Programmed Cell Death Receptor Ligand Research Articles

Abstract 501: PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer

Abstract Background: Programmed cell Death receptor Ligand 1 (PD-L1) is a very promising biomarker for the selection of patients for cancer immunotherapy. We recently developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA. The aim of the present study was to study the expression of PD-L1 in CTCs from breast cancer patients with verified metastasis. Methods: We quantified the expression of PD-L1 mRNA transcripts in EpCAM-positive CTCs, by using our recently developed RT-qPCR assay, based on the following procedure: i) immunomagnetic enrichment of EpCAM-positive CTCs from 20mL of peripheral blood, ii) total RNA isolation iii) cDNA synthesis and iv) RT-qPCR for PD-L1. PD-L1 expression in respect to the expression of B2M as a reference gene, was normalized using the 2-ΔΔCt approach. Peripheral blood samples (20mL) were obtained from 22 breast cancer patients with verified metastasis and 14 healthy donors. Results: According to our results, 11/22 (50%) of metastasis-verified breast cancer patients were found to be positive for PD-L1 overexpression in CTCs. These are preliminary results and these percentages may change, since the number of samples that we are analyzing is continuously increasing. Our results are in concordance with a recent study by Mazel et al (Mol Oncol 2015), that by using the CellSearch(®) system found PD-L1((+)) CTCs in 11/16(68.8%) patients with metastatic breast cancer. Conclusion: This is the first time that a quantitative RT-qPCR molecular assay is used for the evaluation of PD-L1expression levels in EpCAM-positive CTCs in metastatic breast cancer patients. The assay is closed-tube, quantitative, highly specific and sensitive, and high-throughput. We are currently evaluating the assay in a large number of clinical samples. Citation Format: Martha Zavridou, Areti Strati, Nikos Malamos, Vasilis Georgoulias, Evi S. Lianidou. PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 501.

Monocyte programmed death ligand-1 expression after 3-4 days of sepsis is associated with risk stratification and mortality in septic patients: a prospective cohort study.

BackgroundSeptic shock is a major healthcare problem with a high mortality rate that might be caused by immunosuppression. Programmed cell death receptor-1 (PD-1) and programmed cell death receptor ligand-1 (PD-L1), which are co-inhibitory receptor molecules, participate in sepsis-induced immunosuppression. In this study, we investigated which PD-1-related molecules can be used to evaluate the risk stratification and prognosis of septic patients. Furthermore, we explored the prognostic significance of a combination of ideal predictors and conventional clinical risk parameters in septic shock patients.MethodsIn total, 29 healthy controls, 59 septic patients, and 76 septic shock patients were enrolled in this study. Considering that the focus of the research was on the second phase of sepsis, blood samples were obtained at days 3–4 after the onset of systemic inflammatory response syndrome (SIRS). PD-1 and PD-L1 expression were measured on circulating CD4+ T cells, CD8+ T cells, and monocytes (PD-L1 only) by flow cytometry.ResultsOur results showed that only monocyte PD-L1 expression gradually increased, based on the increasing severity of disease (P < 0.001). Similarly, multivariate logistic regression analysis showed that only monocyte PD-L1 expression was an independent predictor of 28-day mortality in septic shock patients. Area under the receiver operating characteristic curve analysis of the combination of monocyte PD-L1 expression and conventional clinical risk parameters indicated a more significant prognostic ability than analysis of each parameter alone.ConclusionOur study demonstrated that, among PD-1-related molecules, only monocyte PD-L1 expression after 3–4 days of sepsis was associated with risk stratification and mortality in septic patients. Furthermore, measurement of monocyte PD-L1 expression was a promising independent prognostic marker for septic shock patients.

Immune regulation of canine tumour and macrophage PD‐L1 expression

Expression of programmed cell death receptor ligand 1 (PD-L1) on tumor cells has been associated with immune escape in human and murine cancers, but little is known regarding the immune regulation of PD-L1 expression by tumor cells and tumor-infiltrating macrophages in dogs. Therefore, 14 canine tumor cell lines, as well as primary cultures of canine monocytes and macrophages, were evaluated for constitutive PD-L1 expression and for responsiveness to immune stimuli. We found that PD-L1 was expressed constitutively on all canine tumor cell lines evaluated, although the levels of basal expression were very variable. Significant upregulation of PD-L1 expression by all tumor cell lines was observed following IFN-γ exposure and by exposure to a TLR3 ligand. Canine monocytes and monocyte-derived macrophages did not express PD-L1 constitutively, but did significantly upregulate expression following treatment with IFN-γ. These findings suggest that most canine tumors express PD-L1 constitutively and that both innate and adaptive immune stimuli can further upregulate PD-L1 expression. Therefore the upregulation of PD-L1 expression by tumor cells and by tumor-infiltrating macrophages in response to cytokines such as IFN-γ may represent an important mechanism of tumor-mediated T-cell suppression in dogs as well as in humans.

A Novel Role for Programmed Cell Death Receptor Ligand-1 (PD-L1) in Sepsis-Induced Intestinal Dysfunction.

Studies imply that intestinal barrier dysfunction is a key contributor to morbid events associated with sepsis. Recently, co-inhibitory molecule, programmed death-ligand1 (PD-L1) has been shown to be involved in the regulation of intestinal immune tolerance and/or inflammation. Our previous studies showed that PD-L1 gene deficiency reduced sepsis-induced intestinal injury morphologically. However, it isn't known how PD-L1 expression impacts intestinal barrier dysfunction during sepsis. Here we tested the hypothesis that PD-L1 expressed on intestinal epithelial cells (IECs) has a role in sepsis-induced intestinal barrier dysfunction. To address this, C57BL/6 or PD-L1 gene knockout mice were subjected to experimental sepsis and PD-L1 expression, intestinal permeability, tissue cytokine levels were assessed. Subsequently, septic or non-septic patient colonic samples (assigned by pathology report) were immunohistochemically stained for PD-L1 I a blinded fashion. Finally, human Caco2 cells were used for in vitro studies. The results demonstrated that PD-L1 was constitutively expressed and sepsis significantly up-regulates PD-L1 in IECs from C57BL/6 mice. Concurrently, we observed an increased PD-L1 expression in colon tissue samples from septic patients. PD-L1 gene deficiency reduced ileal permeability, tissue levels of IL-6, TNF-α and MCP-1, and prevented ileal tight junction protein loss compared to WT after sepsis. Comparatively, while Caco2 cell monolayers responded to inflammatory cytokine stimulation also with elevated PD-L1 expression, increased monolayer permeability and altering/decreasing monolayer tight junction protein morphology/expression; these changes were reversed by PD-L1 blocking antibody. Together these data indicate that ligation of ICE PD-L1 plays a novel role in mediating the pathophysiology of sepsis-induced intestinal barrier dysfunction.

Programmed cell death receptor ligand 1 modulates the regulatory T cells' capacity to repress shock/sepsis-induced indirect acute lung injury by recruiting phosphatase SRC homology region 2 domain-containing phosphatase 1.

We recently reported that adoptively transferred (AT) exogenous CD4+ CD25+ regulatory T cells (Tregs) to wild-type (WT) mice can directly act to repress shock/sepsis-induced experimental indirect acute lung injury (iALI), and this is mediated in part by programmed cell death receptor 1 (PD-1). In this study, we further determine whether recipient mouse lacking PD-L1, one of the primary ligands for PD-1, contributes to the manipulation of the Tregs' capacity to repress lung injury. To do this, Tregs isolated from the spleen of WT mice were AT into PD-L1 mice subjected to hemorrhagic shock and subsequent to cecal ligation and puncture to induce iALI. Samples were collected for analyses 24 h after cecal ligation and puncture. We found that in PD-L1-recipient mice, AT WT-Tregs lost the ability to reverse the development of iALI seen in WT recipient mice (i.e., no reduction of lung injury indices assessed by histology and vascular leakage, failure to decrease the lung neutrophil influx [myeloperoxidase activity], or the rise in lung apoptosis [caspase 3 activity]). Also, a significant increase in interleukin 1β (IL-1β) and keratinocyte-derived chemokine, but no changes in IL-6, IL-10, and IL-17A levels in lung tissues were seen in these mice compared with iALI mice without AT of Tregs. Furthermore, we noted that the lung tissue tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1), but not SHP-2, was activated with the AT of Tregs in PD-L1(-/-) iALI mice. Finally, through local depletion of CD4+ T cells or CD25+ (Tregs) in the lung, prior to inducing iALI, we found that SHP-1 activation was associated with the loss of Tregs' protective effects in vivo. Collectively, our data reveal that PD-L1 is a critical modulator of Tregs' ability to suppress iALI, and this appears to involve SHP-1 activation.