乙型肝炎肝硬化患者外周血T细胞PD-1和PD-L1表达水平及意义

目的探讨乙型肝炎肝硬化患者外周血T淋巴细胞程序性死亡分子-1(PD-1)及其主要配体PD-L1的表达情况。方法在50例乙型肝炎肝硬化患者和25例健康体检者,使用流式细胞仪检测外周血T细胞PD-1和PD-L1表达;采用荧光定量核酸扩增及测序法检测血清HBV DNA载量。结果对照组和肝硬化组外周血T细胞PD-1阳性表达率分别为11.93±1.23%和33.13±3.38%(P<0.05),PD-L1阳性表达率分别为10.59±1.88%和32.47±2.18%(P<0.05);Child-Pugh A级(30.58±2.99%和32.19±1.44%)、B级(34.61±1.43%和33.46±2.58%)和C级(34.2±2.31%和31.76±2.33%)患者外周血T细胞PD-1和PD-L1表达率无显著性相差(P>0.05);在肝硬化患者,T细胞表面PD-l和PD-Ll表达水平与血清HBV DNA载量呈明显正相关(r2=0.8326和:r2=0.643,P<0.05)。结论肝硬化患者外周血T细胞PD-1和PD-L1表达水平明显上调,且与血清HBV DNA载量呈明显正相关,提示T细胞高表达的PD-1可能通过与其配体PD-L1作用而抑制T细胞免疫应答,并导致病毒感染持续。     

肝细胞癌患者CD8＋T淋巴细胞上PD-1高表达的意义研究

目的了解细胞程序性死亡受体1（programmed death1,PD—1）在HBV相关性肝细胞癌（hepatocellular carcinoma,HCC）患者CD8＋T淋巴细胞上的表达及与疾病状态的关系。方法用流式技术检测58例HBV相关性PHC患者外周血及肝组织中CD8^＋T淋巴细胞上PD-1的表达,并与20例HBV相关性肝硬化患者及20例健康对照者相比较。结果HCC患者外周血CD8^＋T淋巴细胞上PD-1的表达明显上调,与肝硬化组及健康组相比,差异有统计学意义,并且肿瘤区的表达高于非肿瘤区,随着HCC病情的进展而增加。结论PD-1在CD8^＋T淋巴细胞上表达具有负性调节CD8^＋T淋巴细胞活化增殖作用,其表达水平对HCC病情有预测作用。     

PD-1/PD-L1信号通路与慢性乙型肝炎

目的：程序性死亡分子-1（programmed death-1,PD-1）是近年来发现的属于B7/CD28家族的重要协同刺激分子,与其配体（programmed death -1 ligand,PD-L）结合后在调节T淋巴细胞的活化、分化及增殖功能方面起着重要作用。在慢性HBV感染不同阶段,PD-1表达水平存在差异,且与肝脏炎症程度、ALT及病毒载量等密切相关。通过不同途径阻断PD-1/PD-L1通路可以使耗竭的T淋巴细胞功能得到改善,提示可能是未来抗病毒治疗的方向之一。     

自身免疫性肝炎患者外周血CD8~+T淋巴细胞PD-1表达及意义

目的研究自身免疫性肝炎患者外周血CD8+T淋巴细胞程序性死亡受体1(PD-1)表达的变化。方法选择自身免疫性肝炎患者22例和健康人20例,使用流式细胞仪检测所有被研究者外周血CD8+T淋巴细胞PD-1分子的表达状况,比较不同分期和不同性别疾病患者PD-1表达水平。结果自身免疫性肝炎患者外周血CD8+T淋巴细胞PD-1分子阳性百分比为2.5±0.5%,显著高于健康对照组(0.5±0.1%,P<0.001);自身免疫性肝炎发病期患者CD8+T淋巴细胞表达PD-1百分比为2.6±0.7%,与缓解期比无统计学差异(3.4±0.8%);16例女性AIH患者外周血CD8+T淋巴细胞PD-1阳性百分比为3.5±0.7%,亦略高于6例男性患者的1.3±0.3%,但无显著统计学差异(P=0.1021),可能与例数较少有关。结论自身免疫性肝炎患者CD8+T淋巴细胞PD-1表达率增加,PD-1可能在自身免疫性肝炎的发病中起了重要作用。     

PD-1/PD-L1信号通路在乙型病毒性肝炎免疫调节作用的研究进展

程序性死亡分子1(programmed death-1,PD-1)是由pdcdl基因编码的一个抑制性共刺激分子,在维持外周耐受中起着关键性的作用,并在慢性病毒感染、肿瘤免疫及自身免疫性疫病的发生过程中发挥重要的生物学作用,受到广泛的关注.本文主要综述PD-1/PD-L1信号通路在乙型病毒性肝炎免疫调节作用的研究进展.     

自身免疫性肝炎患者外周血CD8+T细胞PD-1表达水平变化

目的 探讨自身免疫性肝炎（AIH）患者外周血CD8⁺T细胞程序性死亡因子（PD-1）表达水平的变化。方法 随机选取我院收治的AIH患者25例和健康志愿者25例,使用流式细胞仪进行检测外周血CD8⁺T 淋巴细胞PD-1分子和PD-1分子受体（PD-L1）表达水平。结果 与健康人比,AIH血CD8⁺T细胞PD-1/PD-L1表达阳性百分比分别为（2.6±0.1）%和（2.1±0.8）%,明显高于健康人【（0.5±0.2）%和（0.4±0.1）%,P<0.01）;10例男性AIH患者血CD8⁺T细胞PD-1/PD-L1表达阳性百分比分别为（1.4±0.5）%和（2.3±0.6）%,显著低于15例女性患者【（3.8±0.8）%和（2.5±0.5）%）,P<0.05）;经皮质激素治疗4 w,25例AIH患者获得病情缓解。AIH患者在缓解期PD-1/PD-L1表达水平分别为（3.3±0.2）%和（2.8±0.3）%。结论 AHI患者外周血CD8⁺T细胞PD-1表达水平升高,与疾病活动可能存在密切的关系。     

PD-1分子对慢性丙型肝炎患者T细胞免疫功能的影响

目的 探讨慢性丙型肝炎患者外周血T细胞表面程序性死亡因子-1（PD-1）分子在T细胞免疫应答中的作用。方法受试对象包括40例慢性丙型肝炎患者,10例非病毒性肝炎的肝病患者,以及20名健康对照。取外周血,采用流式细胞术检测CD4^＋及CD8^＋T细胞上PD-1的表达水平;ELIsA法测定混合淋巴细胞培养上清中IFN—γ及IL-2的浓度。结果慢性丙型肝炎患者外周血CD4^＋及CD8^＋T细胞上PD-1阳性表达率[（38．61±4．35）％、（48．17±5．16）％]明显高于其他肝病患者及健康对照（P〈0．01）。慢性丙型肝炎患者产生Ⅰ型细胞因子的能力明显降低,阻断PD-1共刺激信号途径能够增强患者T细胞分泌I型细胞因子的能力。结论慢性丙型肝炎患者外周血T细胞上PD-1表达水平的升高,可能是导致T淋巴细胞应答能力下降的重要原因。     

慢性乙型肝炎患者PD-1/PD-L1信号通路的免疫调节作用研究进展

程序性死亡分子-1（PD-1）是T淋巴细胞膜表面表达的负向协同刺激分子, 与其主要配体（PD-L1）形成通路后,可以减弱T淋巴细胞的免疫反应,甚至导致T淋巴细胞功能衰竭。PD-1/PD-L1信号通路在乙型肝炎病毒感染后的效应T细胞免疫耐受中具有重要作用,阻断该途径可能是抗病毒治疗的方向之一,具有良好的应用前景。     

HIV/AIDS患者外周血程序性死亡受体-1的表达及临床意义

目的 探讨HIV感染及抗病毒治疗对程序性死亡受体-1(programmed death-1, PD-1)表达的影响。方法 根据是否接受抗病毒治疗将61例HIV/AIDS患者分为治疗组及未治疗组,并以35例健康者作为对照。利用逆转录聚合酶链反应(RT-PCR)技术探究基因PD-1 mRNA在人外周血单个核细胞(PBMC)中的表达;通过双夹心抗体ELISA法测定血清中可溶性PD-1(sPD-1)表达水平。并比较不同CD4⁺ T淋巴细胞数的HIV/AIDS患者血清sPD-1的差异。结果 未治疗组、治疗组、健康组研究对象PBMC中PD-1 mRNA的相对表达量均数分别为0.337 8±0.064、0.578 2±0.073和0.771 5±0.124,健康组与未治疗组差异极显著,健康组与治疗组、治疗组与未治疗组之间差异显著(P=0.031、P=0.043);未治疗组、治疗组、健康组血清sPD-1浓度分别为42.22±2.21 ng/mL、38.24±2.79 ng/mL和29.88±1.41 ng/mL。健康组与未治疗组、治疗组,治疗组与未治疗组分别具有显著性差异(P=0.008、P=0.040和P=0.020)。差异性分析结果表明,未治疗组、治疗组CD4⁺T淋巴细胞数<350 个/mm³患者血清sPD-1水平均显著高于CD4⁺T淋巴细胞数>350 个/mm³患者。结论 抗病毒治疗在一定程度上使高水平sPD-1表达下调以促使PD-1/PD-L1通路的恢复,从而促进机体免疫重建。监测PD-1 mRNA及sPD-1的表达在HIV的辅助诊断和推断病情发展上具有一定的价值。     

Hepatoma cells up-regulate expression of programmed cell death-1 on T cells

AIM： To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 （PD-1）, and the function of PD-1 on T cells. METHODS： HepG2 or HepG2.2.1.5 cells were cocultured with a lymphoma cell line-Jurkat cells. PD-1 expression was detected by flow cytometry. IL：2, INF-γ and IL-10 in culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. Cytotoxic action of T cells was determined by MIF reduction assay-direct mononuclear cell cytotoxicity assay. RESULTS： The PD-1 expression on Jurkat cells increased by 16.17% ± 2.5% and 17.43% ± 2.2% after HepG2 or HepG2.2.1.5 cells were co-cultured for 48 h. The levels of IL-2, INF-γ and IL-10 in the culture supernatant were 202.9 ＋ 53.0 pg/mL, 88.6 ± 4.6 pg/mL and 63.7± 13.4 pg/mL respectively, which were significantly higher than those （102.9 ± 53 pg/mL, 39.3 ± 4.2 pg/mL, and 34.6 =E13.7 pg/mL） in the control group （P 〈 0.05）. The OD value for MTT assay in the blocking group （0.29 ± 0.06） was significantly higher than that （0.19 ± 0.09） in the control group （P 〈 0.05）. CONCLUSION： PD-1 expression on Jurkat cells is upregulated by hepatoma cells, cytokines and cytotoxic action are elevated after PD-1/PD-L1 is blocked.     

PD-1 preferentially inhibits the activation of low-affinity T cells

Anti–PD-1 therapies can activate tumor-specific T cells to destroy tumors. However, whether and how T cells with different antigen specificity and affinity are differentially regulated by PD-1 remain vaguely understood. Upon antigen stimulation, a variety of genes is induced in T cells. Recently, we found that T cell receptor (TCR) signal strength required for the induction of genes varies across different genes and PD-1 preferentially inhibits the induction of genes that require stronger TCR signal. As each T cell has its own response characteristics, inducibility of genes likely differs across different T cells. Accordingly, the inhibitory effects of PD-1 are also expected to differ across different T cells. In the current study, we investigated whether and how factors that modulate T cell responsiveness to antigenic stimuli influence PD-1 function. By analyzing TCRs with different affinities to peptide–MHC complexes (pMHC) and pMHCs with different affinities to TCR, we demonstrated that PD-1 inhibits the expression of TCR-inducible genes efficiently when TCR:pMHC affinity is low. In contrast, affinities of peptides to MHC and MHC expression levels did not affect PD-1 sensitivity of TCR-inducible genes although they markedly altered the dose responsiveness of T cells by changing the efficiency of pMHC formation, suggesting that the strength of individual TCR signal is the key determinant of PD-1 sensitivity. Accordingly, we observed a preferential expansion of T cells with low-affinity to tumor-antigen in PD-1–deficient mice upon inoculation of tumor cells. These results demonstrate that PD-1 imposes qualitative control of T cell responses by preferentially suppressing low-affinity T cells.

T cell activation initiated by antigen-dependent signals through T cell receptors (TCRs) is tightly controlled by antigen-independent signals through a variety of stimulatory and inhibitory coreceptors (1–4). Inhibitory coreceptors play especially important roles in the development of self-tolerance as the immune cells learn not to attack host cells. However, such inhibitory coreceptors can be hijacked by tumors and pathogens to escape from the immune system. Anti–PD-1 therapies that aim to activate tumor-specific T cells to destroy cancer by banning such immune escape of tumor cells significantly improved the outcomes of patients with diverse cancer types and revolutionized cancer treatment (5, 6). Nevertheless, response rates are rather low and immune-related adverse events (irAEs) are observed in substantial proportion of patients (7, 8), indicative of the continued need to decipher the complex biology of PD-1 as well as T cell activation.Diversity and antigen specificity are the hallmarks of T cell responses. Each T cell expresses TCRs of a unique amino acid sequence and responds to major histocompatibility complexes presenting cognate peptides (pMHCs) on antigen-presenting cells (APCs). As the antigen specificities of T cells and the amino acid sequences of peptides are diverse, the affinities of TCR:pMHC vary depending on their combinations. A plethora of studies demonstrated that the response characteristics of T cells including the strength of TCR:pMHC affinity qualitatively affect the TCR signal, T cell activation, and ensuing biological outcomes (9, 10). Especially, the affinities of self-reactive TCRs are generally lower compared with those of TCRs reactive to foreign antigens (11), which can partly be explained by the negative selection of highly self-reactive T cells in the thymus. Thus, self-reactive T cells are supposed to have different response characteristics from T cells reactive to foreign antigens. However, it remains to be elucidated whether and how T cells with such diverse response characteristics are differentially regulated.Engagement of PD-1 with either of its two ligands, PD-L1 and PD-L2, during antigen stimulation leads to the recruitment of the protein tyrosine phosphatase SHP-2, which dephosphorylates signaling molecules including CD3ζ, ZAP70, and CD28 (12–14). PD-1 has been postulated to lower the responsiveness of T cells to antigen stimulation and suppress TCR-induced events uniformly because PD-1 inhibits TCR-proximal signaling. However, we have recently found that the suppressive effects of PD-1 on TCR-induced gene expression differ across different genes (15). By quantifying the half-maximal effective concentration (EC₅₀) of antigen as the relationship between change in gene expression and TCR signal strength, we demonstrated that TCR signal strength required for the induction of genes varies across different genes and that PD-1 preferentially inhibits the up-regulations of genes that require stronger TCR signal (15). As each T cell has its own response characteristics, inducibility of genes likely differs across different T cells. Accordingly, the inhibitory effects of PD-1 are also expected to differ across different T cells.In this study, we investigated whether and how factors that alter the responsiveness of T cells to antigenic stimuli influence the inhibitory effects of PD-1. By comparing T cells bearing TCRs with different affinities to pMHC, we demonstrated that PD-1 inhibits the expression of TCR-inducible genes more efficiently in the activation of T cells expressing TCRs with lower affinity to pMHC. We also tested peptides with different affinities to TCR and found that PD-1 inhibits the expression of TCR-inducible genes more efficiently when T cells are stimulated by peptides with lower affinity to TCR. In contrast, the manipulation of the affinity of peptides to MHC and the expression level of MHC did not affect the PD-1 sensitivity at all, although they markedly altered the dose responsiveness of T cells by changing the efficiency of pMHC formation. These results suggest that the strength of individual TCR signal is the critical determinant of PD-1 sensitivity. We further demonstrated that T cells with low-affinity to tumor-antigen were preferentially expanded in PD-1–deficient mice upon inoculation of tumor cells. Our current findings indicate that PD-1 regulates the quality of T cell responses by preferentially suppressing low-affinity T cells.     

T lymphocyte subsets and PD-1 expression on lymphocytes in peripheral blood of patients with non-small cell lung cancer

The incidence rate and mortality rate of lung cancer (LC) are very high. This study aimed to analyze the T lymphocyte subsets and programmed death-1 (PD-1) expression on lymphocytes in the peripheral blood of non-small cell lung cancer (NSCLC) patients and explore whether there were changes in cellular immunity in NSCLC. Peripheral blood samples were collected from newly diagnosed NSCLC patients and healthy individuals. The T lymphocyte subsets and PD-1 expression were evaluated using flow cytometry. Single-sample gene set enrichment analysis (ssGSEA) was performed to explore the correlations of PD-1 expression with infiltration patterns for tumor-infiltrating T immune cells. By flow cytometry, two populations of lymphocytes in NSCLC patients were observed. Apart from a population of normal volume lymphocytes (Lym1), the other population had larger volume and more particles (Lym2). Compared with the healthy group, the proportion of CD4+ T cells and PD-1 expression on Lym1 was higher, and that of CD8+ T cells was lower in the NSCLC group. In the NSCLC group, the proportions of CD3+ T cells, CD8+ T cells, CD4+CD8+ T (DPT) cells, and PD-1 expression were higher on Lym2 than those on Lym1 (P < .05). ssGSEA showed that tumor infiltrating immune T cells were positively correlated with PD-1 expression. The PD-1 expression on lymphocytes increased in recurrent patients who treated with PD-1 inhibitor. Lym2 may be tumor-infiltrating lymphocytes (TILs) which upregulated PD-1 expression in NSCLC. PD-1 expression on lymphocytes may be used as a recurrence indicator for NSCLC patients treated with PD-1 inhibitors.     

Contributions of PD-1/PD-L1 pathway to interactions of myeloid DCs with T cells in atherosclerosis

Although inflammatory cells contribute to immunopathogenesis of atherosclerosis, underlying molecular mechanisms remain largely undefined. Recently, it has been demonstrated in mouse model that Programmed death-1 (PD-1)/PD-1 ligand (PD-L) pathway plays a critical role in proatherogenic immune responses. Here we examined the expression of PD-1 and PD-L1 on peripheral blood mononuclear cells by flow cytometry in 76 patients with coronary artery disease (CAD), and 25 healthy volunteers. The expression of PD-1 and PD-L1 is significantly down-regulated on T cells and myeloid dendritic cells (mDCs) in CAD patients than in healthy individuals, respectively. More importantly, we found that decreased PD-L1 expression on mDCs is related with the increased T cell immune responses in CAD patients. In addition, stimulation of PD-L1 expression in vitro could attenuate the stimulatory ability on allogeneic T cell proliferation and its cytokine production, including IFN-γ and IL-2, and also influence the production of IL-10 and IL-12 by mDCs. Taken together, we can draw a conclusion that PD-1/PD-L1 pathway plays a key role in the regulation of proatherogenic T cell immunity by intervening antigen presenting cell (APC)-dependent T cell activation, which associates with pro-inflammatory or anti-inflammatory cytokine production, and further studies need gain insight into that this pathway represents a strategy of immunotherapy for atherosclerosis.     

The PD-1/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungus Histoplasma capsulatum

The PD-1 costimulatory receptor inhibits T cell receptor signaling upon interacting with its ligands PD-L1 and PD-L2. The PD-1/PD-L pathway is critical in maintaining self-tolerance. In this study, we examined the role of PD-1 in a mouse model of acute infection with Histoplasma capsulatum, a major human pathogenic fungus. In a lethal model of histoplasmosis, all PD-1-deficient mice survived infection, whereas the wild-type mice died with disseminated disease. PD-L expression on macrophages and splenocytes was up-regulated during infection, and macrophages from infected mice inhibited in vitro T cell activation. Of interest, antibody blocking of PD-1 significantly increased survival of lethally infected wild-type mice. Thus, our studies extend the role of the PD-1/PD-L pathway in regulating antimicrobial immunity to fungal pathogens. The results show that the PD-1/PD-L pathway has a key role in the regulation of antifungal immunity, and suggest that manipulation of this pathway represents a strategy of immunotherapy for histoplasmosis.     

晚期消化道肿瘤患者CD8~+T细胞表面程序性凋亡受体表达特点及临床意义

目的探讨晚期消化道肿瘤患者T细胞表面程序性凋亡受体1(programmed death 1,PD-1)的表达水平及其与肿瘤病变程度的关系。方法选取不同类型的消化道肿瘤患者58例(肿瘤组),健康人34名(健康对照组);采用集新鲜血液分离外周血,运用流式细胞仪检测外周血T细胞表面PD-1表达的阳性比例及水平。结果肿瘤组外周血T细胞表面PD-1阳性表达率及水平明显高于健康对照组(P0.05),不同分期消化道肿瘤患者T细胞表面PD-1表达的频率不同,差异有统计学意义(P0.05)。结论消化道肿瘤患者外周血T细胞表面PD-1的表达率较正常人明显上调,且与消化道肿瘤的不同分型呈正相关。