Overexpression Of Programmed Death-1 Research Articles

Knockdown of Programmed Death 1 Inhibited Progression of Papillary Thyroid Carcinoma in Mice.

PD-L1 and PD1 mainly focused on melanoma, lung cancer and other tumors, while the related studies on early lymph node metastasis of papillary thyroid carcinoma were rarely reported. For elucidating the role of programmed death 1 (PD1)/programmed death ligand 1 (PD-L1) pathway in tumor growth of papillary thyroid carcinoma (PTC). Human thyroid cancer cell line and human normal thyroid cell line were obtained and transfected with si-PD1 or pCMV3-PD1 for the construction of PD1 knockdown or overexpression models. BALB/c mice were purchased for in vivo studies. Nivolumab was implemented for in vivo inhibition of PD1. Western blotting was performed for determining protein expression, while RTqPCR was used to measure relative mRNA levels. The PD1 and PD-L1 levels were both significantly upregulated in PTC mice, while the knockdown of PD1 downregulated both PD1 and PD-L1 levels. Protein expression of VEGF and FGF2 was increased in PTC mice, while si-PD1 decreased their expression. Silencing of PD1 using si-PD1 and nivolumab both inhibited tumor growth in PTC mice. Suppressing PD1/PD-L1 pathway significantly contributed to the tumor regression of PTC in mice.

Differential expression of programmed death 1 (PD-1) on various immune cells and its role in human leprosy.

Leprosy is a chronic bacterial disease caused by Mycobacterium leprae. Leprosy patients have been found to have defects in T cells activation, which is critical to the clearance of the bacilli. Treg cell suppression is mediated by inhibitory cytokines such as IL10, IL-35 and TGF-β and its frequency is higher in leprosy patients. Activation and overexpression of programmed death 1 (PD-1) receptor is considered to one of the pathways to inhibit T-cell response in human leprosy. In the current study we address the effect of PD-1 on Tregs function and its immuno-suppressive function in leprosy patients. Flow cytometry was used to evaluate the expression of PD-1 and its ligands on various immune cells T cells, B cells, Tregs and monocytes. We observed higher expression of PD-1 on Tregs is associated with lower production of IL-10 in leprosy patients. PD-1 ligands on T cells, B cells, Tregs and monocytes found to be higher in the leprosy patients as compared to healthy controls. Furthermore, in vitro blocking of PD-1 restores the Tregs mediated suppression of Teff and increase secretion of immunosuppressive cytokine IL-10. Moreover, overexpression of PD-1 positively correlates with disease severity as well as Bacteriological Index (BI) among leprosy patients. Collectively, our data suggested that PD-1 overexpression on various immune cells is associated with disease severity in human leprosy. Manipulation and inhibition of PD-1 signaling pathway on Tregs alter and restore the Treg cell suppression activity in leprosy patients.

Over-expression of programmed death-ligand 1 and programmed death-1 on antigen-presenting cells as a predictor of organ dysfunction and mortality during early sepsis: a prospective cohort study.

This study aimed to explore the changes of programmed death-ligand 1 (PD-L1) and programmed death-1 (PD-1) expression on antigen-presenting cells (APCs) and evaluate their association with organ failure and mortality during early sepsis. In total, 40 healthy controls and 198 patients with sepsis were included in this study. Peripheral blood was collected within the first 24 h after the diagnosis of sepsis. The expression of PD-L1 and PD-1 was determined on APCs, such as B cells, monocytes, and dendritic cells (DCs), by flow cytometry. Cytokines in plasma, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), IL-6, IL-10, and IL-17A were determined by Luminex assay. PD-1 expression decreased significantly on B cells, monocytes, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs) as the severity of sepsis increased. PD-1 expression was also markedly decreased in non-survivors compared with survivors. In contrast, PD-L1 expression was markedly higher on mDCs, pDCs, and monocytes in patients with sepsis than in healthy controls and in non-survivors than in survivors. The PD-L1 expression on APCs (monocytes and DCs) was weakly related to organ dysfunction and inflammation. The area under the receiver operating characteristic curve (AUC) of the PD-1 percentage of monocytes (monocyte PD-1%)+APACHE II model (0.823) and monocyte PD-1%+SOFA model (0.816) had higher prognostic value than other parameters alone. Monocyte PD-1% was an independent risk factor for 28-day mortality. The severity of sepsis was correlated with PD-L1 or PD-1 over-expression on APCs. PD-L1 in monocytes and DCs was weakly correlated with inflammation and organ dysfunction during early sepsis. The combination of SOFA or APACHE II scores with monocyte PD-1% could improve the prediction ability for mortality.

The effect of PD-1 expression on tumor-associated macrophage in T cell lymphoma.

Our study aimed to explore the programmed death 1 (PD-1) expression on tumor-associated macrophage (TAM) in T cell non-Hodgkin lymphoma (T-NHL) and its relationship with lymphoma prognosis. The effect of PD-1 expression on the function of macrophages was also studied. Multispectral image quantitative analysis was applied for detecting PD-1 expression on macrophages in T cell lymphoma tissues. The Kaplan-Meier analysis was performed to evaluate the value of PD-1 expression of TAM in predicting the overall survival of T-NHL. PD-1 overexpression THP-1-derived macrophage was constructed and was cocultured with Jurkat cells to explore the effect of PD-1 on macrophage function. In 17T cell lymphoma cases, the 1-year overall survival rate was significantly lower in patients with higher PD-1 expression on TAMs (0.25 vs 0.86, p < 0.05). After co-cultured with Jurkat cells, classically activated (M1)-related markers on PD-1 overexpressed macrophages were significantly lower than those on controls, while the expressions of alternatively activated (M2) related markers were similar. The PD-1 overexpressed macrophages showed inhibited phagocytosis (4.42% vs 40.7%, p<0.001) and increased IL-10 secretion (144.48pg/ml vs 32.32pg/ml, p<0.001). High PD-1 expression on TAMs in T-NHL may predict poor prognosis. The PD-1 overexpression of macrophages significantly inhibited polarization of M1 macrophages and phagocytosis, and more IL-10 was excreted. These changes may enhance the pro-tumor effects of tumor microenvironment.

PD-1 Expression on Circulating CD8+ T-Cells as a Prognostic Marker for Patients With Gastric Cancer.

Programmed death-1 (PD1) is an immunoinhibitory receptor, and PD1 overexpression on T-cells is involved in immune evasion in cancer. This study investigated the prognostic significance of PD1 expression on T-cells in gastric cancer. PD1 expression on CD4+ and CD8+ T-cells obtained from peripheral blood mononuclear cells was evaluated by multicolor flow cytometry. Based on cutoff values from receiver operating characteristic analysis, patients were sub-grouped according to PD1 positivity as having high or low PD1+CD4+ T-cell (cutoff ≥34.2%) and PD1+CD8+ T-cell (cutoff ≥28.7%) frequencies. Five-year overall survival rates differed significantly between the groups with low and high frequency of PD1+CD4+ T-cells (75.1% vs. 27.2%, respectively; p=0.0008). The 5-year overall survival rates were 78.3% and 37.2%, respectively, for the corresponding PD1+CD8+ T-cell subgroups (p=0.0004). Multivariate analysis revealed that the frequency of PD1+CD8+ T-cells was an independent prognostic indicator. The frequency of PD1+CD8+ T-cells may predict the prognosis of patients with gastric cancer.

PD-1 and PD-L1 Are Overexpressed in the "Intermediate CD14+CD16+" and "Non Classical CD14lowCD16+" but Not in the "Classical CD14+CD16-" Monocytes in the Peripheral Blood of Chronic Myelomonocytic Leukemia

Chronic myelomonocytic Leukemia (CMML) is a heterogeneous clonal disorder highly resistant to the few therapies that are available nowadays.There is increasing evidence to suggest that Programmed Death-1 (PD-1), and its major ligand Programmed Death Ligand-1 (PD-L1), are involved in immune suppression and disease progression, are highly expressed in many hematological malignancies, and can be involved in MDS pathogenesis and resistance mechanisms to hypomethylating agents. However, the expression of PD-1 and PD-L1 is not widely explored in CMML.Different types of monocytes based on CD14 and CD16 expression show different genetic profiles and functions, having different distribution in several conditions, including malignancy.In our study, we studied the expression of PD-1 and PD-L1 in the peripheral blood (PB) monocytic compartment of patients with CMML using flow cytometry , to better understand their potential role in the pathogenesis of the disease, and as a basis for the evaluation of this pathway for the development of future immunotherapy strategies.Peripheral blood samples from 16 CMML, and age matched normal (n=10) and reactive (n=9) monocytosis (>1 x109 /L) were studied. Two hundred µl of each PB sample were stained with an 8-color panel of monoclonal antibodies (CD16-FITC, CD64-PE, PD1-PCP5.5, PDL1-PC7, CD300-APC, CD14-APCH7, HLADR-V450 and CD45-OC515). A minimum of 1x 106 events were acquired by FACSCanto II (BD Biosciences, San Jose, USA) and the data was analyzed with the Infinicyt software (Cytognos SL, Spain). Monocytic population was selected first on the automated population separator plot and confirmed by the expression of CD64 and HLADR expression. Lymphocytes were used as the internal control. Three types of monocytes were defined based on the CD14 and CD16 expression, as previously described.As expected, CMML type 1 patients had higher absolute monocyte counts in PB than reactive and normal cases (p=0.001), and higher percentage of monocytic cells by flow (0.001).The distribution (median) of the monocytic subpopulations based on CD14 and CD16 expression among the monocytic compartment in PB of CMML, reactive and normal cases, respectively, was as follows: "classical"(CD14+CD16-) were of 98%, 90% and 85% (p<0.000); "intermediate" (CD14+CD16+) 1.4%, 3.7% and 2.6% (p=0.01); and "non-classical" (CD14lowCD16+) monocytes 1%, 5% and 12% (p<0.001).The expression of PD-1 in the major population ("classical" monocyte) was similar among CMML (Median MFI 370), reactive (Median MFI 403), and normal cases (Median MFI 265). However, in the "intermediate CD14+CD16+" and in the "non-classical CD14lowCD16+" monocytes, PD-1 was overexpressed in CMML and reactive cases, compared to normal controls. Reactive cases had even a higher overexpression of PD1 in both "intermediate" and "non-classical" monocytes compared to CMML (Median MFI of 312, 529, and 398 for "intermediate" and Median MFI of 185, 465, and 271 for "non- classical" in normal, reactive and CMML cases, respectively-p=0.01, and p<0.01).For PDL1, we did not find differences in their expression in "classical" nor "intermediate" monocytes among CMML, reactive and normal cases (Median MFI in "classical" monocytes of 2415 vs 2086 vs 2003 for CMML, reactive and normal cases -p>0.05-; and Median MFI in "intermediate" monocytes of 3803 vs 2737 vs 3200 for CMML, reactive and normal cases -p>0.05-). However, in the "non-classical" monocytic population, PD-L1 was clearly overexpressed in CMML (Median MFI of 1782) compared to normal controls (Median MFI of 699), and this was also significantly higher than in reactive cases (Median MFI of 1040) (p=0.002).We found that PD-1 and PD-L1 were overexpressed in CMML, but not in the main "classical" monocyte population of the PB, but in the less represented "intermediate" and "non classical" monocytic compartment. Interestingly, the CD16+ monocytes (intermediate and non-classical) were proposed to have a more important role in inflammation and immunomodulation. Therefore, these populations could have an important function in the pathogenesis of the CMML, and the overexpression of PD-1 and PD-L1 could be investigated as a target for immunotherapy in the development of new therapeutical strategies to improve the adverse prognosis of the CMML. DisclosuresDiez Campelo:Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau. Puig:The Binding Site: Consultancy; Janssen: Consultancy.

P8.02 - Overexpression of PD-1 and PD-L1 in renal cell carcinoma is associated with poor prognosis in metastatic patients treated with sunitinib

Anti vascular growth epithelial factor (VEGF) therapies are currently used in first line of metastatic clear cell renal cell carcinoma (ccRCC), but some patients are inherently resistant to these treatments. Immunotherapy based on disruption of immune checkpoints, such as Programmed Death 1 (PD-1) and Programmed Death Ligand 1 (PD-L1), has been showed promising results. PD-1 and PD-L1 are mainly expressed by T-Cells and tumor cells respectively. Interaction between PD-1 and PD-L1 down-regulates antitumor activity and could be involved in resistance to anti-VEGF therapies. This study assessed PD-1 and PD-L1 expressions in primary ccRCC of metastatic patients with sunitinib first-line treatment and its correlation with clinical outcomes. Formalin-fixed paraffin samples were obtained from primary ccRCC before any systemic treatment. Prognostic pathological criteria and clinical data were collected with a median follow up of 27 months after introduction of sunitinib. PD-1 and PD-L1 expressions were evaluated by immunohistochemistry on the most inflammatory and the highest Fuhrman nuclear grade areas respectively, considering tumor heterogeneity. Kaplan Meier survival curves were compared by log rank test. PD-1 and PD-L1 were considered overexpressed when at least a moderate to marked density of positive immune cells and at least 30% of tumor cells were positive with moderate to strong membranous staining intensity, respectively. Overexpression of both PD-1 and PD-L1 was observed in a subgroup of 17/47 primary ccRCC (36%). Patients in this subgroup compared to others had a worse progression free survival (p = 0.001) with medians of 9 months and 14 months respectively. Even if statistical significance was not reached, there was also a trend toward a worse median overall survival (18 months versus 32). This is the first study to assess the PD-1 and PD-L1 expressions in primary ccRCC of metastatic patients with sunitinib first-line treatment. The subgroup with both PD-1 and PD-L1 overexpression should be individualized as having a peculiar immune phenotype. These patients experienced poor prognosis when treated by sunitinib and may be good candidates from anti-PD-1 or anti-PD-L1 immunotherapies.

T Cells in CTCL Have an Exhausted Phenotype While Cutaneous Dendritic Cells Display a Normally Activated Mature Phenotype

Background: Mycosis fungoides (MF) and Sézary syndrome (SS) represent the most common types of cutaneous T-cell lymphoma (CTCL). CTCL develops from clonally-expanded, effector/central memory CD4+ T cells in a background of chronic inflammation. The eliciting Ag(s) and how this inflammation contributes to the development and progression of CTCL remain important unknowns. Despite tumor-infiltrating CD8+ T cells in skin, the malignant CD4+ T-cell population persists, indicating that these CD8+ T cells cannot exert an effective anti-tumor response, potentially due to T-cell exhaustion, which has been observed in chronic viral infections and is associated with disease progression in hematologic malignancies like ATLL and CLL. Exhausted T-cells are characterized by immunoinhibitory molecules, like programmed death-1 (PD-1). Engagement of PD-1 by PD-Ligand (L)-1/L-2 transduces a signal that leads to inhibition of T-cell functions, but the cells are not deleted. PD-L1 is expressed on T cells, dendritic cells (DCs), and many tumor cells, whereas PD-L2 expression is limited to antigen-presenting cells. Although malignant CD4+ T cells in Sézary syndrome lesions express PD-1, the relevance of the PD-1/PD-L1/2 pathway in CTCL and the immune cells in CTCL that express PD-L1/L2 are not known.Methods: Epidermis and dermis of skin biopsies from consented CTCL pts (12) and healthy donors (3) were separated using dispase-II and incubated in RPMI-10% pooled human serum for 24 hrs. Migrated cutaneous T cells and DCs were phenotyped by flow cytometry for a variety of maturation markers and immune checkpoint molecules. In 3 pts skin findings were compared with matched PBMCs.Results: Freshly isolated cutaneous CD8+ T cells displayed distinct effector and memory subsets with a higher frequency of effector-memory (CD28-CD45RA-CCR7-) and terminally differentiated effector cells (CD28-CD45RA+CCR7-). In addition, CD8+ T-cells exhibited enhanced expression of PD-1, Lag-3, Tim-3, and ICOS. PD-1 was the receptor most overexpressed in CD8+ T cells compared with healthy controls. PD-1 expression on circulating CD8+ T cells was lower compared with skin-infiltrating CD8+ T-cells. In contrast, the malignant CD4+ T-cells expressed predominantly a central memory or transient memory subtype (CD28+CD45RA-CCR7+/-), with overexpression of PD-1, CTLA-4, Lag-3, and ICOS. Immune-suppressive CD3+CD4+FoxP3+CD127-Tregs were not increased in PBMCs of CTCL pts. Migrated DCs displayed an activated, mature phenotype expressing HLA-DR+, CD1a+/-, CD207 (Langerin)+/-, CD14+/-, CD11c+, CD83low/med, CD86low/med and PD-L1+/- phenotype.Conclusion: Both the malignant CD4+ T cells and tumor-infiltrating CD8+ T cells display an exhausted phenotype, while DC subsets display a normal activated phenotype. Our preliminary data imply a role for PD-1 and other checkpoint molecules like ICOS, Tim-3, Lag-3 in attenuating the anti-tumor response. Studies are underway in vitro to establish the function of PD-1, Lag-3, Tim-3, and ICOS on T cells in response to DC/LC stimulation to develop a rationale for therapeutic checkpoint blockade inhibitors to reverse the exhausted T cell phenotype and attendant lack of immune reactivity. DisclosuresNo relevant conflicts of interest to declare.

A phase III comparative study of nivolumab (anti-PD-1; BMS-936558; ONO-4538) versus everolimus in patients (pts) with advanced or metastatic renal cell carcinoma (mRCC) previously treated with antiangiogenic therapy.

TPS4592 Background: Standard approved treatments for pts with mRCC include interleukin-2 as well as therapies that target the vascular endothelial growth factor (eg, sunitinib, pazopanib) or mammalian target of rapamycin (eg, temsirolimus, everolimus) pathways. However, most pts may develop resistance, and overall survival (OS) improvement has only been shown in one phase 3 trial in poor-risk pts. Everolimus demonstrated a 3-month improvement in median progression-free survival (PFS) vs placebo, with no OS improvement (Motzer RJ, et al. Lancet. 2008;372:449-56). A phase 1 study of the fully human programmed death-1 (PD-1) receptor blocking monoclonal antibody nivolumab showed encouraging antitumor activity with previously treated mRCC (objective response rate [ORR], 29% (10/34); stable disease at ≥24 weeks [wks], 27% [9/34]). PD-1 is an immune checkpoint receptor that negatively regulates T-cell activation and PD-1 overexpression by tumor infiltrating lymphocytes has been associated with poor prognosis in multiple tumor types. Therefore, an ongoing, randomized, open-label, global phase 3 trial was developed to evaluate the clinical benefit of nivolumab in mRCC pts previously treated with anti-angiogenic therapy. Methods: Approximately 822 pts with advanced or metastatic clear-cell mRCC who have received ≤2 prior anti-angiogenic therapies and ≤3 total prior systemic regimens will be randomized 1:1 to receive nivolumab 3 mg/kg IV every 2 wks (Arm A) or everolimus 10 mg PO daily (Arm B). Pts will be stratified by region, MSKCC risk group, and number of prior anti-angiogenic therapy regimens. Tumor response will be assessed using RECIST 1.1 every 8 wks following randomization for the first year and then every 12 wks until disease progression or treatment discontinuation, whichever occurs later. The primary endpoint is OS. Secondary endpoints include PFS, ORR, OR duration, adverse events, OS in PD ligand 1 (PD-L1) positive or negative subgroups, and patient-reported outcomes. The trial is open and enrolling pts. Clinical trial information: NCT01668784.

CO-INHIBITORY MOLECULE PROGRAMMED DEATH-1 AND ITS LIGANDS: A NEW ALTERNATIVE THERAPY FOR HUMAN IMMUNODEFICIENCY VIRUS INFECTION?

Chronic viral infections are characterized by the up-regulation of a set of immunomodulatory receptors. The over-expression of co-inhibitory molecules on T cells leads to a dysfunctional T cell response with an &#34exhausted&#34 phenotype. Programmed Death-1 (PD-1) is a molecule that exerts an inhibitory signal on the T cell receptor when it binds to the PD-L1 or PD-L2 ligands present on antigen-presenting cells. Also, the expression of these molecules has been associated to the loss of T cell functions as well as clinical markers of the progression of HIV infection. The study of these molecules has gained attention due to reports indicating that blockade of PD-1 pathway could partially reconstitute T cell functions. In fact, this mechanism has been proposed as an alternative treatment for some chronic viral infections such as HIV infection. This review is focused on those mechanisms that might be favouring the over-expression of PD-1 and its ligands during HIV infection and on the possible new approaches that, by reducing its expression, might represent new strategies for the treatment of HIV infection. Knowing the exact mechanism leading to PD-1, PD-L1 and PDL2 expression in physiologic and pathological conditions is essential for the development of successful treatments. Novel molecular mechanisms inhibiting PD-1 activation might have potential therapeutic use not only in HIV infection but also in other diseases.

DNA demethylation in PD-1 gene promoter induced by 5-azacytidine activates PD-1 expression on Molt-4 cells

The expression of the programmed death 1 (PD-1) gene is an indicator of exhausted T-cells with decreased activation and function. It remains unknown, however, whether the methylation status of the PD-1 gene promoter is associated with PD-1 expression level. This study shows the changes of PD-1 expression levels and the demethylation status of the PD-1 promoter region in Molt-4 cells under different concentrations of 5-azacytidine (5-Zac). The result demonstrated that DNA demethylation at PD-1 promoter may contribute to PD-1 overexpression.

Programmed Death-1 (PD-1):PD-Ligand 1 Interactions Inhibit TCR-Mediated Positive Selection of Thymocytes

Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.