PD-L1 Signaling Research Articles

Preclinical Studies of a Novel Human PD-1 B-Cell Peptide Cancer Vaccine PD1-Vaxx From BALB/c Mice to Beagle Dogs and to Non-Human Primates (Cynomolgus Monkeys)

Immunotherapy with monoclonal antibodies to checkpoint inhibitors against the PD-1/PD-L1 signaling pathway is a landmark achievement in cancer therapy. Some anti-PD-1 inhibitors such as nivolumab and pembrolizumab have shown clinical success, in a percentage of patients with prolonged survival rates. However, adverse effects accompany these benefits. In this case, strategies with lower toxicity and increased specificity are urgently required. Cancer vaccines have the ability to stimulate the native immune system and in particular, an engineered B-cell epitope can elicit high-affinity polyclonal antibodies with similar efficacy to PD-1 monoclonal antibodies in murine animal models. We have previously designed and synthesized a unique B-cell vaccine, PD1-Vaxx [MVF-PD-1(92-110)], and we have tested the immunogenicity and antitumor properties in CT26 colon cancer BALB/c syngeneic mice model. This manuscript provides results from comprehensive preclinical pharmacology studies encompassing primary and secondary pharmacodynamics, biodistribution, and safety studies. The results from these preclinical studies support the use of PD1-Vaxx in a first-in-human clinical trial in patients with non-small cell lung cancer (NSCLC). A phase I trial in patients with NSCLC has commenced.

The PD-1 with PD-L1 Axis Is Pertinent with the Immune Modulation of Atrial Fibrillation by Regulating T Cell Excitation and Promoting the Secretion of Inflammatory Factors.

Objective To analyze the role of PD-1/PD-L1 signaling pathway in regulating T cell activation and secretion of proinflammatory factors in atrial fibrillation. Methods Forty-five patients with atrial fibrillation admitted to the cardiology department of our hospital from July 2019 to March 2021 were selected to be included in the atrial fibrillation group, and another 45 healthy volunteers were selected as the control group to compare the changes of T cell CD69 and human leukocyte antigen-DR (HLA-DR) expression in the peripheral blood of the two study groups; compare the changes of programmed death factor-1 on CD4+ and CD8+ lymphocytes in the peripheral blood of the two groups (PD-1) expression changes and PD-L1 and PD-L2 expression changes on peripheral blood myeloid dendritic cells (mDCs) cells; compare the changes of interleukin-2, interleukin-6, interleukin-10, and interleukin-17A (IL-2, IL-6, IL-10, and IL-17), tumor necrosis factor (TNF), and interferon gamma (IFN-γ) concentrations on peripheral blood inflammatory factors in the two groups; and isolate the two groups of peripheral blood mDCs cells; α interferon upregulated PD-L1 expression in the cells and analyzed the effect of PD-L1 expression on the ability of mDCs to stimulate T cells to secrete cytokines.Results The positive expression rates of CD69 and HLA-DR on peripheral blood CD3+ T lymphocytes were significantly higher in patients in the atrial fibrillation group than in the control group, and the differences were statistically significant (P < 0.01). The positive expression rate of PD-1 on CD4+ lymphocytes was significantly lower in patients in the atrial fibrillation group than in the control group (P < 0.01). There was no statistically significant difference between the two groups in terms of PD-1 positive expression rate on CD8+ lymphocytes (P > 0.05). The positive expression rate of PD-L1 on mDCs cells was significantly lower in patients in the atrial fibrillation group than in the control group (P < 0.01), and there was no statistically significant difference between the two groups in the positive expression rate of PD-L2 on mDCs cells, PD-L1, and PD-L2 on CD4+ and CD8+ T cells (P > 0.05). The concentrations of IL-2, IL-6, IL-10, and IFN-γ in peripheral blood were significantly higher in patients in the atrial fibrillation group than in the control group (P < 0.05), and there was no statistically significant difference in the comparison of IL-17A and TNF concentrations in peripheral blood between the two groups (P > 0.05). In the atrial fibrillation group, the ability of mDCs to stimulate T cells to secrete IL-2 and IFN-γ was significantly higher, and the ability to secrete IL-10 was significantly lower compared with the control group (P < 0.05). After α interferon upregulated PD-L1 expression in cells, the ability of mDCs to stimulate T cells to secrete IL-2, IL-10, and IFN-γ cytokines was reversed in patients in the atrial fibrillation group, and the differences compared with the control group were not statistically significant (P > 0.05). Conclusion PD-1/PD-L1 signaling pathway may play an immunomodulatory role in the pathogenesis of atrial fibrillation by promoting increased secretion of inflammatory factors through regulating T cell activation.

Oncolytic Viruses as an Adjunct to Immune Checkpoint Inhibition.

Utilizing viruses in the treatment of cancer, or oncolytic viral therapy (OVT), began in the 1950s with the idea that viruses could invade and destroy cancer cells. Barriers to this approach included a lack of specificity towards cancer cells and intolerable toxicities. However, it was discovered that OVT increases cytokines such as interferon gamma and interleukins within the tumor microenvironment. This "priming" of the tumor microenvironment can lead to an improved innate immunologic response to tumor cells. An "OVT-as-monotherapy" approach has led to modest tumor response rates that have unfortunately not translated well in clinical trials. Currently, only one OVT agent-talimogene laherparevec (TVEC)-has been approved by the FDA for unresectable melanoma with limited visceral metastases. Further advancements in immunotherapy combined with improved viral engineering over the last decade have paved the way for a renewed focus on OVT. For example, various viruses have been modified to infiltrate and upregulate PD-L1 signaling within tumor cells. Upregulation of PD-L1 on tumor cells can increase tumor cell response to immunotherapies that utilize the interaction between PD-L1 on tumor cells and PD-1 on lymphocytes to allow for immune cell destruction of cancer cells. Combining OVT and immunotherapy offers more promise than OVT as monotherapy. Currently, several are actively investigating the combinatorial approach of OVT and immunotherapy in treating non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer, melanoma, pancreatic cancer, multiple myeloma, and head and neck squamous cell carcinoma. In this review, we will discuss the history of OVT including its limitations as a monotherapy. We will also discuss the background of combining OVT and immunotherapy including possible benefits and pitfalls of this approach. Lastly, we will review current clinical trials investigating OVT and immunotherapy in multiple cancers.

Pharmacologic Tumor PDL1 Depletion with Cefepime or Ceftazidime Promotes DNA Damage and Sensitivity to DNA-Damaging Agents.

The interaction between tumor surface-expressed PDL1 and immune cell PD1 for the evasion of antitumor immunity is well established and is targeted by FDA-approved anti-PDL1 and anti-PD1 antibodies. Nonetheless, recent studies highlight the immunopathogenicity of tumor-intrinsic PDL1 signals that can contribute to the resistance to targeted small molecules, cytotoxic chemotherapy, and αPD1 immunotherapy. As genetic PDL1 depletion is not currently clinically tractable, we screened FDA-approved drugs to identify those that significantly deplete tumor PDL1. Among the candidates, we identified the β-lactam cephalosporin antibiotic cefepime as a tumor PDL1-depleting drug (PDD) that increases tumor DNA damage and sensitivity to DNA-damaging agents in vitro in distinct aggressive mouse and human cancer lines, including glioblastoma multiforme, ovarian cancer, bladder cancer, and melanoma. Cefepime reduced tumor PDL1 post-translationally through ubiquitination, improved DNA-damaging-agent treatment efficacy in vivo in immune-deficient and -proficient mice, activated immunogenic tumor STING signals, and phenocopied specific genetic PDL1 depletion effects. The β-lactam ring and its antibiotic properties did not appear contributory to PDL1 depletion or to these treatment effects, and the related cephalosporin ceftazidime produced similar effects. Our findings highlight the rapidly translated potential for PDDs to inhibit tumor-intrinsic PDL1 signals and improve DNA-damaging agents and immunotherapy efficacy.

PD-1 licenses activated Treg for lymphatic migration.

Abstract Rather than serving as fluid transport conduits, the lymphatic vessels have been proved to be critical portals for immune cell migration. As non-immune cell, the lymphatic endothelial cell (LEC) constitutively express high level of programmed death-1 (PD-1) ligand (PD-L1), known as an immune checkpoint protein. We hypothesized that LECs use PD-L1 to regulate PD-1-expressing immune cells during transendothelial migration (TEM). Activated T cells expressed substantially elevated levels of both PD-1 and PD-L1. For human and mouse Tregs PD-1 was in excess of PD-L1, while effector CD4 T cells (Teffs) expressed PD-L1 in excess of PD-1. PD-1−/− but not PD-L1−/− Tregs had reduced migration and motility compared to WT. Engagement by PD-L1 Fc induced rapid activation of Akt but suppressed classical NFkB-P65 signaling in Tregs. WT LECs constitutively expressed high level of surface PD-L1. PD-L1−/− LEC or lymphatic vessels (LV) had increased adhesion and junction proteins VCAM-1 and zonulin-1, and had more zipper junctions. PD-1 triggered PD-L1 signaling in LEC result in strong PI3K/Akt and NFkB-p65 responses that regulated endothelial structure and enhanced TEM. Similar changes to LEC junctions were observed when PD-1high Tregs migrated across LEC layers, resulting in rapidly induced NFkB-p65 translocation into the LEC nucleus. PD-1-induced PD-L1 signaling decreased VE-cadherin expression, which was restored by blocking ERK and PI3K/Akt. Importantly, PD-1-blockade of intra-tumoral Tregs reduced tumor egress and the detained Tregs converted to IFN-g-producing Th1Tregs. These data demonstrate novel roles for Treg PD-1 and LEC PD-L1 in regulation of lymphatic migration and ultimately Treg suppressive function. Supported by grants from NIH (2R37AI062765-17A1)

28P ACLY-mediated breast tumorigenesis is driven by immune escape via PD-L1 signaling

Deregulated lipogenesis is essential for tumor cell survival. Regardless of circulating lipid levels, a wide range of tumors presents accelerated de novo fatty acid synthesis. Oncogenic lipogenesis is manifested by enhanced activity and coordinated expression of multiple lipogenic enzymes including ATP-citrate lyase (ACLY) in tumor cells. Therefore, the present study was aimed at understanding the influence of ACLY on breast tumorigenesis and its role in inducing immune escape.

Brain resident memory CD8+ T cells trigger CNS immune activation and infiltration

Abstract The shifting paradigm regarding immune surveillance of the central nervous system (CNS) has shed light on immune cell networks thought to be absent from healthy brain tissue. Resident memory T cells (TRM) are a unique subset of memory T cells that persist within non-lymphoid tissues to provide rapid onset protection against reinfection. Recently, brain TRM (bTRM) have been identified in humans, with CNS or peripheral infection, or vaccination leading to bTRM establishment in mice. While bTRM are important for protection of the CNS against reinfection, their regulation and involvement in shaping the neuroimmune landscape remains unknown. Here, we take a reductionist approach employing viral-derived peptides to show that CD8+ bTRM reactivation is sufficient to initiate a cascade of innate and adaptive immune activation in the brain. Specifically, bTRM reactivation triggered activation of NK cells, T cells, microglia, and induced dendritic cell (DC) maturation, including upregulation of lymph node homing molecules. Reactivated bTRM also promoted accumulation of DCs in draining lymph nodes, and macrophages, monocyte-derived DCs, T cells and NK cells in the brain. Our preliminary data suggests that PD-1:PD-L1 signaling regulates the magnitude of this response, as PD-L1 blockade led to enhanced bTRM activation and neuroinflammation. We anticipate our results to illuminate roles for PD-1 signaling on bTRM in the context of pathogen clearance, and neurologic toxicities seen in cancer patients following PD-1 inhibitor treatment. This study will also provide insight into the pathologic or protective capacity of bTRM in neurologic diseases where T cells are implicated, such as Alzheimer’s disease, multiple sclerosis, and brain cancer. Supported by grants from NIH (K22 AI148508-02, T32 AI007363)

PD-L1 signaling selectively regulates T cell lymphatic transendothelial migration

Programmed death-1 (PD-1) and its ligand PD-L1 are checkpoint molecules which regulate immune responses. Little is known about their functions in T cell migration and there are contradictory data about their roles in regulatory T cell (Treg) function. Here we show activated Tregs and CD4 effector T cells (Teffs) use PD-1/PD-L1 and CD80/PD-L1, respectively, to regulate transendothelial migration across lymphatic endothelial cells (LECs). Antibody blockade of Treg PD-1, Teff CD80 (the alternative ligand for PD-L1), or LEC PD-L1 impairs Treg or Teff migration in vitro and in vivo. PD-1/PD-L1 signals through PI3K/Akt and ERK to regulate zipper junctional VE-cadherin, and through NFκB-p65 to up-regulate VCAM-1 expression on LECs. CD80/PD-L1 signaling up-regulates VCAM-1 through ERK and NFκB-p65. PD-1 and CD80 blockade reduces tumor egress of PD-1high fragile Tregs and Teffs into draining lymph nodes, respectively, and promotes tumor regression. These data provide roles for PD-L1 in cell migration and immune regulation.

Combination gemcitabine and PD-L1xCD3 bispecific T cell engager (BiTE) enhances T lymphocyte cytotoxicity against cholangiocarcinoma cells

Cholangiocarcinoma (CCA) is a lethal cancer with rapid progression and poor survival. Novel and more effective therapies than those currently available are, therefore, urgently needed. Our research group previously reported the combination of gemcitabine and cytotoxic T lymphocytes to be more effective than single-agent treatment for the elimination of CCA cells. However, gemcitabine treatment of CCA cells upregulates the expression of an immune checkpoint protein (programmed death-ligand 1 [PD-L1]) that consequently inhibits the cytotoxicity of T lymphocytes. To overcome this challenge and take advantage of PD-L1 upregulation upon gemcitabine treatment, we generated recombinant PD-L1xCD3 bispecific T cell engagers (BiTEs) to simultaneously block PD-1/PD-L1 signaling and recruit T lymphocytes to eliminate CCA cells. Two recombinant PD-L1xCD3 BiTEs (mBiTE and sBiTE contain anti-PD-L1 scFv region from atezolizumab and from a published sequence, respectively) were able to specifically bind to both CD3 on T lymphocytes, and to PD-L1 overexpressed after gemcitabine treatment on CCA (KKU213A, KKU055, and KKU100) cells. mBiTE and sBiTE significantly enhanced T lymphocyte cytotoxicity against CCA cells, especially after gemcitabine treatment, and their magnitudes of cytotoxicity were positively associated with the levels of PD-L1 expression. Our findings suggest combination gemcitabine and PD-L1xCD3 BiTE as a potential alternative therapy for CCA.

Adaptive antitumor immune response stimulated by bio-nanoparticle based vaccine and checkpoint blockade

BackgroundInteractions between tumor and microenvironment determine individual response to immunotherapy. Triple negative breast cancer (TNBC) and hepatocellular carcinoma (HCC) have exhibited suboptimal responses to immune checkpoint inhibitors (ICIs). Aspartate β-hydroxylase (ASPH), an oncofetal protein and tumor associated antigen (TAA), is a potential target for immunotherapy.MethodsSubcutaneous HCC and orthotopic TNBC murine models were established in immunocompetent BALB/c mice with injection of BNL-T3 and 4 T1 cells, respectively. Immunohistochemistry, immunofluorescence, H&E, flow cytometry, ELISA and in vitro cytotoxicity assays were performed.ResultsThe ASPH-MYC signaling cascade upregulates PD-L1 expression on breast and liver tumor cells. A bio-nanoparticle based λ phage vaccine targeting ASPH was administrated to mice harboring syngeneic HCC or TNBC tumors, either alone or in combination with PD-1 blockade. In control, autocrine chemokine ligand 13 (CXCL13)-C-X-C chemokine receptor type 5 (CXCR5) axis promoted tumor development and progression in HCC and TNBC. Interactions between PD-L1+ cancer cells and PD-1+ T cells resulted in T cell exhaustion and apoptosis, causing immune evasion of cancer cells. In contrast, combination therapy (Vaccine+PD-1 inhibitor) significantly suppressed primary hepatic or mammary tumor growth (with distant pulmonary metastases in TNBC). Adaptive immune responses were attributed to expansion of activated CD4+ T helper type 1 (Th1)/CD8+ cytotoxic T cells (CTLs) that displayed enhanced effector functions, and maturation of plasma cells that secreted high titers of ASPH-specific antibody. Combination therapy significantly reduced tumor infiltration of immunosuppressive CD4+/CD25+/FOXP3+ Tregs. When the PD-1/PD-L1 signal was inhibited, CXCL13 produced by ASPH+ cancer cells recruited CXCR5+/CD8+ T lymphocytes to tertiary lymphoid structures (TLSs), comprising effector and memory CTLs, T follicular helper cells, B cell germinal center, and follicular dendritic cells. TLSs facilitate activation and maturation of DCs and actively recruit immune subsets to tumor microenvironment. These CTLs secreted CXCL13 to recruit more CXCR5+ immune cells and to lyse CXCR5+ cancer cells. Upon combination treatment, formation of TLSs predicts sensitivity to ICI blockade. Combination therapy substantially prolonged overall survival of mice with HCC or TNBC.ConclusionsSynergistic antitumor efficacy attributable to a λ phage vaccine specifically targeting ASPH, an ideal TAA, combined with ICIs, inhibits tumor growth and progression of TNBC and HCC.

421 Specific cephalosporin antibiotics deplete tumor PD-L1 to inhibit DNA damage sensing and sensitize to Chk1 inhibitors in vivo

OBJECTIVES/GOALS: Tumor PDL1 signals to immune cells for tumor immune evasion but has cell-intrinsic signals that promote tumor virulence. We identify novel tumor PDL1 depleting drugs (PDDs) to interrupt tumor-intrinsic PDL1 signals and sensitize tumors to targeted therapy in vitro and in vivo. METHODS/STUDY POPULATION: We screened the Prestwick and LOPAC libraries for FDA-approved drugs reducing B16 melanoma PDL1 &gt; 2.6-fold. ?-lactam antibiotics were used at 80 ?M and Chk1 inhibitor rabusertib as indicated in T24 human bladder cancer, and murine ID8agg ovarian cancer, 4T1 breast cancer and B16. Genetic PDL1 KO was by CRISPR or shRNA and re-expression by lentivirus. Viability was by MTT and protein by immunoblot. We challenged 5 NSG mice/group with 2x106 T24 (SQ) cells and 5 BALB/c mice/group with 5x105 4T1 cells (mammary fat pad) and treated with cefepime (200 mg/kg), rabusertib (2.5 mg/kg), vehicle, or combo daily from day 3. RESULTS/ANTICIPATED RESULTS: Structurally-related ?-lactam antibiotics cefepime and ceftazidime are tumor PDDs. Cefepime or ceftazidime reduced tumor PD-L1 and thus its cell-intrinsic signals to deplete the DNA damage sensing Chk2 protein and promote rabusertib synthetic lethality in vitro and in vivo in a tumor PDL1-dependent manner independent of immunity. Structurally distinct ?-lactam antibiotics did not sensitize tumor cells to rabusertib, suggesting ?-lactam antimicrobial functions did not promote PDL1 depletion or rabusertib treatment effects in vivo. Although rabusertib effects were immune-independent, both PDDs induced immunogenic tumor STING signaling, suggesting they can improve tumor immunotherapy. DISCUSSION/SIGNIFICANCE: We show a rapidly translatable way to deplete detrimental tumor-intrinsic PDL1 signals, and sensitize tumors to rabusertib. We are testing PDD structure activity relationships to improve PDD effects and testing PDD effects on other treatments, e.g., PARP inhibitors, immunotherapy.

A blood-based miRNA signature with prognostic value for overall survival in advanced stage non-small cell lung cancer treated with immunotherapy

Immunotherapies have recently gained traction as highly effective therapies in a subset of late-stage cancers. Unfortunately, only a minority of patients experience the remarkable benefits of immunotherapies, whilst others fail to respond or even come to harm through immune-related adverse events. For immunotherapies within the PD-1/PD-L1 inhibitor class, patient stratification is currently performed using tumor (tissue-based) PD-L1 expression. However, PD-L1 is an accurate predictor of response in only ~30% of cases. There is pressing need for more accurate biomarkers for immunotherapy response prediction. We sought to identify peripheral blood biomarkers, predictive of response to immunotherapies against lung cancer, based on whole blood microRNA profiling. Using three well-characterized cohorts consisting of a total of 334 stage IV NSCLC patients, we have defined a 5 microRNA risk score (miRisk) that is predictive of overall survival following immunotherapy in training and independent validation (HR 2.40, 95% CI 1.37–4.19; P < 0.01) cohorts. We have traced the signature to a myeloid origin and performed miRNA target prediction to make a direct mechanistic link to the PD-L1 signaling pathway and PD-L1 itself. The miRisk score offers a potential blood-based companion diagnostic for immunotherapy that outperforms tissue-based PD-L1 staining.

Aspirin Suppressed PD-L1 Expression through Suppressing KAT5 and Subsequently Inhibited PD-1 and PD-L1 Signaling to Attenuate OC Development.

Ovarian cancer (OC) is a frequently occurred malignancy with high incidence and poor survival worldwide. In recent years, immune checkpoint inhibition that targets PD-1/PD-L1 axis has become an efficient and popular therapy for cancers. Aspirin (ASP), an anti-inflammatory drug, exhibits a wide spectrum of biological functions including anticancer property. However, the role of ASP treatment in ovarian cancer treatment remains unclear. In this work, we explored the role of ASP in modulating PD-L1 signaling during OC development. Notably, in vitro experiments showed that ASP treatment caused repressed proliferation of OC cells. The results from in vivo xenograft model suggested suppressed tumor growth and tumor weight under ASP treatment. ASP treatment also caused downregulated PD-L1 and Ki-67 levels in mice tumors. Moreover, the IFN-γ-caused PD-L1 accumulation was inhibited by ASP treatment. The administration of ASP decreased the expression of PD-L1 of OC cells in a coculture system with activated T cell or unstimulated PBMCs, along with decreased expression of PD-1 by activated T cells. ASP reversed PD-L1 expression caused by coculture with activated T cells and abolished the suppressed T cells activation and proliferation. Analysis on molecular mechanisms revealed that KAT5 bonded to the promoter region of PD-L1 and upregulated its expression via enhancing histone H3 lysine 27 acetylation (H3K27ac), whereas ASP downregulated KAT5 expression and blocked this phenomenon. Moreover, ASP enhanced the effect of antiPD-L1 therapy in the in vivo tumor model. Hence, we proposed that ASP decreased expression of PD-L1 protein via inhibiting the epigenetic regulation by KAT5 and suppressed the PD-1/PD-L1 signaling to attenuate tumor growth. ASP may be a promising adjuvant in OC immunotherapy.

Biological Characteristics and Clinical Significance of Soluble PD-1/PD-L1 and Exosomal PD-L1 in Cancer.

The immune checkpoint pathway consisting of the cell membrane-bound molecule programmed death protein 1 (PD-1) and its ligand PD-L1 has been found to mediate negative regulatory signals that effectively inhibit T-cell proliferation and function and impair antitumor immune responses. Considerable evidence suggests that the PD-1/PD-L1 pathway is responsible for tumor immune tolerance and immune escape. Blockage of this pathway has been found to reverse T lymphocyte depletion and restore antitumor immunity. Antagonists targeting this pathway have shown significant clinical activity in specific cancer types. Although originally identified as membrane-type molecules, several other forms of PD-1/PD-L1 have been detected in the blood of cancer patients, including soluble PD-1/PD-L1 (sPD-1/sPD-L1) and exosomal PD-L1 (exoPD-L1), increasing the composition and functional complications of the PD-1/PD-L1 signaling pathway. For example, sPD-1 has been shown to block the PD-1/PD-L immunosuppressive pathway by binding to PD-L1 and PD-L2, whereas the role of sPD-L1 and its mechanism of action in cancer remain unclear. In addition, many studies have investigated the roles of exoPD-L1 in immunosuppression, as a biomarker for tumor progression and as a predictive biomarker for response to immunotherapy. This review describes the molecular mechanisms underlying the generation of sPD-1/sPD-L1 and exoPD-L1, along with their biological activities and methods of detection. In addition, this review discusses the clinical importance of sPD-1/sPD-L1 and exoPD-L1 in cancer, including their predictive and prognostic roles and the effects of treatments that target these molecules.

PD-1/PD-L1 inhibitor ameliorates silica-induced pulmonary fibrosis by maintaining systemic immune homeostasis

Pulmonary fibrosis induced by silica particles is defined as silicosis, which is an incurable disease. The pathogenesis of silicosis is not completely clear, but it’s certain that immune system dysfunction is closely related to it. Immune checkpoint inhibitors (ICIs) are emerging immunotherapeutic agents that mainly target adaptive immune cells, and there is abundant evidence that ICIs are of great value in cancer treatment. However, whether these attractive agents can be implemented in silicosis treatment is unclear. In this study, we explored the efficacy of small molecule inhibitors targeted PD-1/PD-L1 and CTLA-4 on silica-induced pulmonary fibrosis in mice. ICIs were injected intraperitoneally into mice that received silica instillation twice a week. The mice were sacrificed 7 and 28 days after the injection. The lungs, spleen, hilar lymph nodes, thymus, and peripheral blood of mice were collected and subjected to histological examination, flow cytometry analysis, and mRNA and protein quantification. Our results demonstrated that silica exposure caused damage to multiple immune organs in mice, leading to an imbalance in systemic immune homeostasis. Specifically, proportions and subtypes of T and B cells were significantly altered, and the expressions of PD-1, PD-L1 and CTLA-4 were abnormal on these cells. Both PD-1/PD-L1 and CTLA-4 inhibitor administration modulated silica-induced immune system disruption, however, only PD-1/PD-L1 signaling inhibition showed significant amelioration of silicosis. Our findings confirmed for the first time the potential value of ICIs for the treatment of silica-induced pulmonary fibrosis, and this may provide new ideas for the treatment of other fibrosis-related diseases.

Combination cancer immunotherapy targeting TNFR2 and PD-1/PD-L1 signaling reduces immunosuppressive effects in the microenvironment of pancreatic tumors

BackgroundsIn advanced pancreatic ductal adenocarcinoma (PDAC), immune therapy, including immune checkpoint inhibitors, has limited efficacy, encouraging the study of combination therapy.MethodsTumor necrosis factor receptor 2 (TNFR2) was analyzed via immunohistochemistry,...

Genomic sequencing for bladder urothelial carcinoma and its clinical implications for immunotherapy.

551 Background: To explore the genomic and transcriptomic landscape of bladder cancer (BC) and its implication for treatment with an immune checkpoint inhibitor (ICI). Methods: We analyzed whole-exome and -transcriptome sequences of tumor samples from 64 BC patients who underwent surgical resection with either transurethral resection or radical cystectomy. For exploratory purposes, PD-L1 expression was evaluated in a subset of patients (n = 57) including those treated with ICI (n = 8). Results: We identified frequent molecular dysregulations in chromatin regulatory genes ( KDM6A, ARID1A, MLL2, and STAG2) and recurrent copy number alterations. Thirty-five samples (54.7%) were PD-L1-positive (PD-L1 combined positive score ≥ 1) with a significantly higher exonic tumor mutational burden (TMB) compared to PD-L1-negative BC samples ( P= 0.010). We observed that various immune-responsive pathways, including the PD-L1 signaling pathway, were enriched significantly in PD-L1-positive BCs. Interestingly, genes in the CTLA4 pathway were enriched significantly in PD-L1-positive BC as well. Among 8 patients who received ICI, progressive disease was confirmed in one patient, whose tumor had low exonic TMB, negative PD-L1 status, and a relatively colder microenvironment. Conclusions: Gaining new insights into the molecular landscape of BC will improve treatment strategies. Our analysis suggests a rationale for studying dual checkpoint inhibition against BC.

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.

PD-L1 signaling in reactive astrocytes counteracts neuroinflammation and ameliorates neuronal damage after traumatic brain injury

BackgroundTissue damage and cellular destruction are the major events in traumatic brain injury (TBI), which trigger sterile neuroimmune and neuroinflammatory responses in the brain. While appropriate acute and transient neuroimmune and neuroinflammatory responses facilitate the repair and adaptation of injured brain tissues, prolonged and excessive neuroimmune and neuroinflammatory responses exacerbate brain damage. The mechanisms that control the intensity and duration of neuroimmune and neuroinflammatory responses in TBI largely remain elusive.MethodsWe used the controlled cortical impact (CCI) model of TBI to study the role of immune checkpoints (ICPs), key regulators of immune homeostasis, in the regulation of neuroimmune and neuroinflammatory responses in the brain in vivo.ResultsWe found that de novo expression of PD-L1, a potent inhibitory ICP, was robustly and transiently induced in reactive astrocytes, but not in microglia, neurons, or oligodendrocyte progenitor cells (OPCs). These PD-L1+ reactive astrocytes were highly enriched to form a dense zone around the TBI lesion. Blockade of PD-L1 signaling enlarged brain tissue cavity size, increased infiltration of inflammatory Ly-6CHigh monocytes/macrophages (M/Mɸ) but not tissue-repairing Ly-6CLowF4/80+ M/Mɸ, and worsened TBI outcomes in mice. PD-L1 gene knockout enhanced production of CCL2 that is best known for its ability to interact with its cognate receptor CCR2 on Ly-6CHigh M/Mϕ to chemotactically recruit these cells into inflammatory sites. Mechanically, PD-L1 signaling in astrocytes likely exhibits dual inhibitory activities for the prevention of excessive neuroimmune and neuroinflammatory responses to TBI through (1) the PD-1/PD-L1 axis to suppress the activity of brain-infiltrating PD-1+ immune cells, such as PD-1+ T cells, and (2) PD-L1 intrinsic signaling to regulate the timing and intensity of astrocyte reactions to TBI.ConclusionsPD-L1+ astrocytes act as a gatekeeper to the brain to control TBI-related neuroimmune and neuroinflammatory responses, thereby opening a novel avenue to study the role of ICP–neuroimmune axes in the pathophysiology of TBI and other neurological disorders.

Influence of Helicobacter pylori infection on PD-1/PD-L1 blockade therapy needs more attention.

The tumor microenvironment and tumor immunity are crucially involved in tumor therapy. Immune checkpoint inhibitors targeting PD-1/PD-L1 signal transduction have been widely used in tumor therapy and have shown ideal clinical efficacy. However, some kinds of cancers still do not respond to PD-1/PD-L1 blockade therapy effectively, including gastric cancer. The related factors should be explored. This review summarizes the recent progression of understanding the influence of Helicobacter pylori infection on PD-1/PD-L1 blockade therapy. Current pieces of evidence have indicated that H.pylori infection might affect the curative effect of tumor therapy associating with the induced immunomodulation. It is necessary to understand the overall integration of PD-1/PD-L1 blockade therapy, the tumor microenvironment, and H.pyloriinfection. Much attention on the influence of H.pylori infection on the efficacy of tumor immunotherapy should be paid.