Programmed Death Ligand 1 Interaction Research Articles

Shedding Light on the Role of Exosomal PD-L1 (ExoPD-L1) in Cancer Progression: an Update.

Exosomes are the primary category of extracellular vesicles (EVs), which are lipid-bilayer vesicles with biological activity spontaneously secreted from either normal or tansformed cells. They serve a crucial role for intercellular communication and affect extracellular environment and the immune system. Tumor-derived exosomes (TEXs) enclose high levels of immunosuppressive proteins, including programmed death-ligand 1 (PD-L1). PD-L1 and its receptor PD-1 act as crucial immune checkpoint molecules, thus facilitating tumor advancement by inhibiting immune responses. PDL-1 is abundantly present on tumor cells and interacts with PD-1 on activated T cells, resulting in T cell suppression and allowing immune evasion of cancer cells. Various FDA-approved monoclonal antibodies inhibiting the PD-1/PD-L1 interaction are commonly used to treat a diverse range of tumors. Although the achieved results are significant, some individuals have a poor reaction to PD-1/PD-L1 blocking. PD-L1-enriched TEXs may mimic the impact of cell-surface PD-L1, consequently potentiating tumor resistance to PD1/PD-L1 based therapy. In light of this, a strong correlation between circulating exosomal PD-L1 levels and response rate to anti-PD-1/PD-L1 antibody treatment has been evinced. This article inspects the function of exosomal PDL-1 in developing resistance to anti-PD-1/PD-L1 therapy for opening new avenues for overcoming tumor resistance to such modalities and development of more favored combination therapy.

Design, Synthesis, and Biological Evaluation of Chroman Derivatives as PD-1/PD-L1 Antagonists.

Programmed death-ligand 1 (PD-L1) has emerged as a promising therapeutic target for various cancers due to its crucial role in promoting tumor immune evasion. Here, we report a novel class of chroman-like small-molecule PD-L1 inhibitors exhibiting significant activity in inhibiting the PD-1/PD-L1 interaction. Employing a "ring-close" strategy for conformational restriction, we have achieved compound C27, which demonstrates superior PD-1/PD-L1 inhibitory activity compared to the positive control. Molecular dynamics simulation and binding free energy calculation predict that (R)-C27 with inhibitory activity surpassed (S)-C27. The experimental results from bioassay and X-ray structural analysis corroborate these findings. All these results collectively indicate that (R)-C27 is a promising lead compound deserving further exploration.

Polymer-based antibody mimetics (iBodies) target human PD-L1 and function as a potent immune checkpoint blocker

Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability.Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.

Regulation of PD-L1 Expression by YY1 in Cancer: Therapeutic Efficacy of Targeting YY1.

During the last decade, we have witnessed several milestones in the treatment of various resistant cancers including immunotherapeutic strategies that have proven to be superior to conventional treatment options, such as chemotherapy and radiation. This approach utilizes the host's immune response, which is triggered by cancer cells expressing tumor-associated antigens or neoantigens. The responsive immune cytotoxic CD8+ T cells specifically target and kill tumor cells, leading to tumor regression and prolongation of survival in some cancers; however, some cancers may exhibit resistance due to the inactivation of anti-tumor CD8+ T cells. One mechanism by which the anti-tumor CD8+ T cells become dysfunctional is through the activation of the inhibitory receptor programmed death-1 (PD-1) by the corresponding tumor cells (or other cells in the tumor microenvironment (TME)) that express the programmed death ligand-1 (PD-L1). Hence, blocking the PD-1/PD-L1 interaction via specific monoclonal antibodies (mAbs) restores the CD8+ T cells' functions, leading to tumor regression. Accordingly, the Food and Drug Administration (FDA) has approved several checkpoint antibodies which act as immune checkpoint inhibitors. Their clinical use in various resistant cancers, such as metastatic melanoma and non-small-cell lung cancer (NSCLC), has shown significant clinical responses. We have investigated an alternative approach to prevent the expression of PD-L1 on tumor cells, through targeting the oncogenic transcription factor Yin Yang 1 (YY1), a known factor overexpressed in many cancers. We report the regulation of PD-L1 by YY1 at the transcriptional, post-transcriptional, and post-translational levels, resulting in the restoration of CD8+ T cells' anti-tumor functions. We have performed bioinformatic analyses to further explore the relationship between both YY1 and PD-L1 in cancer and to corroborate these findings. In addition to its regulation of PD-L1, YY1 has several other anti-cancer activities, such as the regulation of proliferation and cell viability, invasion, epithelial-mesenchymal transition (EMT), metastasis, and chemo-immuno-resistance. Thus, targeting YY1 will have a multitude of anti-tumor activities resulting in a significant obliteration of cancer oncogenic activities. Various strategies are proposed to selectively target YY1 in human cancers and present a promising novel therapeutic approach for treating unresponsive cancer phenotypes. These findings underscore the distinct regulatory roles of YY1 and PD-L1 (CD274) in cancer progression and therapeutic response.

Discovery of Novel PD-L1 Small-Molecular Inhibitors with Potent In Vivo Anti-tumor Immune Activity.

Programmed death-ligand 1 (PD-L1) has surfaced as a promising therapeutic target for various cancers due to its pivotal role in facilitating tumor immune evasion. Herein, we report a series of novel small-molecule PD-L1 inhibitors exhibiting remarkable inhibitory activity against the PD-1/PD-L1 interaction (X18: IC50 = 1.3 nM) and reinstating the suppressive effect of PD-L1 on T cells (X18: EC50 = 152.8 nM). Crystallographic studies revealed the binding mode of X18 and PD-L1. Through a rational prodrug design approach, we have successfully optimized the oral pharmacokinetic properties of X22, effectively addressing the poor oral pharmacokinetic profile of PD-L1 small-molecule inhibitors. Notably, X22 demonstrated significant antitumor efficacy in murine models of MC38 and CT26 colon cancer through the upregulation of tumor infiltration and cytotoxicity of CD8+ T cells partially. These findings offer promising prospects for the advancement of PD-L1 inhibitors as innovative agents in cancer immunotherapy.

PD-1/PD-L1 Interaction Upregulates YAP1 Expression in HepG2 Cells Through MAPK/ERK Pathway

Background Antibodies, which target programmed cell death protein-1 (PD-1) or its ligand programmed death ligand-1 (PD-L1), can rescue T cells from an exhausted state and resume their immune response to cancer cells. Clinically, the purpose of blocking the PD-1/PD-L1 signaling pathway is to induce immune cells to play an anti-tumor role. However, the effect of intertumor PD-1/PD-L1 signal blocking on tumor cells remains unclear. Methods HepG2 cells were treated with DHA, IFN-γ, BSA, DDP, PD-1-Fc (1 μg/ml), IgG-Fc, nivolumab, or human IgG for 24 h, respectively. GEPIA, cBioPortal, and TIMER databases were used to analyze the correlation between YAP1, PD-1, and PD-L1 and ERK, ERK-5, JNK, and p38. Western blot was used to detect the expression of YAP1 and p-ERK. Results GEPIA, cBioPortal, and TIMER databases analysis showed that YAP1 was positively correlated with ERK. After HepG2 cells were treated with PD98059 (ERK inhibitor), the expression of YAP1 was decreased. In this study, we investigated the inhibitory effect of PD98059 on PD-1/PD-L1 signaling. Our study found that PD-1-Fc (PD-1 fusion protein) promoted the expression of p-ERK/ERK and YAP1 in HepG2 cells. In contrast, nivolumab (PD-1 blocking antibody) reduced the expression of p-ERK/ERK and YAP1 in IFN-γ-pretreated HepG2 cells. In addition, the application of DHA also inhibited the expression of p-ERK/ERK to inhibit YAP1. Furthermore, treatment of HepG2 cells with DHA alone or DHA combined with cisplatin (DDP) both inhibited the expression of p-ERK/ERK and YAP1. Conclusions These results suggested that PD-1/PD-L1 interactions between tumor cells could promote the expression of ERK or YAP1 within tumors. Moreover, the conduction of PD-1/PD-L1 could be reversed using ERK inhibitors.

Discovery of a novel dual-targeting D-peptide to block CD24/Siglec-10 and PD-1/PD-L1 interaction and synergize with radiotherapy for cancer immunotherapy

BackgroundAside from immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1), intervention of CD47/Sirpα mediated ‘don’t eat me’ signal between macrophage and tumor...

IL-8 concurrently promotes idiopathic pulmonary fibrosis mesenchymal progenitor cell senescence and PD-L1 expression enabling escape from immune cell surveillance.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease. We discovered fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that display cell-autonomous fibrogenicity and drive fibrotic progression. In a study of the IPF MPC nuclear proteome, we identified DNA damage as one of the most altered functions in IPF MPCs. In prior work we found that IL-8 drives IPF MPC self-renewal. IL-8 can promote replicative stress and DNA damage and induce senescence through the CXCR2 receptor. We hypothesized that IL-8 promotes DNA damage-mediated senescence in IPF MPCs. We show that IL-8 induces DNA damage and promotes IPF MPC senescence. We discovered that IL-8 concurrently promotes senescence and upregulation of the programmed death ligand 1 (PD-L1) in a CXCR2-dependent manner. Disruption of programmed cell death protein-1 (PD-1)-PD-L1 interaction promotes natural killer (NK) cell killing of IPF MPCs in vitro and arrests IPF MPC-mediated experimental lung fibrosis in vivo. Immunohistochemical (IHC) analysis of IPF lung tissue identified PD-L1-expressing IPF MPCs codistributing with NK cells and β-galactosidase-positive cells. Our data indicate that IL-8 simultaneously promotes IPF MPC DNA damage-induced senescence and high PD-L1 expression, enabling IPF MPCs to elude immune cell-targeted removal. Disruption of PD-1-PD-L1 interaction may limit IPF MPC-mediated fibrotic progression.NEW & NOTEWORTHY Here we show that IL-8 concurrently promotes senescence and upregulation of PD-L1 in IPF MPCs. IHC analysis identifies the presence of senescent IPF MPCs intermingled with NK cells in the fibroblastic focus, suggesting that senescent MPCs elude immune cell surveillance. We demonstrate that disruption of PD-1/PD-L1 interaction promotes NK cell killing of IPF MPCs and arrests IPF MPC-mediated experimental lung fibrosis. Disruption of PD-1/PD-L1 interaction may be one means to limit fibrotic progression.

Discovery of potential PD-L1 small molecule inhibitors as novel cancer therapeutics using machine learning-based QSAR models: A virtual drug repurposing study.

Binding of programmed death 1 (PD-1) to programmed death ligand-1 (PD-L1) triggers downstream signaling pathways and results in inhibition of new cytotoxic T-cell activation. Cancer cells can “trick” the immune system with the T-cell suppression ability of the PD-1/PD-L1 signaling mechanism. Blocking the PD-1/PD-L1 interaction through target-specific small molecules can recruit cytotoxic T cells and reactivate the immune response, is a potential therapy in many types of cancer. In spite of that, there are currently no approved small molecule inhibitors targeting the PD-1/PD-L1 pathway. In this study, we aim to discover target-specific and effective small molecules with anti-cancer activity. With this objective, we performed a virtual drug repurposing study on PD1 protein, PD1-PDL1 structure and PDL1-PDL1 homodimer complexes. The results revealed that PDL1-dimer structure is the most suitable target model for virtual screening of small molecule inhibitors. Three different approaches with different algorithms were used in virtual screening to discover the best hit molecules: (i) structure-based, (ii) structure-based pharmacophore (hybrid) and (iii) machine learning-based QSAR. At the end of the study, which included three different approaches, promising PDL1 small molecule inhibitors such as anidulafungin, bemotrizinol, deferoxamine, dequalinium, neratinib, ubiquinol and vilanterol were discovered.

PD-L1: expression regulation.

Programmed death-ligand 1 (PD-L1), expressed on the surface of tumor cells, can bind to programmed cell death-1 (PD-1) on T cells. The interaction of PD-1 and PD-L1 can inhibit T-cell responses by decreasing T-cell activity and accelerating their apoptosis. Various cancers express high levels of PD-L1 and exploit PD-L1/PD-1 signaling to evade T-cell immunity, and immunotherapies targeting the PD-1/PD-L1 axis have been shown to exert remarkable anti-tumor effects; however, not all tumor patients benefit from these therapies. Therefore, study of the mechanisms regulating PD-L1 expression are imperative. In this review, we explore regulation of PD-L1 expression in the contexts of gene transcription, signaling pathways, histone modification and remodeling, microRNAs, long noncoding RNAs, and post-translational modification. Current developments in studies of agents that block PD-L1 and correlations between immunotherapies targeting PD-1/PD-L1 and PD-L1 expression are also summarized. Our review will assist in understanding of PD-L1 expression regulation and discusses the implications of reported findings in cancer diagnosis and immunotherapy.

Gallium-68-labeled Peptide PET Quantifies Tumor Exposure of PD-L1 Therapeutics.

Immune checkpoint therapy (ICT) is currently ineffective in a majority of patients. Tumor drug exposure measurements can provide vital insights into mechanisms involved in the resistance of solid tumors to those therapeutics; however, tools to quantify in situ drug exposure are few. We have investigated the potential of programmed death-ligand 1 (PD-L1) pharmacodynamics, quantified using PET, to inform on the tumor exposure of anti-PD-L1 (aPD-L1) therapeutics. To noninvasively quantify PD-L1 levels, we first developed a novel peptide-based gallium-68-labeled binder, [68Ga]Ga-DK223, and evaluated its in vivo distribution, pharmacokinetics, and PD-L1 specificity in preclinical models of triple-negative breast cancer and urothelial carcinoma with variable PD-L1 expression. We then quantified baseline and accessible PD-L1 levels in tumors as a noninvasive pharmacodynamic measure to assess tumor exposure to two aPD-L1 antibodies (avelumab and durvalumab). DK223 exhibited a KD of 1.01±0.83 nmol/L for PD-L1 and inhibited the PD-1:PD-L1 interaction in a dose-dependent manner. [68Ga]Ga-DK223 provides high-contrast PET images within 60 minutes of administration and detects PD-L1 in an expression-dependent manner in xenograft models. PD-L1 pharmacodynamics measured using [68Ga]Ga-DK223-PET revealed that avelumab and durvalumab had similar exposure early during therapy, but only durvalumab exhibited sustained exposure at the tumor. [68Ga]Ga-DK223 detected variable PD-L1 levels and exhibited salient features required for clinical translation. [68Ga]Ga-DK223-PET could be useful for quantifying total PD-L1 levels at baseline and accessible PD-L1 levels during therapy to understand drug exposure at the tumor, thus supporting its use for guiding and optimizing ICT.

Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells

Background aimsCholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA. MethodsTo overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28. ResultsInitially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8+ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed. ConclusionTaken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.

Aptamer-conjugated nano-liposome for immunogenic chemotherapy with reversal of immunosuppression.

Cancer cells have various immune evasion mechanisms that resist the immune cells by reprogramming the tumor microenvironment (TME), such as programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase-1 (IDO1) overexpression. One of the approaches to restore antitumor immune response by T-cells is through induction of immunogenic cell death (ICD). Thus, drug carrier containing IDO1 siRNA and ICD inducer would be effective anticancer regimen to modulate the immunosuppressive TME by reversing the IDO1-mediated immunosuppression in a synergistic combination with ICD induction. However, numerous nanocarrier platforms for co-delivery of multiple drugs mostly depend on the enhanced permeation and retention (EPR), which is insufficient to achieve selectivity in tumor sites harboring various types of cells. We designed a targeted drug delivery system using nano-sized liposomes functionalized with anti-CD44 and anti-PD-L1 DNA aptamers, which target breast cancer cells and inhibit PD-1/PD-L1 interaction between cancer cells and T-cells. To reverse immunosuppressive TME and reactivate immune response, cancer-targeting nano-liposomes were prepared to contain immunogenic cell death inducer (Doxorubicin, DOX) and IDO1 siRNA, namely Aptm[DOX/IDO1]. The Aptm[DOX/IDO1] specifically delivered the loaded DOX and IDO1 siRNA into target breast cancer cells through aptamer-mediated endocytosis. Cancer-targeted DOX/IDO1 siRNA delivery enhanced ICD and suppressed IDO1 expression with significantly high toxicity in cancer cells. We demonstrated that Aptm[DOX/IDO1] could achieve synergistic antitumor effects by facilitating ICD response and simultaneous reversal of the immunosuppressive TME with IDO1 knockdown in the subcutaneous breast cancer model mice, thus reducing tumor size. These antitumor effects were exerted with intratumoral infiltration of CD8+ cytotoxic T lymphocyte as well as attenuation of regulatory T-cell recruitment in the tumor sites. We further proved that our Aptm[DOX/IDO1] strategy significantly reduced tumor metastasis in tumor-xenograft mice through a synergistic combination of cancer cell-targeted ICD induction and reversal of the IDO1-mediated immunosuppressive TME. Our nanocarrier platform based on cationic liposomes containing DOX and IDO1 siRNA, which are conjugated with two DNA aptamers targeting the cancer cell surface, accomplished synergistic chemoimmunotherapy through tumor-specific immune modulation into immune-favorable TME in vivo.

A Generic Strategy to Generate Bifunctional Two-in-One Antibodies by Chicken Immunization.

Various formats of bispecific antibodies exist, among them Two-in-One antibodies in which each Fab arm can bind to two different antigens. Their IgG-like architecture accounts for low immunogenicity and also circumvents laborious engineering and purification steps to facilitate correct chain pairing. Here we report for the first time the identification of a Two‐in‐One antibody by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibody simultaneously targets the epidermal growth factor receptor (EGFR) and programmed death‐ligand 1 (PD-L1) at the same Fv fragment with two non-overlapping paratopes. The dual action Fab is capable of inhibiting EGFR signaling by binding to dimerization domain II as well as blocking the PD-1/PD-L1 interaction. Furthermore, the Two-in-One antibody demonstrates specific cellular binding properties on EGFR/PD-L1 double positive tumor cells. The presented strategy relies solely on screening of combinational immune-libraries and obviates the need for any additional CDR engineering as described in previous reports. Therefore, this study paves the way for further development of therapeutic antibodies derived from avian immunization with novel and tailor-made binding properties.

Pyrazolones as inhibitors of immune checkpoint blocking the PD-1/PD-L1 interaction

Immuno-therapy has become a leading strategy to fight cancer. Over the past few years, immuno-therapies using checkpoint inhibitor monoclonal antibodies (mAbs) against programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) have demonstrated improved survival compared with chemotherapy. We describe the microwave-assisted synthesis and the characterization of an innovative series of synthetic compounds endowed with nanomolar activity against PD-L1. The properties of the compounds were characterized using several biophysical techniques including microscale thermophoresis (MST) and fluorescence resonance energy transfer (FRET) measurements. A few small molecules demonstrated a high affinity for human PD-L1, potently disrupted the PD-L1:PD-1 interaction and inhibited Src homology region 2 domain-containing phosphatase (SHP2) recruitment to PD-1. More than 30 molecules from the pyrazolone family have been synthesized and 5 highly potent “PD-L1 silencing compounds” have been identified, based on in vitro measurements. Structure-activity relationships have been defined and ADME properties were evaluated. The phenyl-pyrazolone unit offers novel perspectives to design PD-L1-targeting agents, potentially useful to combat cancer and other pathologies implicating the PD-1/PD-L1 checkpoint.

A novel PD-L1-targeted shark VNAR single-domain-based CAR-T cell strategy for treating breast cancer and liver cancer

Chimeric antigen receptor (CAR)-T cell therapy shows excellent potency against hematological malignancies, but it remains challenging to treat solid tumors, mainly because of a lack of appropriate antigenic targets and an immunosuppressive tumor microenvironment (TME). The checkpoint molecule programmed death-ligand 1 (PD-L1) is widely overexpressed in multiple tumor types, and the programmed death-ligand 1 (PD-1)/PD-L1 interaction is a crucial mediator of immunosuppression in the TME. Here we constructed a semi-synthetic shark VNAR phage library and isolated anti-PD-L1 single-domain antibodies. Among these VNARs, B2 showed cross-reactivity to human, mouse, and canine PD-L1, and it partially blocked the interaction of human PD-1 with PD-L1. CAR (B2) T cells specifically lysed human breast cancer and liver cancer cells by targeting constitutive and inducible expression of PD-L1 and hindered tumor metastasis. Combination of PD-L1 CAR (B2) T cells with CAR T cells targeted by GPC3 (a liver cancer-specific antigen) regresses liver tumors in mice. We concluded that PD-L1-targeted shark VNAR single-domain-based CAR-T therapy is a novel strategy to treat breast and liver cancer. This study provides a rationale for potential use of PD-L1 CAR-T cells as a monotherapy or in combination with a tumor-specific therapy in clinical studies.

ICAM-1-mediated adhesion is a prerequisite for exosome-induced Tcell suppression.

Tumor-derived extracellular vesicles (TEVs) suppress the proliferation and cytotoxicity of CD8+ Tcells, thereby contributing to tumor immune evasion. Here, we report that the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) co-localizes with programmed death ligand 1 (PD-L1) on the exosomes; both ICAM-1 and PD-L1 are upregulated by interferon-γ. Exosomal ICAM-1 interacts with LFA-1, which is upregulated in activated Tcells. Blocking ICAM-1 on TEVs reduces the interaction of TEVs with CD8+ Tcells and attenuates PD-L1-mediated suppressive effects of TEVs. During this study, we have established an extracellular vesicle-target cell interaction detection through SorTagging (ETIDS) system to assess the interaction between a TEV ligand and its target cell receptor. Using this system, we demonstrate that the interaction of TEV PD-L1 with programmed cell death 1 (PD-1) on Tcells is significantly reduced in the absence of ICAM-1. Our study demonstrates that ICAM-1-LFA-1-mediated adhesion between TEVs and Tcells is a prerequisite for exosomal PD-L1-mediated immune suppression.

Ezrin and Radixin Differentially Modulate Cell Surface Expression of Programmed Death Ligand-1 in Human Pancreatic Ductal Adenocarcinoma KP-2 Cells

Immune checkpoint blockade (ICB) therapies, such as immune checkpoint inhibitors against programmed death ligand-1 (PD-L1), have not been successful in treating patients with pancreatic ductal adenocarcinoma (PDAC). Despite the critical role of PD-L1 in various types of cancers, the regulatory mechanism of PD-L1 expression on the cell surface of PDAC is poorly understood. Therefore, uncovering potential modulators of cell surface localisation of PD-L1 may provide a new strategy to improve ICB therapy in patients with PDAC. Here, we examined the role of ezrin/radixin/moesin (ERM) family scaffold proteins that crosslink transmembrane proteins with the actin cytoskeleton in the surface localisation of PD-L1 in KP-2 cells, a human PDAC cell line. Our results demonstrated the abundant protein expression of PD-L1, ezrin, and radixin, but not moesin, as well as their colocalisation in the plasma membrane. Interestingly, immunoprecipitation analysis detected the molecular interaction of PD-L1 with ezrin and radixin. Moreover, gene silencing of ezrin moderately decreased the mRNA and cell surface expression of PD-L1, while that of radixin greatly decreased the surface expression of PD-L1 without altering the mRNA levels. Thus, radixin and ezrin differentially modulate the cell surface localisation of PD-L1 in KP-2 cells, highlighting a potential therapeutic target to improve the current ICB therapy in PDAC.

Disruption of Her2-Induced PD-L1 Inhibits Tumor Cell Immune Evasion in Patient-Derived Gastric Cancer Organoids.

Simple SummaryHER2 may contribute to immune evasion in gastric cancer that is associated with PD-L1 expression. Autologous organoid/immune cell co-cultures serve as an appropriate in vitro model to study the effects of anti-HER2 targeted therapy in combination with anti-PD1 immune checkpoint inhibition and may be used as an ex vivo tool for precision medicine.(1) Background: The expression of programmed death-ligand 1 (PD-L1), which interacts with programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTLs), enables tumors to escape immunosurveillance. The PD-1/PD-L1 interaction results in the inhibition of CTL proliferation, and effector function, thus promoting tumor cell evasion from immunosurveillance and cancer persistence. Despite 40% of gastric cancer patients exhibiting PD-L1 expression, only a small subset of patients responds to immunotherapy. Human epidermal growth factor receptor2 (HER2) is one of the critical regulators of several solid tumors, including metastatic gastric cancer. Although half of PD-L1-positive gastric tumors co-express HER2, crosstalk between HER2 and PD-1/PD-L1 in gastric cancer remains undetermined. (2) Methods: Human gastric cancer organoids (huTGOs) were generated from biopsied or resected tissues and co-cultured with CTLs and myeloid-derived suppressor cells (MDSCs). Digital Spatial Profiling (DSP) was performed on FFPE tissue microarrays of numerous gastric cancer patients to examine the protein expression of immune markers. (3) Results: Knockdown of HER2 in PD-L1/HER2-positive huTGOs led to a concomitant decrease in PD-L1 expression. Similarly, in huTGOs/immune cell co-cultures, PD-L1 expression decreased in huTGOs and was correlated with an increase in CTL proliferation which enhanced huTGO death. Treatment with Nivolumab exhibited similar effects. However, a combinatorial treatment with Mubritinib and Nivolumab was unable to inhibit HER2 expression in co-cultures containing MDSCs. (4) Conclusions: Our study suggested that co-expression of HER2 and PD-L1 may contribute to tumor cell immune evasion. In addition, autologous organoid/immune cell co-cultures can be exploited to effectively screen responses to a combination of anti-HER2 and immunotherapy to tailor treatment for gastric cancer patients.

Potential PD-L1 expressing cytotoxic T-cell immunopathology in Alzheimer disease.

Growing evidence suggested that adaptive immunity involved Alzheimer's disease (AD). Peripheral blood mononuclear cell (PBMC) subpopulations and expression of immune checkpoints such as programmed death-ligand 1(PD-L1) and programmed cell death protein-1 (PD-1) could play roles in Alzheimer disease. The interaction of PD-L1/PD-1 lead PD-1 expressing T cells to attenuate T cell-activating signals. Recent studies had demonstrated the blockade of PD-L1/PD-1 axis modified brain pathology and restored cognitive performance in tauopathy murine model. However similar human experiment suggested controversial results in AD patients. To observe the changes of PBMC subpopulations and PD-L1/PD-1 expression in AD and healthy controls, we utilized flow cytometry, and applied t-distributed stochastic neighbor embedding (t-SNE) algorithm as a new approach to analyze results. AD patients (n=16) with different disease severity (CDR= 1 - 2) and healthy controls (n=16) were enrolled. Expression levels of PD-L1/PD-1 in AD patients and healthy controls were derived by flow cytometric analysis parameters. Moreover, a dimensionality reduction algorithm for visualized t-SNE, was implemented to analyze the PD-L1/PD-1 expressing cell clusters between AD patients and healthy controls. We found no significant PD-1 expression intensity difference between cytotoxic T lymphocytes (CTL) of AD patients and healthy controls. In AD patients, subpopulation of CTL expressing PD-L1 was elevated by 85.53%. Further, PD-L1 expressing CTL clusters between AD patients and healthy controls were compared. We observed that densities of PD-L1high CTL clusters are significantly higher than the healthy controls. Upregulation of PD-L1 and PD-L1 expressing CTL were increased in peripheral blood of AD patients, linking to newly found strong correlation between the level of PD-L1 positive CTL and markers which indicated negative immune climate. Abnormal adaptive immune response of CTL might associate with concurrent viral infection, intracellular bacterial infection, and underlying cancer in AD patients. Our results suggested a novel strategy to prevent AD progression with regulation of PD-L1 expressing CTL, and notation of underlying intracellular infection and malignancy for which could relate to dementia progression.