Regulation Of PD-L1 Research Articles

Tumor-specific T cell-mediated upregulation of PD-L1 in myelodysplastic syndrome cells does not affect T-cell killing.

The PD-1:PD-L1 axis is a binary interaction that delivers inhibitory signals to T cells, impeding both immune surveillance and response to immunotherapy. Here we analyzed a phenomenon whereby tumor-specific T cells induce PD-L1 upregulation in autologous MDS cells in short-term culture, through a mechanism that is cell-contact-independent and partially IFNγ-dependent. After investigating a panel of small-molecule inhibitors, we determined that PD-L1 upregulation was attributed to the PKR-like ER kinase (PERK) branch of the unfolded protein response. Interestingly, we found that the cytotoxic capacity of tumor-specific T cells was not impaired by the expression of PD-L1 on MDS target cells. These results highlight a little appreciated aspect of PD-1:PD-L1 regulation in hematologic cancers and indicate that this phenomenon, while likely to hinder autochthonous immune surveillance, may not be an obstacle to immunotherapies such as personalized adoptive T-cell therapy.

Beyond Cancer: Regulation and Function of PD-L1 in Health and Immune-Related Diseases.

Programmed Cell Death 1 Ligand 1 (PD-L1, CD274, B7-H1) is a transmembrane protein which is strongly involved in immune modulation, serving as checkpoint regulator. Interaction with its receptor, Programmed Cell Death Protein 1 (PD-1), induces an immune-suppressive signal, which modulates the activity of T cells and other effector cells. This mediates peripheral tolerance and contributes to tumor immune escape. PD-L1 became famous due to its deployment in cancer therapy, where blockage of PD-L1 with the help of therapeutic antagonistic antibodies achieved impressive clinical responses by reactivating effector cell functions against tumor cells. Therefore, in the past, the focus has been placed on PD-L1 expression and its function in various malignant cells, whereas its role in healthy tissue and diseases apart from cancer remained largely neglected. In this review, we summarize the function of PD-L1 in non-cancerous cells, outlining its discovery and origin, as well as its involvement in different cellular and immune-related processes. We provide an overview of transcriptional and translational regulation, and expression patterns of PD-L1 in different cells and organs, and illuminate the involvement of PD-L1 in different autoimmune diseases as well as in the context of transplantation and pregnancy.

Immunorthodontics: PD-L1, a Novel Immunomodulator in Cementoblasts, Is Regulated by HIF-1α under Hypoxia.

Recent studies have revealed that hypoxia alters the PD-L1 expression in periodontal cells. HIF-1α is a key regulator for PD-L1. As hypoxia presents a hallmark of an orthodontically induced microenvironment, hypoxic stimulation of PD-L1 expression may play vital roles in immunorthodontics and orthodontically induced inflammatory root resorption (OIIRR). This study aims to investigate the hypoxic regulation of PD-L1 in cementoblasts, and its interaction with hypoxia-induced HIF-1α expression. The cementoblast (OCCM-30) cells (M. Somerman, NIH, NIDCR, Bethesda, Maryland) were cultured in the presence and absence of cobalt (II) chloride (CoCl2). Protein expression of PD-L1 and HIF-1α as well as their gene expression were evaluated by Western blotting and RT-qPCR. Immunofluorescence was applied to visualize the localization of the proteins within cells. The HIF-1α inhibitor (HY-111387, MedChemExpress) was added, and CRISPR/Cas9 plasmid targeting HIF-1α was transferred for further investigation by flow cytometry analysis. Under hypoxic conditions, cementoblasts undergo an up-regulation of PD-L1 expression at protein and mRNA levels. Silencing of HIF-1α using CRISPR/Cas9 indicated a major positive correlation with HIF-1α in regulating PD-L1 expression. Taken together, these findings show the influence of hypoxia on PD-L1 expression is modulated in a HIF-1α dependent manner. The HIF-1α/PD-L1 pathway may play a role in the immune response of cementoblasts. Thus, combined HIF-1α/PD-L1 inhibition could be of possible therapeutic relevance for OIIRR prevention.

HOXA11-AS1 Promotes PD-L1-Mediated Immune Escape and Metastasis of Hypopharyngeal Carcinoma by Facilitating PTBP1 and FOSL1 Association.

Simple SummaryThe metastasis of hypopharyngeal squamous cell carcinoma (HSCC) is the main reason for the poor prognosis of patients. Increasing studies have shown that abnormally expressed lncRNAs play crucial roles in HSCC, providing new perspectives for exploring cancer pathogenesis and matastasis. The expressions of HOXA11-AS1 and PD-L1 were found to be closely related to the overall survival of HSCC patients. Subsequently, the potential target genes, namely PBTP1 and FOSL1, were identified by expression correlation analysis. Finally, HOXA11-AS1/FOSL1/PTBP1/PD-L1 axis was identified to be a novel pathway provided a feasible preliminary basis for the future application of immunotherapy or targeted therapies in HSCC. Background: The metastatic characteristics of hypopharyngeal squamous cell carcinoma (HSCC) lead to many diagnostic and therapeutic challenges, while functional long non-coding RNAs (lncRNAs) can provide effective strategies for its diagnosis and treatment. Methods: RT-qPCR, Western blot, immunohistochemistry, and an immunofluorescence assay were used to detect the related gene expression. Flow cytometry was used to measure the percentage of CD8+ and CD4+ T cells. CCK-8 and transwell assays were performed to analyze the role of HOXA11-AS1. The targeted relationship of the FOSL1/PD-L1 promoter was measured by ChIP and dual-luciferase reporter assays. RNA pulldown and RIP assays were used to measure the interaction between HOXA11-AS1, FOSL1, and PTBP1. A tumor xenograft study was used to analyze HOXA11-AS1 function in vivo. Results: HOXA11-AS1, PD-L1, and FOSL1 were upregulated in HSCC, and HOXA11-AS1 positively correlated with PD-L1. HOXA11-AS1 knockdown upregulated CD8+ T cells through an increase in IFN-γ concentration while decreasing the proliferation, migration, and invasion of HSCC cells. FOSL1 bound the PD-L1 promoter, increasing gene expression. HOXA11-AS1 enhanced the stability of FOSL1 mRNA by binding to PTBP1. HOXA11-AS1 or PTBP1 overexpression increased FOSL1 and PD-L1 expression. PD-L1 knockdown arrested the inhibiting function of HOXA11-AS1 overexpression on CD8+ T cell content. HOXA11-AS1 knockdown inhibited immune escape and metastasis through PD-L1 regulation by downregulating FOSL1 in vivo. Conclusion: HOXA11-AS1 promoted PD-L1 expression by upregulating FOSL1 levels through PTBP1, thereby facilitating immune escape, proliferation, and metastasis of HSCC cells.

CMTM6 and CMTM4 as two novel regulators of PD-L1 modulate the tumor microenvironment.

The tumor microenvironment (TME) plays crucial roles in regulating tumor occurrence, progress, metastasis and drug resistance. However, it remains largely elusive how the components of TME are regulated to govern its functions in tumor biology. Here, we discussed how the two novel functional proteins, chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 6 (CMTM6) and CMTM4, which involved in the post-translational regulation of PD-L1, modulate the TME functions. The roles of CMTM6 and CMTM4 in regulating TME components, including immune cells and tumor cells themselves were discussed in this review. The potential clinical applications of CMTM6 and CMTM4 as biomarkers to predict therapy efficacy and as new or combined immunotherapy targets are also highlighted. Finally, the current hot topics for the biological function of CMTM6/4 and several significant research directions for CMTM6/4 are also briefly summarized in the review.

Inflammation-Driven Regulation of PD-L1 and PD-L2, and Their Cross-Interactions with Protective Soluble TNFα Receptors in Human Triple-Negative Breast Cancer.

Simple SummaryImmune checkpoint blockades (ICBs) to PD-L1 have led to major breakthroughs in cancer therapy, but in triple-negative breast cancer (TNBC) success rates are rather limited. Following studies suggesting that chronic inflammation may limit ICB efficacy, we found that pro-inflammatory cytokines up-regulated the proportion of TNBC cells co-expressing the inhibitory immune checkpoint PD-L1 and its cognate PD-L2 molecule. Moreover, we demonstrated that in the context of inflammation-driven signals, PD-L1 down-regulated the cell-derived levels of sTNFR1 and sTNFR2, the soluble receptors of tumor necrosis factor α (TNFα); these soluble receptors were found to exert protective/anti-metastatic effects in TNBC cells, manifested by their ability to down-regulate TNFα-induced production of pro-metastatic chemokines by TNBC cells. Our findings possibly testify for a novel mechanism of PD-L1-mediated tumor progression in which PD-L1 prevents the anti-metastatic effects of sTNFR1 and sTNFR2 in TNBC cells. This mechanism may also act in vivo, in parallel to immune suppression under inflammatory conditions.Pro-inflammatory cytokines play key roles in elevating cancer progression in triple-negative breast cancer (TNBC). We demonstrate that specific combinations between TNFα, IL-1β and IFNγ up-regulated the proportion of human TNBC cells co-expressing the inhibitory immune checkpoints PD-L1 and PD-L2: TNFα + IL-1β in MDA-MB-231 cells and IFNγ + IL-1β in BT-549 cells; in the latter cells, the process depended entirely on STAT1 activation, with no involvement of p65 (CRISPR-Cas9 experiments). Highly significant associations between the pro-inflammatory cytokines and PD-L1/PD-L2 expression were revealed in the TCGA dataset of basal-like breast cancer patients. In parallel, we found that the pro-inflammatory cytokines regulated the expression of the soluble receptors of tumor necrosis factor α (TNFα), namely sTNFR1 and sTNFR2; moreover, we revealed that sTNFR1 and sTNFR2 serve as anti-metastatic and protective factors in TNBC, reducing the TNFα-induced production of inflammatory pro-metastatic chemokines (CXCL8, CXCL1, CCL5) by TNBC cells. Importantly, we found that in the context of inflammatory stimulation and also without exposure to pro-inflammatory cytokines, elevated levels of PD-L1 have down-regulated the production of anti-tumor sTNFR1 and sTNFR2. These findings suggest that in addition to its immune-suppressive activities, PD-L1 may promote disease course in TNBC by inhibiting the protective effects of sTNFR1 and sTNFR2.

Generation, secretion and degradation of cancer immunotherapy target PD-L1.

Cancer immunotherapy is a rapidly developing and effective method for the treatment of a variety of malignancies in recent years. As a significant immune checkpoint, programmed cell death 1 ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) play the most significant role in cancer immune escape and cancer immunotherapy. Though PD-L1 have become an important target for drug development and there have been various approved drugs and clinic trials targeting it, and various clinical response rate and adverse reactions prevent many patients from benefiting from it. In recent years, combination trials have become the main direction of PD-1/PD-L1 antibodies development. Here, we summarized PD-L1 biofunctions and key roles in various cancers along with the development of PD-L1 inhibitors. The regulators that are involved in controlling PD-L1 expression including post-translational modification, mRNA level regulation as well as degradation and exosome secretory pathway of PD-L1 were focused. This systematic summary may provide comprehensive understanding of different regulations on PD-L1 as well as a broad prospect for the search of the important regulator of PD-L1. The regulatory factors of PD-L1 can be potential targets for immunotherapy and increase strategies of immunotherapy in combination.

The miR-199a-5p/PD-L1 axis regulates cell proliferation, migration and invasion in follicular thyroid carcinoma

BackgroundFollicular thyroid carcinoma (FTC) is the second most common cancer of the thyroid and easily develops into distant metastasis. PD-L1 is known to be associated with the carcinogenesis and progression of thyroid carcinoma. Our study aimed to investigate the biological functions of PD-L1 and to identify miRNAs that were responsible for modulating the activity of PD-L1.MethodsA total of 72 patients with FTC at The Second Affiliated Hospital of Fujian Medical University were enrolled in this retrospective study. Immunohistochemical (IHC) assay was used to measure PD-L1 expression in FTC. The association between PD-L1 expression and clinicopathologic characteristics was evaluated. Bioinformatics analysis, RT–qPCR and western blotting were used to examine the relationships between miR-199a-5p, PD-L1 and Claudin-1. Cell proliferation, migration and invasion were evaluated by using CCK8 and Transwell migration and invasion assays. Target prediction and luciferase reporter assays were performed to verify the binding between miR-199a-5p and PD-L1. Rescue assay was performed to confirm whether PD-L1 downregulation abolished the inhibitory effect of miR-199a-5p.ResultsAmong 72 pairs of tumor and normal specimens, the proportion of PD-L1 positive samples was higher in FTC tissues than in normal tissues. The results of ESTIMATE and CIBERSORT illustrated that there was a positive correlation between PD-L1 expression and immune infiltration, especially regulatory T cells and M1 macrophages. Prediction of immunotherapy revealed that patients with high PD-L1 expression might benefit from immune checkpoint inhibitors. Transwell migration and invasion assays showed that PD-L1 downregulation in FTC cells could significantly inhibit cell migration and invasion. The bioinformatics analysis and luciferase activity results indicated that PD-L1 was a potential target of miR-199a-5p. Knockdown of PD-L1 reversed the miR-199a-5p inhibitor mediated promotion effect. In addition, we found that PD-L1 expression was positively correlated with Claudin-1 expression and that miR-199a-5p affected the progression of FTC cells through the negative regulation of PD-L1 and Claudin-1.ConclusionsOur study revealed that PD-L1 expression was elevated in FTC and was closely associated with tumor aggressiveness and progression. MiR-199a-5p has a functional role in the progression and metastasis of FTC by regulating PD-L1 and Claudin-1 expression.

Abstract 1349: Hypomethylating agents modulate the PD-1/PD-L1 signaling in a murine breast cancer model and show additive effects in the combination with PD-1 blockage in vitro and in vivo

Abstract With epigenetic therapy emerging as a potential therapy for solid tumors and recent reports that indicate an influence of hypomethylating agents on the PD-1/PD-L1 expression in tumor tissue, epigenetic therapy in combination with immune-modulatory compounds might open-up new avenues for cancer treatment. To investigate the influence of hypomethylating agents on the PD-1/PD-L1 signaling cascade in breast cancer and its implication on possible treatment options, we determined the effect of 5-Azacytidine (5-AZA), decitabine (DAC) and anti-PD1 in mono- and combined therapy on the mouse breast cancer model 4T1 in vitro and in vivo. In a life cell imaging based 2D system we evaluated the influence of the different treatment arms on tumor growth, methylation status and PD-L1 expression. Furthermore, we determined the effect of 5-AZA and DAC on autologous T cells in the presence and absence of 4T1 cancer cells. The combination of 5-AZA and anti-PD1 treatment was further evaluated in an in vivo setting. 24 4T1 bearing balb/c mice were treated with either an isotype control, 5-AZA, anti-PD1 or a combination of the latter. Tumor growth was monitored by caliper measurement. At the end of the experiment tumor and hematopoietic organs were analyzed by flow cytometry. A panel of 36 murine cytokines was determined in the murine serum at different time points. Low dose 5-AZA (IC10) and DAC (IC2.5) induced a down regulation of PD-L1 on 4T1 cells in vitro. Interestingly, this effect was most prominent at the dose level and time point when the hypomethylation effect of the respective compound was most pronounced (day 7). 5-AZA and DAC induced an upregulation of PD-1 on autologous CD4+ as well as CD8+ T cells. Of note, 4T1 cells reduced the expression of PD-1 on autologous CD4+ and CD8+ T cells. Antitumoral activity in vitro was determined in all treatment arms. An additive effect of the combination was specifically determined when combining 5-AZA and anti PD-1. Hence, 5-AZA and anti PD-1 were investigated in vivo more in detail. 4T1 is characterized by an immune suppressive tumor microenvironment as indicated by the in vitro results: we determined a reduced CD4/CD8 ratio and a high percentage of MDSC. These features were reverted by the applied treatments. Along the same line, 5-AZA in mono- and in combination therapy with anti-PDL1 specifically increased the G-CSF levels in murine serum. In addition, the PDL-1 downregulation on the tumor cells was reproduced in vivo. Finally, the combination arm displayed the most prominent antitumoral activity. In summary, we have strong evidence that low dose hypomethylating agents influence the immune landscape of breast cancer via the modulation of PD-1/PD-L1 signaling. The combination with checkpoint inhibitors was superior to the monotherapy and might lead to new treatment options for breast cancer patients. Citation Format: Julia Schüler, Agon Kolica, Hung Ngyuen, Anna Edinger, Kanstantsin Lashuk, Eva Oswald. Hypomethylating agents modulate the PD-1/PD-L1 signaling in a murine breast cancer model and show additive effects in the combination with PD-1 blockage in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1349.

Abstract CT527: A phase I open-label study of FGFR/VEGFR inhibitor FH-2001 in patients with advanced solid tumors

Abstract Purpose: FH-2001 is a FGFR/VEGFR targeted drug that can reduce the expression level of PD-L1 in tumor cells by down-regulating the cMYC gene. The anti-tumor activity of FH-2001 has been confirmed in preclinical studies. Apart from exerting potent anti-proliferative effect on FGFR-driven tumors, FH-2001 may be able to overcome primary and secondary resistance of conventional immune checkpoint inhibitors or increase the response rate through PD-L1 regulation. This first-in-human study evaluated the safety, tolerability, pharmacokinetics and anti-tumor activity of FH-2001 in patients with advanced solid tumors. Methods: This open-label, non-randomized, multicenter phase I study has two parts. The first part comprised a dose escalation and a dose expansion phase to evaluate the safety, tolerability and determine the maximum tolerated dose(MTD)and phase II recommended dose (RP2D) of FH-2001. The dose escalation phase enrolled patients with advanced solid tumors with planned dose levels for 1, 2, 4, 8, 12 or 16 mg. The accelerated titration design was used in the dose escalation phase for 1 and 2 mg dose, and if grade 2 or higher drug-related adverse events (TRAEs) occurred during the dose-limiting toxicity (DLT) period between cycle 0 and 1 with 25 days, dose escalation would subsequently follow the 3+3 design. All other doses utilized the 3+3 design. Patients first received a single dose of FH-2001, and if no DLT or other unacceptable occurred, patients subsequently receive FH-2001 once daily in 21-day cycles. FH-2001 at a dose where objective efficacy was observed or reached effective exposure were selected for dose expansion. In the dose expansion phase, patients with advanced solid tumors harboring FGFR gene aberrations were enrolled, with 6-12 patients at each dose level, and they received FH-2001 once-a-day continuously in 21-day cycles. RP2D was determined based on the safety, pharmacokinetic/pharmacodynamic and preliminary efficacy data that were available in the first part of the study. The second part of the study commenced after RP2D was confirmed, which is a two-cohort study to evaluate the efficacy and safety of FH-2001. The first cohort comprised patients with advanced solid tumors who had progressed on prior treatment with an immune checkpoint inhibitor. The second cohort included patients with locally advanced or metastatic solid tumors with FGFR gene aberrations. Approximately 30 patients were enrolled in each cohort. Safety was assessed throughout the study and for 30 days after the last dose. Tumor response was assessed by the investigators according to RECIST v1.1. Pharmacokinetics and pharmacodynamics were evaluated at prespecified time points. Citation Format: Zhengbo Song, Ai-min Hui, Zhuli Wu, Huilong Liu, Xiangdong Cheng. A phase I open-label study of FGFR/VEGFR inhibitor FH-2001 in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT527.

Abstract 1285: Chemotherapy induces PD-L1 overexpression in CRC through NF-κBp65

Abstract Background: The programmed cell death ligand (PD-L1) is an immune-inhibitory molecule that suppresses the activation of T cells, leading to the survival and growth of tumors. Overexpression of PD-L1 on cancer cells is associated with poor clinical outcomes in many types of cancers. PD-L1 upregulation contributes to chemoresistance in several types of cancer, but little is known about the mechanisms for alterations in PD-L1 expression associated with chemotherapy, especially in patients with CRC. Therefore, a better understanding of the mechanisms underlying changes in PD-L1 levels in response to standard of care chemotherapeutics in CRC is important. In this study, we examined the effects of thechemotherapeutics that are approved for the treatment of patients with CRC (oxaliplatin, 5FU, and irinotecan whose metabolite is SN38) in inducing PD-L1 expression and determining the molecular pathwaysinvolved in PD-L1 regulation. Methods: PD-L1 expression in CRC cell lines was determined in vitro using western blotting and FACS analysis. Changes in PD-L1 transcript levels were measured using real time PCR. RPPA analyses were performed to identify proteins/pathways affected by chemotherapeutics. Pharmacological inhibitors and siRNAs were used to target specific proteins in determining their role in PD-L1 expression. Results: We observed that oxaliplatin, 5FU, and SN38, resulted in induction of PD-L1 to various degrees. Interestingly this induction was observed in KRAS or BRAF mutated CRC cells but not in CRC cells with wild-type KRAS and BRAF. Increased levels of PD-L1 were due to increased transcription of PD-L1 resulting from exposure to chemotherapeutics. Chemotherapeutics lead to the activation of the DNA damage signaling mediator Chk1 and the downstream transcription activator NF-κBp65. Inhibition of Chk1 or NF-κBp65 resulted in abrogated PD-L1 induction in response to chemotherapeutic treatment. Conclusion: PD-L1 can be induced following treatment with chemotherapy in CRC cells harboring mutations in BRAF or KRAS. Our findings provide a rationale for studies combining inhibitors of NF-κBp65 with standard of care chemotherapeutics in CRC to block PD-L1 expression and enhance the efficacy of chemotherapy damaged-tumor cell clearance by innate immunity that, in turn, should improve patient outcomes. Citation Format: Fan Fan, Susmita Ghosh, Lee Ellis, Rajat Bhattacharya. Chemotherapy induces PD-L1 overexpression in CRC through NF-κBp65 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1285.

Self-Sustained Regulation or Self-Perpetuating Dysregulation: ROS-dependent HIF-YAP-Notch Signaling as a Double-Edged Sword on Stem Cell Physiology and Tumorigenesis.

Organ development, homeostasis, and repair often rely on bidirectional, self-organized cell-niche interactions, through which cells select cell fate, such as stem cell self-renewal and differentiation. The niche contains multiplexed chemical and mechanical factors. How cells interpret niche structural information such as the 3D topology of organs and integrate with multiplexed mechano-chemical signals is an open and active research field. Among all the niche factors, reactive oxygen species (ROS) have recently gained growing interest. Once considered harmful, ROS are now recognized as an important niche factor in the regulation of tissue mechanics and topology through, for example, the HIF-YAP-Notch signaling pathways. These pathways are not only involved in the regulation of stem cell physiology but also associated with inflammation, neurological disorder, aging, tumorigenesis, and the regulation of the immune checkpoint molecule PD-L1. Positive feedback circuits have been identified in the interplay of ROS and HIF-YAP-Notch signaling, leading to the possibility that under aberrant conditions, self-organized, ROS-dependent physiological regulations can be switched to self-perpetuating dysregulation, making ROS a double-edged sword at the interface of stem cell physiology and tumorigenesis. In this review, we discuss the recent findings on how ROS and tissue mechanics affect YAP-HIF-Notch-PD-L1 signaling, hoping that the knowledge can be used to design strategies for stem cell-based and ROS-targeting therapy and tissue engineering.

Lipopolysaccharide facilitates immune escape of hepatocellular carcinoma cells via m6A modification of lncRNAMIR155HG to upregulate PD-L1 expression.

Recent studies have suggested that the initiation and progression of hepatocellular carcinoma (HCC) are closely associated with lipopolysaccharide (LPS) of intestinal bacteria. However, the role of LPS in immune regulation of HCC remains largely unknown. An orthotopic Hepa1-6 tumor model of HCC was constructed to analyze the effect of LPS on the expression of immune checkpoint molecules PD-1 and PD-L1. Then we verified the regulation of PD-L1 by LPS in HCC cells. Based on the previous finding that lncRNA MIR155HG regulates PD-L1 expression in HCC cells, we analyzed the relationship of LPS signaling pathway molecules with PD-L1 and MIR155HG by bioinformatics. The molecular mechanism of MIR155HG regulating PD-L1 expression induced by LPS was investigated by RNA pull-down followed by mass spectrometry, RNA immunoprecipitation, fluorescence in situ hybridization, and luciferase reporter assay. Finally, the HepG2 xenograft model was established to determine the role of MIR155HG on PD-L1 expression in vivo. We showed that LPS induced PD-1 and PD-L1 expression in mouse tumor tissues and induced PD-L1 expression in HCC cells. Mechanistically, upregulation of METTL14 by LPS promotes the m6A methylation of MIR155HG, which stabilizes MIR155HG relying on the "reader" protein ELAVL1 (also known as HuR)-dependent pathway. Moreover, MIR155HG functions as a competing endogenous RNA (ceRNA) to modulate the expression of PD-L1 by miR-223/STAT1 axis. Our results suggested that LPS plays a critical role in immune escape of HCC through METTL14/MIR155HG/PD-L1 axis. This study provides a new insight for understanding the complex immune microenvironment of HCC. 1. LPS plays a critical role in immune escape of HCC, especially HCC with cirrhosis. 2. Our study reveals that LPS regulates PD-L1 by m6A modification of lncRNA in HCC. 3. MIR155HG plays an important role in LPS induced PD-L1 expression. 4. LPS-MIR155HG-PD-L1 regulatory axis provides a new target for the treatment of HCC.

Dissection of PD-L1 promoter reveals differential transcriptional regulation of PD-L1 in VHL mutant clear cell renal cell carcinoma

AbstractProgrammed death-ligand 1 (PD-L1) is constitutively expressed by hypoxia-inducible factor 2α (HIF2α). It can be induced by interferon gamma (IFNγ) signaling in clear cell renal cell carcinoma (ccRCC). Clinical trials of metastatic ccRCCs have suggested that a canonical IFNγ signature is a better biomarker for therapeutic response to immune checkpoint inhibitors (ICIs) than PD-L1 expression levels in tumor cells. To understand the therapeutic response to ICIs according to PD-L1 expression levels, we analyzed transcriptional regulation of the PD-L1 promoter by HIF2α and IFNγ-inducible interferon regulatory factor-1 (IRF-1) in ccRCC cells. Here, we present two ccRCC cell models showing differential PD-L1 expression levels in response to IFNγ and hypoxia. Analysis of The Cancer Genome Atlas RNA-sequencing data revealed that PD-L1 expression correlated with JAK2 and STAT1 expression of the canonical IFNγ signature in ccRCC tissues. Upon IFNγ stimulation, PD-L1 was induced by sequential activation of JAK2/STAT1/IRF-1 signaling in both WT- and Mut- VHL ccRCC cells. IFNγ activated the IRF-1α site of the PD-L1 promoter. The IFNγ-mediated increase of PD-L1 expression in Mut-VHL cells was 4.8-fold greater than that in WT-VHL cells. Under normoxia condition, PD-L1 expression in Mut-VHL cells was significantly higher than that in WT-VHL cells due to high basal HIF2α expression. Under hypoxia condition, PD-L1 expression in WT-VHL cells was induced up to 1.8-fold through activation of hypoxia-response elements 2 and 3. In contrast, although PD-L1 in Mut-VHL cells was already highly expressed in the basal state through activation of hypoxia-response elements 2, 3, and 4, it was no longer induced by hypoxia. In conclusion, Mut-VHL ccRCC cells displayed higher PD-L1 expression due to high basal HIF2α expression and a stronger response to IFNγ stimulation than WT-VHL cells. The fact that HIF2α antagonists can potentially reduce PD-L1 expression levels should be considered in ICI combination therapy.Through analysis of the PD-L1 promoter, the authors describe two clear cell renal cell carcinoma (ccRCC) models that exhibit differential PD-L1 expression levels upon IFNγ and hypoxia stimulation. Mutant-VHL ccRCC cells display higher PD-L1 expression than wild-type-VHL ccRCC cells due to high basal HIF2α expression and a stronger response to IFNγ stimulation. Thus, mutant-VHL ccRCC cells are expected to respond well to immune checkpoint inhibitors.

CMTM6 as a master regulator of PD-L1.

Immune checkpoint proteins, such as programmed cell death receptor 1 (PD-1) and its ligand (PD-L1), play critical roles in the pathology of chronic inflammatory pathological conditions, particularly cancer. In addition, the activation of PD-1/PD-L1 pathway is involved in mediating resistance to certain anti-cancer chemo- and immuno-therapeutics. Unfortunately, targeting the PD-1/PD-L1 pathway by the available anti-PD-1/PD-L1 drugs can benefit only a small proportion of cancer patients. Thus, studying the factors that regulate the expression of these immune checkpoint proteins is of central importance in this context. Recent investigations have identified CMTM6 and, to a lesser extent, CMTM4, as master regulators of PD-L1 expression in various cancer cells. Understanding the mechanisms by which such proteins upregulate the expression of PD-L1 in tumor cells, and determining the potential regulators of CMTM6 expression in different types of cancers will accelerate the development of new therapeutic targets and/or lead to the enhancement of the currently available PD-1/PD-L1 blockade therapies.

Programmed death-ligand 1 signaling and expression are reversible by lycopene via PI3K/AKT and Raf/MEK/ERK pathways in tongue squamous cell carcinoma

BackgroundCancer therapy targeting programmed death receptor-1 (PD-1 or CD279) or programmed death-ligand 1 (PD-L1 or CD274) gives hope to Tongue Squamous Cell Carcinoma (TSCC) treatment. However, the tumor-intrinsic mechanism of PD-L1 is not fully elucidated in TSCC. On the other hand, lycopene showed antitumor effects and chemotherapy/radiotherapy-enhancing effects by mechanisms closely correlated with PD-L1.PurposeWe aimed to explore whether the mechanisms of PD-L1 signaling and regulation are reversible by lycopene treatment in TSCC.MethodsWe collected TSCC tissues and normal tissues for assessment of PD-L1 expression by immunohistochemical technique and western blotting. We measured the expression of PD-L1 in three TSCC cell lines and constructed cell lines with knockdown and overexpression of PD-L1. Then, we measured the proliferation by CCK-8 assay, migration and invasion by Transwell assay, and apoptosis by TUNEL assay in five groups with treatment of blank control, negative control with vector transfection, PD-L1 knockdown/overexpression, 4 μM lycopene, and combined 4 μM lycopene and PD-L1 knockdown/overexpression. We also systematically analyzed the PD-L1 constitutive signaling pathways and their effect EMT pathways. In order to bring out the mechanism underlying PI3K/AKT depressing Raf/MEK/ERK, we used PI3K inhibitor LY294002.ResultsWe detected significant PD-L1 upregulation in biopsies by western blot and immunohistochemistry. Our study demonstrated that PD-L1 upregulation elevated IGF-1R to activate the PI3K/AKT pathway but inactivated the Raf/MEK/ERK pathway in TSCC cell line CAL27, while PD-L1 knockdown decreased IGF-1R to inactivate both PI3K/AKT and Raf/MEK/ERK pathways in cell line SCC9, to increase/decrease p-FOXOs and decrease/increase p-GSK-3β, producing further changes in EMT, proliferation, migration, invasion, and apoptosis. Lycopene reversed PD-L1 signaling and expression by mechanisms opposite to PD-L1 upregulation but similar to PD-L1 knockdown.ConclusionTaken together, this study firstly confirmed PD-L1 expression and signaling are reversible by lycopene via PI3K/AKT and Raf/MEK/ERK pathways in TSCC. Our study provides a sounder basis for comprehending PD-L1 signaling and expression and prevention and treatment of TSCC.

Genetic analysis of cancer drivers reveals cohesin and CTCF as suppressors of PD-L1

Immune evasion is a significant contributor to tumor evolution, and the immunoinhibitory axis PD-1/PD-L1 is a frequent mechanism employed to escape tumor immune surveillance. To identify cancer drivers involved in immune evasion, we performed a CRISPR-Cas9 screen of tumor suppressor genes regulating the basal and interferon (IFN)-inducible cell surface levels of PD-L1. Multiple regulators of PD-L1 were identified, including IRF2, ARID2, KMT2D, and AAMP. We also identified CTCF and the cohesin complex proteins, known regulators of chromatin architecture and transcription, among the most potent negative regulators of PD-L1 cell surface expression. Additionally, loss of the cohesin subunit RAD21 was shown to up-regulate PD-L2 and MHC-I surface expression. PD-L1 and MHC-I suppression by cohesin were shown to be conserved in mammary epithelial and myeloid cells. Comprehensive examination of the transcriptional effect of STAG2 deficiency in epithelial and myeloid cells revealed an activation of strong IFN and NF-κB expression signatures. Inhibition of JAK-STAT or NF-κB pathways did not result in rescue of PD-L1 up-regulation in RAD21-deficient cells, suggesting more complex or combinatorial mechanisms at play. Discovery of the PD-L1 and IFN up-regulation in cohesin-mutant cells expands our understanding of the biology of cohesin-deficient cells as well as molecular regulation of the PD-L1 molecule.

Vitamin D can reduce severity in COVID-19 through regulation of PD-L1.

COVID-19 is a highly contagious viral infection that has killed millions of people around the world. The most important diagnostic feature of COVID-19 is lymphocyte depletion, particularly the depletion of T cells. In COVID-19 infections, there is a link between destruction of T cells and increased expression of inhibitory immune checkpoint molecules (PD-1/PD-L1) on T cell surfaces. It was shown that PD-1/PD-L1 levels increase in severely COVID-19 infected individuals. Higher proinflammatory cytokine levels cause increased PD-1/PD-L1 expression. In severe COVID-19, higher proinflammatory cytokine levels may increase PD-1/PD-L1. Vitamin-D is an important immune regulator. It is known that the numbers of CD4+ and CD8+ T lymphocytes decrease in vitamin D deficiency while vitamin D supplementation increases CD + 4 lymphocytes. Vitamin D can increase regulatory T cell (Treg) activity. Vitamin D also has a diminishing effect on proinflammatory cytokines. In severe COVID-19 cases, vitamin D supplementation may inhibit the increase of PD-L1 expression through reducing proinflammatory cytokine levels. Thus, vitamin D supplementation could eliminate the suppressive effect of PD-L1 on CD4+ and CD8+ T cells, preventing lymphopenia and reducing disease severity and mortality in patients infected with COVID-19. Besides, vitamin D supplementation can reduce inflammation by increasing Treg activity. The aim of this letter is to discuss the functions of inhibitory immune checkpoint molecules and their effects on dysfunction and depletion of T-cells as well as to explain the possible modulatory effect of vitamin D on these checkpoints and T cells.

COX-2 Inhibitors Decrease Expression of PD-L1 in Colon Tumors and Increase the Influx of Type I Tumor-infiltrating Lymphocytes.

Nonsteroidal anti-inflammatories (NSAID) are an essential component of any combination chemoprevention of colon cancer. We show NSAID treatment reduces PD-L1 expression on intestinal tumor cells. NSAID regulation of PD-L1 is dependent on COX-2 expression. These data underscore an important immunologic mechanism of action for NSAID in colon cancer prevention. See related Spotlight, p. 209.

4-Octyl itaconate regulates immune balance by activating Nrf2 and negatively regulating PD-L1 in a mouse model of sepsis.

Introduction: Sepsis is a major global health challenge with high mortality rates and no effective treatment. Recent studies have suggested that sepsis may be associated with immune system dysfunction. Itaconate may exert anti-inflammatory effects via Nrf2 signaling. Although Nrf2 regulates oxidative/exogenous stress responses and inhibits inflammatory responses, the mechanism via which Nrf2 regulates immune checkpoints in sepsis remains unclear. Objectives: This study aimed to investigate the role of the Nrf2 signaling pathway in sepsis immunosuppression injury by exploring Nrf2 target genes in inflammatory macrophages in a mouse model of sepsis. Methods: We evaluated the effects of 4-octyl itaconate (OI) on pro-inflammatory and anti-inflammatory cytokines in a mouse model of sepsis and RAW264.7 cells. In addition, we investigated if OI could inhibit LPS-induced oxidative stress by activating Nrf2 signaling in vitro and in vivo. Results: OI reduced the release of pro-inflammatory cytokines and increased the release of anti-inflammatory cytokines, thereby inhibiting inflammation. OI increased glutathione synthase (GSS) expression by activating the Nrf2 signaling pathway to promote GSH synthesis, thus, inhibiting oxidative stress. OI inhibited the early release of inflammatory and oxidative stress-related factors to reduce tissue and organ injury in mice with sepsis, while Nrf2 interfered with PD-L1 induction and inhibited PD-L1 expression at an advanced stage to reduce the occurrence of sepsis immunosuppression. Conclusions: This study indicates that Nrf2 is a novel negative regulator of PD-L1 that functions at immune checkpoints and suggests an underlying mechanism for the anti-inflammatory process mediated by Nrf2.