Programmed Death Protein-1 Expression Research Articles

159 PD-1 Agonism as a Strategy to Prevent Neuroinflammation After Acute Large Vessel Occlusion

INTRODUCTION: Stroke is a leading cause of death and long-term disability worldwide. Acute ischemia results in a profound inflammatory response that is counterproductive to short- and long-term recovery. Programmed Death Protein 1 (PD-1) is an immune checkpoint that has been shown to be upregulated after acute cerebral injury, and—upon binding its ligand PD-L1—negatively regulates immune cells to limit the inflammatory response. METHODS: C57BL/6J mice underwent middle cerebral artery occlusion (MCAO) and were randomized into three arms: (1)sham surgery, (2)MCAO control, and (3)MCAO+PD-L1. Survival, brain water content, and volumetric MRI analysis of cerebral edema were measured. Brains were analyzed by flow cytometry to quantify PD-1 expression on infiltrating immune cells. Peripheral blood was harvested for single-cell RNA-sequencing. Blood from stroke patients was collected, and the frequency of PD-1+ monocytes was correlated to clinical outcomes. RESULTS: PD-L1 significantly decreased brain edema as measured by brain water content (p = 0.0277) and high-resolution MRI (p < 0.0001), and improved overall survival (p = 0.0022). PD-1+ monocytes are upregulated in the ischemic hemisphere, and appear to be critical mediators of edema. PD-L1 treatment decreased PD-1+ monocyte infiltration , while global PD-1 (PD-1-/-) and myeloid-specific knockout of PD-1 (PD-1f/fLysMcre) abrogated the treatment effect on edema. Single-cell RNA sequencing and differential expression analysis of peripheral monocytes revealed downregulation of multiple pro-inflammatory pathways in the treated mice versus the controls. Human correlative studies confirmed that PD-1+ monocytes were present in the peripheral blood of MCA stroke patients, and the frequency of this cell population correlated with post-stroke cerebral edema. CONCLUSIONS: Our results support PD-L1 as a novel therapeutic strategy for neuroinflammation and cerebral edema after acute ischemic stroke.

PD-L1 and PD-1 expression in pediatric post-transplant Burkitt lymphoma and other monomorphic post-transplant lymphoproliferative disorders.

Post-transplant lymphoproliferative disorders (PTLD) develop as a consequence of immune suppression. Programmed death protein 1 (PD-1), a regulator of host immune activation, binds to programmed death-ligand 1 (PD-L1) to suppress the T-cell immune response. PD-1/PD-L1 pathway may play a role in PTLD. The objective was to describe intratumoral expression of PD-L1 and PD-1 in pediatric monomorphic PTLD, and assess if density of these cells is associated with progression-free survival (PFS) and overall survival (OS). Clinical variables and outcome data were collected on B-cell monomorphic PTLD treated in Toronto, Canada between 2000 and 2017. Diagnostic area from tumor tissue was identified to count CD3-positive or PD-1-positive cells and CD3-negative lymphoma B cells or PD-L1-positive cells. CD3+ , PD-1+ , and PD-L1+ cell densities were compared between cases of PTLD. OS and PFS were analyzed. We identified 25 cases of B-cell monomorphic PTLD; majority Burkitt lymphoma (32%) and diffuse large B-cell lymphoma (56%). All cases had CD3+ cells infiltrating the tumor, and median percentage of CD3+ cells was 14% (interquartile range: 6.2%-25%). Twelve cases (48%) had PD-1+ cell infiltrating (range: 1%-83%) and 13 cases (52%) had no PD-1+ cells infiltrating. Sixteen cases (64%) had PD-L1+ cells present; however, there was no PD-L1 expression on any Burkitt lymphoma tissue. When comparing PD-1 and PD-L1 expression, there was no difference in OS or PFS. Intratumoral presence of PD-1+ and PD-L1+ cells varied in pediatric patients with monomorphic PTLD; however, no relationship to OS and PFS was identified.

Dynamics of PD-1 expression are associated with treatment efficacy and prognosis in patients with intermediate/high-risk myelodysplastic syndromes under hypomethylating treatment

Hypomethylating agents (HMAs) are widely used in patients with higher-risk MDS not eligible for stem cell transplantation. However, the general response rate by HMAs is lesser than 50% in MDS patients, while the relapse rate is high. Emerging evidence indicates that demethylating effects committed by HMAs may facilitate the up-regulation of a range of immune checkpoints or cancer suppressor genes in patients with MDS, among which the programmed death protein 1 (PD-1) and its ligands are demonstrated to be prominent and may contribute to treatment failure and early relapse. Although results from preliminary studies with a limited number of enrolled patients indicate that combined administration of PD-1 inhibitor may yield extra therapeutic benefit in some MDS patients, identifications of this subgroup of patients and optimal timing for the anti-PD-1 intervention remain significant challenges. Dynamics of immune checkpoints and associated predictive values during HMA-treatment cycles remained poorly investigated. In this present study, expression levels of immune checkpoints PD-1 and its ligands PD-L1 and PD-L2 were retrospectively analyzed by quantitative PCR (Q-PCR) in a total of 135 myelodysplastic syndromes (MDS) cohort with higher-risk stratification. The prognostic value of dynamics of these immune checkpoints during HMA cycles was validated in two independent prospective cohorts in our center (NCT01599325 and NCT01751867). Our data revealed that PD-1 expression was significantly higher than that in younger MDS patients (age ≤ 60) and MDS with lower IPSS risk stratification (intermediate risk-1). A significantly up-regulated expression of PD-1 was seen during the first four HMA treatment cycles in MDS patients, while similar observation was not seen concerning the expression of PD-L1 or PD-L2. By utilizing binary logistic regression and receiver operating characteristic (ROC) models, we further identified that higher or equal to 75.9 PD-1 expressions after 2 cycles of HMA treatment is an independent negative prognostic factor in predicting acute myeloid leukemia (AML) transformation and survival. Collectively, our data provide rationales for monitoring the expression of PD-1 during HMA treatment cycles, a higher than 75.9 PD-1 expression may identify patients who will potentially benefit from the combined therapy of HMA and PD-1 inhibitors.

Upregulation of PD-1 Expression and High sPD-L1 Levels Associated with COVID-19 Severity

COVID-19 has several mechanisms that can lead to lymphocyte depletion/exhaustion. The checkpoint inhibitor molecule programmed death protein 1 (PD-1) and its programmed death-ligand 1 (PDL-1) play an important role in inhibiting cellular activity as well as the depletion of these cells. In this study, we evaluated PD-1 expression in TCD4+, TCD8+, and CD19+ lymphocytes from SARS-CoV-2-infected patients. A decreased frequency of total lymphocytes and an increased PD-1 expression in TCD4+ and CD19+ lymphocytes were verified in severe/critical COVID-19 patients. In addition, we found a decreased frequency of total monocytes with an increased PD-1 expression on CD14+ monocytes in severe/critical patients in association with the time of infection. Moreover, we observed an increase in sPD-L1 circulant levels associated with the severity of the disease. Overall, these data indicate an important role of the PD-1/PDL-1 axis in COVID-19 and may provide a severity-associated biomarker and therapeutic target during SARS-CoV-2 infection.

Macrophage PD-1 associates with neutrophilia and reduced bacterial killing in early cystic fibrosis airway disease

BackgroundMacrophages are the major resident immune cells in human airways coordinating responses to infection and injury. In cystic fibrosis (CF), neutrophils are recruited to the airways shortly after birth, and actively exocytose damaging enzymes prior to chronic infection, suggesting a potential defect in macrophage immunomodulatory function. Signaling through the exhaustion marker programmed death protein 1 (PD-1) controls macrophage function in cancer, sepsis, and airway infection. Therefore, we sought to identify potential associations between macrophage PD-1 and markers of airway disease in children with CF. MethodsBlood and bronchoalveolar lavage fluid (BALF) were collected from 45 children with CF aged 3 to 62 months and structural lung damage was quantified by computed tomography. The phenotype of airway leukocytes was assessed by flow cytometry, while the release of enzymes and immunomodulatory mediators by molecular assays. ResultsAirway macrophage PD-1 expression correlated positively with structural lung damage, neutrophilic inflammation, and infection. Interestingly, even in the absence of detectable infection, macrophage PD-1 expression was elevated and correlated with neutrophilic inflammation. In an in vitro model mimicking leukocyte recruitment into CF airways, soluble mediators derived from recruited neutrophils directly induced PD-1 expression on recruited monocytes/macrophages, suggesting a causal link between neutrophilic inflammation and macrophage PD-1 expression in CF. Finally, blockade of PD-1 in a short-term culture of CF BALF leukocytes resulted in improved pathogen clearance. ConclusionTaken together, these findings suggest that in early CF lung disease, PD-1 upregulation associates with airway macrophage exhaustion, neutrophil takeover, infection, and structural damage.

Expression of immune checkpoint molecules programmed death protein 1, programmed death-ligand 1 and inducible T-cell co-stimulator in mycosis fungoides and Sézary syndrome: association with disease stage and clinical outcome.

BackgroundThe relationship between immune checkpoint status and disease outcome is a major focus of research in cutaneous T‐cell lymphoma (CTCL), a disfiguring neoplastic dermatological disorder. Mycosis fungoides (MF) and Sézary syndrome (SS) are the two most common types of CTCL.ObjectivesThe aim was to evaluate the immune checkpoint markers programmed death protein 1 (PD1), inducible T‐cell co‐stimulator (ICOS) and programmed death‐ligand 1 (PD‐L1) in skin biopsies from patients with CTCL relative to disease stage and overall survival.MethodsThis consecutive case series enrolled 47 patients: 57% had stage IA–IIA disease and 43% had stage IIB–IVA2 disease (including seven with SS).ResultsPD1, PD‐L1 and ICOS expression was seen in all biopsies. Notably, PD‐L1 was predominantly expressed on histiocytes/macrophages, but focal expression on CTCL cells was seen. High expression of either ICOS or PD‐L1 was associated with advanced‐stage disease (P = 0·007 for both) and with the appearance of large‐cell transformation (LCT), a histopathological feature associated with a poor prognosis (ICOS: P = 0·02; PD‐L1: P = 0·002). PD1 expression was not significantly associated with disease stage (P = 0·12) or LCT (P = 0·49), but expression was high in SS biopsies. A high combined checkpoint marker score (PD1, PD‐L1 and ICOS) was associated with advanced‐stage disease (P = 0·001), LCT (P = 0·021) and lower overall survival (P = 0·014).ConclusionsThese findings demonstrate the existence of a complex immunoregulatory microenvironment in CTCL and support the development of immunotherapies targeting ICOS and PD‐L1 in advanced disease.

Programmed cell death protein-1 (PD-1) protects liver damage by suppressing IFN-\u03b3 expression in T cells in infants and neonatal mice

BackgroundBiliary atresia (BA) is a severe cholangiopathy possibly resulting from virus-induced and immune-mediated injury of the biliary system. IFN-γ, secreted from CD4+ Th1 cells and CD8+ cytotoxic T cells, is a major mediator of liver pathology. Programmed death protein-1 (PD-1) signaling suppresses T cell function. However, how PD-1 modify T cell function in BA remains incompletely understood.MethodsFrequencies of PD-1 expressing CD4+ and CD8+ T cells were analyzed in the liver and blood from BA and control subjects. Associations of PD-1+CD4+/CD8+T cell abundances with liver function indices were measured. Function of PD-1 was measured by administration of an anti-PD-1 antibody in a Rhesus Rotavirus (RRV)-induced BA model. Survival, histology, direct bilirubin, liver immune cell subsets and cytokine production were analyzed.ResultsPD-1 was significantly upregulated in CD4+ and CD8+ T cells in patients with BA compared with control subjects. PD-1 expression in T cells was negatively associated with IFN-γ concentration in liver (PD-1+CD4+T cells in liver vs. IFN-γ concentration, r = − 0.25, p = 0.05; PD-1+CD8+T cells in liver vs. IFN-γ concentration, r = − 0.39, p = 0.004). Blockade of PD-1 increased IFN-γ expression in CD4+ T and CD8+ T cells (RRV vs. anti-PD-1 treated RRV mice: 11.59 ± 3.43% vs. 21.26 ± 5.32% IFN-γ+ in hepatic CD4+T cells, p = 0.0003; 9.33 ± 4.03% vs. 22.55 ± 7.47% IFN-γ+ in hepatic CD8+T cells, p = 0.0001), suppressed bilirubin production (RRV vs. anti-PD-1 treated RRV mice: 285.4 ± 47.93 vs. 229.8 ± 45.86 μmol/L total bilirubin, p = 0.01) and exacerbated liver immunopathology.ConclusionsPD-1 plays a protective role in infants with BA by suppressing IFN-γ production in T cells. Increasing PD-1 signaling may serve as a therapeutic strategy for BA.

PD-1 Expression and its Correlation With Prognosis in Clear Cell Renal Cell Carcinoma.

Programmed death ligand-1 (PD-L1) and programmed death protein 1 (PD-1) expression levels in many tumors and their correlation with prognosis have been actively studied. However, studies on PD-1 expression and its prognostic value in clear cell renal cell carcinoma (ccRCC) are limited and controversial. In this study, we describe the expression of PD-1 and its prognostic significance and association with clinical features in patients with ccRCC. We obtained clinicopathological data from 166 patients with ccRCC who were treated at Gyeongsang National University Hospital, Jinju, Korea between January 2000 and December 2009. Tissue microarray blocks were made using representative paraffin blocks of ccRCC specimens. Two pathologists analyzed PD-L1 and PD-1 expression in both tumor and inflammatory cells. PD-1 expression in tumor-infiltrating inflammatory cells was significantly correlated with unfavorable disease-free survival (DFS) (p<0.001) and disease-specific survival (DSS) (p=0.002) in the univariate analysis. A statistically significant correlation between PD-1 expression and unfavorable DFS (p=0.025) was observed in the multivariate analysis. PD-1 expression in tumor-infiltrating inflammatory cells serves as an independent prognostic factor for unfavorable DSS in patients with ccRCC.

Expression of PD-1, TIM-3, LAG-3 and BTLA in diffuse large B-cell lymphoma and its effect on prognosis

Objective: To investigate the expression levels of programmed death protein 1 (PD-1)、T cell immunoglobulin domain and mucin domain 3(TIM-3)、lymphocyte activating gene 3 (LAG-3) and B and T lymphocyte attenuator (BTLA) in Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) and their effects on prognosis. Methods: The paraffin specimens of 30 patients with DLBCL, NOS newly diagnosed in People's Hospital of Zhengzhou University were stained with immunohistochemistry. The effects of single positive and co-expression of the above molecules on progression-free survival (PFS) phase and overall survival (OS) phase were analyzed. Results: There was no significant difference in prognosis between PD-1, TIM-3, LAG3, BTLA single positive group and single negative group. The median PFS phase of PD-1 and TIM-3 co-expression group and TIM3 and BTLA co-expression group were 26 and 24 months respectively, which were both lower than the 54 months (P=0.021) and 47 months (P=0.037) in non-co-expression group. The median PFS phase and OS phase of PD-1, TIM-3 and LAG-3 co-expression group were 17 and 25 months respectively, which were significantly lower than the 41 months (P=0.024) and 60 months (P=0.015) of non-co-expression group. The median PFS phase and OS phase of PD-1, TIM-3, LAG-3 and BTLA co-expression group were 18 and 26 months respectively, which were significantly lower than the 40 months (P=0.038) and 57 months (P=0.041) of non-co-expression group. Conclusions: In patients with DLBCL, NOS, those with PD-1 and TIM-3 co-expression as well as those with TIM-3 and BTLA co-expression have poor PFS phase. Patients with PD-1, TIM-3 and LAG-3 co-expression and patients with PD-1, TIM-3, LAG-3 and BTLA co-expression have poor PFS and OS phase.

PD-1 and PD-L1 expression in pulmonary carcinoid tumors and their association to tumor spread.

Pulmonary carcinoid (PC) tumors are rare tumors that account for approximately 1% of all lung cancers. The primary treatment option is surgery, while there is no standard treatment for metastatic disease. As the number of PCs diagnosed yearly is increasing, there is a need to establish novel therapeutic options. This study aimed to investigate programmed death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) expression in PC tumors since blocking of the PD-1/PD-L1 pathway is a promising therapeutic option in various other malignancies. A total of 168 PC patients treated between 1990 and 2013 were collected from the Finnish biobanks. After re-evaluation of the tumors, 131 (78%) were classified as typical carcinoid (TC) and 37 (22%) as atypical carcinoid (AC) tumors. Primary tumor samples were immunohistochemically labeled for PD-1, PD-L1 and CD8. High PD-1 expression was detected in 16% of the tumors. PD-L1 expression was detected in 7% of TC tumors; all AC tumors were PD-L1 negative. PD-L1 expression was associated with mediastinal lymph-node metastasis at the time of diagnosis (P = 0.021) as well as overall metastatic potential of the tumor (P = 0.010). Neither PD-1 expression, PD-L1 expression nor CD8+ T cell density was associated with survival. In conclusion, PD-1 and PD-L1 were expressed in a small proportion of PC tumors and PD-L1 expression was associated with metastatic disease. Targeting of the PD-1/PD-L1 pathway with immune checkpoint inhibitors may thus offer a treatment option for a subset of PC patients.

Tumor cell-intrinsic programmed death protein 1 expression and induction in human cancer cell lines

Abstract The programmed death protein 1 (PD-1) and ligand (PD-L1) axis inhibits T cell activation and reduces anti-tumor immunity. Most studies of the PD-1/PD-L1 axis test extrinsic, immune-dependent effects. However, tumor cell-intrinsic PD-L1 regulates immune-independent tumor mTOR signals, autophagy, and tumor initiating cell generation. Melanoma cell-intrinsic PD-1 regulates melanoma growth and mTOR. We tested PD-1 expression and induction in multiple human tumor cell lines. We detected PD-1 in human cell lines in vitro from ES2 ovarian cancer, VMM122 melanoma, and RT4 bladder cancer by flow cytometry, Western blot and RT-PCR. All lines were negative for the other PD-L1 receptor, CD80, by flow cytometry. Incubating lines with IFNα or IFNγ for 48 hours further augmented PD-1 expression as detected by flow, Western blot and RT-PCR. αPD-1 antibody (50 μg/ml) reduced proliferation of RT4 bladder cancer cells in vitro, demonstrating a direct PD-1 signal effect in these cells, and αPD-L1 similarly reduced in vitro proliferation. Interestingly, anti-PD-1 effects on reducing proliferation were similar: 41.5% reduction; p&amp;lt;0.0001. We used CRISPR/Cas9 to generate PD-L1KO RT4 cells. αPD-L1 did not reduce in vitro proliferation as expected, but unexpectedly, αPD-1 also had no anti-proliferative effect on PD-L1KO RT4 cells, suggesting a PD-L1/PD-1 interaction. In murine B16 melanoma, we generated PD-L1KO and PD-1KO cells using CRISPR/Cas9. Cell-intrinsic PD-L1 and PD-1 downregulate chemokine production. Among chemokines analyzed, PD-L1 suppressed CCL2 and CCL5, PD-1 suppressed CCL4 and CCL7, and both suppressed CCL3 and CXCL2. Understanding intrinsic PD-1 and PD-L1 effects on tumor cells can be exploited to improve cancer immunotherapy

Evaluating the significance of density, localization, and PD-1/PD-L1 immunopositivity of mononuclear cells in the clinical course of lung adenocarcinoma patients with brain metastasis.

Management of lung cancer patients who suffer from brain metastases represents a major challenge. Considering the promising results with immune checkpoint inhibitor treatment, evaluating the status of immune cell (IC) infiltrates in the prognosis of brain metastasis may lead to better therapeutic strategies with these agents. The aim of this study was to characterize the distribution of ICs and determine the expression of the checkpoint molecules programmed death protein 1 (PD-1) and its ligand, PD-L1, in brain metastasis of lung adenocarcinoma (LUAD) patients and to analyze their clinicopathological correlations. We determined the presence of peritumoral mononuclear cells (mononuclear ring) and the density of intratumoral stromal mononuclear cells on brain metastasis tissue sections of 208 LUAD patients. PD-L1/PD-1 expressions were analyzed by immunohistochemistry. Mononuclear rings were significantly associated with better survival after brain metastasis surgery. Cases with massive stromal IC infiltration also showed a tendency for better overall survival. Lower expression of PD-1 and PD-L1 was associated with better survival in patients who underwent surgery for the primary tumor and had multiple brain metastases. Steroid administration and chemotherapy appear not to influence the density of IC in brain metastasis. This is the first study demonstrating the independent prognostic value of mononuclear rings in LUAD cases with brain metastasis. Our results also suggest that the density of tumor-associated ICs in addition to PD-L1 expression of tumor cells and ICs as well as PD-1 expression of ICs may hold relevant information for the appropriate selection of patients who might benefit from anti-PD-L1 or anti-PD-1 therapy.

Pembrolizumab for the treatment of non-small cell lung cancer

ABSTRACTIntroduction: Immune checkpoint inhibitors targeting programmed death protein 1 (PD-1) receptor and its ligand, PD-L1, have recently led to significant and durable improvements in the clinical outcomes of some types of cancers including lung cancer.Areas covered: Pembrolizumab was approved by the US FDA for the treatment of advanced or metastatic NSCLC whose disease has progressed after other treatments and with tumors that express PD-L1. In the phase I KEYNOTE-001 trial, the overall response rate (ORR) was 19.4%, the median progression-free survival (PFS) and overall survival (OS) were 3.7 months and 12.0 months for 495 unselected NSCLC patients. Strong PD-L1 expression (≥ 50%) was associated with higher ORR, longer PFS, and longer OS. The phase II/III randomized KEYNOTE-010 trial demonstrated that pembrolizumab improved OS versus docetaxel in patients with previously treated NSCLC.Expert opinion: Pembrolizumab, demonstrated durable response and prolonged OS especially in NSCLC patients with high expression of PD-1, thereby suggests a new treatment paradigm. However, many issues remain to be explored, including the identification of other robust biomarkers that can accurately predict the immune-responsiveness of tumors. Along with the identification of predictive biomarkers, further understanding of the tumor microenvironment is necessary to improve treatment outcomes through combinations of immunotherapy or combined with other targeted therapies.

Changes in T cell immunity in patients with metastatic castration resistant prostate treated with Radium-223 treatment.

295 Background: The bone seeking calcium mimetic, Radium223 (Ra223), emits high energy alpha particle that cause irreparable double strand DNA breaks in osseous metastases. Ra223 improves survival in patients with bone-predominant metastatic castration resistant prostate cancer (mCRPC). The effect of Ra223 on the host immune system, and T cell immunity in particular, is unknown. Methods: Eligible patients include men with mCRPC, osseous metastases and a clinical indication for Ra223 were eligible. Blood samples were collected at the following time points: prior to the first, second, and fourth treatments (tx) and 4 weeks after the sixth dose. Peripheral blood mononuclear cells (PBMCs) were purified and stained with a cocktail of antibodies to surface and effector immune molecules. Some PBMCs were stimulated and stained for intracellular cytokines (IFN-g, TNF-a, IL-13, IL-17, and IL-21). Stained cells were analyzed by flow cytometry. Results: A total seven patients completed at least two sample collections. The median number of prior lines of therapy was three. Median age was 71 (55-79). Median PSA was 78 (2-351). Effector memory (EM) CD8+ T cells were divided into two subsets expressing high and low levels of the IL-7 receptor alpha chain (IL-7Ra) with distinct cellular characteristics including perforin expression. The frequency of IL-7Rahigh and low EM CD8+ T cells expressing programmed death protein 1 (PD-1) was decreased after Ra223 treatment (pre vs. post, median (%) ± interquartile range, 14.3 ± 12.9 vs. 7.2 ± 20.9, P = 0.03; 16.7 ± 28.8 vs. 14.1 ± 37.8, P = 0.015 by Wilcoxon matched-pairs signed rank test). A similar finding was observed in the frequency of memory CD4+ T cells expressing PD-1 after the treatment (median (%) ± interquartile range, 7.2 ± 7.7 vs. 3.9 ± 3.4, P= 0.03). Conclusions: In this preliminary cohort of patients, Ra223 tx was associated with decrease in PD-1 expression by effector T cells. Further investigations of an immune mechanism of Ra223 mediated antitumor activity are warranted.