Mechanisms of PD-L1 Regulation in Malignant and Virus-Infected Cells

Programmed cell death protein 1 (PD-1), a receptor on T cells, and its ligand, PD-L1, have been a topic of much interest in cancer research. Both tumour and virus-infected cells can upregulate PD-L1 to suppress cytotoxic T-cell killing. Research on the PD-1/PD-L1 axis has led to the development of anti-PD-1/PD-L1 immune checkpoint blockades (ICBs) as promising cancer therapies. Although effective in some cancer patients, for many, this form of treatment is ineffective due to a lack of immunogenicity in the tumour microenvironment (TME). Despite the development of therapies targeting the PD-1/PD-L1 axis, the mechanisms and pathways through which these proteins are regulated are not completely understood. In this review, we discuss the latest research on molecules of inflammation and innate immunity that regulate PD-L1 expression, how its expression is regulated during viral infection, and how it is modulated by different cancer therapies. We also highlight existing research on the development of different combination therapies with anti-PD-1/PD-L1 antibodies. This information can be used to develop better cancer immunotherapies that take into consideration the pathways involved in the PD-1/PD-L1 axis, so these molecules do not reduce their efficacy, which is currently seen with some cancer therapies. This review will also assist in understanding how the TME changes during treatment, which will provide further rationale for combination therapies.     

Focus on PD-1/PD-L1 as a Therapeutic Target in Ovarian Cancer

Ovarian cancer is considered one of the most aggressive and deadliest gynecological malignancies worldwide. Unfortunately, the therapeutic methods that are considered the gold standard at this moment are associated with frequent recurrences. Survival in ovarian cancer is associated with the presence of a high number of intra tumor infiltrating lymphocytes (TILs). Therefore, immunomodulation is considered to have an important role in cancer treatment, and immune checkpoint inhibitors may be useful for restoring T cell-mediated antitumor immunity. However, the data presented in the literature until now are not sufficient to allow for the identification and selection of patients who really respond to immunotherapy among those with ovarian cancer. Although there are some studies with favorable results, more prospective trials are needed in this sense. This review focuses on the current and future perspectives of PD-1/L1 blockade in ovarian cancer and analyzes the most important immune checkpoint inhibitors used, with the aim of achieving optimal clinical outcomes. Future studies and trials are needed to maximize the efficacy of immune checkpoint blockade therapy in ovarian cancer, as well as in all cancers, in general.     

Improving Anti-PD-1/PD-L1 Therapy for Localized Bladder Cancer

In high-risk non-muscle invasive bladder cancer (HR-NMIBC), patient outcome is negatively affected by lack of response to Bacillus-Calmette Guérin (BCG) treatment. Lack of response to cisplatin-based neoadjuvant chemotherapy and cisplatin ineligibility reduces successful treatment outcomes in muscle-invasive bladder cancer (MIBC) patients. The effectiveness of PD-1/PD-L1 immune checkpoint inhibitors (ICI) in metastatic disease has stimulated its evaluation as a treatment option in HR-NMIBC and MIBC patients. However, the observed responses, immune-related adverse events and high costs associated with ICI have provided impetus for the development of methods to improve patient stratification, enhance anti-tumorigenic effects and reduce toxicity. Here, we review the challenges and opportunities offered by PD-1/PD-L1 inhibition in HR-NMIBC and MIBC. We highlight the gaps in the field that need to be addressed to improve patient outcome including biomarkers for response stratification and potentially synergistic combination therapy regimens with PD-1/PD-L1 blockade.     

PD-L1 Expression in 65 Conjunctival Melanomas and Its Association with Clinical Outcome

Conjunctival melanoma (CM) iss a rare and aggressive tumour that is increasing in frequency. The prognostic value of PD-L1 expression, alone or in combination with CD8 and PD-1 expression and the BRAF and NRAS status, has not been determined in CM to date. We evaluated the expression of PD-L1, CD8, PD-1 in CM and investigated whether there was an association between the expression of these markers and the BRAF and NRAS molecular profile as well as some clinico-pathological criteria. A total of sixty-five CM were assessed for PD-L1, PD-1, and CD8 expression by immunohistochemistry (IHC) and for BRAF and NRAS genomic alterations using molecular biology techniques and anti-BRAF and anti-NRAS antibodies. PD-L1 expression in tumour cells (TC) was very low or absent but detected in tumour-infiltrating immune cells (IC). A correlation was observed between the expression of PD-L1, CD8, and PD-1 in IC. No correlation between PD-L1 expression (in tumour and/or immune cells) and BRAF or NRAS mutations was observed. PD-L1 expression in IC correlated with a higher pTNM stage and PD-L1 expression in TC with worse disease-specific survival. PD-L1 expression is a potential prognostic biomarker that correlates with poor prognosis in CM patients.     

PD-L1 Expression in Monocytes Correlates with Bacterial Burden and Treatment Outcomes in Active Pulmonary Tuberculosis

The PD-1/PD-L1 pathway is critical in T cell biology; however, the role of the PD-1/PD-L1 pathway in clinical characteristics and treatment outcomes in pulmonary tuberculosis (PTB) patients is unclear. We prospectively enrolled PTB, latent TB infection (LTBI), and non-TB, non-LTBI subjects. The expression of PD-1/PD-L1 on peripheral blood mononuclear cells (PBMCs) was measured and correlated with clinical characteristics and treatment outcomes in PTB patients. Immunohistochemistry and immunofluorescence were used to visualize PD-1/PD-L1-expressing cells in lung tissues from PTB patients and from murine with heat-killed MTB (HK-MTB) treatment. A total of 76 PTB, 40 LTBI, and 28 non-TB, non-LTBI subjects were enrolled. The expression of PD-1 on CD4+ T cells and PD-L1 on CD14+ monocytes was significantly higher in PTB cases than non-TB subjects. PTB patients with sputum smear/culture unconversion displayed higher PD-L1 expression on monocytes. PD-L1-expressing macrophages were identified in lung tissue from PTB patients, and co-localized with macrophages in murine lung tissues. Mycobacterium tuberculosis (MTB) whole cell lysate/EsxA stimulation of human and mouse macrophages demonstrated increased PD-L1 expression. In conclusion, increased expression of PD-L1 on monocytes in PTB patients correlated with higher bacterial burden and worse treatment outcomes. The findings suggest the involvement of the PD-1/PD-L1 pathway in MTB-related immune responses.     

Current Immunotherapeutic Strategies Targeting the PD-1/PD-L1 Axis in Non-Small Cell Lung Cancer with Oncogenic Driver Mutations

Treatment strategies targeting programed cell death 1 (PD-1) or its ligand, PD-L1, have been developed as immunotherapy against tumor progression for various cancer types including non-small cell lung cancer (NSCLC). The recent pivotal clinical trials of immune-checkpoint inhibiters (ICIs) combined with cytotoxic chemotherapy have reshaped therapeutic strategies and established various first-line standard treatments. The therapeutic effects of ICIs in these clinical trials were analyzed according to PD-L1 tumor proportion scores or tumor mutational burden; however, these indicators are insufficient to predict the clinical outcome. Consequently, molecular biological approaches, including multi-omics analyses, have addressed other mechanisms of cancer immune escape and have revealed an association of NSCLC containing specific driver mutations with distinct immune phenotypes. NSCLC has been characterized by driver mutation-defined molecular subsets and the effect of driver mutations on the regulatory mechanism of PD-L1 expression on the tumor itself. In this review, we summarize the results of recent clinical trials of ICIs in advanced NSCLC and the association between driver alterations and distinct immune phenotypes. We further discuss the current clinical issues with a future perspective for the role of precision medicine in NSCLC.     

Reconstitution of Monocyte Subsets and PD-L1 Expression but Not T Cell PD-1 Expression in Obstructive Sleep Apnea Patients upon PAP Therapy

Obstructive sleep apnea (OSA) is characterized by nocturnal breathing intermissions resulting in oxidative stress and eventually, a low-grade systemic inflammation. The study aimed to investigate the impact of positive airway pressure (PAP) therapy on the inflammatory milieu as measured by monocyte and T cell phenotypic alterations. Participants were assessed for their OSA severity before PAP therapy and about six months later, including patient-reported outcome and therapy usage by telemetry readout. The distributions of the CD14/CD16-characterized monocyte subsets as well as the CD4/CD8-characterized effector T cell subsets with regard to their PD-1 and PD-L1 expression were analyzed by flow cytometry from blood samples. Data of 25 patients revealed a significant reconstitution of the monocyte subset distribution and a decrease in PD-L1 expression on pan-monocytes and CD8⁺ T cells without an association to initial AHI and overweight. The PD-1 expression was still increased on T cell subsets, especially on CD4⁺ TH17/22 cells. We conclude that PAP therapy might have a rapid effect on the monocyte phenotype and overall PD-L1 expression levels. However, T cell immune alterations especially on TH17/22 cells persist longer, indicating an ongoing disturbance of the adaptive immune system.     

Identification of the FDA-Approved Drug Pyrvinium as a Small-Molecule Inhibitor of the PD-1/PD-L1 Interaction

Immune checkpoint blockade involving inhibition of the PD-1/PD-L1 interaction has provided unprecedented clinical benefits in treating a variety of tumors. To date, a total of six antibodies that bind to either PD-1 or PD-L1 protein and in turn inhibit the PD-1/PD-L1 interaction have received clinical approvals. Despite being highly effective, these expensive large biotherapeutics possess several inherent pharmacokinetic limitations that can be successfully overcome through the use of low-molecular-weight inhibitors. One such promising approach involves small-molecule induced dimerization and sequestration of PD-L1, leading to effective PD-1/PD-L1 inhibition. Herein, we present the discovery of such potential bioactive PD-L1 dimerizers through a structure- and ligand-based screening of a focused library of approved and investigational drugs worldwide. Pyrvinium, an FDA-approved anthelmintic drug, showed the highest activity in our study with IC₅₀ value of ∼29.66 μM. It is noteworthy that Pyrvinium, being an approved drug, may prove especially suitable as a good starting point for further medicinal chemistry efforts, leading to design and development of even more potent structural analogs as selective PD-1/PD-L1 inhibitors. Furthermore, the adopted integrated virtual screening protocol may prove useful in screening other larger databases of lead- and drug-like molecules for hit identification in the domain of small-molecule PD-1/PD-L1 inhibitors.     

PD-L1 Expression in High-Risk Early-Stage Colorectal Cancer—Its Clinical and Biological Significance in Immune Microenvironment

Programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule that can regulate immune responses in the tumor microenvironment (TME); however, the clinical applications of PD-L1 in early-stage colorectal cancer (CRC) remain unclear. In this study, we aimed to investigate the relationship between PD-L1 expression and survival outcome and explore its relevant immune responses in CRC. PD-L1 expression was evaluated by immunohistochemical staining to determine the tumor proportion score and combined positive score (CPS) in a Taiwanese CRC cohort. The oncomine immune response research assay was conducted for immune gene expression analyses. CRC datasets from the TCGA database were reappraised for PD-L1-associated gene enrichment analyses using GSEA. The high expression of PD-L1 (CPS ≥ 5) was associated with longer recurrence-free survival (p = 0.031) and was an independent prognostic factor as revealed by multivariate analysis. High PD-L1 expression was related to six immune-related gene signatures, and CXCL9 is the most significant overexpressed gene in differential analyses. High CXCL9 expression correlated with increased infiltration levels of immune cells in the TME, including CD8+ T lymphocytes and M1 macrophages. These findings suggest that high PD-L1 expression is a prognostic factor of early-stage CRC, and CXCL9 may play a key role in regulating PD-L1 expression.     

The Role of microRNAs in Cholangiocarcinoma

Cholangiocarcinoma (CCA), an aggressive malignancy, is typically diagnosed at an advanced stage. It is associated with dismal 5-year postoperative survival rates, generating an urgent need for prognostic and diagnostic biomarkers. MicroRNAs (miRNAs) are a class of non-coding RNAs that are associated with cancer regulation, including modulation of cell cycle progression, apoptosis, metastasis, angiogenesis, autophagy, therapy resistance, and epithelial–mesenchymal transition. Several miRNAs have been found to be dysregulated in CCA and are associated with CCA-related risk factors. Accumulating studies have indicated that the expression of altered miRNAs could act as oncogenic or suppressor miRNAs in the development and progression of CCA and contribute to clinical diagnosis and prognosis prediction as potential biomarkers. Furthermore, miRNAs and their target genes also contribute to targeted therapy development and aid in the determination of drug resistance mechanisms. This review aims to summarize the roles of miRNAs in the pathogenesis of CCA, their potential use as biomarkers of diagnosis and prognosis, and their utilization as novel therapeutic targets in CCA.     

Genetic LGALS1 Variants Are Associated with Heterogeneity in Galectin-1 Serum Levels in Patients with Early Arthritis

Galectin 1 (Gal1) exerts immunomodulatory effects leading to therapeutic effects in autoimmune animal models. Patients with rheumatoid arthritis have been reported to show higher Gal1 serum levels than the healthy population. Our study aimed to find genetic variants on the Gal1 gene (LGALS1) modulating its expression and/or clinical features in patients with early arthritis (EA). LGALS1 was sequenced in 53 EA patients to characterize all genetic variants. Then, we genotyped rs9622682, rs929039, and rs4820293, which covered the main genetic variation in LGALS1, in 532 EA patients. Gal1 and IL-6 serum levels were measured by ELISA and Gal1 also by western blot (WB) in lymphocytes from patients with specific genotypes. Once disease activity improved with treatment, patients with at least one copy of the minor allele in rs9622682 and rs929039 or those with GG genotype in rs4820293 showed significantly higher Gal1 serum levels (p < 0.05). These genotypic combinations were also associated with higher Gal1 expression in lymphocytes by WB and lower IL-6 serum levels in EA patients. In summary, our study suggests that genetic variants studied in LGALS1 can explain heterogeneity in Gal1 serum levels showing that patients with higher Gal1 levels have lower serum IL-6 levels.     

Rationale and Clinical Research Progress on PD-1/PD-L1-Based Immunotherapy for Metastatic Triple-Negative Breast Cancer

Metastatic triple-negative breast cancer (mTNBC), a highly aggressive and malignant tumor, currently lacks an effective treatment. There has been some progress in the treatment of mTNBC with programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) immunotherapy in recent years. The combination of PD-1/PD-L1 inhibitors with other therapies is a noteworthy treatment strategy. Immunotherapy in combination with chemotherapy or small-molecule inhibitors still faces many challenges. Additionally, there are some new immunotherapy targets in development. We aimed to further evaluate the effectiveness and usefulness of immunotherapy for treating mTNBC and to propose new immunotherapy strategies. This review explains the rationale and results of existing clinical trials evaluating PD-1/PD-L1 inhibitors alone or in combination for the treatment of mTNBC. For patients with aggressive tumors and poor health, PD-1/PD-L1 inhibitors, either alone or in combination with other modalities, have proven to be effective. However, more research is needed to explore more effective immunotherapy regimens that will lead to new breakthroughs in the treatment of mTNBC.     

Programmed Death-Ligand 1 as a Regulator of Tumor Progression and Metastasis

Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint has long been implicated in modeling antitumor immunity; PD-1/PD-L1 axis inhibitors exert their antitumor effects by relieving PD-L1-mediated suppression on tumor-infiltrating T lymphocytes. However, recent studies have unveiled a distinct, tumor-intrinsic, potential role for PD-L1. In this review, we focus on tumor-intrinsic PD-L1 signaling and delve into preclinical evidence linking PD-L1 protein expression with features of epithelial-to-mesenchymal transition program, cancer stemness and known oncogenic pathways. We further summarize data from studies supporting the prognostic significance of PD-L1 in different tumor types. We show that PD-L1 may indeed have oncogenic potential and act as a regulator of tumor progression and metastasis.     

Molecular Mechanism of Food-Derived Polyphenols on PD-L1 Dimerization: A Molecular Dynamics Simulation Study

In cancer immunotherapy, an emerging approach is to block the interactions of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) using small-molecule inhibitors. The food-derived polyphenols curcumin (CC), resveratrol (RSV) and epigallocatechin gallate (EGCG) have anticancer immunologic functions, which, recently, have been proposed to act via the downregulation of PD-L1 expression. However, it remains unclear whether they can directly target PD-L1 dimerization and, thus, interrupt the PD-1/PD-L1 pathway. To elucidate the molecular mechanism of such compounds on PD-L1 dimerization, molecular docking and nanosecond molecular dynamics simulations were performed. Binding free energy calculations show that the affinities of CC, RSV and EGCG to the PD-L1 dimer follow a trend of CC > RSV > EGCG. Hence, CC is the most effective inhibitor of the PD-1/PD-L1 pathway. Analysis on contact numbers, nonbonded interactions and residue energy decomposition indicate that such compounds mainly interact with the C-, F- and G-sheet fragments of the PD-L1 dimer, which are involved in interactions with PD-1. More importantly, nonpolar interactions between these compounds and the key residues Ile54, Tyr56, Met115, Ala121 and Tyr123 play a dominant role in binding. Free energy landscape and secondary structure analyses further demonstrate that such compounds can stably interact with the binding domain of the PD-L1 dimer. The results provide evidence that CC, RSV and EGCG can inhibit PD-1/PD-L1 interactions by directly targeting PD-L1 dimerization. This provides a novel approach to discovering food-derived small-molecule inhibitors of the PD-1/PD-L1 pathway with potential applications in cancer immunotherapy.     

Decreased Levels of Microfibril-Associated Glycoprotein (MAGP)-1 in Patients with Colon Cancer and Obesity Are Associated with Changes in Extracellular Matrix Remodelling

Objective: The protein microfibril-associated glycoprotein (MAGP)-1 constitutes a crucial extracellular matrix protein. We aimed to determine its impact on visceral adipose tissue (VAT) remodelling during obesity-associated colon cancer (CC). Methods: Samples obtained from 79 subjects (29 normoponderal (NP) (17 with CC) and 50 patients with obesity (OB) (19 with CC)) were used in the study. Circulating concentrations of MAGP-1 and its gene expression levels (MFAP2) in VAT were analysed. The impact of inflammation-related factors and adipocyte-conditioned media (ACM) on MFAP2 mRNA levels in colon adenocarcinoma HT-29 cells were further analysed. The effects of MAGP-1 in the expression of genes involved in the extracellular matrix (ECM) remodelling and tumorigenesis in HT-29 cells was also explored. Results: Obesity (p < 0.01) and CC (p < 0.001) significantly decreased MFAP2 gene expression levels in VAT whereas an opposite trend in TGFB1 mRNA levels was observed. Increased mRNA levels of MFAP2 after the stimulation of HT-29 cells with lipopolysaccharide (LPS) (p < 0.01) and interleukin (IL)-4 (p < 0.01) together with a downregulation (p < 0.05) after hypoxia mimicked by CoCl₂ treatment was observed. MAGP-1 treatment significantly enhanced the mRNA levels of the ECM-remodelling genes collagen type 6 α3 chain (COL6A3) (p < 0.05), decorin (DCN) (p < 0.01), osteopontin (SPP1) (p < 0.05) and TGFB1 (p < 0.05). Furthermore, MAGP-1 significantly reduced (p < 0.05) the gene expression levels of prostaglandin-endoperoxide synthase 2 (COX2/PTGS2), a key gene controlling cell proliferation, growth and adhesion in CC. Interestingly, a significant decrease (p < 0.01) in the mRNA levels of MFAP2 in HT-29 cells preincubated with ACM from volunteers with obesity compared with control media was observed. Conclusion: The decreased levels of MAGP-1 in patients with obesity and CC together with its capacity to modulate key genes involved in ECM remodelling and tumorigenesis suggest MAGP-1 as a link between AT excess and obesity-associated CC development.     

CD73 and PD-L1 as Potential Therapeutic Targets in Gallbladder Cancer

Gallbladder cancer (GBC) is one of the most common and aggressive biliary tract cancers with a dismal prognosis. Ongoing clinical trials are evaluating a few selected immune checkpoint inhibitors (ICIs) as monotherapy for the treatment of GBC patients. However, only a subset of patients benefits from these treatments. To improve ICI therapy response, molecular mechanisms that confer resistance to immune checkpoint (IC) blockade needs to be explored. Epithelial-to-mesenchymal transition (EMT) program and cancer stem cells (CSCs) have been implicated as key processes that confer ICI treatment resistance. However, in GBC the EMT-CSC-IC axis has not yet been clearly elucidated. This study aims to examine the aberrant expression of ICs associated with CSC and EMT. We successfully enriched CSCs by utilizing a 3-dimensional culture system and established a reversible EMT model with human GBC NOZ cell line. Notably, ICs CD73 and PD-L1 were closely associated with both CSC and EMT phenotypes. Knockdown of CD73 or PD-L1 reduced the proliferative and motile abilities of both adherent monolayers and anchorage-free spheroids. In conclusion, blocking CD73 and PD-L1 offer a promising therapeutic strategy for targeting highly aggressive populations with CSC and EMT phenotype to improve GBC patient prognosis.     

Tumor Microenvironment in Male Breast Carcinoma with Emphasis on Tumor Infiltrating Lymphocytes and PD-L1 Expression

Male breast cancer (MBC) is rare and usually presents as a locally advanced disease. Stromal tumor-infiltrating lymphocytes (sTILs) are associated with a better response to neoadjuvant chemotherapy and improved prognosis in all molecular subtypes of female breast cancer, but their role in MBC is less clear. We studied sTILs and the expression of programmed cell death ligand 1 (PD-L1) and pan-TRK in MBC. We retrospectively studied 113 cases of MBC surgically treated between 1988 and 2015. The tumors were evaluated for histological type and grade, stage, intrinsic subtype and sTILs. We performed immunohistochemistry for PD-L1 (clone SP142) and pan-TRK (clone {"type":"entrez-protein","attrs":{"text":"EPR17341","term_id":"523383444","term_text":"EPR17341"}}EPR17341) on tissue microarrays. Pan-TRK positive cases were further analyzed by next-generation sequencing. The median age was 69 years (range 60–77). Invasive carcinoma of no special type was found in 94.7% of cases, of which 53.1% were grade 2. Estrogen receptor was positive in 92% of the tumors, progesterone receptor in 85.8%, androgen receptor in 70.8%; 4.4% were human epidermal growth factor receptor 2 (HER2)-positive, and 55.8% HER2-low. 40.7% of tumors were luminal A and 51.3% luminal B, 4.4% HER2-enriched and 3.5% triple negative carcinoma. sTILs density was <50% in 96.4% of the tumors, >50% in 3.6% of the tumors. PD-L1 immune cell score >1% was found in 7.1% of the tumors (all of luminal subtype). A weak focal cytoplasmic pan-TRK staining was present in 8.8% but without NTRK fusion. Neither sTILs nor PD-L1 had statistically significant outcomes. Our findings suggest that a subset of MBC patients harbors an immunological environment characterized by increased sTILs with PD-L1 expression. These patients may potentially benefit from immune checkpoint inhibitor therapy. Frequent HER2-low may offer novel anti-HER2 treatment options.     

Role of PD-L1 in Gut Mucosa Tolerance and Chronic Inflammation

The gastrointestinal (GI) mucosa is among the most complex systems in the body. It has a diverse commensal microbiome challenged continuously by food and microbial components while delivering essential nutrients and defending against pathogens. For these reasons, regulatory cells and receptors are likely to play a central role in maintaining the gut mucosal homeostasis. Recent lessons from cancer immunotherapy point out the critical role of the B7 negative co-stimulator PD-L1 in mucosal homeostasis. In this review, we summarize the current knowledge supporting the critical role of PD-L1 in gastrointestinal mucosal tolerance and how abnormalities in its expression and signaling contribute to gut inflammation and cancers. Abnormal expression of PD-L1 and/or the PD-1/PD-L1 signaling pathways have been observed in the pathology of the GI tract. We also discuss the current gap in our knowledge with regards to PD-L1 signaling in the GI tract under homeostasis and pathology. Finally, we summarize the current understanding of how this pathway is currently targeted to develop novel therapeutic approaches.