Research progress of PD-1 and PD-L1 in the immune of sepsis / 国际生物医学工程杂志

Sepsis is currently the leading cause of death in the intensive care unit, and its survivors also experience long-term immunosuppression and high rates of recurrent infections. At present, the clinical treatment of sepsis is still based on antibiotics, intravenous rehydration, and vasopressors, and there is no targeted drug treatment. However, as the rate of antibiotic resistance continues to increase, immunotherapy is highly anticipated as a new treatment. Patients with sepsis are often accompanied by acute leukocyte immune dysfunction and immunosuppression, which may be an important risk factor for the increasing morbidity and mortality of patients. Targeted inhibition of specific cell surface inhibitory immune checkpoint receptors and ligands, such as programmed death receptor-1 (PD-1), programmed death-ligand 1 (PD-L1), and other targets, can improve the host’s resistance to infection. In this paper, the research progress of PD-1 and PD-L1 in the immune response to sepsis was summarized to provide a theoretical basis for their further application in the treatment of sepsis in the future.

Efficacy and safety of PD-1/PD-L1 inhibitors for neoadjuvant treatment of bladder cancer：meta-analysis of single-group rates / 中国药房

OBJECTIVE To evaluate the efficacy and safety of PD-1/PD-L1 inhibitors for neoadjuvant treatment of bladder cancer， and to provide evidence-based reference for clinical treatment. METHODS Retrieved from PubMed， Cochrane Library， Embase， American Society of Clinical Oncology Meeting Library， CNKI， VIP and Wanfang database， etc.， the randomized controlled trials （RCTs）， non-RCT， case-control studies， cohort studies， etc. about PD-1/PD-L1 inhibitors for neoadjuvant treatment of bladder cancer were collected from the inception to Jan 31st， 2023. After literature screening， data extraction and quality evaluation， RevMan 5.3 software was used to perform meta-analysis of single-group rates； sensitivity analysis and publication bias analysis were conducted using Stata12 software. RESULTS A total of 25 studies were included in this discussion， involving 940 patients. The results of meta-analysis showed that the pathologic complete response （pCR） rate was 32% [OR＝0.32， 95%CI （0.22， 0.45）， P＝0.006]， downstaging rate was 52% [OR＝0.52， 95%CI （0.45， 0.60）， P＝0.55]， and the incidence of ≥grade 3 immune-related adverse events （irAEs） was 16% [OR＝0.16， 95%CI （0.11， 0.22）， P＜0.000 01]. Subgroup analysis showed that the patients receiving PD-1/PD-L1 inhibitors alone had a pCR rate of 25% and a incidence of Grade≥3 irAEs of 9%； the patients receiving combined immunotherapy had a pCR rate of 29% and a incidence of Grade≥3 irAEs of 28%； the patients receiving PD-1/PD-L1 inhibitors combined with chemotherapy had a pCR rate of 43% and a incidence of Grade≥3 irAEs of 12%； PD-L1 positive patients had a pCR rate of 44%， and PD-L1 negative patients had a pCR rate of 25%. The results of the sensitivity analysis showed that the study was robust. The results of the publication bias analysis showed that there was no significant publication bias. CONCLUSIONS PD-1/PD-L1 inhibitors are effective and safe for adjuvant treatment of bladder cancer.

Progress on application of PD⁃1／PD⁃L1 monoclonal antibodies in oncotherapy / 中国生物制品学杂志

@#Abstract：Programmed death receptor⁃ 1（PD⁃1）belongs to the family of immunoglobulin B7⁃CD28，which plays an important role in regulating immune response in human body. Since the first PD⁃1／PD⁃ligand 1（PD⁃L1）monoclonal antibody was approved for marketing in China in 2018，the value of PD⁃1／PD⁃L1 immunotherapy in oncotherapy has attracted wide attention. Based on the introduction of the action mechanism of PD⁃1／PD⁃L1 mAbs，this paper reviews the application progress of 8 on ⁃ market PD ⁃ 1／PD ⁃ L1 mAbs in China in oncotherapy from the perspectives of approved indications，clinical trials，usage and dosage，and adverse reactions，in order to provide reference for the rational appli⁃ cation of PD⁃1／PD⁃L1 monoclonal antibodies in clinic.

Effect of MiR-424-5p on the Drug Resistance of Diffuse Large B-Cell Lymphoma Cells by Regulating PD-1/PD-L1 Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of microRNA-424-5p (miR-424-5p) on the drug resistance of diffuse large B-cell lymphoma (DLBCL) cells by regulating the programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway.@*METHODS@#Human DLBCL cell line CRL2631 cells were induced to construct CRL2631-CHOP resistant cell line. RT-qPCR and Western blot were used to detect the expression levels of MiR-424-5p, PD-L1 mRNA and protein, and multidrug resistance gene-1 (MDR-1) protein in CRL2631 cells and CRL2631-CHOP cells, respectively. The target genes of MiR-424-5p was verified by dual luciferase reporter assay. The miRNA simulation/interference technology and thiazole blue (MTT) method were used to detect the resistance of CRL2631 cells and CRL2631-CHOP cells to CHOP.@*RESULTS@#Compared with CRL2631 cells, the drug resistance of CRL2631-CHOP cells to CHOP and the levels of MDR-1 protein (P<0.05), PD-L1 mRNA and protein in the cells were significantly increased (both P<0.001), while the relative level of MiR-424-5p was significantly reduced (P<0.001). The result of the dual luciferase reporter assay showed that PD-L1 was the direct downstream target gene of MiR-424-5p (P<0.001). After transfection of MiR-424-5p inhibitor, the resistance of CRL2631 cells to CHOP drugs increased, and the expression level of MDR-1 protein (P<0.01), PD-L1 mRNA and protein also increased significantly (both P<0.01). After transfection of MiR-424-5p mimics, the resistance of CRL2631-CHOP cells to CHOP drugs decreased, and the expression level of MDR-1 protein (P<0.001), PD-L1 mRNA and protein also decreased significantly (both P<0.001). Overexpression of PD-L1 could reverse the inhibitory effect of upregulating MiR-424-5p on PD-L1 (P<0.001).@*CONCLUSION@#Down-regulation of MiR-424-5p enhances the drug resistance of DLBCL cells by regulating the PD-1/PD-L1 signaling pathway.

Effects and molecular mechanism of histone methyltransferase enhancer of zeste homolog 2 on regulating sepsis-induced T cell dysfunction / 中华危重病急救医学

Objective:To investigate the effect and mechanism of histone methyltransferase enhancer of zeste homolog 2 (EZH2) on sepsis-induced T cell dysfunction.Methods:Twenty-four male C57BL/6 mice were divided into three groups randomly: sham operated group, sepsis model group [cecum ligation and puncture (CLP)+dimethyl sulfoxide (DMSO) group] and EZH2 selective inhibitor treated group (CLP+GSK126 group), with 8 mice in each group. Sepsis murine model was reproduced by CLP. CLP+DMSO group and CLP+GSK126 group were treated with DMSO or GSK126 (10 mg/kg) respectively right after surgery through intraperitoneal injection. The mice were sacrificed 24 hours after operation, and the mesenteric lymph nodes were collected. The expression of EZH2, apoptosis rates, cell proliferation marker ki-67 antigen positive T lymphocytes (ki-67 + cell), interferon-γ positive T lymphocytes (IFN-γ + cell), programmed death receptor-1 positive T lymphocytes (PD-1 + cell) and programmed death-ligand 1 positive T lymphocytes (PD-L1 + cell) were determined by flow cytometry. Results:Compared with sham operated group, the expression of EZH2 in T lymphocytes was up-regulated on mesenteric lymph nodes of CLP+DMSO group. Compared with CLP+DMSO group, the ratio of CD3 + T lymphocytes in CLP+GSK126 group was up-regulated (0.70±0.02 vs. 0.50±0.07, P < 0.01), indicating that the EZH2 inhibitor could increase the number of T lymphocytes in lymph nodes of septic mice; the ratio of ki-67 + cells in CD4 + and CD8 + T lymphocytes in CLP+GSK126 group was increased (CD4 +: 0.74±0.05 vs. 0.63±0.04, CD8 +: 0.82±0.06 vs. 0.70±0.04, both P < 0.05), indicating that the EZH2 inhibitor could increase the ratio of T lymphocytes with high proliferative activity in lymph nodes of septic mice. However, no significant difference was found on both CD4 + and CD8 + T lymphocytes apoptosis rates in the mesenteric lymph nodes of mice between CLP+GSK126 group and CLP+DMSO group [CD4 +: (21.53±2.87)% vs. (20.48±3.21)%, CD8 +: (8.34±1.02)% vs. (7.71±1.38)%, both P > 0.05], indicating that no extra T lymphocytes apoptosis was induced by EZH2 inhibitor. Compared with CLP+DMSO group, the ratios of IFN-γ + CD4 + and IFN-γ + CD8 + T lymphocytes were increased in CLP+GSK126 group (IFN-γ +CD4 +: 0.31±0.11 vs. 0.14±0.06, IFN-γ +CD8 +: 0.30±0.10 vs. 0.13±0.06, both P < 0.05), suggesting that secretion of IFN-γ in lymph nodes by sepsis T lymphocytes was augmented after EZH2 inhibitor administration. Furthermore, compared with CLP+DMSO group, the ratio of PD-1 + cell in CD8 + T lymphocyte was down-regulated in CLP+GSK126 group (0.092±0.006 vs. 0.135±0.004, P < 0.01), suggesting that EZH2 inhibitor restrained the PD-1 expression on sepsis lymphoid node CD8 + T lymphocytes, however, it had no significant effect on PD-L1 + cells. Conclusion:EZH2, regulates sepsis-induced T lymphocyte dysfunction, possibly through modulating the expression of PD-1.

Refractory primary central nervous system lymphoma treated with programmed death-1 monoclonal antibody and Bruton tyrosine kinase inhibitor combined with rituximab: report of 1 case and review of literature / 白血病·淋巴瘤

Objective:To investigate the effect and safety of rituximab, programmed death 1 (PD-1) monoclonal antibody, and Bruton tyrosine kinase (BTK) inhibitor on elderly refractory primary central nervous system lymphoma (PCNSL).Methods:The clinical data of an elderly patient with refractory PCNSL treated with the combination of rituximab, PD-1 monoclonal antibody and BTK inhibitor in the First Hospital of Jilin University in February 2020 were retrospectively analyzed. The relevant literature was reviewed.Results:The patient had primary central nervous system diffuse large B-cell lymphoma (high-risk group), and the Memorial Sloan Kettering Cancer Center (MSKCC) score was 2 (estimated overall survival time was 7 months). Disease progressed after 1 course of treatment. Complete remission was achieved after the therapy of rituximab, PD-1 monoclonal antibody combined with BTK inhibitor. PD-1 monoclonal antibody maintenance therapy was performed and patient was followed up until November 17, 2021. The patient's condition was stable. The second progression-free survival (PFS) time was 20 months, and the overall survival time was 21 months. The patient well tolerated the new drug treatment, and no adverse reactions of grade 3 or above occurred.Conclusions:The new targeted combination therapy can be used as a treatment option for elderly PCNSL patients, which can further improve the curative effect and significantly improve the prognosis.

Expression and Significance of PD-1, PD-L1 and PD-L2 in T Cell Subsets and Myeloma Cells in Newly Diagnosed Multiple Myeloma Patients / 肿瘤防治研究

Objective To investigate the expression of PD-1 and PD-L1/2 in T cell subsets and myeloma cells in the bone marrow from newly diagnosed multiple myeloma (NDMM) patients and their relation with clinical features. Methods We collected the bone marrow and clinical data of 22 NDMM patients and 18 cases of healthy controls. We sorted CD4+T cells, CD8+T cells and myeloma cells by flow cytometry, and observed the expression of PD-1 and PD-L1/2. Results Compared with the control group, the proportion of CD8+T cells in the NDMM group was significantly higher, while the ratio of CD4+/CD8+ was significantly lower (both P < 0.05). The expression levels of PD-1 and PD-L2 in CD4+T cells in the NDMM group were significantly higher than those in the control group (both P < 0.05). The expression levels of PD-1, PD-L1 and PD-L2 in T cell subsets and myeloma cells of NDMM patients were not correlated with the gender, age, immune typing, Durie-Salmon stage and subtypes, ISS stage or mSMART3.0 stratification (both P > 0.05). Conclusion Most of MM patients suffering immune abnormality, which may be associated with the mutual immunosuppressive effects between T lymphocytes and plasma cells which expressing PD-1 and PD-L1/2.

Expression of PD-L1 and PD-1 in Pathological Tissue of Patients Newly Diagnosed with Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of PD-L1 and PD-1 in pathological tissue of patients newly diagnosed with diffuse large B-cell lymphoma (DLBCL).@*METHODS@#Data of DLBCL patients who visited the Department of Hematology, Peking University Third Hospital from May 2014 to March 2017 were collected, and a total of 21 patients with pathological tissue sections which were still available at the initial treatment were selected. The patients were divided into complete remission (CR) group and refractory relapse (RR) group according to clinical outcome. The expression and proportion of PD-1 and PD-L1 in pathological tissue sections were detected by multiplex fluorescence immunohistochemical staining, and the differences in the expression of different molecular markers in different clinical characteristics and different prognosis were compared using non-parametric test.@*RESULTS@#The ratio of PD-L1+ cells to PD-1+ cells (PD-L1+ : PD-1+) was 5.14±3.825 in increased lactate dehydrogenase (LDH) group, which was significantly higher than 0.76±0.563 in non-increased LDH group (P=0.001). The ratio of PD-L1+ : PD-1+ in increased Treg cells group was 1.41±1.454, which was lower than 6.42±4.426 in decreased Treg cells group (P=0.023).@*CONCLUSION@#The increased expression ratio of PD-L1 to PD-1 in pathological tissue sections of newly diagnosed DLBCL patients is associated with poor prognostic clinical characteristics.

Relationship between CD8 +FoxP3 +CD25 + T cell subsets and the therapeutic effect of pembrolizumab in patients with uterine cervical cancer / 肿瘤研究与临床

Objective:To investigate the relationship between CD8 +FoxP3 +CD25 + T cell subsets and the therapeutic effect of programmed death receptor 1 (PD-1) inhibitor pembrolizumab in treatment of uterine cervical cancer. Methods:The data of 105 patients with uterine cervical cancer who received pemblizumab therapy based on chemotherapy in the First Hospital of Qinhuangdao from January 2018 to January 2020 were retrospectively analyzed. Flow cytometry was used to detect the ratio of CD8 +FoxP3 +CD25 + T cell in peripheral blood of patients. The efficacy and safety were analyzed. According to the efficacy, all patients were divided into remission group (complete remission + partial remission) and non-remission group (stable disease + progressive disease). The clinical characteristics and CD8 +FoxP3 +CD25 + T cell ratio of the two groups were compared. Multivariate logistic regression model was used to analyze the influencing factors for the efficacy. The efficacy of CD8 +FoxP3 +CD25 + T cell ratio predicting the therapeutic effect of patients was analyzed by using receiver operating characteristic (ROC) curve. Results:The objective remission rate of all patients was 17.14% (18/105), and the incidence of adverse reaction was 39.05% (41/105). The proportion of patients with a family history of cervical cancer in the remission group was lower than that than in the non-remission group [5.56% (1/18) vs. 34.48% (30/87)], and the difference was statistically significant ( χ2=6.00, P=0.014). The proportion of CD8 +FoxP3 +CD25 + T cell of 105 patients before and after treatment was (0.83±0.21)% and (0.77±0.10)%, respectively; the proportion of CD8 +FoxP3 +CD25 + T cell before and after treatment in the remission group was (0.55±0.26)%, (0.31±0.12)%, respectively; the proportion of CD8 +FoxP3 +CD25 + T cell before and after treatment in the non-remission group was (0.89±0.30)%, (0.87±0.28)%, respectively. The proportion of CD8 +FoxP3 +CD25 + T cell after treatment in the remission group was lower than that before treatment ( P < 0.05); there was no statistically significant difference in the proportion of CD8 +FoxP3 +CD25 + T cell before and after treatment in the non-remission group ( P>0.05). The proportion of CD8 +FoxP3 +CD25 + T cell before and after treatment in the non-remission group was higher than that in the remission group (all P<0.001). The proportion of CD8 +FoxP3 +CD25 + T cell higher than the mean value of both groups before treatment and the proportion of CD8 +FoxP3 +CD25 + T cell higher than the mean value of both groups after treatment were independent risk factor of disease remission ( OR=2.542, 95% CI 1.649-3.918, P<0.001; OR=2.936, 95% CI 2.154-4.002, P<0.001). ROC curve analysis showed that the area under the curve of CD8 +FoxP3 +CD25 + T cell ratio predicting the disease remission before treatment was 0.720, and its best cut-off value was 0.77%, the senfitivity was 77.78%, the specificity was 70.11%. Conclusions:Early detection of CD8 +FoxP3 +CD25 + T cell ratio helps to predict the effect of PD-1 inhibitor pembrolizumab therapy for uterine cervical cancer.

Correlation Study on Expression of PD-1 and PD-L1 in Non-small Cell Lung Cancer and Epidermal Growth Factor Receptor Mutations / 中国肺癌杂志

BACKGROUND@#The treatment mode of lung cancer is epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) as a first-line treatment for patients with EGFR mutant in non-small cell lung cancer (NSCLC). At the same time programmed death receptor 1 (PD-1) and its programmed death receptor ligand 1 (PD-L1) inhibitors therapy as the representative immune checkpoint inhibitors (ICIs) has a significant effect in the treatment of lung cancer. The aim of this study was to investigate the correlation between the expression of PD-1 and PD-L1 in NSCLC and clinicopathologic feature, EGFR gene mutation.@*METHODS@#The protein expression of PD-1 and PD-L1 was detected by immunohistochemistry from 127 patients with NSCLC and EGFR gene mutation was detected by quantitative polymerase chain reaction (qPCR) to analyze its relation with clinicopathologic feature. Also, the correlation between protein expression of PD-1 and PD-L1 and EGFR mutation.@*RESULTS@#The PD-1 positive expression in NSCLC tumor cells and tumor infiltrating immune cells is 53.5% (68/127), PD-L1 is 57.5% (73/127). The PD-1 and PD-L1 expression significantly higher in well-differentiated and moderately-differentiated carcinoma than poorly differentiated carcinoma, I+II than III+IV in clinical staging (P<0.05). The EGFR mutation rate was 46.5% (59/127), correlate with female, without smoking history, adenocarcinoma and well-differentiated and moderately-differentiated patients respectively higher than male, smoking history, squamous carcinoma and poorly differentiated patients (P<0.05). The protein expression of PD-L1 and PD-1 had the consistency in NSCLC patients (kappa=0.107,5, P=0.487). There was a negative correlation between the EGFR mutation and PD-1 and PD-L1 expression (Φ=-0.209, Φ=-0.221, P<0.05). Follow-up of NSCLC patients, the median total survival in under the age of 65, adenocarcinoma, well-differentiated and moderately-differentiated, with PD-L1 expression patients respectively higher than over the age of 65, squamous carcinoma, poorly differentiated, without PD-L1 expression patients (P<0.05). The median survival of hypo expression patients of PD-L1 significantly higher than hyper expression patient (P=0.04).@*CONCLUSIONS@#According to the Chinese Expert Consensus on Standards of PD-L1 immunohistochemistry testing for NSCLC, we tested the PD-L1 expression in NSCLC and then the dominant population of anti-PD-1/PD-L1 treatment was screened out. Patients with EGFR mutation were also detected and EGFR mutation was negatively correlated with the expression of PD-1 and PD-L1 as well. On the basis of PD-L1 expression and EGFR mutation status, it may benefit NSCLC patients from individualized treatment. Meanwhile, patients who were under the age of 65, adenocarcinoma, well-differentiated and moderately-differentiated, hypo expression of PD-L1 have a relatively good prognosis, to provide reference for the prognosis evaluation of NSCLC.

Research progress on programmed death receptor 1/ ligand 1 inhibitor in immunotherapy of head and neck squamous cell carcinoma / 口腔疾病防治

@#Head and neck squamous cell carcinoma (HNSCC) is a common malignant tumor that seriously threatens human health and life. With increasing studies on the mechanism of tumor immune escape, programmed death receptor 1 (PD-1) and programmed death ligand receptor 1 (PD-L1) have been proven to be involved in tumor immune escape. The primary mechanism is that PD-1 recruits protein tyrosine phosphatase (SHP-2) to dephosphorylate downstream tyrosine kinase (SyK) and phosphatidylinositol 3-kinase (PI3K), thereby inhibiting downstream protein kinase B (AKT), extracellular regulated protein kinases (ERK) and other important signaling pathways, ultimately inhibiting T cell activation. In recent years, PD-1/PD-L1 inhibitors have become popular immunotherapies. Pembrolizumab and nivolumab have been approved for HNSCC patients by the U.S. Food and Drug Administration. Both durvalumab and atezolizumab are still in clinical trials, and published data show that both have certain safety and efficacy but still need much clinical data to support them. Meanwhile, the combination of PD-1/PD-L1 inhibitors with radiotherapy, chemotherapy and immunotherapy is still controversial in terms of clinical efficacy and adverse events, and further research is needed. However, serious immune-related adverse reactions limit the clinical application of PD-1/PD-L1 inhibitors, despite promising curative effects. Therefore, developing novel inhibitors and investigating stable and effective biomarkers and upstream and downstream signaling mechanisms are urgent issues.

Correlation of cervical cancer with vaginal flora disorders and high expression of PD-1 molecules / 西安交通大学学报(医学版)

Objective: To investigate the correlation of the occurrence and development of cervical cancer with the disturbance of vaginal flora and the expression of programmed cell death receptor-1 (PD-1) molecules on the surface of T cells in order to provide new direction for PD-1 immune checkpoint blocking therapy. Methods: Twenty-four female SPF Kunming mice were randomly divided into two groups: normal control group (CON) (n=12) and cervical cancer model group (CCM) (n=12). We established the mouse cervical cancer U14 cell subcutaneous transplantation tumor model. After 21 days of rearing under the same conditions, vaginal secretions and blood samples of mice were collected. 16S rRNA gene amplification and sequencing technology were used to analyze the vaginal flora in the two groups. Flow cytometry was used to detect the subsets of T lymphocytes in the peripheral blood of mice and the expression of PD-1 on its surface. Results: Beta diversity analysis showed that the vaginal flora of the CON group and the CCM group were significantly different in species composition and relative abundance of each species. The dominant bacteria in the CON group were Lactobacillus, while the figure in the CCM group converted to Pasteurella and Helicobacter. The mean expression of PD-1 on the surface of T cells in mice in the CCM group was 12.78%, which was significantly higher than that in the CON group (10.45%) (P<0.05). The mean of the total number of T cells (CD3+T cells) and the number of CD8+T cells in the CCM group were 38.78% and 11.98%, respectively, which were significantly lower than those in the CON group (51.37% and 20.08%) (P<0.05). Conclusion: Cervical cancer can cause vaginal flora disorder. There may be a certain correlation between the vaginal flora disorder and the high expression of PD-1 on the surface of T cells, suggesting that there may be a new regulatory mechanism for the immune escape in cervical cancer.

Development of programmed death receptor-1 and programmed death receptor-1 ligand in oral squamous cell carcinoma / 华西口腔医学杂志

Oral squamous cell carcinoma (OSCC) is a common malignant tumor in the oral and maxillofacial region. At present, the treatment of OSCC is mainly based on surgical oriented comprehensive sequence therapy, especially the triple therapy of surgery, radiotherapy, and chemotherapy. However, the overall five-year survival rate is relatively low. Therefore, researching the pathogenesis and treatment methods of OSCC is important. The immune checkpoint of programmed death receptor-1 (PD-1) and programmed death receptor-1 ligand (PD-L1) have been the focus of research in recent years. Several studies have shown that the high expression of PD-1/PD-L1 in most OSCC microenvironments may contribute to the immune escape of tumors. In this study, the research status of immune checkpoint of PD-1/PD-L1 and its relevant inhibitors in OSCC were reviewed.

Effect of CRISPR/Cas9-mediated PD-1 gene knockout on proliferation, phenotype,IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects of CRISPR/Cas9 gene editing mediated PD-1 knockdown on the proliferation, phenotype, IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys. Methods: gRNA targeting PD-1 gene of Cynomolgus monkey was designed, and the corresponding plasmid was constructed and extracted. peripheral blood mononuclear cells (PBMCs) of Cynomolgus monkeys were isolated, and plasmid DNAs were added for transfection by using Lonza 4D electrorotometer. FACS analysis and fluorescence microscopy were used to detect transfection efficiency at 48 h after transfection. Genomic DNA of T cells was extracted for PCR amplification and T7E1 digestion identification. The proliferation of T cells was induced under the stimulation of human CD3 antibody and IL-2, and the cell growth curve was drawn. PI staining flow cytometry was used to detect cell cycle and the expression levels of CD4 and CD8, and ELISA was used to detect the secretion of IFN- γ and IL-2. Results: At 48 h after transfection, the cells with green fluorescent protein expression in experimental group were observed under fluorescence microscopy with a transfection efficiency of (21.6±3.2)%. T7E1 enzyme digestion results showed that the PCR product of genomic DNA of cells in experimental group showed 3 bands after digestion, including the target cleavage bands(243，197 bp). Compared with non-transfected cells, the cells in experimental group exhibited slow proliferation, delayed colony formation, with small volume and weak refraction; the number of T cells at G0/G1 phase of the experimental group was significantly increased (P<0.05), while the number of cells at G2/M phase was significantly reduced (P<0.05); and the secretion levels of IFN-γ and IL-2 in the cells of the experimental group increased significantly (both P<0.05). However, the difference in the expression levels of CD4 and CD8 was not statistically significant between the two groups (both P>0.05). Conclusion: PD-1 gene knockout can arrest T cells in Cynomolgus monkey at G0/G1 phase, thereby inhibiting its proliferation and increasing the secretion of IFN-γ and IL-2 in the meanwhile.

Expression of programmed death receptor-1 and its prediction of postoperative infectious complications in elderly patients with hip fracture / 中华创伤骨科杂志

Objective@#To investigate the expression of programmed death receptor-1 (PD-1) and its value in predicting the infectious complications in elderly patients with hip fracture.@*Methods@#A total of 119 elderly patients with intertrochanteric fracture(observation group) were prospectively enrolled for this study from August 2015 to August 2016 at Department of Orthopaedics, The Seventh Medical Center of PLA. They were 86 males and 33 females with an age of 80.7±8.1 years. Another 20 elderly out-patients with gonarthritis but without trauma or major medical disease were enrolled as a control group. The observation group received closed reduction and internal fixation with proximal femoral intramedullary nails. The PD-1 expression in peripheral blood T cells was detected at preoperation, 1, 3 and 5 days postoperation in the patients of observation group. They were further divided into an infection group and a non-infection group depending on whether infectious complications occurred during the follow-up period. The association between PD-1 and infectious complications was analyzed and its predictive value was calculated.@*Results@#The observation group and the control group, as well as the infection group and the non-infection group, were compatible due to their insignificant differences in baseline data(P>0.05).Compared with the control group, the expression of PD-1 in the peripheral blood T cells in the observation group was all significantly increased at preoperation, 1, 3 and 5 days postoperation (P<0.05). The PD-1 expression in patients of the infection group (24 cases) increased significantly higher at 1, 3 and 5 days postoperation (33.60%±2.44%, 35.68%±4.05% and 30.36%±4.42%) than in those of the non-infection group (29.57%±3.20%, 21.45%±2.20% and 20.94%±2.30%) (P<0.05). The PD-1 expression in the infection group reached its peak at 3 days postoperation and did not fall to the preoperative level at 5 days postoperation. However, the PD-1 expression in patients of the non-infection group (95 cases) showed a transient rise only on the first day postoperation. The cutoff value of the difference in PD-1 expression between 1 and 3 days postoperation that could predict the infectious complications was 3.4%, with a sensitivity of 95.6% and a specificity of 92.6%.@*Conclusions@#The expression of PD-1 may significantly increase after intramedullary fixation of hip fracture in elderly patients. The patients with infectious complications may exhibit a greater and longer increase in PD-1 expression than those without infectious complications. The difference in PD-1 expression between 1 and 3 days postoperation has a good predictive value for infectious complications because its high sensitivity and specificity.

Expression of PD-1 in T-cell acute lymphoblastic leukemia cells and its clinical significance / 中国肿瘤生物治疗杂志

@# Objective: To investigate the expression and clinical significance of PD-1 molecule in tumor cells (T-ALL cells) derived from the patient with T-cell acute lymphoblastic leukemia (T-ALL). Methods: T-ALL cells from one patient and PBMCs from four healthy volunteers provided by the Department of Hematology in Jiangsu Provincial Hospital of Traditional Chinese Medicine in December 2015, and human 293T/PD-1 cells provided by Persongen Bio Therapeutics (Suzhou) Co., Ltd. were used for this study. The mouse T-ALL xenograft model was constructed by injecting T-ALL cells into tail vein of B-NDG mice, and flow cytometry was used to verify whether the cells obtained from the spleen of transplanted mice were mainly consisted of T-ALL cells. Flow cytometry was used to study the protein expression of PD-1 in T-ALL cells, and RT-PCR was applied to further verify the mRNA expression of PD-1 in T-ALL cells. The PD-1 gene in T-ALL cells was sequenced by SNP genotyping to detect whether the DNA sequence of PD-1 gene changed. PD-1 inhibitor was used in vitro to study their effects on proliferation, apoptosis, and the mRNA expression levels of related factors in T-ALL cells. Results: The mouse T-ALL xenograft model was successfully constructed and verified by flow cytometry as T-ALL. PD-1 was highly expressed at both mRNA and protein levels in T-ALL cells (all P<0.01). A C-to-T mutation was detected in the fifth exon of the PD-1 gene. PD-1 inhibitor had no significant effect on proliferation and apoptosis of T-ALL cells in vitro; PD-1 inhibitor up-regulated the mRNA expression of tumor-suppressor protein IGFBP3 and decreased the mRNA expression of oncoprotein SULT1A3 (all P<0.01). Conclusion: PD-1 is highly expressed in T-ALL cells, and PD-1 could be used as a target for clinical diagnosis and treatment for T-ALL.

PD-S15 fusion protein specifically target PD-1 and rapidly expands NK/T cells / 中国肿瘤生物治疗杂志

@#Objective: To investigate the function of anti-PD-1 (scFv)/IL-15/IL-15Rα-sushi fusion protein (PD-S15) to specifically bind to PD-1 in vitro and to explore its effect on NK/T cell proliferation. Methods: The human anti-PD-1 (scFv) gene sequence and human IL-15/IL-15Rα-sushi fusion gene sequence were synthesized chemically. The recombinant expression plasmid pUC57-PD-S15 was constructed by enzyme digestion and ligation of the two target genes, and then transiently transfected into HEK293T cells by lipofectamineTM 2000. The supernatants of cell culture medium were acquired, and the expression of PD-S15 fusion protein in cell culture supernatants was detected by Wb assay. PBMCs and TILs were cultured in mediums with different proportion of PD-S15/X-VIVOTM15, respectively. Then, the capacity of PD-S15 fusion protein to bind to PD-1 in vitro and its effect on the proliferation of PBMCs and the proportion of CD3+CD8+, CD3+CD4+ and CD3-CD56+ subsets were detected by flow cytometry. The effect of PD-S15 fusion protein on the proliferation of TILs was detected by cytometry. Results: The successful construction of pUC57-PD-S15 eukaryotic expression plasmid was confirmed by double enzyme digestion and sequencing, and then successfully transfected into HEK293T cells. The relative molecular weight of the target protein was approximately 55 000, and was in line with expectations. PD-S15 fusion protein could specifically combine with PD-1 in vitro (P<0.05) and stimulate NK/T cell proliferation (P<0.05). Compared with classical TILs culture method, the efficiency of activation and amplification of T cells in vitro by PD-S15 culturemethodwasbetter (P<0.01). Conclusion: PD-S15 fusion protein can specifically target PD-1 and rapidly expand NK/T cells in vitro, which lays a foundation for the selective expansion of CD8+PD-1+ antigen-specific T lymphocytes from tumor tissues and even peripheral blood.

Expression of programmed death receptor-1 and its prediction of postoperative infectious complications in elderly patients with hip fracture / 中华创伤骨科杂志

Objective To investigate the expression of programmed death receptor-1 (PD-1) and its value in predicting the infectious complications in elderly patients with hip fracture.Methods A total of 119 elderly patients with intertrochanteric fracture (observation group) were prospectively enrolled for this study from August 2015 to August 2016 at Department of Orthopaedics,The Seventh Medical Center of PLA.They were 86 males and 33 females with an age of 80.7 ± 8.1 years.Another 20 elderly out-patients with gonarthritis but without trauma or major medical disease were enrolled as a control group.The observation group received closed reduction and internal fixation with proximal femoral intramedullary nails.The PD-1 expression in peripheral blood T cells was detected at preoperation,1,3 and 5 days postoperation in the patients of observation group.They were further divided into an infection group and a non-infection group depending on whether infectious complications occurred during the follow-up period.The association between PD-1 and infectious complications was analyzed and its predictive value was calculated.Results The observation group and the control group,as well as the infection group and the non-infection group,were compatible due to their insignificant differences in baseline data(P ＞ 0.05).Compared with the control group,the expression of PD-1 in the peripheral blood T cells in the observation group was all significantly increased at preoperation,1,3 and 5 days postoperation (P ＜ 0.05).The PD-1 expression in patients of the infection group (24 cases) increased significantly higher at 1,3 and 5 days postoperation (33.60％ ± 2.44％,35.68％ ± 4.05％ and 30.36％ ±4.42％) than in those of the non-infection group (29.57％ ±3.20％,21.45％ ±2.20％ and 20.94％ ± 2.30％) (P ＜ 0.05).The PD-1 expression in the infection group reached its peak at 3 days postoperation and did not fall to the preoperative level at 5 days postoperation.However,the PD-1 expression in patients ofthe non-infection group (95 cases) showed atransient rise only on the first daypostoperation.The cutoff value of the difference in PD-1 expression between 1 and 3 days postoperation that could predict the infectious complications was 3.4％,with a sensitivity of 95.6％ and a specificity of 92.6％.Conclusions The expression of PD-1 may significantly increase after intramedullary fixation of hip fracture in elderly patients.The patients with infectious complications may exhibit a greater and longer increase in PD-1 expression than those without infectious complications.The difference in PD-1 expression between 1 and 3 days postoperation has a good predictive value for infectious complications because its high sensitivity and specificity.

Relationship between preoperative programmed death receptor 1,programmed death ligand 1 and clinical pathological parameters, early postoperative recurrence and metastasis in Patients with Esophageal Squamous Cell Carcinoma / 中国综合临床

Objective To investigate the relationship between programmed death 1 ( PD?1), programmed death receptor?1 ligand ( PD?L1 ) and clinical pathological parameters, early postoperative recurrence and metastasis in patients with esophageal squamous cell carcinoma.MethodsThe retrospectively analyze of Paraffin tissue specimens and clinical pathology data in 58 Patients undergoing radical esophageal squamous cell carcinoma surgery from January 2015 to January 2017 in the 910 hospital of PLA Joint Service Support force were performed.Expression of PD?1 and PD?L1 in esophageal squamous cell carcinoma and normal esophageal mucosa were detected by SP immunohistochemical staining.The positive expression rates of PD?1 and PD?L1 in normal esophageal mucosa and esophageal squamous cell carcinoma were compared.the relationship between PD?1 and PD?L1 and gender, age, family history, depth of tumor invasion, degree of differentiation, lymph node metastasis, and TNM staging were analyzed.Follow?up was performed by outpatient consultation and telephone consultation.The recurrence and metastasis of early postoperative (≤1 year) was analyzed.The PD?1 and PD?L1 in esophageal squamous cell carcinoma were analyzed in patients with recurrent metastasis and non?relapsing and metastasis.Results The positive expression rate of PD?1 in esophageal squamous cell carcinoma was 37.93%( 22／58 ), which was significantly higher than that in normal esophageal mucosa 15.52%( 9／58).The difference was statistically significant (χ2＝7.440,P＝0.006).The positive expression rate of PD?L1 in esophageal squamous cell carcinoma was 43.10%( 25／58), which was significantly higher than that of normal esophageal mucosa 18.97%(11／58).The difference was statistically significant (χ2＝7.894,P＝0.005).There was a difference in the positive expression rate of PD?L1 between different infiltration depth and TNM stage, P＜0.05.58 patients who underwent radical esophageal squamous cell carcinoma had been followed up for 6?12 months.A total of 14 patients had recurrence and metastasis,the incidence rate was 24.14%.The positive expression rate of PD?1 in the recurrence group was 42.86%(6／14),and that in the non?recurrent group was 36.36%(16／44).The difference was not statistically significant,(χ2＝0.190,P＞0.05).The positive expression rate of PD?L1 in the recurrence group was 71.43%(10／14),and that in the non?recurrent group was 34.09%(15／44).The difference was statistically significant,(χ2＝6.037,P＜0.05).Conclusion The expression of PD?1 and PD?L1 in cancer tissues of patients with esophageal squamous cell carcinoma is highly expressed.PD?L1 is closely related to the occurrence and progression of esophageal squamous cell carcinoma,and it is also an important index affecting early recurrence and metastasis.Which can be selected as a new target for early diagnosis and treatment.

Characteristics of expressions of programmed death receptor 1 and its ligand in colorectal metastases / 肿瘤研究与临床

The combination of programmed death receptor 1 (PD-1) and its ligand 1 (PD-L1) can inhibit and deplete T lymphocyte function, thereby inducing immune tolerance, enabling tumor cells to escape the attack of immune cells and promoting the growth and metastasis of tumors. At the same time, the high expression of PD-1/PD-L1 can enhance metastatic ability of tumors and lead to increased mortality, and the treatment of inhibiting PD-1/PD-L1 signaling pathway has been proved effective in many tumors. However, the traditional treatment methods are limited for the treatment of colorectal metastatic cancer. The emergence of immunotherapy may bring new breakthroughs for the treatment of colorectal metastatic cancer. This article reviews the characteristics of expressions of PD-1/PD-L1 in colorectal metastases.