC、BCL-2及BCL-6基因断裂；采用一代测序法检测MYD88 L265P、CD79B Y196F的突变；并复习相关文献。结果 18例CD5⁺ DLBCL中有3例(16.7%)CD30阳性；13例(72.2%)C-MYC阳性，16例(88.9%)BCL-2阳性，其中C-MYC和BCL-2双阳性12例(66.7%);15例(83.3%)BCL-6阳性；14例(81.8%)伴p53突变(13例错义突变、1例无义突变)。10例(55.6%)肿瘤细胞PD-1阳性，阳性率5%～80%;肿瘤浸润淋巴细胞PD-1阳性，阳性率5%～60%。8例(44.4%)肿瘤细胞PD-L1阳性，阳性率2%～8...     

PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients

Programmed death-1 (PD-1) is demonstrated to have an increased expression on antigen-specific T cells during chronic virus infections, and the blockage of PD-1/PD-ligand (PD-L1) pathway could restore the function of exhausted T cells. We measured the PD-1 expression levels on HBV-specific CD8 T cells and investigated the role of PD-1/PD-L1 pathway in T-cell responses of patients with different HBV infection statuses. Compared to the patients with convalescent acute hepatitis B, PD-1 expression on total CD8 T cells from chronic hepatitis B (CHB) patients was significantly upregulated, especially on the HBV pentamer-positive CD8 T cells. And PD-L1, but not PD-L2, was also significantly upregulated on PBMC from CHB patients. In CHB patients, HBV-specific T cells and cellular proliferation could be observed under the recombinant HBV-Ag stimulation in vitro, and blockade of PD-1 pathway significantly enhanced the IFN-gamma production and cellular proliferation of PBMC. Furthermore, PD-1 expression level on HBV-pentamers positive CD8 T cells was positively associated with plasma viral load in CHB patients. Thus, PD-1 upregulation on HBV-specific CD8 T cells is engaged in the dysfunction of T cells and high viraemia in CHB patients, and the antiviral T-cell responses could be improved by the blockade of this inhibitory PD-1/PD-L1 pathway.     

Intrahepatic Expression of Programmed Death-1 and its Ligands in Patients with HBV-Related Acute-on-Chronic Liver Failure

Hepatitis B virus (HBV) infection is a major public health problem, and HBV-related acute-on-chronic liver failure (HBV-ACLF) has an extremely poor prognosis due to a lack of understanding of pathogenesis as well as a lack of effective treatments. Signals from the inhibitory receptor programmed death-1 (PD-1) have been demonstrated to be involved in regulating the pathogenesis of infectious diseases. However, the expression of PD-1 and its ligands in HBV-ACLF patients has yet to be evaluated. In this study, the expression of PD-1 and its ligands, PD-L1 and PD-L2, in liver biopsies from HBV-ACLF as well as chronic hepatitis B (CHB) patients were analyzed by immunohistochemistry. The results showed that all three molecules were observed in the HBV-ACLF samples and their levels were significantly higher than they were in CHB. Immunofluorescence double-staining showed that PD-1 was found on CD3⁺, CD8⁺ T cells, CD56⁺ NK cells, CD68⁺ macrophages, CK-18⁺ epithelial cells, and CD16⁺ monocytes. The PD-L1 expression was observed on all cell types detected and the PD-L2 was chiefly on CK-18⁺ epithelial cells and CD31⁺ endothelial cells. Interestingly, high levels of virus-induced procoagulant molecule fibrinogen-like protein 2 (FGL2) were observed in liver sections from HBV-ACLF, and PD-L1 and PD-L2 expression was also observed on FGL2⁺ cells in these patients. Our combined results suggest that the expression of PD-L1 and PD-L2 may be biomarkers to identify and diagnose ACLF, and a clear understanding of their functional roles should further elucidate the pathogenesis of this disease.     

Programmed death 1 is a marker of angioimmunoblastic T-cell lymphoma and B-cell small lymphocytic lymphoma/chronic lymphocytic leukemia

Programmed death 1 (PD-1) is a lymphoid receptor that negatively regulates immune responses. PD-1 expression was recently reported in some T-cell non-Hodgkin lymphoma (NHL) subtypes, but the expression profile of PD-1 and its ligands (PD-L1 and PD-L2) in B-NHLs remains largely to be characterized. To investigate this issue, monoclonal antibodies against PD-1, PD-L1, and PD-L2 were generated by immunization of balb-c mice. A series of 161 lymphoma tissue and 11 blood samples was analyzed using either immunohistochemistry or flow cytometry. In reactive lymph nodes, PD-1 was mainly expressed in follicular T cells. In B-NHLs, PD-1 was mainly expressed in reactive T cells; but expression was also noted in neoplastic B cells from small lymphocytic lymphoma (SLL, 12/13), grade III follicular lymphoma (3/3), and diffuse large cell lymphoma (2/25). In contrast, neoplastic B cells from mantle cell lymphoma (0/11), marginal zone lymphoma (0/12), Burkitt lymphoma (0/3), and grade 1 to 2 follicular lymphoma (0/40) were PD-1 negative. PD-L1 and PD-L2 were negative in small B-cell lymphomas, including B-SLL. Flow cytometry showed that blood cells from chronic lymphocytic leukemia (B-CLL) also displayed PD-1 expression, which could be increased by CD40 stimulation. PD-1 expression in T-NHLs was restricted to the angioimmunoblastic subtype (8/8). These results show that PD-1 expression among B-NHLs is mainly associated with SLL/CLL and is influenced by activation of the CD40/CD40L pathway. Because the anti-PD-1.6.4 antibody works on paraffin sections, it represents a useful tool to differentiate SLL/CLL from other small B-cell lymphomas.     

Fine-tuned expression of programmed death 1 ligands in mature dendritic cells stimulated by CD40 ligand is critical for the induction of an efficient tumor specific immune response

During maturation, murine myeloid dendritic cells （DCs） upregulated the expressions of CDllc, CD25, CD40, CD80, CD86, MHC Ⅱ and programmed death 1 ligands 1 and 2 （PD-L1 and PD-L2）. Differential expression patterns of PD-L1 and PD-L2 were found when DCs were triggered by CD40 ligand and TNF-α. PD-L1 expression was repressed and PD-L2 expression remained unchanged in mature CD40-ligated DCs, whereas TNF-α stimulated DCs kept high expression of PD-L1 and significantly enhanced PD-L2 expression on DCs. Proliferations of T lymphocytes stimulated by immature DCs were enhanced by blockade of the PD-1 and PD-1 ligand interaction. But inhibitive effects were found in T lymphocytes stimulated by CD40-ligated DCs. With the fine-tuned expressions of PD-L1 and PD-L2, CD40-1igated DCs could sustain a longer activation period and elicit a more efficient T lymphocyte activation. Cellular ＆ Molecular Immunology.     

慢性乙型肝炎患者血清HBV DNA水平与细胞毒性T淋巴细胞表面PD-1表达的关系和意义

目的 探讨慢性乙型肝炎（CHB）患者血清HBV DNA水平与HBV特异性CTL表面程序性死亡受体-1（PD-1）表达的关系和意义.方法 将133例CHB患者根据血清HBV DNA水平分为甲、乙两组,甲组70例（52.63％）,HBV DNA水平为104～105拷贝/ml,乙组63例（47.37％）,HBVDNA水平分107 ～ 108拷贝/ml,对两组患者作HBV特异性CTL、非特异性CTL,HBV特异性CTL表面PD-1表达和肝功能的比较.结果 甲组HBV DNA水平为（5.03±1.01）log10拷贝/ml,低于乙组（7.59±0.99） log10拷贝/ml,t=11.23,P＜0.01,HBV特异性CTL表面PD-1表达（26.32±2.56）％,低于乙组（39.35 ±2.86）％,t=18.69,P＜0.01,HBV特异性CTL （0.37±0.02）％,高于乙组（0.22±0.02）％,t=25.80,P＜0.01,非特异性CTL（16.26 ±2.17）％,低于乙组（19.94±2.47）％,t=3.19,P＜0.01,ALT （259.16 ±85.11） U/L,低于乙组（501.16±86.15） U/L,t=15.10,P＜0.01,TBil（27.99±6.13）μmol/L,低于乙组（44.86±9.51） μmol/L,t=3.92,P＜0.01.结论 CHB患者HBV DNA水平低者,HBV特异性CTL表面PD-1表达也低,HBV特异性CTL水平高,HBV DNA水平高者,HBV特异性CTL表面PD-1表达也高,导致HBV特异性CTL水平降低,并启动非特异性CTL,引起肝功能损害加重.     

Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways

Summary: Classically, the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA-4) and B7 families of cell surface molecules regulate complex signaling pathways that profoundly affect T-cell responses. The recent identification and characterization of additional CD28 and B7 family members including programmed death-1 (PD-1), programmed death ligand-1 (PD-L1) (B7-H1), and PD-L2 (B7-DC) has added to the complexity and greater appreciation of how surface molecules control T-cell activation and peripheral tolerance. CD28/B7 interactions mediate co-stimulation and significantly enhance peripheral T-cell responses. CTLA-4, in contrast, interacting with the same B7 molecules, results in decreased T-lymphocyte activity and regulates the immune response. Similarly, PD-1 interactions with PD-L1 and PD-L2 downmodulate T-cell immune responses. Despite these similarities, the regulatory roles of the CTLA-4 and PD-1 pathways are distinct. This may be due, at least in part, to the differential expression patterns of the CTLA-4 and PD-1 ligands both temporally and spatially. This article examines the role of CTLA-4 and PD-1 in limiting autoreactivity and establishing peripheral self-tolerance with the hypothesis that CTLA-4 signals are required early in the lymph node during initiation of an immune response and PD-1 pathways act late at the tissue sites to limit T-cell activity.     

慢性乙型肝炎患者HBV C、B基因型与特异性细胞毒性T淋巴细胞表面PD-1表达的关系

目的 探讨慢性乙型肝炎（CHB）患者HBV C、B基因型与HBV特异性细胞毒性T淋巴细胞（CTL）表面程序性死亡受体-1（PD-1）表达的关系和意义.方法 共研究CHB患者71例,人白细胞抗原（HLA）-A2阳性,HBV DNA ＞103拷贝/ml,其中C基因型34例（47.89％）,B基因型36例（50.70％）.比较C、B基因型感染者外周血HBV特异性CTL表面PD-1的表达水平、HBV特异性CTL水平、HBV DNA水平、ALT和TBil的水平.结果 CHB C基因型感染者的HBV特异性CTL表面PD-1的表达水平（37.30±3.05％）高于B基因型感染者（26.19±3.06％）,t=15.47,P＜0.001,HBV特异性CTL水平（0.25±0.03％）低于B基因型感染者（0.45±0.13％）,t=21.54,P＜0.001,HBV DNA水平（6.75±0.77lo910拷贝/ml）高于B基因型感染者（4.96±1.12 log10拷贝/ml）,t=7.93,P＜0.001,ALT水平（487.39±87.36IU/L）高于B基因型感染者（235.25±90.91IU/L）,t=12.32,P＜0.001,TBil水平（49.73±6.45）高于B基因型感染者（28.48±5.89％）,t=9.01,P＜0.001.结论 CHB C基因型感染者外周血HBV特异性CTL表面PD-1表达水平高于B基因型感染者,导致C基因型感染者HBV特异性CTL水平低于B基因型感染者,HBV DNA水平高于B基因型感染者,因此,肝功能损害比B基因型感染者重.