Programmed Cell Death Ligand 1 mRNA Research Articles

Prognostic effect of programmed cell death ligand 1/programmed cell death 1 expression in cancer stem cells of human oral squamous cell carcinoma.

The relationship between cancer stem cells (CSCs) in oral squamous cell carcinoma (OSCC) and programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) remains unclear. Therefore, the present study aimed to clarify the association between the CD44v3high/CD24low immunophenotype of CSCs in OSCC and PD-L1/PD-1 co-expression, and to assess the prognostic effect of CSCs in terms of immune checkpoint molecules. Formalin-fixed, paraffin-embedded tissue samples and clinicopathological data from 168 patients with OSCC were retrospectively retrieved. Immunohistochemical staining and reverse transcription quantitative polymerase chain reaction were applied to a tissue microarray of the invasive front of each case. Semi-automated cell counting was used to assess CD44v3, CD24, PD-L1 and PD-1 expression by immunohistochemistry (IHC) using a digital image analysis program. Associations between immunological markers and clinicopathological variables were estimated. Patients with the CSC immunophenotype CD44v3high/CD24low, and patients with a high PD-L1/PD-1-positive cell density in the tumor parenchyma and stroma had significantly lower survival rates. Furthermore, patients with the CSC immunophenotype (CD44v3high/CD24low) and high PD-L1/PD-1 co-expression had even lower survival rates (P<0.01, log-rank test). Notably, there was a positive correlation between CD44v3 and PD-L1 expression (τ=0.1096, P=0.0366, Kendall rank correlation coefficient) and a negative correlation between CD24 and PD-1 expression (τ=-0.1387, P=0.0089, Kendall rank correlation coefficient). Additionally, the high CD44v3 expression group, as determined by IHC, exhibited significantly decreased expression of U2 small nuclear RNA auxiliary factor 1 (U2AF1) at the mRNA level compared with that in the low CD44v3 expression group (P<0.001, Mann-Whitney U test), and U2AF1 and PD-L1 mRNA expression exhibited a significant negative correlation (τ=-0.3948, P<0.001, Kendall rank correlation coefficient). In conclusion, CSCs in OSCC may evade host immune mechanisms and maintain CSC stemness via PD-L1/PD-1 co-expression, resulting in unfavorable clinical outcomes.

Expression of PD-1/PD-L1 axis in mediastinal lymph nodes and lung tissue of human and experimental lung fibrosis indicates a potential therapeutic target for idiopathic pulmonary fibrosis

BackgroundMediastinal lymph node enlargement is prevalent in patients with idiopathic pulmonary fibrosis (IPF). Studies investigating whether this phenomenon reflects specific immunologic activation are lacking.MethodsProgrammed cell death-1 (PD-1)/ programmed cell death ligand-1 (PD-L1) expression in mediastinal lymph nodes and lung tissues was analyzed. PD-1, PD-L1 mRNA expression was measured in tracheobronchial lymph nodes of mice following bleomycin-induced injury on day 14. Finally, the effect of the PD-1 inhibitor, pembrolizumab, in bleomycin-induced pulmonary fibrosis was investigated.ResultsWe analyzed mediastinal lymph nodes of thirty-three patients (n = 33, IPF: n = 14, lung cancer: n = 10, concomitant IPF and lung cancer: n = 9) and lung tissues of two hundred nineteen patients (n = 219, IPF: 123, controls: 96). PD-1 expression was increased, while PD-L1 expression was decreased, in mediastinal lymph nodes of patients with IPF compared to lung cancer and in IPF lungs compared to control lungs. Tracheobronchial lymph nodes isolated on day 14 from bleomycin-treated mice exhibited increased size and higher PD-1, PD-L1 mRNA levels compared to saline-treated animals. Pembrolizumab blunted bleomycin-induced lung fibrosis, as indicated by reduction in Ashcroft score and improvement in respiratory mechanics.ConclusionsMediastinal lymph nodes of patients with IPF exhibit differential expression profiles than those of patients with lung cancer indicating distinct immune-mediated pathways regulating fibrogenesis and carcinogenesis. PD-1 expression in mediastinal lymph nodes is in line with lung tissue expression. Lower doses of pembrolizumab might exert antifibrotic effects. Clinical trials aiming to endotype patients based on mediastinal lymph node profiling and accordingly implement targeted therapies such as PD-1 inhibitors are greatly anticipated.

High glucose-upregulated PD-L1 expression through RAS signaling-driven downregulation of PTRH1 leads to suppression of T cell cytotoxic function in tumor environment

BackgroundNearly 80% of patients with pancreatic cancer suffer from glucose intolerance or diabetes. Pancreatic cancer complicated by diabetes has a more immunosuppressive tumor microenvironment (TME) and is associated with a worse prognosis. The relationship between glucose metabolism and programmed cell death-Ligand 1 (PD-L1) is close and complex. It is important to explore the regulation of high glucose on PD-L1 expression in pancreatic cancer and its effect on infiltrating immune effectors in the tumor microenvironment.MethodsDiabetic murine models (C57BL/6) were used to reveal different immune landscape in euglycemic and hyperglycemic pancreatic tumor microenvironment. Bioinformatics, WB, iRIP [Improved RNA Binding Protein (RBP) Immunoprecipitation]-seq were used to confirm the potential regulating role of peptidyl-tRNA hydrolase 1 homolog (PTRH1) on the stability of the PD-L1 mRNA. Postoperative specimens were used to identify the expression of PD-L1 and PTRH1 in pancreatic cancer. Co-culturing T cells with pancreatic cancer cells to explore the immunosuppressive effect of pancreatic tumor cells.ResultsOur results revealed that a high dose of glucose enhanced the stability of the PD-L1 mRNA in pancreatic tumor cells by downregulating PTRH1 through RAS signaling pathway activation following epidermal growth factor receptor (EGFR) stimulation. PTRH1 overexpression significantly suppressed PD-L1 expression in pancreatic cells and improved the proportion and cytotoxic function of CD8+ T cells in the pancreatic TME of diabetic mice.ConclusionsPTRH1, an RBP, plays a key role in the regulation of PD-L1 by high glucose and is closely related to anti-tumor immunity in the pancreatic TME.

Regulation of PD-L1 Expression by Nuclear Receptors.

The suppression of excessive immune responses is necessary to prevent injury to the body, but it also allows cancer cells to escape immune responses and proliferate. Programmed cell death 1 (PD-1) is a co-inhibitory molecule that is present on T cells and is the receptor for programmed cell death ligand 1 (PD-L1). The binding of PD-1 to PD-L1 leads to the inhibition of the T cell receptor signaling cascade. PD-L1 has been found to be expressed in many types of cancers, such as lung, ovarian, and breast cancer, as well as glioblastoma. Furthermore, PD-L1 mRNA is widely expressed in normal peripheral tissues including the heart, skeletal muscle, placenta, lungs, thymus, spleen, kidney, and liver. The expression of PD-L1 is upregulated by proinflammatory cytokines and growth factors via a number of transcription factors. In addition, various nuclear receptors, such as androgen receptor, estrogen receptor, peroxisome-proliferator-activated receptor γ, and retinoic-acid-related orphan receptor γ, also regulate the expression of PD-L1. This review will focus on the current knowledge of the regulation of PD-L1 expression by nuclear receptors.

Mutual connected IL-6, EGFR and LIN28/Let7-related mechanisms modulate PD-L1 and IGF upregulation in HNSCC using immunotherapy.

The development of techniques and immunotherapies are widely applied in cancer treatment such as checkpoint inhibitors, adoptive cell therapy, and cancer vaccines apart from radiation therapy, surgery, and chemotherapy give enduring anti-tumor effects. Minority people utilize single-agent immunotherapy, and most people adopt multiple-agent immunotherapy. The difficulties are resolved by including the biomarkers to choose the non-responders' and responders' potentials. The possibility of the potential complications and side effects are examined to improve cancer therapy effects. The Head and Neck Squamous Cell Carcinoma (HNSCC) is analyzed with the help of programmed cell death ligand 1 (PD-L1) and Insulin-like growth factor (IGF). But how IGF and PD-L1 upregulation depends on IL-6, EGFR, and LIN28/Let7-related mechanisms are poorly understood. Briefly, IL-6 stimulates gene expressions of IGF-1/2, and IL-6 cross-activates IGF-1R signaling, NF-κB, and STAT3. NF-κB, up-regulating PD-L1 expressions. IL-6/JAK1 primes PD-L1 for STT3-mediated PD-L1 glycosylation, stabilizes PD-L1 and trafficks it to the cell surface. Moreover, ΔNp63 is predominantly overexpressed over TAp63 in HNSCC, elevates circulating IGF-1 levels by repressing IGFBP3, and activates insulin receptor substrate 1 (IRS1).TP63 and SOX2 form a complex with CCAT1 to promote EGFR expression. EGFR activation through EGF binding extends STAT3 activation, and EGFR and its downstream signaling prolong PD-L1 mRNA half-life. PLC-γ1 binding to a cytoplasmic motif of elevated PD-L1 improves EGF-induced activation of inositol 1,4,5-tri-phosphate (IP3), and diacylglycerol (DAG) subsequently elevates RAC1-GTP. RAC1-GTP was convincingly demonstrated to induce the autocrine production and action of IL-6/IL-6R, forming a feedback loop for IGF and PD-L1 upregulation. Furthermore, the LIN28-Let7 axis mediates the NF-κB-IL-6-STAT3 amplification loop, activated LIN28-Let7 axis up-regulates RAS, AKT, IL-6, IGF-1/2, IGF-1R, Myc, and PD-L1, plays pivotal roles in IGF-1R activation and Myc, NF-κB, STAT3 concomitant activation. Therefore, based on a detailed mechanisms review, our article firstly reveals that IL-6, EGFR, and LIN28/Let7-related mechanisms mediate PD-L1 and IGF upregulation in HNSCC, which comprehensively influences immunity, inflammation, metabolism, and metastasis in the tumor microenvironment, and might be fundamental for overcoming therapy resistance.

Elevated PD-L1 Expression and Microsatellite Instability in Elderly Patients With Gastric Cancer.

Immunotherapy in combination with chemotherapy is the current treatment of choice for frontline programmed cell death ligand 1 (PD-L1)-positive gastric cancer. However, the best treatment strategy remains an unmet medical need for elderly or fragile patients with gastric cancer. Previous studies have revealed that PD-L1 expression, Epstein-Barr virus association, and microsatellite instability-high (MSI-H) are the potential predictive biomarkers for immunotherapy use in gastric cancer. In this study, we showed that PD-L1 expression, tumor mutation burden, and the proportion of MSI-H were significantly elevated in elderly patients with gastric cancer who were older than 70 years compared with patients younger than 70 years from analysis of The Cancer Genome Atlas gastric adenocarcinoma cohort [≥70/<70: MSI-H: 26.8%/15.0%, P =0.003; tumor mutation burden: 6.7/5.1 Mut/Mb, P =0.0004; PD-L1 mRNA: 5.6/3.9 counts per million mapped reads, P =0.005]. In our real-world study, 416 gastric cancer patients were analyzed and showed similar results (≥70/<70: MSI-H: 12.5%/6.6%, P =0.041; combined positive score ≥1: 38.1%/21.5%, P <0.001). We also evaluated 16 elderly patients with gastric cancer treated with immunotherapy and revealed an objective response of 43.8%, a median overall survival of 14.8 months, and a median progression-free survival of 7.0 months. Our research showed that a durable clinical response could be expected when treating elderly patients with gastric cancer with immunotherapy, and this approach is worth further study.

USP7 Enables Immune Escape of Glioma Cells by Regulating PD-L1 Expression

ABSTRACT Background Glioma is the most common primary intracranial tumor. This study investigated the mechanism of ubiquitin-specific processing protease 7 (USP7) on the immune escape of glioma cells via the regulation of programmed cell death ligand-1 (PD-L1). Methods USP7 and PD-L1 expressions in glioma and normal brain tissues were detected using quantitative reverse transcriptase polymerase chain reaction and Western blot. The glioma cells U-251 MG were transfected with si-USP7 and pcDNA3.1-PD-L1, or treated with anti-PD-L1, after which the cell viability, colony-forming ability, and apoptosis rate were evaluated using cell counting kit-8, colony formation, and TdT-mediated dUTP nick-end labeling assays. Then, CD8+ T cells were purified, extracted, then co-cultured with U-251 MG cells. CD8+ T cell proliferation and the concentrations of interferon (IFN-γ), tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, interleukin (IL)-1β, and IL-10 were verified using carboxyfluorescein succinimidyl amino ester proliferation assay and enzyme-linked immunosorbent assay. Afterwards, the interaction between USP7 and PD-L1 and the ubiquitination level of PD-L1 were also assessed. Results USP7 was highly-expressed and PD-L1 mRNA levels did not change, while PD-L1 protein levels were up-regulated in the glioma cells. Silencing USP7 in U-251 MG cells limited the growth of the glioma cells, promoted glioma cell apoptosis, and facilitated the proliferation of CD8+ T cells, thus inhibiting immune escape. USP7 stabilized PD-L1 expression through deubiquitination. PD-L1 overexpression reversed the inhibitory effect of silencing USP7 on the immune escape of glioma cells. Conclusion USP7 stabilized PD-L1 through deubiquitination and accelerated the immune escape of glioma cells.

Genetic Variation of PD-L1 Gene Affects its Expression and Is Related to Clinical Outcome in Epithelial Ovarian Cancer.

ObjectiveThis study aims to investigate the effect of polymorphisms of programmed cell death-ligand 1 (PD-L1) on the risk and patient’s outcomes of epithelial ovarian cancer (EOC).MethodsTotally, 568 patients and 532 healthy women were included. Three polymorphisms in the PD-L1 gene, rs2297136, rs4143815 and rs4742098, were genotyped by the polymerase chain reaction/ligase detection reaction (PCR-LDR). Survival analysis was performed in 234 patients (received primary debulking surgery followed by platinum-based chemotherapy).ResultsPatients with the rs2297136 AG + GG genotypes had shorter progression-free survival (PFS) (hazard ratio (HR)=1.44, 95% CI=1.03-2.01) and overall survival (OS) (HR=1.55, 95% CI=1.06-2.27) than those with the AA genotype. Moreover, the mRNA and protein expression levels of PD-L1 in EOC tissues with the rs2297136 AG + GG genotypes were remarkably higher than those with the AA genotype (P=0.032 and P=0.047, respectively). Survival analysis showed that high expression of PD-L1 mRNA was remarkably associated with worse 10-year PFS (HR=1.55, 95% CI=1.28-1.88) and OS (HR=1.51, 95% CI=1.00-2.28) in EOC patients.ConclusionsThe rs2297136 may not only effectively influence the expression of PD-L1, but also is significantly associated with EOC patients’ outcomes.

Deep Downregulation of PD-L1 by Caged Peptide-Conjugated AIEgen/miR-140 Nanoparticles for Enhanced Immunotherapy.

Downregulating programmed cell death ligand 1(PD-L1) protein levels in tumor cells is an effective way to achieve immune system activation for oncology treatment, but current strategies are inadequate. Here, we design a caged peptide-AIEgen probe (GCP) to self-assemble with miR-140 forming GCP/miR-140 nanoparticles. After entering tumor cells, GCP/miR-140 disassembles in the presence of Cathepsin B (CB) and releases caged GO203 peptide, miR-140 and PyTPA. Peptide decages in the highly reductive intracellular environment and binds to mucin 1 (MUC1), thereby downregulating the expression of PD-L1. Meanwhile, miR-140 reduces PD-L1 expression by targeting downregulation of PD-L1 mRNA. Under the action of PyTPA-mediated photodynamic therapy (PDT), tumor-associated antigens are released, triggering immune cell attack on tumor cells. This multiple mechanism-based strategy of deeply downregulating PD-L1 in tumor cells activates the immune system and thus achieves effective immunotherapy.

Cellular and soluble immune checkpoint signaling forms PD-L1 and PD-1 in renal tumor tissue and in blood

Immune checkpoint blockade therapy is a treatment option of various metastatic cancer diseases including renal cell carcinoma (RCC). Approved antibody drugs target the co-inhibitory signaling of Programmed Cell Death Ligand-1 (PD-L1) and its receptor Programmed Cell Death-1 (PD-1). The combined evaluation of PD-L1 and PD-1 at the mRNA and protein levels in tumor tissue with differentiation of tumor and immune cells as well as of soluble forms (sPD-L1) and (sPD-1) in blood is of basic interest in assessing biomarker surrogates. Here, we demonstrate that PD-L1 determined as fraction of stained tumor cells (TPS-score) correlates with PD-L1-mRNA in tumor tissue, reflecting the predominant expression of PD-L1 in tumor cells. Conversely, PD-1 in immune cells of tumor tissue (IC-score) correlated with PD-1-mRNA tissue levels reflecting the typical PD-1 expression in immune cells. Of note, sPD-L1 in blood did not correlate with either the TPS-score of PD-L1 or with PD-L1-mRNA in tumor tissue. sPD-L1 released into the supernatant of cultured RCC cells closely followed the cellular PD-L1 expression as tested by interferon γ (IFNG) induction and siRNA knockdown of PD-L1. Further analysis in patients revealed that sPD-L1 significantly increased in blood following renal tumor resection. In addition, sPD-L1 correlated significantly with inflammation marker C-reactive protein (CRP) and with PD-L1 mRNA level in whole blood. These results indicate that the major source of sPD-L1 in blood may be peripheral blood cells and not primarily tumor tissue PD-L1.

Programmed Cell Death Ligand 1 Is Enriched in Mammary Stem Cells and Promotes Mammary Development and Regeneration.

Programmed cell death ligand 1 (PD-L1) is widely expressed in a variety of human tumors, and inhibition of the PD-L1/PD-1 pathway represents one of the most promising therapy for many types of cancer. However, the physiological function of PD-L1 in tissue development is still unclear, although PD-L1 mRNA is abundant in many tissues. To address this puzzle, we investigated the function of PD-L1 in mammary gland development. Interestingly, we found that PD-L1 is enriched in protein C receptor (Procr)-expressing mammary stem cells (MaSCs), and PD-L1-expressing mammary basal cells (PD-L1+ basal cells) exhibit robust mammary regeneration capacity in transplantation assay. The lineage tracing experiment showed that PD-L1+ cells can differentiate into all lineages of mammary epithelium cells, suggesting that PD-L1+ basal cells have the activities of MaSCs. Furthermore, PD-L1 deficiency significantly impairs mammary development and reduces mammary regeneration capacity of mammary basal cells, suggesting that PD-L1 is not only enriched in MaSCs but also improves activities of MaSCs. In summary, these results demonstrated that PD-L1 is enriched in MaSCs and promotes mammary gland development and regeneration. Mechanistically, our data indicated that PD-L1 expression is induced by continuous activation of Wnt/ß-catenin signaling. In conclusion, these results demonstrated that PD-L1 is a marker of MaSCs, and PD-L1 is essential for mammary development. Our study provides novel insight into the physiological functions of PD-L1 in tissue development.

Abstract 72: Bifidobacterium longum RAPO enhances efficacy of anti-PD-1 immunotherapy in a mouse model of triple-negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) represents approximately 15-20% of all breast cancers and 35% of breast cancer death. TNBC has a highly aggressive behavior with the increased risk of early metastasis and relapse and poor survival rate. Given that TNBC is associated with higher levels of tumor-infiltrating lymphocytes and programmed cell death ligand 1 (PD-L1), immune checkpoint inhibitors (ICI) are considered as the appropriate treatment of patients with TNBC. However, it has been reported that ICI monotherapy is less effective compared with combination therapy. Recent several studies suggest that gut microbiome plays a critical role in the efficacy of ICI therapy. Thus, we investigated the effects of Bifidobacterium longum RAPO supplementation on anti-PD-1 therapy against TNBC and the underlying mechanisms in vivo. Methods: The 4T1 murine metastatic breast cancer cells were injected s.c into the flank of female BALB/c mice. When tumors reached 50-150 mm3, mice were randomly assigned to one of four groups; tumor control (TC), Anti-PD-1, B. longum RAPO (RAPO), or Anti-PD-1+RAPO (n=10 per group). Anti-PD-1 antibody was injected i.p. 5 times at 3 day intervals. B. longum RAPO was orally administered daily from 2 days before the first injection of anti-PD-1. Spleen and tumor tissues were analyzed by flow cytometry, qRT-PCR, and immunohistochemistry, and fecal samples were collected for 16S rRNA gene sequencing. Results: On day 12, tumor volume was significantly inhibited in Anti-PD-1+RAPO group compared with TC and Anti-PD-1 groups. Higher levels of PD-L1 IHC score and PD-L1 mRNA expression were observed in tumor tissues of Anti-PD-1+RAPO group. Anti-PD-1+RAPO group was also associated with the increased levels of spleen NKT cells and tumor NK cells. The proportion of pro-tumor M2 macrophages and M2/M1 ratio were significantly decreased in tumor tissues of Anti-PD-1+RAPO group than those of Anti-PD-1 group. Similarly, combination therapy of Anti-PD-1 with RAPO significantly reduced mRNA expression of M2 markers such as IL10 and Arg1, while it augmented that of anti-tumor cytokines including IFNγ and TNFα compared with Anti-PD-1 treatment alone. In addition, the relative abundance of the genus Bifidobacterium was remarkably enriched in Anti-PD-1+RAPO group compared with other groups. In Anti-PD-1+RAPO group, the relative abundance of the genus Akkermansia and Lachnospiraceae NK4A136 group were significantly increased after administration for 2 weeks. Conclusions: Our data suggests that combination therapy of B. longum RAPO with anti-PD-1 modulates anti-tumor immunity and gut microbiota, contributing to the better response to anti-PD-1 immunotherapy in mice with TNBC. This study provides evidence that B. longum RAPO might be a potential candidate for immunomodulatory pharmabiotics in cancer immunotherapy. Citation Format: Hyeyoon Kim, Rira Oh, Sangjun Park, Geun Eog Ji, Myeong Soo Park, Sung-Eun Kim. Bifidobacterium longum RAPO enhances efficacy of anti-PD-1 immunotherapy in a mouse model of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 72.

Combination Therapy of Bifidobacterium longum RAPO With Anti-PD-1 Treatment Enhances Anti-tumor Immune Response in Association With Gut Microbiota Modulation

ObjectivesTriple negative breast cancer (TNBC) has a poor prognosis with a high risk of metastasis and relapse, and accounts for approximately 35% of breast cancer death. Immune checkpoint inhibitors (ICI) have been in the spotlight recently as a novel treatment of TNBC due to the higher expressions of programmed cell death ligand 1 (PD-L1) and tumor-infiltrating lymphocytes in TNBC. Considering that gut microbiome is associated with immune response and ICI efficacy, we investigated whether Bifidobacterium longum RAPO supplementation would affect the efficacy of anti-PD-1 therapy against TNBC in vivo. MethodsFemale BALB/c mice bearing 4T1 breast cancer cells were randomly divided into four groups; tumor control (TC), Anti-PD-1, B. longum RAPO (RAPO), or Anti-PD-1 + RAPO (Combi). Anti-PD-1 antibody was injected i.p. 5 times at 3-day intervals and B. longum RAPO was orally administered daily from 2 days before the first injection of anti-PD-1. Spleen and tumor tissues were analyzed by flow cytometry, IHC, and qRT-PCR. Fecal samples were analyzed by 16S rRNA gene sequencing. ResultsTumor volume was reduced in the Combi group than TC and Anti-PD-1 groups on day 12. PD-L1 IHC score and PD-L1 mRNA expression were significantly increased in tumors of the Combi group. The levels of the spleen CD8/CD4 ratio and tumor NK cells were also increased in the Combi group. Compared with the Anti-PD-1 group, the pro-tumor M2 macrophages and related cytokines (IL10, Arg1) were significantly decreased, while anti-tumor M1 cytokines (IFNγ, TNFα) were elevated in tumors of the Combi group. Consistently, immunogenic cell death-associated markers were significantly higher in the Combi group. We found that the relative proportion of the genus Bifidobacterium, Lachnoclostridium, Lachnospiraceae NK4A136 group, and Clostridium sensu stricto 1, which are known to be associated with the better response to ICI therapy, were significantly increased, while the genus Mucispirillum and Dubosiella were decreased in the Combi group than the Anti-PD-1 group. ConclusionsOur data indicates that combination therapy of B. longum RAPO with anti-PD-1 enhances anti-tumor immune response in association with modulation of gut microbiota, suggesting that B. longum RAPO might be a potential candidate for pharmabiotics in ICI treatment. Funding SourcesBIFIDO CO, the National Research Foundation of Korea grant.

Effects of transarterial chemoembolization on the immunological function of patients with hepatocellular carcinoma.

The present study aimed to investigate the effects of transarterial chemoembolization (TACE) on the immune function of patients with hepatocellular carcinoma (HCC). A total of 114 patients with HCC were selected and their peripheral blood was collected before and 1 month after TACE treatment. Flow cytometry and reverse transcription-quantitative PCR were performed to analyze the changes in immune function in patients before and after treatment. Kaplan-Meier curves were plotted for survival analysis. The programmed cell death ligand 1 (PD-L1) and programmed cell death protein 1 (PD1) expression before TACE treatment were significantly higher in patients with poor TACE response compared with those patients with well response. Higher PD-L1 mRNA expression in the peripheral blood mononuclear cells after TACE predicted a superior prognosis. After TACE treatment, the proportion of CD4+/CD8+ cells were decreased while the expression levels of programmed cell death protein 1 (PD1) were significantly increased. To conclude, TACE could reduce the proportion of CD4+/CD8+ cells and improve the mRNA expression levels of PD1 in patients with HCC. The expression levels of PD1 and PD-L1 were closely related to the therapeutic effect of TACE and the prognosis of patients with HCC. TACE combined with immunotherapy may have potential clinical value for patients with HCC.

Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples.

Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/μL with a range of 768-7510 copies/μL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection.

The Clinical Significance of HPV L1/PD-L1 Tests Combined with Colposcopy for Cervical Precancerous Lesions and Cervical Cancer

To explore the expression of human papilloma virus (HPV) L1 protein and programmed cell death ligand-1 (PD-L1) protein in cervical precancerous lesions and cervical cancer, to analyze the correlation between HPV L1 and PD-L1 expression tests combined with colposcopy and the occurrence and development of of cervical lesions, and to determine the significance of the combined examination for auxiliary differential diagnosis. 260 patients with high-risk HPV (HR-HPV) infection who were treated at West China Second University Hospital, Sichuan University from January, 2018 to January, 2020 were included in the study. 260 cervical cytology specimens were collected, of which 218 cervical histology specimens were collected, of which 202 cases underwent colposcopy. Among the 260 cervical cytology specimens, 40 were of cervical inflammatory cells, 40 were of low-grade squamous intraepithilia lesions (LSIL) with mild atypical hyperplasia, 80 were of high-grade squamous intraepithilia lesions (HSIL) with moderate and severe atypical hyperplasia, and 100 were of cervical carcinoma cells (CCC). Among the 218 cervical histology specimens, 15 were of chronic cervicitis tissue, 20 were of cervical intraepithelial neoplasia (CIN) 1, 32 were of CIN 2, 51 were of CIN 3, and 100 were of cervical cancer (CC). Among the 260 patients, 202 underwent colposcopy. Immunocytochemistry and immunohistochemistry were used to assess the expression of HPV L1 protein and PD-L1 protein, and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the level of PD-L1 mRNA in chronic cervicitis tissues and CC. The sensitivity, specificity, positive predictive value and negative predictive value of HPV L1 and PD-L1 tests combined with colposcopy in the detection of cervical lesions were studied. ①In the cervical cytology group, the positive rate of HPV L1 expression was 82.50%, 57.50%, 11.25%, and 3.00% in cervical inflammatory cells, low-grade squamous intraepithilia lesions (LSIL), high-grade squamous intraepithilia lesions (HSIL) and CCC, respectively, showing decreasing levels of positive expression rate ( P<0.05). In the cervical histology group, the positive rate of HPV L1 expression was 86.67%, 65.00%, 34.38%, 11.76% and 4.00% in chronic cervicitis tissues, CIN 1 group, CIN 2 group, CIN 3 group and CC group, respectively, showing decreasing levels of positive expression ( P<0.05). ②In the cervical cytology group, the average positive expression scores of PD-L1 in the cervical inflammatory cells, LSIL group, HSIL group, and CCC group were 0.25±0.12, 1.05±0.67, 1.39±0.11 and 2.14±0.17, respectively, showing increasing levels of positive expression scores ( P<0.05). In the cervical histology group, the average positive expression scores of PD-L1 were 0.28±0.24, 1.21±0.79, 1.56±0.26, 1.80±0.24, and 2.10±0.19 in the chronic cervicitis tissue, CIN 1 group, CIN 2 group, CIN 3 group and CC group, respectively, showing increasing levels of positive expression scores ( P<0.05). The relative expression of PD-L1 mRNA in chronic cervicitis tissue and CC is 1.02±0.04 and 1.81±0.22, respectively ( P<0.05). ③The sensitivity and specificity of diagnosis of cervical tissue CIN2 and abovelesions, HPV L1 detection alone was 95.8%, 47.2%, PD-L1 detection alone was 96.5%, 32.8%, colposcopy alone was 77.5%, 70.8%, HPV L1/PD-L1 tests combined detection was 92.4%, 64.5%, HPV L1/PD-L1 detection combined colposcopy was 71.6% and 89.6%, respectively. The sensitivity and specificity of the HPV L1/PD-L1 combined test for the diagnosis of CIN3 and above cervical lesions were 71.9% and 86.1%, HPV L1/PD-L1 combined with colposcopy were 50.5% and 100.0%, respectively. The specificity of HPV L1/PD-L1 detection combined with colposcopy for CIN2 and above lesions is higher than that of HPV L1 detection alone, PD-L1 detection alone and colposcopy alone. HPV L1/PD-L1 detection combined with colposcopy detection for CIN3 and above lesions has an important auxiliary diagnostic value.

Campylobacter concisus upregulates PD-L1 mRNA expression in IFN-γ sensitized intestinal epithelial cells and induces cell death in esophageal epithelial cells

ABSTRACT Introduction: Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) and Barrett’s esophagus (BE). Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that is used by tumor cells for immune evasion and has increased expression in patients with IBD and BE. We examined whether C. concisus upregulates PD-L1 expression in intestinal and esophageal epithelial cells. Methods: Human intestinal epithelial HT-29 cells and esophageal epithelial FLO-1 cells with and without interferon (IFN)-γ sensitization were incubated with C. concisus strains. The level of PD-L1 mRNA was quantified using quantitative real-time PCR. Cytokines were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Apoptosis of HT-29 and FLO-1 cells were measured using caspase 3/7 assay. Results: We found that intestinal epithelial cells with IFN-γ sensitization incubated with C. concisus significantly upregulated PD-L1 expression and significantly increased the production of interleukin (IL)-8. Whereas, PD-L1 expression was significantly inhibited in IFN-γ sensitized FLO-1 cells incubated with C. concisus strains. Furthermore, FLO-1 cells with and without IFN-γ sensitization incubated with C. concisus strains both had significantly higher levels of cell death. Conclusion: C. concisushas the potential to cause damage to both intestinal and esophageal epithelial cells, however, with different pathogenic effects.

Programmed Cell Death Protein 1/Programmed Cell Death Ligand-1 Axis activates Intracellular ERK Signaling in Tumor Cells to Mediate Poor Prognosis in T-cell Lymphoma.

Purpose: To investigate the prognostic significance of programmed cell death ligand-1 (PD-L1) and phosphorylated ERK (p-ERK) and their interactions in T-cell lymphoma (TCL).Methods: The mRNA levels of PD-L1 and ERK in TCL samples were analyzed. Formalin-fixed paraffin-embedded tissues from 69 TCL patients were collected to detect the expression of PD-L1 and p-ERK by multiplexed immunofluorescence staining. The total PD-L1 and p-ERK was measured by western blotting, and membrane PD-L1 was determined using flow cytometry.Results: PD-L1 and ERK mRNA levels were significantly upregulated in TCL. The expression rates of PD-L1 and p-ERK were 52.2% and 27.5%, respectively. PD-L1 expression correlated with stage (R=0.304, P=0.011) and IPI score (R=0.313, P=0.009), and p-ERK expression correlated with stage (R=0.330, P=0.006) and IPI score (R=0.376, P=0.002). PD-L1 expression positively correlated with p-ERK expression (R=0.355, P=0.003). Patients with co-expression of PD-L1 and p-ERK had the worst overall survival (P=0.007). In three TCL cell lines with PD-L1 expression, we demonstrated that the expression of p-ERK was upregulated after stimulation with PD-1, suggesting that ERK signaling was activated.Conclusions: The PD-1/PD-L1 axis activates intracellular ERK signaling in tumor cells and that PD-L1, p-ERK or their combination are potential biomarkers for predicting the prognosis in TCL patients.

Blocking IL-17A enhances tumor response to anti-PD-1 immunotherapy in microsatellite stable colorectal cancer

BackgroundImmune checkpoint inhibitors (ICIs), including anti-PD-1 therapy, have limited efficacy in patients with microsatellite stable (MSS) colorectal cancer (CRC). Interleukin 17A (IL-17A) activity leads to a protumor microenvironment, dependent on...

Escherichia coli K12 Upregulates Programmed Cell Death Ligand 1 (PD-L1) Expression in Gamma Interferon-Sensitized Intestinal Epithelial Cells via the NF-κB Pathway.

Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein which is used by tumor cells for immune evasion. PD-L1 is upregulated in inflamed intestinal tissues. The intestinal tract is colonized by millions of bacteria, most of which are commensal bacterial species. We hypothesized that under inflammatory conditions, some commensal bacterial species contribute to increased PD-L1 expression in intestinal epithelium and examined this hypothesis. Human intestinal epithelial HT-29 cells with and without interferon (IFN)-γ sensitization were incubated with six strains of four enteric bacterial species. The mRNA and protein levels of PD-L1 in HT-29 cells were examined using quantitative real-time PCR and flow cytometry, respectively. The levels of interleukin (IL)-1β, IL-18, IL-6, IL-8, and tumor necrosis factor (TNF)-α secreted by HT-29 cells were measured using enzyme-linked immunosorbent assay. Apoptosis of HT-29 cells was measured using a caspase 3/7 assay. We found that Escherichia coli K12 significantly upregulated both PD-L1 mRNA and protein in IFN-γ-sensitized HT-29 cells. E. coli K12 induced the production of IL-8 in HT-29 cells, however, IL-8 did not affect HT-29 PD-L1 expression. Inhibition of the nuclear factor-kappa B pathway significantly reduced E. coli K12-induced PD-L1 expression in HT-29 cells. The other two E. coli strains and two enteric bacterial species did not significantly affect PD-L1 expression in HT-29 cells. Enterococcus faecalis significantly inhibited PD-L1 expression due to induction of cell death. Data from this study suggest that some gut bacterial species have the potential to affect immune function under inflammatory conditions via upregulating epithelial PD-L1 expression.