PD-L1 Protein Research Articles

Development of polyclonal heavy chain antibodies targeting programmed death ligand-1.

Programmed death ligand-1 (PD-L1, CD274 and B7-H1) has been described as a ligand for immune inhibitory receptor programmed death protein 1 (PD-1). With binding to PD-1 on activated T cells, PD-L1 can prevent T cell responses via motivating apoptosis. Consequently, it causes cancers immune evasion and helps the tumor growth; hence, PD-L1 is regarded as a therapeutic target for malignant cancers. The anti-PD-L1 monoclonal antibody targeting PD-1/PD-L1 immune checkpoint has attained remarkable outcomes in clinical application and has turned to one of the most prevalent anti-cancer drugs. The present study aimed to develop polyclonal heavy chain antibodies targeting PD-L1via Camelus dromedarius immunization. The extra-cellular domain of human PD-L1 (hPD-L1) protein was cloned, expressed, and purified. Afterwards, this recombinant protein was utilized as an antigen for camel immunization to acquire polyclonal camelid sera versus this protein. Our outcomes showed that hPD-L1 protein was effectively expressed in the prokaryotic system. The antibody-based techniques, such as enzyme-linked immunosorbent assay, western blotting, and flow cytometry displayed that the hPD-L1 protein was detected by generated polyclonal antibody. Due to the advantages of multi-epitope-binding ability, our study exhibited that camelid antibody is effective to be applied significantly for detection of PD-L1 protein in essential antibody-based studies.

Status ofPD-1 and PD-L1 expression in invasive urothelial carcinoma ofthebladder with mismatch repair protein deficiency.

It has been reported that mismatch repair deficient (d-MMR) tumors show sensitivity to immune checkpoint inhibitors. We aimed to evaluate thecorrelation ofd-MMR and PD-1/PD-L1 expression in invasive urothelial carcinoma ofthebladder. Tissue microarray (TMA) tissues were stained PD-1/PD-L1 and MMR proteins. Theexpression ratio ofthese markers has been compared with histopathologic parameters. d-MMR tumors were more superficial muscle invasive (p=0.012). When thed-MMR, and PD-1/PD-L1 expression ratios were examined, asignificant correlation was obtained between thed-MMR and PD-L1 expression ratio of> 5% in both thetumor and immune cells (p=0.02 and p=0.004, respectively). Theexpression ratio was higher in thepatients without MMR loss. PD-1 and PD-L1expression in those with MSH6 loss was one or none. When PD1/PDL1 expression was compared with histopathological parameters, asignificant relationship was observed between tumor grade and depth ofmuscle invasion. PD-L1 expression was not observed in thesuperficial muscle invasive tumors. This study was shown thestatus ofd-MMR and PD-1/PD-L1 in invasive urothelial cancers and their correlation with prognostic markers. PD-1/PD-L1 expression may contribute to theprogression and poor prognosis ofbladder cancer. However, further studies are required to research theclinical utility.

Correlation of expression of Major Vault Protein with androgen receptor and immune checkpoint protein B7-H3, and with poor prognosis in prostate cancer.

Prostate cancer diagnosis and early stratification is an important aspect to avoid undertreatment of high-risk prostate cancer patients. Major Vault Protein (MVP) has been proposed as a prognostic biomarker in prostate cancer. PTEN and the immune checkpoint protein B7-H3 interact with MVP and are important in prostate cancer progression and therapy response. We evaluated the expression of MVP by immunohistochemistry of tissue microarray samples from a retrospective cohort consisting of 119 prostate cancer patients. We correlated the protein expression of MVP with clinicopathological characteristics, and protein expression of androgen receptor (AR), PTEN, immune checkpoint proteins B7-H3 and PD-L1. We found MVP to be expressed in 53% of prostate tumors, and correlated positively with biochemical recurrence (ρ=0.211/p=0.021). Furthermore, we found positive correlation of MVP expression with expression of AR (ρ=0.244/p=0.009) and the immune checkpoint protein B7-H3 (ρ=0.200/p=0.029), but not with PD-L1 (ρ=0.152/p=0.117) or PTEN expression (ρ=-0.034/p=0.721). Our findings support the notion that expression of MVP is associated with poor prognosis in prostate cancer. The correlation between MVP and immune checkpoint protein B7-H3 in prostate cancer suggests a role for MVP in immunoregulation and drug resistance.

Virtual screening of drugs targeting PD-L1 protein

Monoclonal antibody inhibitors targeting PD-1/PD-L1 immune checkpoints are gradually entering the market and have achieved certain positive effects in the treatments of various types of tumors. However, with the expansion of application, the limitations of antibody drugs and problems such as excessive homogenization of research gradually appear, making small-molecule inhibitors the new focus of researchers. This study aims to use ligand-based and structure-based binding activity prediction methods to achieve virtual screening of small-molecule inhibitors targeting PD-L1, thereby helping to accelerate the development of small molecule drugs. A dataset of PD-L1 small-molecule inhibitory activity from relevant research literature and patents is collected and activity judgment classification models with intensity prediction regression models are constructed based on different molecular featurization methods and machine learning algorithms. The two types of models filter 68 candidate compounds with high PD-L1 inhibitory activity from a large drug-like small molecule screening pool (ZINC15). Ten of these compounds not only have good drug similarities and pharmacokinetics, but also exhibit comparable binding affinities and similar mechanisms of action with previous reported hotspot compounds in molecular docking. This phenomenon is further verified in subsequent molecular dynamics simulation and the estimation of binding free energy. In this study, a virtual screening workflow integrating ligand-based method and structure-based method is developed, and potential PD-L1 small-molecule inhibitors are effectively screened from large compound databases, which is expected to help accelerate the application and expansion of tumor immunotherapy.

High expression of PD-L1 mainly occurs in non-small cell lung cancer patients with squamous cell carcinoma or poor differentiation.

Lung cancer is one of the most lethal cancers worldwide, but studies have shown that the higher the expression of programmed cell death protein 1 ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC), the more likely it will benefit from anti-PD-L1 immunotherapy. The purpose of our study was to collect and analyze abundant clinical samples in order to provide evidence for clinicians and patients who might consider anti-PD-L1 immunotherapy while jointly formulating treatment plans. On the one hand, we obtained cases from The Cancer Genome Atlas (TCGA) database, including 498 lung squamous cell cancer (LUSC) patients and 515 lung adenocarcinoma (LUAD) patients. We studied the lung caner driver gene in LUSC and LUAD. On the other hand, PD-L1 expression was detected in lung cancer tissues of 1,008 NSCLC patients with immunohistochemistry staining (IHC), and we studied the correlation between PD-L1 protein expression and clinicopathological characteristics. PD-L1 expression was higher in LUSC than in LUAD at the mRNA level. In univariate analysis, PD-L1 expression at the protein level was higher in patients who were males, were LUSC, were smokers, had a tumor diameter >3 cm, had poor differentiation, or had stages III~IV disease. In multivariate analysis, PD-L1 expression was higher in patients who were LUSC or in poor differentiation. In term of protein level, PD-L1 expression was higher in NSCLC patients who were LUSC or in poor differentiation. We recommend that PD-L1 IHC detection can be routinely performed in such populations that are likely to benefit most from PD-L1 immunotherapy.

High PD-L2 Predicts Early Recurrence of ER-Positive Breast Cancer.

T-cell-mediated cytotoxicity is suppressed when programmed cell death-1 (PD-1) is bound by PD-1 ligand-1 (PD-L1) or PD-L2. Although PD-1 inhibitors have been approved for triple-negative breast cancer, the lower response rates of 25%-30% in estrogen receptor-positive (ER+) breast cancer will require markers to identify likely responders. The focus of this study was to evaluate whether PD-L2, which has higher affinity than PD-L1 for PD-1, is a predictor of early recurrence in ER+ breast cancer. PD-L2 protein levels in cancer cells and stromal cells of therapy-naive, localized or locoregional ER+ breast cancers were measured retrospectively by quantitative immunofluorescence histocytometry and correlated with progression-free survival (PFS) in the main study cohort (n = 684) and in an independent validation cohort (n = 273). All patients subsequently received standard-of-care adjuvant therapy without immune checkpoint inhibitors. Univariate analysis of the main cohort revealed that high PD-L2 expression in cancer cells was associated with shorter PFS (hazard ratio [HR], 1.8; 95% CI, 1.3 to 2.6; P = .001), which was validated in an independent cohort (HR, 2.3; 95% CI, 1.1 to 4.8; P = .026) and remained independently predictive after multivariable adjustment for common clinicopathological variables (HR, 2.0; 95% CI, 1.4 to 2.9; P < .001). Subanalysis of the ER+ breast cancer patients treated with adjuvant chemotherapy (n = 197) revealed that high PD-L2 levels in cancer cells associated with short PFS in univariate (HR, 2.5; 95% CI, 1.4 to 4.4; P = .003) and multivariable analyses (HR, 3.4; 95% CI, 1.9 to 6.2; P < .001). Up to one third of treatment-naive ER+ breast tumors expressed high PD-L2 levels, which independently predicted poor clinical outcome, with evidence of further elevated risk of progression in patients who received adjuvant chemotherapy. Collectively, these data warrant studies to gain a deeper understanding of PD-L2 in the progression of ER+ breast cancer and may provide rationale for immune checkpoint blockade for this patient group.

ALKBH5 promotes PD-L1-mediated immune escape through m6A modification of ZDHHC3 in glioma

N6-methylation of adenosine (m6A) is one of the most frequent chemical modifications in eukaryotic RNAs and plays a vital role in tumorigenesis and progression. Recently, emerging studies have shown that m6A modification by ALKBH5 was associated with immunotherapy response in various types of cancer. However, whether m6A demethylases ALKBH5 participate in regulating the tumor immune microenvironment and the efficacy of immunotherapy in glioblastoma remain unknown. Here, we found that deletion of ALKBH5 significantly inhibited the growth of glioma allografts, rescued the antitumoral immune response, and increased cytotoxic lymphocyte infiltration and proinflammatory cytokines in CSF while significantly suppressing PD-L1 protein expression. m6A-methylated RNA immunoprecipitation sequencing and RNA sequencing identify ZDDHC3 as the direct target of ALKBH5. Mechanically, ALKBH5 deficiency impairs the YTHDF2-mediated stability of ZDHHC3 mRNA, thereby suppressing PD-L1 expression by accelerating PD-L1 degradation in glioma. In addition, genetic deletion or pharmacological inhibition of ALKBH5 with IOX1 enhances the therapeutic efficacy of anti-PD-1 treatment in preclinical mice models. These data suggest that the combination of anti-PD-1 therapy and ALKBH5 inhibition may be a promising treatment strategy in glioma.

Discovery of small-molecule PD-1/PD-L1 antagonists through combined virtual screening and experimental validation

Inhibition of the interaction between the PD-1 protein on activated lymphocytes and the PD-L1 protein on tumors represents a novel therapeutic approach for selective activation of the innate immune response against a variety of cancers. Therefore, the present study utilized a combined virtual and experimental screening approach to screen databases of both lead-like and larger molecules for identification of novel inhibitors of PD-1/PD-L1 interaction. First, high-throughput virtual screening of ∼3.7 million lead-like molecules using a rigid-receptor docking approach against both human PD-1 and PD-L1 proteins revealed possible small-molecule tractability of PD-1, but not PD-L1, binding interface. The subsequent work, therefore, involved screening of the National Cancer Institute (NCI) compound database against the PD-1 pocket. Several NCI compounds were identified with potential to bind to the PD-1 pocket and in turn inhibit the PD-1/PD-L1 interaction. The dynamic binding behavior of these molecules was further investigated using long 100 ns molecular dynamics (MD) stimulation revealing NSC631535 to be a potentially stable binder at PD-1 interface pocket. In support of these MD data, the experimental testing of NSC631535 exhibited 50% inhibition at ∼15 μM test concentration. The observed activity of this compound is promising as despite its relatively low molecular weight (415.5 g/mol) it is still capable of inhibiting the PD-1/PD-L1 interaction having a large interface area (∼1970 Å2). In summary, our integrated computational and experimental screening led to identification of a novel PD-1 antagonist that may serve as a starting point for further optimization into more potent small-molecule PD-1/PD-L1 inhibitors for cancer immunotherapy.

Potential Role of Tumor-Derived Exosomes in Non-Small-Cell Lung Cancer in the Era of Immunotherapy.

Lung cancer, of which non-small-cell lung cancer (NSCLC) represents about 80% of all cases, is the second most common cancer diagnosed in the general population and one of the major causes of cancer-related deaths worldwide. Overall, the outcomes of patients with advanced NSCLC are still disappointing despite advances in diagnosis and treatment. In recent years immune-checkpoint inhibitors (ICIs), administered alone or in combination with chemotherapy, have revolutionized the treatment landscape of patients with advanced non-small-cell lung cancer. However, until now, tissue expression of PD-L1 and tumor mutation burden represent the only available biomarkers for NSCLC patients treated with ICIs. A growing body of evidence showed that tumor-derived exosomes (TDEs) have the PD-L1 protein on their surface and that they are involved in angiogenesis, tumor growth, invasion, metastasis and immune escape. This review focused on the potential clinical applications of TDEs in NSCLC, including their possible role as a biomarker for prognosis and disease monitoring in patients undergoing immunotherapy.

In silico mapping of the dynamic interactions and structure-activity relationship of flavonoid compounds against the immune checkpoint programmed-cell death 1 pathway

Flavonoids are a class of natural products widely available in medicinal and dietary plants. Their pharmacological use has shown the potential to reduce the risk of different types of cancer, among other prevalent diseases. Their molecular scaffold inhibits the PD-1/PD-L1 axis, an important pathway related to the adaptive immune resistance of cancer cells already targeted for developing new cancer immunotherapy. However, despite the availability of kinetic and thermodynamic experimental data on the flavonoid–PD-1/PD-L1 interaction, there is still a lack of reliable information about their binding mode at the atomic level. Thus, we aimed to computationally predict the binding mode of flavonoid molecules with PD-1 and/or PD-L1 proteins using unbiased computational methodologies such as blind docking and supervised molecular dynamics simulation. The molecular interactions and dynamics of these predicted poses of protein-flavonoid complexes were further analyzed through multiple molecular dynamics simulations. This information, corroborated with the IC50 and KD values from available literature, was used to perform molecular matched-pair analysis to comprehensively describe the main interactions governing the inhibition of the complex PD-1/PD-L1 by the flavonoid scaffold. By analyzing the effect of substitutions in such a scaffold, we observed a clear correspondence with literature binding assays. Thus, we propose, for dimeric PD-L1, that the 7-O-glucoside forces the molecule displacement in the dimer interface. Furthermore, the 3-OH plays an essential role in stabilizing the buried binding mode by water-bridged hydrogen bonds with Asp122 and Gln66 in both extremities of the pocket. In PD-1, we suggest that flavonoids could bind through the BC loop by inducing a flip of Phe56 after a conformational change of the Asn58 glycosylation. Hence, our results introduced unprecedented information on flavonoid interaction and dynamics when complexed with PD-1 checkpoint pathway proteins and can pave the road for developing new flavonoid derivatives with selective anticancer activity.

Inhibition of EZH2 Ameliorates Sepsis Acute Lung Injury (SALI) and Non-Small-Cell Lung Cancer (NSCLC) Proliferation through the PD-L1 Pathway.

(1) Background: Both sepsis acute lung injury (SALI) and non-small-cell lung cancer (NSCLC) are life-threatening diseases caused by immune response disorders and inflammation, but the underlining linking mechanisms are still not clear. This study aimed to detect the shared gene signature and potential molecular process between SALI and NSCLC. (2) Methods: RNA sequences and patient information on sepsis and NSCLC were acquired from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) was used to build a co-expression network associated with sepsis and NSCLC. Protein-protein interaction (PPI) analysis of shared genes was intuitively performed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The involvement of EZH2 in the tumor immune microenvironment (TIME) and sepsis immune microenvironment (IME) was assessed by R software. Western blot, flow cytometry, and other in vitro assays were performed to further confirm the function and mechanism of EZH2 in NSCLC and SALI. (3) Results: WGCNA recognized three major modules for sepsis and two major modules for NSCLC, and there were seven shared genes identified for the two diseases. Additionally, the hub gene EZH2 was screened out. It was shown that EZH2 was closely related to the IME in the two diseases. In the validation assay, our data showed that EZH2 was expressed at a higher level in peripheral blood mononuclear cells (PBMCs) of septic patients than those of healthy donors (HDs), and EZH2 was also expressed at a higher level in lipopolysaccharide (LPS)-induced PBMCs and non-small cell lung cancer (A549) cells. EZH2 inhibitor (GSK343) downregulated the proliferation ability of A549 cells in a concentration-dependent manner, parallel with the decreased expression level of PD-L1. Similarly, GSK343 inhibited PD-L1 protein expression and downregulated the level of proinflammatory factors in LPS-induced PBMCs. In the co-culture system of PBMCs and human type II alveolar epithelial cells (ATIIs), the addition of GSK343 to PBMCs significantly downregulated the apoptosis of LPS-induced ATIIs. (4) Conclusions: This study illustrated that EZH2 inhibition could ameliorate A549 cell proliferation and LPS-induced ATII apoptosis in parallel with downregulation of PD-L1 protein expression, which provided new insights into molecular signaling networks involved in the pathogenetics of SALI and NSCLC.

A Mathematical Model for the Treatment of Melanoma with the BRAF/MEK Inhibitor and Anti-PD-1

Skin cancer treatment is a combination of BRAF and MEK kinase inhibitors administered as tablets, along with immunotherapy treatment (treatment into the vein) with a group of drugs that inhibit the activity of the immune barrier proteins PD-1 and PDL1. Here, we propose a new approach to the therapy for melanoma with the BRAF/MEK inhibitor and anti-PD-1. With the help of explicit analytical functions, we were able to model this combined treatment and present the treatment in a mathematical model described by a system of differential equations including variables, such as Treg, IL12, Il10, TGF-β, and cytokine, which are significant variables that are all critical factors which determine the effectiveness of therapies. The most significant advantage of a treatment described by a mathematical model with explicit analytical functions is the control of parameters, such as time and dose, which are variable critical parameters in the treatment, that is, these parameters can be adapted to the patient’s personalized treatment. In the current study, we showed that by simultaneously changing and combining these two parameters, we could decrease the tumor volume. To validate the numerical results, we computed the relative error between the results obtained from the mathematical model and clinical data.

YB-1 promotes cell proliferation and metastasis by targeting cell-intrinsic PD-1/PD-L1 pathway in breast cancer.

Programmed cell death 1 (PD-1) suppresses T effector functions by inhibiting signaling downstream of the T cell receptor and helping tumor cells escape the immune response. However, the effect and mechanism of cell-intrinsic PD-1 in cancer cells are still unknown. Here, we found that PD-1 is aberrantly upregulated in TNBC patients and cell lines. Cell-intrinsic PD-1 in TNBC cells significantly facilitated tumor growth and metastasis in vitro and in vivo. Further studies indicated that PD-1 effect on TNBC cell growth depends on the cell-intrinsic-PD-1/PD-L1 pathway independent of adaptive immunity. In addition, we further found that the activation of cell-intrinsic PD-1/PD-L1 pathway in TNBC cells is regulated by the gene expression regulator YB-1. Mechanistically, the results of protein degradation analysis, mRNA translationally active analysis, CLICK chemistry and L-azidohomoalanine (AHA) incorporation assays, immunoprecipitation assay and Dual-Luciferase reporter assay showed that YB-1 promotes PD-1 expression through the translational activation pathway. We provide in vitro and in vivo evidence that silencing YB-1 expression in TNBC cells inhibits cell proliferation, tumorigenesis, and metastasis. However, this inhibition can be rescued by simultaneous exogenous expression of PD-1 and PD-L1 proteins. In conclusion, our results identify TNBC cell-intrinsic functions of the PD-1/PD-L1 axis in tumor growth and metastasis; and revealed PD-1/PD-L1 is a critical effector of YB-1-mediated TNBC proliferation and metastasis in vitro and in vivo.

A cohort study using IL-6/Stat3 activity and PD-1/PD-L1 expression to predict five-year survival for patients after gastric cancer resection.

The expression/activation of IL-6, p-Stat3, PD-1 and PD-L1 in gastric cancer (GC) tissues were examined to evaluate their abilities in predicting the survival prognosis in postoperative patients with GC. The clinicopathological data and paraffin-embedded tissues of 205 patients who underwent gastric cancer resection were collected at the First Affiliated Hospital of Shihezi University School of Medicine, and the patients were followed-up annually after surgery. Immunohistochemistry (IHC) was used to detect the expression of IL-6, p-Stat3, PD-1 and PD-L1 proteins using tissue microarrays derived from these patients. Statistical analyses were performed using non-parametric tests, Spearman's correlation, ROC curves, Kaplan-Meier survival analysis, Cox single-factor and multifactor regression models. In comparison, the analyses were also performed for GC patients from public databases (407 patients from TCGA and 433 patients from GEO, respectively). (1) The expression levels of IL-6, p-Stat3, PD-1 and PD-L1 in GC tissues were significantly higher than adjacent normal tissues (ANT) (81.01% vs. 52.78%, P<0.001; 100% vs. 93.41%, P<0.001; 58.58% vs. 40.12%, P<0.001; 38.20% vs. 26.90%, P = 0.025, respectively). The mean optical density (MOD) values of IL-6, p-Stat3, PD-1 and PD-L1 were significantly higher in GC tissues. (2) The higher the levels of IL-6 (P<0.001), p-Stat3 (P<0.001), and PD-L1 (P = 0.003) were, the worse the survival prognoses were observed, respectively, among GC patients. The expression of PD-1 was not correlated with the prognosis of GC patients (P>0.05). The lower the degree of cell differentiation (P<0.001) was, the worse the survival prognoses were observed among GC patients. (3) Independent risk factors for postoperative prognosis in GC patients included age (≥60 years old), poor cell differentiation, invasion depth (T3/T4), lymph node metastasis (N1-3), distant metastasis (M1), and high levels of IL-6 (2+/3+). (4) A multi-factor combination (cell differentiation+IL-6+p-Stat3+PD-1+PD-L1) appeared to be the best survival predictor for GC patients as indicated by AUC (AUC 0.782, 95% CI = 0.709, 0.856, P<0.001). This combination may be the optimal predictor for postoperative survival of GC patients. (5) The levels of IL-6, p-Stat3, PD-1 and PD-L1 correlated with the infiltration levels of various tumor-infiltrating immune cells. (6) The analyses of ROC curves, calibration, DCA and Kaplan-Meier (KM) survival curves in TCGA dataset confirmed that the nomogram model could accurately predict the prognosis in GC patients. (1) The expressed levels of IL-6, p-Stat3, PD-1 and PD-L1 are higher in GC tissues than in adjacent normal tissues. (2) The high levels of IL-6, p-Stat3 and PD-L1 are correlated with poor survival in GC patients. (3) The high levels of IL-6, p-Stat3, PD-1 and PD-L1 have influences in GC tumor microenvironment. (4) The multi-predictor combination of "IL-6+p-Stat3+PD-1+cell differentiation" serves as an optimal survival predictor for postoperative GC patients and better than the TNM staging system. As these molecules can be examined in preoperative biopsies, these observations may provide a useful guide for clinicians to strategize individualized surgical plans for GC patients before surgery.

Investigation of prognostic biomarkers in patients with urothelial carcinoma treated with platinum-based regimens.

Bladder cancer (BC) is a heterogeneous malignancy with dismal outcome. Mutations in genes, altered or linked to platinum sensitivity in BC, were examined in 66 patients' tumors along with tumor infiltrating lymphocytes (TILs) density and MMR, PD-L1 and CD8 protein expression, as well as basal and luminal subtypes, defined by protein expression of markers, including CK5/6 and GATA3 or CK20, respectively. 41 tumors harbored mutations, mainly in TP53 (38%), ARID1A (17%) and the DNA damage response and repair (DDR) genes ERCC2 (17%) and BRCA2 (15%). Mutations in other DDR relevant genes were also present. Age showed unfavorable prognosis for overall survival (HR=1.07, P = 0.026); no benefit was seen for patients with TP53, ARID1A, ERCC2 or BRCA2 mutations or mutations in 1 or more DDR genes. PD-L1 status positively correlated with stromal (rho=0.46, P < 0.001) and intratumoral (rho=0.53, P < 0.001) CD8 expression or TILs (rho=0.29, P = 0.018); none associated with overall survival (OS). A statistically significant difference was observed between PD-L1 status and immunohistochemistry (IHC)‑based subtypes, with tumors classified as luminal (GATA3+ and/or CK20+ and CK5/6-) showing lower PD-L1 expression relative to basal (CK5/6+ and GATA3- and/or CK20-) (median value 0 vs. 2.5, P = 0.029). Concerning OS, no statistically significant difference was seen among patients with basal or luminal tumors. No association was seen herein between DDR mutations, TILs, PD-L1, CD8 expression or IHC-based subtypes and patient survival; these observations warrant validation within a larger cohort.

Effect of HDAC Inhibitor Chidamide on PD-L1 Expression in Peripheral T-Cell Lymphoma

To explore the role of chidamide in the regulatory mechanism of PD-1/PD-L1 immune escape signaling pathway in peripheral T-cell lymphoma. Jurkat cell line was treated with different concentrations of chidamide. The changes of PD-L1 and JAK/STAT pathway gene mRNA expression and PD-L1 protein expression on cell surface were detected by fluorescence quantitative PCR and flow cytometry after treatment. Chidamide upregulated PD-L1 mRNA expression in Jurkat cell line in a dose-dependent manner (r=0.989). The mRNA expression of PD-L1 in 5.0 μmol/L group was 15.4 times higher than that in the control group. The proportion of PD-L1 positive cells in Jurkat cell line was less than 0.5%. Chidamide upregulated PD-L1 protein expression on Jurkat cell surface. Chidamide upregulated the mRNA expression of JAK2, STAT1 and STAT3 in Jurkat cell line. The level of up-regulation was obvious in high concentration group (5.0 μmol/L group). Meanwhile, the mRNA expression of SOCS1 and SOCS3, the negative regulatory genes upstream of the JAK/STA T pathway, were up-regulated. In peripheral T-cell lymphoma, chidamide may up-regulate the expression of cell surface PD-L1 and induce T-cell chemokines by upregulation of STAT1 expression, thus improving the reaction rate of PD-1 monoclonal antibody and T-cell toxicity.

The tumor microenvironment and immune targeting therapy in pediatric renal tumors.

This review highlights the role of several immunomodulating elements contributing to the tumor microenvironment of various pediatric renal tumors including Wilms tumor. The roles of innate and adaptive immune cells in renal tumors are summarized as well as immunomodulatory cytokines and other proteins. The expression and the predictive role of checkpoint modulators like PD-L1 and immunomodulating proteins like glypican-3, B7-H3, COX-2 are highlighted with a translational view toward potential therapeutic innovations. We further discuss the current state of preclinical models in advancing this field of study. Finally, examples of clinical trials of immunomodulating strategies such as monoclonal antibodies and chimeric antigen receptor T (CAR-T) cells for relapsed/refractory/progressive pediatric renal tumors are described.

Reprogramming of T cell-derived small extracellular vesicles using IL2 surface engineering induces potent anti-cancer effects through miRNA delivery.

T cell-derived small extracellular vesicles (sEVs) exhibit anti-cancer effects. However, their anti-cancer potential should be reinforced to enhance clinical applicability. Herein, we generated interleukin-2-tethered sEVs (IL2-sEVs) from engineered Jurkat T cells expressing IL2 at the plasma membrane via a flexible linker to induce an autocrine effect. IL2-sEVs increased the anti-cancer ability of CD8+ T cells without affecting regulatory T (Treg ) cells and down-regulated cellular and exosomal PD-L1 expression in melanoma cells, causing their increased sensitivity to CD8+ T cell-mediated cytotoxicity. Its effect on CD8+ T and melanoma cells was mediated by several IL2-sEV-resident microRNAs (miRNAs), whose expressions were upregulated by the autocrine effects of IL2. Among the miRNAs, miR-181a-3p and miR-223-3p notably reduced the PD-L1 protein levels in melanoma cells. Interestingly, miR-181a-3p increased the activity of CD8+ T cells while suppressing Treg cell activity. IL2-sEVs inhibited tumour progression in melanoma-bearing immunocompetent mice, but not in immunodeficient mice. The combination of IL2-sEVs and existing anti-cancer drugs significantly improved anti-cancer efficacy by decreasing PD-L1 expression in vivo. Thus, IL2-sEVs are potential cancer immunotherapeutic agents that regulate both immune and cancer cells by reprogramming miRNA levels.

RBM10 Is a Biomarker Associated with Pan-Cancer Prognosis and Immune Infiltration: System Analysis Combined with In Vitro and Vivo Experiments.

RNA binding motif protein 10 (RBM10) is a splicing factor that has been reported to be involved in the occurrence and progression of multiple malignancies. However, the RBM10 involvement in pan-cancer immunotherapy is not clear. In here, we provide the first comprehensive assessment of the prognostic value and immunological function of RBM10 in human pan-cancer utilizing multiple public databases. Data reveal the aberrant RBM10 expression in most tumors, and its expression is positively or negatively linked with the clinical prognosis of various cancers, depending on the different types and subtypes of cancers. In most tumors, RBM10 mutations are frequently occurred, which is closely related to tumor progression. Moreover, our results also show that RBM10 is considerably linked with most of the immune checkpoint genes, tumor immune cell infiltration, tumor mutation burden, and microsatellite instability. Additionally, RBM10 is significantly positively correlated with the sensitivity of trametinib, 17-AAG, PD-0325901, RDEA119, cetuximab, and afatinib, indicating potential antagonism between RBM10 inhibitors and these antitumor drugs, and more likely to develop drug resistance. We also verify that downregulation of RBM10 enhances the malignant phenotype of lung adenocarcinoma cells using in vitro cell experiments, and in vivo animal experiments show that the overexpression of RBM10 reduces the growth of tumors. Furthermore, upregulating RBM10 greatly reduces the PD-L1 protein levels, while silencing RBM10 considerably enhances PD-L1 protein levels. Moreover, the overexpression of RBM10 decreases the protein stability of PD-L1. To sum up, our pan-cancer analysis indicates that RBM10 is a promising biomarker for prognosis and immunotherapy, which provides a new insight for cancer immunotherapy.

Construction of stable membranal CMTM6-PD-L1 full-length complex to evaluate the PD-1/PD-L1-CMTM6 interaction and develop anti-tumor anti-CMTM6 nanobody.

CKLF (chemokine-like factor)-MARVEL transmembrane domain containing protein 6 (CMTM6) is a novel regulator to maintain the stability of PD-L1. CMTM6 can colocalize and interact with PD-L1 on the recycling endosomes and cell membrane, preventing PD-L1 from lysosome-mediated degradation and proteasome-mediated degradation thus increasing the half-life of PD-L1 on the cell membrane. The difficulties in obtaining stable full-length PD-L1 and CMTM6 proteins hinder the research on their structures, function as well as related drug development. Using lauryl maltose neopentyl glycol (LMNG) as the optimized detergent and a cell membrane mimetic strategy, we assembled a stable membrane-bound full-length CMTM6-PD-L1 complex with amphipol A8-35. When the PD-1/PD-L1-CMTM6 interactions were analyzed, we found that CMTM6 greatly enhanced the binding and delayed the dissociation of PD-1/PD-L1, thus affecting immunosuppressive signaling and anti-apoptotic signaling. We then used the CMTM6-PD-L1 complex as immunogens to generate immune repertoires in camels, and identified a functional anti-CMTM6 nanobody, called 1A5. We demonstrated that the anti-CMTM6 nanobody greatly decreased T-cell immunosuppression and promoted apoptotic susceptibility of tumor cells in vitro, and mainly relied on the cytotoxic effect of CD8+ T-cells to exert tumor growth inhibitory effects in CT26 tumor-bearing mice. In conclusion, the stable membrane-bound full-length CMTM6-PD-L1 complex has been successfully used in studying PD-1/PD-L1-CMTM6 interactions and CMTM6-targeting drug development, suggesting CMTM6 as a novel tumor immunotherapy target.