Co-expression Of PD-L1 Research Articles

PD-L1 binds with B7-1 only in cis on the same cell surface

Abstract Programmed death ligand 1 (PD-L1)-mediated immune suppression is a major regulator of peripheral tolerance and is often co-opted by tumors to evade immune attack. PD-L1 binds to PD-1 but in addition binds to B7-1 (CD80) to regulate T cell function. The binding interactions of the PD-L1-B7-1 pathway and its functional role need further investigation in order to understand differences between PD-1 and PD-L1 mediated tumor immunotherapy. We examined the molecular orientation for PD-L1 binding to B7-1 using cell to cell binding assays, ELISA, and flow cytometry. Surprisingly, we found that PD-L1 transfected cells did not bind to B7-1 transfected cells, whereas PD-L1 transfected cells bound to PD-1 transfected cells. As expected, B7-1 transfected cells bound to CD28 or cytotoxic T-lymphocyte antigen 4 (CTLA-4) transfected cells. By ELISA and flow cytometry with purified proteins, we found that PD-L1 and B7-1 had a strong binding interaction only when the PD-L1 molecule was accessible and flexible. On decreasing its accessibility, the binding was greatly reduced. We observed the PD-L1-B7-1 interaction competed for the binding of PD-L1 to PD-1. Further, we co-transfected PD-L1 and B7-1 on the same cell surface and confirmed the cis binding using a Nanobit Assay system. These results indicate that the PD-L1-B7-1 interaction can occur in cis when the molecules are on the same cell but not in trans when the molecules are on different cells. This binding orientation emphasizes the functional importance of co-expression of B7-1 and PD-L1 on the same cell. Interaction in cis competes for binding to other receptors and modulates their function. Our findings help in further understanding PD-L1 mediated immune suppression.

Programmed death-1 ligands 1 and 2 expression in cutaneous squamous cell carcinoma and their relationship with tumour- infiltrating dendritic cells.

The programmed death-1 (PD-1) receptor ligands, PD-L1 and PD-L2, are co-stimulatory molecules that contribute to the negative regulation of T lymphocyte activation. It is still unclear whether there is correlation between PD-L1 or PD-L2 and tumour-infiltrating dendritic cells (TIDCs) in cutaneous squamous cell carcinoma (CSCC). The aim of this study was to analyse PD-L1 and PD-L2 expression and dendritic cells infiltration in tumour tissue of CSCC patients and investigate their clinical significance. Immunohistochemical analysis was used to evaluate the expression of PD-L1, PD-L2, CD1a and CD83 in 61 CSCC tissues. The immunofluoresence double-labelling technique was performed to detect the co-expression of PD-L1 or PD-L2 and CD1a or CD83 in tumour tissues. We found that 25 of 61 cases CSCC (40·98%) exhibited positivity for PD-L1, whereas 37 of 61 cases CSCC (60·66%) exhibited positivity for PD-L2. A higher percentage of CD1a-positive cases were observed on both PD-L1-positive and PD-L2-positive specimens compared with that of CD83-positive cases (92·29% versus 37·60%, 83·20% versus 33·16%). The expression of PD-L1 and PD-L2 on CD1a+ cells was significantly higher than that on CD83+ cells in tumour tissues of CSCC patients. Furthermore, the expression rate of PD-L1 was associated with UICC stage, and the expression rate of PD-L2 was associated with predominant differentiation and tumour size in CSCC. Our results indicated that higher expression of PD-L1 and PD-L2 on CD1a+ cells than that on CD83+ cells in CSCC tumour tissues may contribute to negative regulation in anti-tumour immune responses.

A Safety Checkpoint to Eliminate Cancer Risk of the Immune Evasive Cells Derived from Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) hold great promise in the regenerative therapy of many currently untreatable human diseases. One of the key bottlenecks is the immune rejection of hESC-derived allografts by the recipient. To overcome this challenge, we have established new approaches to induce immune protection of hESC-derived allografts through the coexpression of immune suppressive molecules CTLA4-Ig and PD-L1. However, this in turn raises a safety concern of cancer risk because these hESC-derived cells can evade immune surveillance. To address this safety concern, we developed a safety checkpoint so that the immune evasive hESC-derived cells in the graft can be effectively eliminated if any cellular transformation is detected. In this context, we knock-in the suicidal gene herpes simplex virus thymidine kinase (HSVTK) into the constitutive HPRT locus of CP hESCs (knock-in hESCs expressing CTLA4-Ig and PD-L1), denoted CPTK hESCs. Employing humanized mice (Hu-mice) reconstituted with human immune system, we demonstrated that the CPTK hESC-derived cells are protected from immune rejection. In addition, CPTK hESC-derived cells can be efficiently eliminated in vitro and in vivo with FDA approved TK-targeting drug ganciclovir. Therefore, this new safety checkpoint improves the feasibility to use the immune evasive hESC-derived cells for regenerative medicine. Stem Cells 2017;35:1154-1161.

P2.01-046 Quantitative Measurement of B7-H3 Protein Expression and Its Association with B7-H4, PD-L1 and TILs in NSCLC

B7-H3 (CD276) is a type I transmembrane protein that belongs to the B7 immunoregulatory family including PD-L1 (B7-H1) and is upregulated in multiple malignancies including Non-Small Cell Lung Cancer (NSCLC). Clinical activity of monoclonal B7-H3 blocking antibodies such as Enoblituzumab are under investigation. In this study we measured the levels of B7-H3 protein in NSCLC and studied its association with major tumor infiltrating lymphocyte (TIL) subsets, levels of PD-L1, B7-H4 and clinico-pathological characteristics in three independent NSCLC cohorts. We used automated quantitative immunofluorescence (QIF) to assess the levels of B7-H3 (clone D9M2L, CST), PD-L1 (clone SP142, Spring), B7-H4 (Clone D1M8I, CST) CD3, CD8 and CD20 in 634 NSCLC cases from 3 retrospective cohorts represented in tissue microarray format. The targets were selectively measured in the tumor and stromal compartments using co-localization with cytokeratin. Associations between the marker levels, major clinic-pathological variables and survival were analyzed. Expression of B7-H3 protein was found in 80.4% (510/634) of the cases and the levels were higher in the tumor than in the stromal compartment. High B7-H3 protein expression level (top 10 percentile) was associated with poor survival in two out of three of the cohorts (p <0.05). Elevated B7-H3 was consistently associated with smoking history across the 3 cohorts, but not with sex, age, clinical stage and histology. Co-expression of B7-H3 and PD-L1 was found in 17.6% of the cases (112/634) and with B7-H4 in 10% (63/634). B7-H4 and PD-L1 were simultaneously detected only in 1.8% of NSCLCs (12/634). The expression of B7-H3 was not associated with the levels of CD3, CD8 and CD20 positive TILs. B7-H3 protein is expressed in the majority of NSCLCs and is associated with smoking history. High B7-H3 protein levels may have a prognostic effect in lung carcinomas. Elevated levels of B7-H3 are not associated with lymphocyte infiltration. Co-expression of B7-H3 with PD-L1 and B7-H4 is relatively low, suggesting a non-redundant biological role of these targets and possibilities for combination therapies using monoclonal antibodies.

P1.05-003 Coexpression of CD8a and PD-L1 Frequently Observed in Resected NSCLC Tumors from Smokers

With the approval of anti-programmed cell death-1 (PD-1) therapy in advanced non-small cell lung cancer (NSCLC), identifying patients with early stage disease most likely to benefit from therapy has become a priority. It has been hypothesized that patients whose tumors show evidence of PD-1 mediated T cell exhaustion, via the presence of both tumor infiltrating lymphocytes (TILs) and PD-L1 expression, are more likely to respond to anti-PD-1 therapy (Teng et al, 2015). The current study utilized microarray analysis to evaluate the relationship between both clinicopathologic features and overall survival (OS) with tumor microenvironment (TME) composition. Gene expression microarray analysis was performed using the Agilent Whole Human Genome 4x44K 2-color platform for 319 NSCLC and 15 normal resection specimens. The reference sample was an equal mixture of 258 of the NSCLC samples. Rosetta Resolver and Statistica 13.0 were used for analysis. Samples with PD-L1 expression levels greater or unchanged from reference level were classified as positive, while those significantly lower [log (ratio) <0 and p<0.01] than the reference were classified as negative. CD8a expression was used as a surrogate for TILs as previously described by Ock et al. (2016), and categorized in the same manner as PD-L1. Relationships between TME composition and clinicopathologic features were evaluated with the chi-square test. Survival analysis was performed using the Kaplan-Meier method and compared using the log-rank test. In the 319 NSCLC samples the incidence of a Type I TME (+CD8a/+PD-L1) was 45%, Type II TME (-CD8a/-PD-L1) 12%, Type III (-CD8a/+PD-L1) 25%, and Type IV (+CD8a/-PD-L1) 18%. When assessing for survival, patients with a PD-L1 negative/CD8a positive (Type IV) TME had improved OS compared to patients with PD-L1 negative/CD8a negative (Type II) TME (p=0.02). When assessed for smoking, ever smokers were more likely to evidence a PD-L1 positive/CD8a positive (type I) TME compared to never smokers, 49% vs 32%, while never smokers more frequently evidenced a PD-L1 positive/CD8a negative (Type III) TME compared to ever smokers, 37% vs 22% (P=0.05). Interestingly, 75% of normal lung samples evidenced a PD-L1 positive/CD8a positive microenvironment. Evidence of both TILs and PD-L1 expression was observed in the majority of normal lung specimens and also more frequently in tumors from smokers compared to non-smokers. Patients whose tumors showed evidence of CD8a, but not PD-L1, had improved OS compared to patients without evidence of either. Future studies will utilize immunohistochemistry to corroborate these findings and investigate other components of the TME.

MTE11.01 The Clinical Impact of the 2015 WHO Classification of Lung Tumors

MTE11.01 The Clinical Impact of the 2015 WHO Classification of Lung Tumors

332O Co-expression of PD-L1 and p-AKT is associated with poor prognosis in diffuse large B-cell lymphoma via PD-1/PD-L1 axis activating intracellular AKT/mTOR pathway in tumor cells

332O Co-expression of PD-L1 and p-AKT is associated with poor prognosis in diffuse large B-cell lymphoma via PD-1/PD-L1 axis activating intracellular AKT/mTOR pathway in tumor cells

10PD - Assessment of PD-L1 and CD47 expression together with tumor-associated TILs in resectable early stage NSCLC

Aim/Background: Solid tumors have been shown to evade host antitumor immunity through upregulation of the immune checkpoint PD-1/PD-L1 pathway. However, CD47, an antiphagocytic ligand expressed on tumor cells, represents another cell surface molecule promoting tumor immune evasion via targeting the innate immune system. We investigated whether PD-L1 and CD47 are co-expressed in early stage non-small cell lung cancer (NSCLC). Methods: Resected tumor and matched adjacent normal tissue from 84 stage I-III NSCLCs (42 adenocarcinomas [Adeno]; 42 squamous cell carcinomas [SqCC]) were processed to single cell suspensions, stained with a panel of antibodies (CD45, CD31, CD14, EpCAM, CD73, CD90, PD-L1, CD47) and subjected to multicolor flow cytometric analysis. In parallel, the phenotype of tumor infiltrating lymphocytes (TILs) was also performed using CD19, CD45RO, CD3, CD4, CD8, PD-1, CD127 and CD107a. In a subset of patients, expression of PD-L1 and CD47 was confirmed via immunohistochemistry. Results: PD-L1 expression on tumor epithelium (EpCAM+) was increased in both Adeno (p = 0.0295) and SqCC (p = 0.0016) patients. CD47 was also upregulated on the tumor EpCAM+ fraction in SqCC (p = 0.05) but not in Adeno patients (p = 0.124). An increase in the tumor mesenchymal fraction in both Adeno (p = 0.015) and SqCC (p = 0.027) patients was found that showed an enhanced expression of PD-L1 but not CD47. Immunohistochemical analysis confirmed coexpression of PD-L1 and CD47 in a subset of patients. Finally, there was an increase in CD4+PD-1hi TILs in Adeno patients, whereas a decrease in CD127 expression was found on CD8+ TILs (p < 0.0001) with a CD107aloPD1hi phenotype in both histological subtypes. Conclusions: We detected PD-L1 expression in different compartments in a subset of patients with early stage Adeno and SqCC, whereas CD47 expression was restricted to the tumor epithelial compartment in SqCC patients only. These differences may be related to the intragraft immune priming inside the tumor microenvironment. Further study is required to determine whether dual targeting of PD-L1 and CD47 in the perioperative setting represents a promising therapeutic strategy to reinvigorate TILs in affected patients. Legal entity responsible for the study: N/A Funding: Inselspital, University Hospital of Bern, Division of Thoracic Surgery Disclosure: All authors have declared no conflicts of interest.

Coexpression of TrkB and PD-L1 on human placenta

Objective: We performed comprehensive miRNA and gene expression profiling of maternal peripheral blood natural killer (pNK) cells during pregnancy. Methods: Samples of pNK cells at different time points of pregnancy (first, second, and third trimester of gestation, and postdelivery) were obtained from healthy pregnant females with their informed consent. We performed real-time PCR-based array analysis to examine the expression levels of 756 miRNAs in maternal pNK cells using TaqMan Array MicroRNA Cards. Moreover, we performed DNA microarray analysis of gene expression levels in firstand third-trimester pNK cells using Agilent Microarray Kits. Results: PCR-based array analysis showed transfer of placentaderivedmiRNAs [chromosome19miRNAcluster (C19MC)miRNAs] into maternal pNK cells occurred during pregnancy. Rapid clearance of C19MC miRNAs also occurred in pNK cells after delivery. DNA microarray analysis identified NK cell function-related genes (e.g., KIR genes) that were differentially expressed between firstand third-trimester pNK cells. Conclusions: Maternal pNK cells may adjust to different gestational conditions via the uptake of exogenousmiRNAs (e.g., C19MC miRNAs) during pregnancy.

Multiplatform biomarker analysis on non-sun exposed mucosal melanoma.

9042 Background: Mucosal melanoma is a rare malignancy, notoriously resistant to conventional chemotherapy, with few treatment options. Because of their origin, they do not receive screening and, hence, are detected in advanced stages where prognosis and curative rates are poor. The purpose of this study is to identify novel, potential targets and therapeutic options for this disease, utilizing a multiplatform approach. Methods: In total, 93 mucosal melanoma specimenswere tested via a multiplatform profiling service (Caris Life Sciences, Phoenix, AZ) consisting of gene sequencing (Sanger or next generation sequencing [NGS]), protein expression (immunohistochemistry [IHC]) and/or gene amplification (CISH or FISH). Conjunctival melanoma was excluded. Results: Notable protein overexpression rates included co-expression of PD-1 and PD-L1 (71.4%, 5/7) and cKIT (42.4%, 14/33). Percent agreement between c-KIT by IHC and sequencing was 62% (16/26). Overall, sequencing revealed the highest mutation rates in TP53 (...

IT-22 * TARGETING THE IMMUNE CHECKPOINT NETWORK WITH miR-138 EXERTS THERAPEUTIC EFFICACY IN MURINE MODELS OF GLIOMA

Antibody therapeutic targeting of the immune checkpoints CTLA-4 and PD-1 has demonstrated marked tumor regression in clinical trials; however, this requires multimodality treatment and has known toxicity. Furthermore, the targeting of one checkpoint often results in the up-regulation of another. MicroRNAs (miRNAs) can modulate gene transcripts, including those of tumor-mediated immune suppressive pathways and networks. Additionally, miRNAs can be delivered to the systemic immune compartment, which can initiate anti-tumor immune response, including within the central nervous system, thereby overcoming the confounding issue of miRNA delivery to the tumor. To identify potential immune modulatory miRNA therapeutics, we exploited human glioblastoma miRNA expression assays followed by a screening process of predicted binding sites in the 3' UTR of immune suppressive pathways and immune checkpoints. MiR-138 emerged as a leading candidate with three predicted binding sites in the 3' UTR of CTLA-4 and one within PD-1, which was functionally confirmed with luciferase expression assays. The miR-138 targeting of CTLA-4 and PD-1 was further validated by expression analysis of transfected human CD4+ T cells. Using human glioma tumor microarrays and in situ hybridization, heterogeneous expression of miR-138 was found amongst all glioma grades and pathological subtypes. The PD-1 ligand, PD-L1, was frequently expressed in glioblastomas based on immunohistochemistry and ex vivo flow cytometry from fresh human glioblastoma. Analysis of TCGA database revealed that co-expression of PD-1 and PD-L1 impacts glioblastoma survival. Given the previously documented role of CTLA-4 as an operational mechanism of glioblastoma-mediated immune suppression, this cumulative data suggests that dual targeting of CTLA-4 and PD-1 with miR-138 in glioblastoma patients may have anti-tumor activity. In vivo treatment with miR-138 in immune-competent mice with GL261 gliomas demonstrated marked tumor regression, a 43% increase in median survival (P = 0.011), and an associated decrease in intratumoral FoxP3+ Tregs, CTLA-4, and PD-1 expression. Conversely, in an immune-incompetent animal background, miR-138 failed to exert any therapeutic effect, indicating that miR-138 mediates in vivo activity via the immune system. Cumulatively, our data indicate that miR-138 may be an active and novel immunotherapeutic agent for human glioblastoma patients.

LPS and HIV-1 induce a mature phenotype in human plasmacytoid dendritic cells and enhance their programmed death ligand-1 expression (VIR1P.970)

Abstract Aberrant immune activation and immune exhaustion are features characteristic of chronic HIV infection. Elevated plasma LPS levels resulting from disruption of gut mucosal integrity during HIV infection have been implicated as a major factor contributing to immune activation. In chronic HIV-1 infection immune exhaustion molecules PD-1 and its ligand PD-L1 negatively regulate the function of exhausted CD8 T cells. The pDC constitute only a minor portion of the PBMC population, but are crucial contributors to the innate as well as adaptive immune response. We have confirmed our previous results that pDC express low levels of TLR4 and respond to LPS. We undertook this study to examine the influence of LPS on virally stimulated pDC maturation and PD-L1 expression and mechanisms thereof. In order to do so we primed pDC with LPS prior to stimulation with HIV. We observed that LPS enhanced HIV-1-induced pDC maturation (CD83 and CD40 expression) and PD-L1 expression. PD-L1 was up-regulated on mature pDC specifically by LPS and HIV combination treatment versus individual treatment with LPS or HIV. The combination treatment specifically induced co-expression of PD-L1 with positive co-stimulatory molecule CD86 but not CD80. Additionally, LPS enhanced CD4 expression on pDC but depressed HIV-1 induced CXCR4 and CCR5 expression. Together, these results indicate that in response to HIV stimulation, pDC adopt a tolerogenic phenotype (i.e. PD-L1-positive) and LPS helps drive this.

Immune checkpoint targets on circulating epithelial cells in early and late breast cancer.

e22201 Background: Our ongoing investigation of circulating epithelial cells(CEC) in early and late breast cancer with the MAINTRAC method (1ml blood, RBC lysis and fluorometric detection) has shown that CEC evade by hypoxia driven EMT (epithelial mesenchymal transition).We saw CEC with EMT and stemcellmarkers in early and late breast cancer. These cells can be found also in non cancer conditions. So far no differences in marker expression of CEC in cancer and noncancer conditions were found. This underlines the need for extended(e.g four color) analysis and allows searching for new promising targets like the immune checkpoint target PD-L1. Methods: We slightly adapted the basic MAINTRAC methodology to allow four color detection on the AMNIS FlowSight device.The IDEAS software allows comprehensive analysis of the populations. Beside four color analysis of single cells, TCA (tumor cell aggregates ) can be detected and characterized. The basic panel (EPCAMAF660.CD45PE,ALDH1FITC,DAPI) allows detection of living and death CEC and expression of the stemcell marker ALDH1. A second panel allows detection of other targets like PD-L1. Results: With the three color based original method the blood of 188 patients with early and 62 with late breast cancer were examined. No differences between conventional subgroups nor molecular subtype groups were found. In a large non cancer (n=256) control group we found high CEC counts in a group of patients with liver affections (mostly NAFLD). In these patients CEC showed the same marker ecpression .In late breast cancer only 60% of patients showed detctable cell counts.In patients with agressive disease (triple negative or HER2 enriched) mostly none or small CEC numbers were detectable.With our recently started (so far n=48) four color assay we detected coexpression of ALDH1 and PD-L1 which are more specific markers then CD44high or vimentin. Conclusions: Extended four color analysis on CEC allows monitoring of living CEC with stemcellmarker expression. Detection of immune checkpoint determinants like PD-L1on CEC is possible, further unraveling the biology of early and late breast cancer.