Expression Levels Of Programmed Death-1 Research Articles

Tumor Microenvironmental Features and Outcome in Post-Transplant Lymphoproliferative Disorder

Background: PTLD comprises a diverse spectrum of hematological conditions, ranging from early lesions, characterized by reactive-like proliferations, to monomorphic lesions, resembling overt lymphoma. Â In most cases, the lesions are believed to arise as the result of reduced immune surveillance secondary to the use of immunosuppressive drugs post-transplant. This view is supported by the observation that PTLDs, particularly those characterized by early or polymorphic lesions, may regress spontaneously upon reduction of the immunosuppressive treatment. Studies in sporadic lymphomas have identified distinct microenvironmental characteristics, predictive of the clinical behavior, but data is scarce in the immunocompromised setting. Therefore, the aim of this study was to investigate the tumor microenvironment in a population-based cohort of PTLD.Methods: We identified 108 PTLD patients diagnosed in the period 1994-2011. Of these, 62 cases had adequate tissue for tissue microarray construction. All biopsies were reviewed and classified according to the WHO 2008 criteria. Immunohistochemically stained sections were digitally quantified using Tissuemorph (Visiopharm Integrator System 4.0.3.0, Visiopharm, Denmark). Determination of optimal cut-off values of the area fraction (AF) was established by a ROC-curve. ROC analyses were performed in every disease entity and used for endpoint analyses.Results: Themedian age was 45 yrs (range 2-77 yrs) with a M/F ratio of 3:1. The majority presented in stage I-II (61%), and B symptoms and extranodal disease were common features (40% and 42%, respectively). Most tissue samples were EBV-positive (85%). The EBV latency pattern was predominantly latency II (41%) or III (43%). The AF of galectin-1 (gal-1) positive cells was clearly correlated to latency type (p=0.0001), whereas FOXP3 and programmed death-1 (PD-1) did not show such correlation (fig 1).Overall survival (OS) was significantly higher in cases with high levels of PD-1 expression, with 5-yrs OS of 39% (23-55%) and 69% (47-83%) for low and high levels, respectively (p=0.01). Expression levels of FOXP3 and gal-1 had no impact on OS in the total cohort (fig 1).In the monomorphic setting, a low AF of FOXP3 positive cells was associated with an increased risk of progression (OR 6.1; 95% CI: 1.4-26.0). This did not, however, translate into an inferior OS (p=0.163).To specifically characterize the tumor microenvironmental features of DLBCL-type PTLD with respect to cell of origin (COO), 30 evaluable cases were analyzed according to the Hans classifier; 10 (33%) were germinal center (GC) type and 20 (67%) of non-GC type. Cases of non-GC subtype had a significantly shorter time to PTLD of 1.16 yrs (CI: 0.64- 2.11), than those of GC-subtype (3.66 yrs; CI: 1.64- 8.16) (p=0.023) and a lower AF of gal-1 positive cells (p=0.042). The 5-yrs OS was 58% (32-77%) and 0% for non-GC versus GC-tumors, respectively (p=0.075). High levels of FOXP3 expression were associated with superior OS in the non-GC sub-group (p=0.04); gal-1 and PD-1 had no influence in the COO setting.Conclusion: The present study is one of the few attempts to describe tumor microenvironmental features in PTLD and relate them to outcome. In a population-based PTLD cohort, we found that specific immune cell subsets were variably expressed in different PTLD subtypes, and that high expression of PD-1 (whole cohort) and FOXP3 (non-GC DLBCL only) correlated with significantly better outcome. ÂN(%) pts in TMAOrgan tx(%)Kidney Heart Lung47(75.8) 9(14.5) 6(9.7)Type PTLD(%)Early/polymorphic lesions Monomorphic PTLD Diffuse large B-cell lymphomaPeripheral T-cel lymphomal, NOS*T-Anaplastic large cell lymphomaBurkitt lymphomaHodgkin lymphoma-type PTLD Marginal zone lymphoma18(29.0) 38(61.2) 32(51.6) 1(1.6) 3(4.8) 2(3.2) 4(6.5) 2(3.2) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load.

Objectives:Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells.Design and methods:Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets.Results:Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells.Conclusion:Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure.

Programmed death-1 expression on CD4⁺ and CD8⁺ T cells in treated and untreated HIV disease.

There is intense interest in the role of programmed death 1 (PD-1) in causing persistent T-cell dysfunction in HIV infection. However, the impact of HIV infection and antiretroviral treatment (ART) on the expression of PD-1 on T cells is still poorly defined. PD-1 was measured longitudinally in a cohort of recently HIV-infected individuals (n = 121) who started ART early (<6 months after infection) vs. later (≥2 years after infection). PD-1 was also measured cross-sectionally in a diverse cohort of chronically HIV-infected adults (n = 206). PD-1 expression levels were high on CD8⁺ T cells during early HIV infection. PD-1 levels increased on both CD4⁺ and CD8⁺ T cells populations in those who delayed therapy (11 and 10%/year, respectively). PD-1 levels declined and were similar in those treated early vs. late after 1 year of ART. In both cohorts, PD-1 expression on CD4⁺ T cells was associated with CD4⁺ T-cell activation (CD38⁺HLA-DR⁺) and inversely with CD4⁺ cell count. In contrast, PD-1 expression on CD8⁺ T cells was most strongly associated with CD8⁺ T-cell activation and with plasma viral load in viremic individuals. Across two large cohorts of untreated and treated individuals, we found consistent associations between HIV RNA levels, CD8⁺ T-cell activation and PD-1 expression on CD8⁺ T cells. In contrast, CD4⁺ T-cell counts and CD4⁺ T-cell activation were more consistent correlates of PD-1 expression on CD4⁺ T cells. PD-1 expression appears to be driven by both direct antigen and homeostatic pathways.

Upregulated PD-1 Expression Is Associated with the Development of Systemic Lupus Erythematosus, but Not the PD-1.1 Allele of the PDCD1 Gene.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complicated genetic inheritance. Programmed death 1 (PD-1), a negative T cell regulator to maintain peripheral tolerance, induces negative signals to T cells during interaction with its ligands and is therefore a candidate gene in the development of SLE. In order to examine whether expression levels of PD-1 contribute to the pathogenesis of SLE, 30 patients with SLE and 30 controls were recruited and their PD-1 expression levels in peripheral blood mononuclear cells (PBMCs) were measured via flow cytometry and quantitative real-time-reverse transcription polymerase chain reaction (RT-PCR). Also, whether PD-1 expression levels are associated with the variant of the SNP rs36084323 and the SLE Disease Activity Index (SLEDAI) was studied in this work. The PD-1 expression levels of SLE patients were significantly increased compared with those of the healthy controls. The upregulated PD-1 expression levels in SLE patients were greatly associated with SLEDAI scores. No significant difference was found between PD-1 expression levels and SNP rs36084323. The results suggest that increased expression of PD-1 may correlate with the pathogenesis of SLE, upregulated PD-1 expression may be a biomarker for SLE diagnosis, and PD-1 inhibitor may be useful to SLE treatment.

PD-1 Analysis on CD28−CD27− CD4 T Cells Allows Stimulation-Independent Assessment of CMV Viremic Episodes in Transplant Recipients

Expression of the inhibitory receptor programmed death 1 (PD-1) on cytomegalovirus (CMV)-specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28−CD27− double negativity is known as a typical phenotype of CMV-specific CD4 T cells. Therefore, the co-expression of inhibitory receptors on CD28−CD27− CD4 T cells was assessed as a rapid, stimulation-independent parameter for monitoring CMV complications after transplantation. Ninety-three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross-sectional and longitudinal manner. CMV-specific CD4 T cell levels quantified after stimulation were compared to levels of CD28−CD27− CD4 T cells. PD-1 and cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) expression on CD28−CD27− CD4 T cells were related to viremia. A percentage of ≥0.44% CD28−CD27− CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV-specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD-1 expression levels on CD28−CD27− CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA-4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD-1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD-1 expression on CD28−CD27− CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation-independent parameter to guide duration of antiviral therapy.

Expression Of Immune Checkpoints PD-L1, PD-L2, PD-1 and CTLA4 Predict For Prognosis and Resistance To Hypomethylating Agents (HMAs) In Myelodysplastic Syndromes (MDS)

The pathogenesis of MDS is multifactorial including both cell intrinsic alterations, such as mutations, and cell extrinsic stimuli such as immune deregulation. The prognosis of patients that lose response to hypomethylating agents (HMAs) is very poor and the mechanisms are not understood. Leukemia cells develop multiple resistance mechanisms to escape host immune response. Programmed death-1 (PD-1) is a negative costimulatory receptor on activated T lymphocytes. Expression of PD-1 ligands on tumor cells can induce immunosuppressive T-cells. Recent studies have indicated that demethylation of PD-1 leads to exhausted CD8+ T cells after chronic virus infection. We hypothesized that dysregulated PD-1 immune checkpoint signaling may be involved in pathogenesis of MDS and resistance to HMAs. We first evaluated the mRNA expression levels of PD-L1, PD-L2, PD-1 and CTLA4 by Q-PCR in bone marrow CD34+ cells from 124 patients which include 69 with MDS, 46 with CMML and 9 with AML. Seventy two (58%) patients were previously untreated. We observed aberrant up-regulated mRNA expression of PD-L1 in 39 patients (34% with fold 0-843.3), PD-L2 in 17 patients (14% with fold 0-22.5), PD-1 in 18 patients (15% with fold 0-76.7) and CTLA4 in 10 patients (8% with fold 0-25.1). No significant differences in gene expression were observed when comparing patients that had and had not received prior therapy. Statistically significant relationships were identified between elevated PD-1 expression and increased age (p=0.008), while elevated PD-L2 expression correlated significantly with female gender (p=0.005). Both elevated PD-L1 and CTLA4 expression correlated with MDS subtype, (p=0.034) and (p=0.012), respectively. Additionally, elevated CTLA4 expression correlated with higher white blood cell count (p=0.021), lack of prior therapy (p=0.02) and lower MDS IPSS score (p=0.027). We then performed an analysis of the impact on survival of the 4 gene expression in patients that had not received prior therapy. Patients with lower expression of PD-L1 had a non-significant trend towards better survival (31.5 months versus 16.2, p=0.24). Forty six of 124 patients analyzed received HMAs, and within these 46 patients lower PD-L1 expression correlated with a significantly improved overall response rate (67% vs 25%, p=0.038). Aberrant up-regulation of these 4 genes was also observed in peripheral blood mononuclear cell (PBMNC) from 61 MDS, CMML and AML patients. The relative expression of PD-L1 was significantly higher in MDS (p=0.018) and CMML (p=0.0128) compared to AML. Of interest, mRNA expression of these 4 genes was significantly higher in PBMNC than in CD34+ cells except PD-L1. By immunohistochemical (IHC) analysis, a strong correlation was observed between protein and mRNA expression. By IHC, we observed that leukemia blasts were positive for PD-L1 whereas stroma/non-blast cellular compartment was positive for PD-1 in bone marrow biopsies from MDS, CMML and AML patients. We then analyzed effect of HMAs on these four gene expression in cohort of 61 patients treated with different trials of epigenetic therapy. Treatment resulted in up regulated expression of PD-L1 in 57% of the patients (with maximum induction fold of 4.8), PD-L2 in 57% (maximum fold of 15.77), PD-1 in 58% (maximum fold of 50.26) and CTLA4 in 66% (maximum fold of 29.9). Of importance, patients resistant to therapy had increased gene expression compared to patients that achieved response (fold 5.3 vs 0.4 for PD-L1, 6.2 vs 0.4 for PD-L2, 3.0 vs 0.4 for PD-1 and 5.4 vs 0.7 for CTLA4). Comparing patients without and with expression induction, the median survival was 11.7 and 6.6 months (p=0.122) for PD-L1, 12.5 and 4.7 months (p=0.029) for PD-L2, indicating a better prognosis in patients without PD-L1 and PD-L2 induction treated with HMAs. To model this observation, we treated KG-1 and THP1 cells with different concentrations of decitabine (0 to 10 uM) and observed a dose dependent up-regulation of PD-L1 and PD-L2 in THP1 cells, and CTLA4, PD-1, PD-L1 in KG1 cells. Exposure to decitabine resulted in demethylation of PD-1 in these cell lines, and the demethylation effect was also observed in HMAs treated MDS and AML patients. This study suggests immune checkpoint PD-1/PD-1 ligands signaling may be involved in MDS pathogenesis and resistance mechanisms to HMAs. Blockade of this pathway can be a potential therapy in MDS and AML. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Strength of PD-1 signaling differentially affects T-cell effector functions

High surface expression of programmed death 1 (PD-1) is associated with T-cell exhaustion; however, the relationship between PD-1 expression and T-cell dysfunction has not been delineated. We developed a model to study PD-1 signaling in primary human T cells to study how PD-1 expression affected T-cell function. By determining the number of T-cell receptor/peptide-MHC complexes needed to initiate a Ca(2+) flux, we found that PD-1 ligation dramatically shifts the dose-response curve, making T cells much less sensitive to T-cell receptor-generated signals. Importantly, other T-cell functions were differentially sensitive to PD-1 expression. We observed that high levels of PD-1 expression were required to inhibit macrophage inflammatory protein 1 beta production, lower levels were required to block cytotoxicity and IFN-γ production, and very low levels of PD-1 expression could inhibit TNF-α and IL-2 production as well as T-cell expansion. These findings provide insight into the role of PD-1 expression in enforcing T-cell exhaustion and the therapeutic potential of PD-1 blockade.

Rapid downregulation of programmed death‐1 and interferon‐γ‐inducible protein‐10 expression is associated with favourable outcome during antiviral treatment of chronic hepatitis B

The dynamics of programmed death-1 (PD-1) as well as cytokine/chemokine expression and its correlation with virological response in patients with chronic hepatitis B (CHB) is unclear. This study was conducted in 29 treatment-naïve patients undergoing telbivudine treatment for 52 weeks. The results showed that PD-1 expression on both CD4+ and CD8+ T cells was positively correlated with hepatitis B virus (HBV) DNA levels (r = 0.621, P < 0.0001; r = 0.548, P = 0.002, respectively), and in virological responders, this decrease was directly correlated with a decrease in HBV DNA levels (r = 0.664, P = 0.002; r = 0.572, P = 0.01, respectively). Furthermore, at the end of 52 weeks, in virological responders, the decreased rate in the frequency of PD-1+ CD8+ T cells was significantly higher than in non-virological responders (58.3% vs 25.7%, P = 0.001), and at weeks 24 and 52, in virological responders, PD-1 expression on CD4+ and CD8+ T cells was lower than in non-virological responders (P = 0.01 and P = 0.035; P < 0.0001 and P < 0.0001, respectively). In 34 cytokines/chemokines detected in serum, IP-10 expression was positively correlated with viral load, level of ALT and PD-1 expression on CD8+ and CD4+ T cells at baseline (r = 0.36, P = 0.055, r = 0.635, P < 0.0001, r = 0.414, P = 0.026, and r = 0.402, P = 0.030, respectively). Moreover, the decrease in IP-10 in serum directly correlated with a decrease in ALT levels (r = 0.751, P < 0.0001). At weeks 24 and 25, IP-10 expression was significantly lower than baseline in virological responders (both P = 0.005); however, this was not observed in nonresponders. Based on the above findings, PD-1 and IP-10 may be used as predictors for virological response, and blockade of their pathway may improve the outcome of patients with CHB.

PD-1 expression in HBcAg-specific CD8+ T cells of adolescents with chronic HBV infection

To investigate the differential expression of programmed death-1 (PD-1) in the hepatitis B core antigen (HBcAg)17-28-specific CD8+ T cell subsets of adolescent patients with chronic hepatitis B virus (HBV) infection during the immune tolerant phase and the immune clearance phase. A total of 105 patients between the ages of 12-28 years old (mean age 17.20+/-6.35) with chronic HBV infection and 15 healthy age-matched individuals were enrolled in the study. The patients were divided into two groups according to their current status in immune clearance phase (n = 55) or immune tolerant phase (n = 50), as determined by hepatic biopsy pathology. Flow cytometry was used to detect HLA-A2 type and PD-1 expression on peripheral blood mononuclear cells (PBMC) and HBcAg17-28-specific CD8+ T cells. PD-1 mRNA levels in PBMCs were measured by reverse transcription-polymerase chain reaction (RT-PCR). Independent samples t-test was used to compare means between the two groups, and one-way ANOVA was used to compare means among multiple groups. Pearson's correlation coefficient was used to assess the significance of correlation. The frequency of HBcAg18-27-specific CD8+ T cells was significantly higher in the immune clearance phase group than in the immune tolerant phase group (t = 18.08, P less than 0.01), but the expression of PD-1 on the HBcAg18-27 specific CD8+ T cells was significantly lower in the immune clearance phase group than in the immune tolerant phase group (t = 4.72, P less than 0.01). A negative correlation existed between the frequency of HBcAg18-27-specific CD8+ T cells and PD-1 expression (r = -0.463, P less than 0.01). A positive correlation existed between HBV viral load and PD-1 expression on the HBcAg18-27-specific CD8+ T cells in chronic HBV infection patients (r = 0.882, P less than 0.01), and there was a negative correlation between PD-1 expression levels on HBcAg18-27-specific CD8+ T cells and hepatic tissue inflammation score (r = -0.76, P less than 0.01). PD-1 mRNA in PBMCs was significantly higher in the immune tolerant phase group than in the immune clearance phase group (t = 30.89, P less than 0.01). Up-regulated expression of PD-1 is associated with HBV-specific CD8+ T cells and may play a crucial role in inhibiting their function during the immune tolerance phase of chronic HBV infection in adolescents.

T Cell Exhaustion Contributes to Immune Evasion in Chronic Lymphocytic Leukaemia

Abstract 1773T cell exhaustion is a state of T cell dysfunction in response to chronic antigen exposure, marked by impaired effector function and the continued expression of inhibitory receptors such as Programmed Death 1 (PD-1) (Wherry EJ. Nat Immunol. 2011 Jun;12(6):492–9.). Because tumour growth in chronic lymphocytic leukaemia (CLL) occurs over a long period of time, we hypothesized that the continued exposure of T cells to a CLL-derived antigen could also lead to a state of T cell exhaustion. We therefore investigated whether T cell exhaustion is induced in CLL by using the Eμ-TCL1 transgenic (tcl1tg) tumour transfer mouse model for this disease (Hofbauer JP, et al. Leukemia. 2011 Sep;25(9):1452-8) and by analyzing primary samples from CLL patients. We found that the number of PD-1+ T cells was increased in both CD4+ and CD8+ populations and in all lymphoid compartments examined of the Eμ-TCL1 transgenic (tcl1tg) tumour recipient mice, but not in recipient mice receiving wildtype (WT) splenocytes showing that leukemic mice have an increased number of T cells displaying an exhausted phenotype that is induced by the presence of CLL cells. We next assessed the expression of the ligands for PD-1 on the surface of murine CLL cells. Peripheral CLL tumour cells showed only a modest increase in PD-L1 expression as compared to WT B cells. However, lymph node and spleen residing tumour cells showed a marked increase in PD-L1 expression, which suggests a microenvironment-induced upregulation of PD-L1 on tumour cells, e.g. by their close contact to accessory cells.To validate our results on primary human CLL samples, we collected peripheral blood from 89 unselected CLL patients and 18 healthy donors and observed an increase in surface expression of PD-1 on the CD4+ and CD8+ T cell populations. While the percentage of PD-1+ CD4+ T cells in chemonaive patients was comparable to healthy donors, chemotherapy drastically increased the number of PD-1-expressing CD4+ T cells (63.81% ±19.75 vs 35.70% ±19.22; p<.001). In contrast, treatment apparently had little impact on the PD-1+ CD8+ population, indicating that PD1 induction on CD8+ T cells might be a more general CLL specific phenomenon. PD-1 expression in chemonaive patients did not correlate with the prognostic markers CD38, Zap-70 and IgVH mutations, however, it significantly correlated with Rai stage (CD4+: p<.001; CD8+: p=.003; spearman correlation). Our immune phenotyping analyses also included markers to distinguish between naïve, memory, and effector T cell subsets. We observed that PD-1 expressing T cells belong primarily to the memory compartment, characterized by the absence of CD45RA expression. PD-1 expression levels in T cells was independent from chronic human cytomegalovirus (HCMV) infection, indicating that increased PD-1+ T cell numbers in CLL do not simply reflect a higher incidence of HCMV infections, which has been described in this patient population.As we speculated that PD1/PDL1 pathway might be exploited by CLL to evade a T cell dependent cytotoxic attack, we next wanted to know whether blocking this pathway by recombinant PD1 (rPD1) or PD-L1 specific Fab fragments in vivo would lead to reinvigoration of CLL specific T cells reflected in CLL clearance. We found that inhibition of PD1/PD-L1 binding in vivo in the tcl1tg tumour transfer model led to a decrease in CLL load in the peripheral blood and in all analysed organs (lymph node, spleen, bone marrow, liver, lung) while in vitro treatment with rPD-1 or PD-L1 Fab fragment did not affect the viability of mouse tumour cells. Our results imply that in tumour bearing mice, a vast number of tumour-specific exhausted CTLs exist that can be immediately reactivated by PD1/PD-L1 blockage leading to cytolysis of the tumour, thereby representing a promising target for therapeutic intervention in CLL patients. Disclosures:No relevant conflicts of interest to declare.

Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in chickens infected with Marek’s disease virus

BackgroundAn immunoinhibitory receptor, programmed death-1 (PD-1), and its ligand, programmed death-ligand 1 (PD-L1), are involved in immune evasion mechanisms for several pathogens causing chronic infections and for neoplastic diseases. However, little has been reported for the functions of these molecules in chickens. Thus, in this study, their expressions and roles were analyzed in chickens infected with Marek’s disease virus (MDV), which induces immunosuppression in infected chickens.ResultsA chicken T cell line, Lee1, which constitutively produces IFN-γ was co-cultured with DF-1 cells, which is a spontaneously immortalized chicken fibroblast cell line, transiently expressing PD-L1, and the IFN-γ expression level was analyzed in the cell line by real-time RT-PCR. The IFN-γ expression was significantly decreased in Lee1 cells co-cultured with DF-1 cells expressing PD-L1. The expression level of PD-1 was increased in chickens at the early cytolytic phase of the MDV infection, while the PD-L1 expression level was increased at the latent phase. In addition, the expression levels of PD-1 and PD-L1 were increased at tumor lesions found in MDV-challenged chickens. The expressions levels of PD-1 and PD-L1 were also increased in the spleens and tumors derived from MDV-infected chickens in the field.ConclusionsWe demonstrated that the chicken PD-1/PD-L1 pathway has immunoinhibitory functions, and PD-1 may be involved in MD pathogenesis at the early cytolytic phase of the MDV infection, whereas PD-L1 could contribute to the establishment and maintenance of MDV latency. We also observed the increased expressions of PD-1 and PD-L1 in tumors from MDV-infected chickens, suggesting that tumor cells transformed by MDV highly express PD-1 and PD-L1 and thereby could evade from immune responses of the host.

HBcAg induces PD-1 upregulation on CD4+T cells through activation of JNK, ERK and PI3K/AKT pathways in chronic hepatitis-B-infected patients

Hyper-expression of programmed death-1 (PD-1) is a hallmark of exhausted T cells. In chronic hepatitis-B virus (HBV)-infected patients, PD-1 upregulation on T cells was often observed. The mechanism of it has not been fully understood. In this study, we examined the dynamic changes of PD-1 expression on T cells during the natural history of chronic HBV infection and explored the signaling pathway of PD-1 upregulation by the hepatitis-B core antigen (HBcAg). Sixty-seven chronic HBV-infected patients were categorized into an immune tolerance group, an immune clearance group and an inactive virus carrier group, and 20 healthy volunteers were chosen as normal control group. Peripheral blood mononuclear cells from patients and healthy volunteers, and T lymphocytes from healthy volunteers were separated. Results showed that the PD-1 expression level on CD4+T cells in every phase of chronic HBV infection was significantly higher than that in healthy volunteers, whereas such effects were not observed on CD8+T cells. In the immune clearance phase, a positive correlation was found between serum HBV DNA level and the PD-1 expression level on CD4+T cells. In all phases, no correlation was shown between serum alanine amino transferase (ALT) level and PD-1 expression level. Phosphorylation of JNK, ERK and AKT was induced by HBcAg, and inhibitors of JNK, ERK and PI3K/AKT significantly decreased the HBcAg-induced PD-1 upregulation on CD4+T cells. In conclusion, the PD-1 expression level on CD4+T cells was upregulated in every phase of chronic HBV infection, which was induced by HBcAg through JNK, ERK and PI3K/AKT signaling pathways.

T-cell Immune Abnormality and Novel Immunotherapeutic Approach by Blocking the B7-H1-PD-1 Pathway Combined with WT1 Tumor Vaccine in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are hematologic malignancies caused by the neoplastic transformation of hematopoietic stem or myeloid progenitor cells and characterized by cytopenias, excessive apoptosis of hematopoietic cells, and a high risk of progression to acute myeloid leukemia. During disease progression, clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Stem cell transplantation is the only curative treatment for MDS, but it is not suitable for elderly patients. B7-H1 (CD274) molecules, which are expressed on antigen-presenting cells and inhibit T-cell responses through programmed death-1 (PD-1) expressed on activated T cells, are expressed on various tumor cells, and B7-H1-expressing tumor cells evade attack by tumor-specific cytotoxic T lymphocytes (CTLs). We reported immune dysfunction associated with T cells in MDS, i.e., an increase in T-cell apoptosis, higher expression levels of PD-1 on circulating T cells, and higher levels of plasma-soluble interleukin-2 receptor. Furthermore, we demonstrated that MDS blasts overexpress Wilms tumor gene WT1 mRNA, suggesting that the anti-WT1 immune response elicited by WT1 peptide vaccine may induce tumor regression in some patients. In the current study, we investigated B7-H1 expression on MDS blasts and analyzed the characteristics and inhibitory effects on T-cell immune responses of B7-H1 blasts. Finally, to develop new strategies, we are now analyzing a novel immunotherapy involving blockade of the B7-H1-PD-1 pathway in combination with WT1 tumor vaccine and or inhibition of regulatory T cells. First, we analyzed B7-H1 expression in 3 MDS cell lines, i.e., F-36P, OIH-1, and SKM-1, and on MDS blasts from 29 MDS patients, 32 patients with acute myeloid leukemia transformed from MDS (AL-MDS), and 10 hematologically normal individuals. A high level of B7-H1 expression at the mRNA and protein levels was detected only in F-36P cells using reverse transcription-PCR and flow cytometry (FCM), respectively. B7-H1 protein was not detectable on OIH-1 and SKM-1. Blasts from patients with high-risk MDS and AL-MDS expressed B7-H1 molecules more often compared with those from low-risk MDS patients. B7-H1 molecules were expressed in fewer than 5% of blasts in normal individuals. Furthermore, we found that the cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which may be associated with MDS pathophysiology, induced B7-H1 expression on SKM-1 cells or blasts from MDS patients, and that B7-H1 induction by these cytokines was mediated by nuclear factor (NF)-κB activation, whose activation was observed in MDS patients, in particular in advanced disease. Second, to investigate the characteristics of B7-H1 blasts, we analyzed proliferative advantage, i.e., cell cycle and colony formation, in B7-H1 and B7-H1 cell fractions in MDS blasts using FCM and the methylcellulose assay, respectively. B7-H1 MDS blasts had greater intrinsic proliferative capacity than B7-H1 MDS blasts when examined in both assays. Third, to investigate the immunomodulatory effects of B7-H1 MDS blasts on T-cells, T-cell apoptosis and

PD-1 Is Elevated on the Peripheral Blood T Cell Subsets of Patients with Relapsed/Refractory Hodgkin Lymphoma Compared to Normal Volunteers

Abstract 4860 Background:Programmed death-1(PD-1) and programmed death-1 ligand (PD-L) signaling are involved in the functional impairment and “exhaustion” of T cells in conditions such as chronic viral infection and in tumor immune evasion. The interaction of PD-1 with its ligand PD-L suppresses T cell function. Up-regulation of PD-L has been demonstrated both in Hodgkin lymphoma (HL) cell lines, and in primary Hodgkin Reed Sternberg (HRS) cells. PD-1 is markedly elevated in the tumor infiltrating lymphocytes of HL patients, leading to the hypothesis that T cell exhaustion and deficient anti-tumor immunity induced by the activation of the PD-1-PD-L signaling pathway may play a key role in creating a permissive milieu for HL. In this setting, elevated PD-1 expression in HL patient T cells may be a biomarker of disease activity. We investigated this hypothesis by examining PD-1 expression in the peripheral T cells of patients with relapsed/ refractory HL. Methods:Patients with relapsed or refractory HL who enrolled in a clinical trial of an HDAC inhibitor in combination with Niacinamide between December of 2010 and July of 2011 were eligible to participate. PD-1 levels were assessed pre-treatment, and at selected timepoints during therapy. The levels of PD-1 expression for 5 relapsed/refractory HL patients and 4 healthy control subjects were evaluated by flow cytometry. An aliquot of cells (1 × 106/mL) was washed and stained with: CD3 APC-H7, CD8 PerCP-Cy5.5, CD4 FITC and PD-1-APC, for 20 minutes at 4°C. Dead cells were excluded using the Live/Dead Fixable Staining Kit (Invitrogen), and stained cells were acquired using the LSR II flow cytometer (BD). Compensation (parallel controls using cells singly stained for each color) and data analysis were performed using FlowJo flow cytometry analysis software (TreeStar). The percentage and/or mean fluorescence intensity (MFI) of PD-1+ cells within the live CD3+CD4+ and CD3+CD8+ populations was compared to isotype controls to establish baseline values and to normal control subjects. Results:The median age of the 5 HL patients was 32 (range 25–73). The median age of the healthy control subjects was 43 (range 39–49). Three of the HL subjects were male; all 4 of the normal controls were female. All 5 of the HL patients were heavily pretreated with an average number of prior regimens of 7.8 (range 5–13). Three of the 5 patients underwent previous autologous stem cell transplant, and 2 had a prior allogeneic stem cell transplant. There was a clear shift of PD1+ cells within the CD4+ T cell population in the HL patients compared to normal controls. PD-1 was significantly elevated in the peripheral blood CD4+ cells of HL patients compared to normal volunteers (mean MFI 1034 vs 123, p < 0.025). PD-1 was also elevated on the CD8+ T cells of the HL patients compared to normal volunteers (MFI 808 vs 221), but did not reach significance. There was no correlation between the level of PD-1 elevation on CD4+ or CD8+ cells, and prior autologous or allogeneic transplantation. Conclusion:PD-1 expression in peripheral blood CD4+ T cells may be a potential biomarker of systemic immune dysregulation in heavily pre-treated HL patients. PD-1 warrants further exploration in relapsed/refractory HL both as a potential biomarker, and as a target for directed immunotherapy. Additional studies by our group, investigating the role PD-1 as a biomarker in relapsed/refractory HL are ongoing. Disclosures:O'Connor:Spectrum: Research Funding; Novartis: Research Funding; Merck: Research Funding; Celgene: Consultancy, Research Funding.

Responsiveness of HIV-specific CD4 T cells to PD-1 blockade

AbstractDefining the T helper functions impaired by programmed death–1 (PD-1) is crucial for understanding its role in defective HIV control and determining the therapeutic potential of targeting this inhibitory pathway. We describe here the relationships among disease stage, levels of PD-1 expression, and reversibility of CD4 T-cell impairment. PD-L1 blockade in vitro enhanced HIV-specific production of Th0 (IL-2), Th1 (IFN-γ), Th2 (IL-13), and TFH (IL-21) cytokines by CD4 T cells. PD-L1 blockade caused an early increase in cytokine transcription and translation that preceded cell proliferation. Although the impact of PD-L1 blockade on cytokine expression and, to a lesser extent, cell proliferation was associated with markers of disease progression, restoration of cytokine secretion was also observed in most subjects with undetectable viremia. PD-L1 blockade restored cytokine secretion in both PD-1intermediate and PD-1high sorted CD4 T-cell subsets. Compared with PD-1high HIV-specific CD8 T cells, PD-1high HIV-specific CD4 T cells showed lower expression of the inhibitory molecules CD160 and 2B4, demonstrating marked differences in expression of inhibitory receptors between T-cell subsets. These data show that PD-1 impairs HIV-specific T helper responses both by limiting expansion of these cells and by inhibiting effector functions of multiple differentiated CD4 T-cell subsets.

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.

Exhaustion of CMV Specific T Cells with Enhanced PD-1 Expression In Persistent Cytomegalovirus Infection After Allogeneic Stem Cell Transplantation

AbstractAbstract 3912Cytomegalovirus (CMV) is one of the most common pathogens causing morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT), despite preemptive treatments employing antiviral drugs. Cytotoxic T cells are indispensable to control CMV infections. Chronic viral infections with human immunodeficiency virus or hepatitis C virus were shown to be associated with exhausted T cells with high expression of the inhibitory molecule programmed death 1 (PD-1). Recently, it has been reported that PD-1 up-regulation on CMV specific T cells was associated with CMV infection after renal and liver transplantation. PD-1 expression on CMV specific T cells after HSCT has not been well examined. We evaluated the involvements of exhausted CMV specific T cells characterized by high PD-1 expression in persistent CMV infection after allogeneic HSCT. Peripheral blood mononuclear cells (PBMC) and serum were obtained from an HLA-A*2402-positive patient who had received bone marrow transplantation from an HLA-A, B, C and DR matched unrelated donor. This patient failed to eliminate CMV for more than one year after transplantation despite intermittent administration of ganciclovir and foscarnet. Control PBMC and serum were obtained from an HLA-A*2402-positive healthy volunteer because the Japan Marrow Donor Program prohibits blood collection for research use from donor. All blood was collected with written informed consent. We at first analyzed frequencies of CMV-specific CD8+ T cells in patient and control PBMC by flow cytometer using QYDPVAALF/A*2402-specific tetramer and CD8 antibodies. QYDPVAALF is derived from CMV pp65 protein and presented by the HLA-A*2402 molecule. Tetramer stained cells were detected in the patient PBMC but control PBMC (0.11% versus undetectable). Patient and control PBMC were stimulated by a synthetic peptide QYDPVAALF in culture media containing IL-2 for 14 days, and stained with QYD/A*2402-specific tetramer. Remarkably, post-stimulated patient PBMC contained only 0.54% of tetramer stained CD8+ T cells, whereas a more dramatic increase (14.1%) in control PBMC. We analyzed frequencies of IFN-g secreting CD8+ T cells in PBMC after stimulation with a peptide pool covering the whole CMV pp65 protein for 4 hours. Less patient CD8+ T cells produced IFN-g, compared with the control CD8+ T cells (0.5% versus 1.1%) These data demonstrate dysfunction of CMV-specific CD8+ T cells in the patient with persistent CMV infection. To examine the mechanism of dysfunction of CMV-specific CD8+ T cells, we analyzed the expression of PD-1 on CMV-specific CD8+ T cells 14 days after stimulation with QYDPVAALF peptide. Multiparameter flow cytometry and tetramer assay exhibited higher expression of PD-1 on CMV-specific CD8+ T cells generated from patient PBMC, compared with CMV-specific CD8+ T cells generated from control PBMC. To find out whether the engagement of PD-1 to its ligand (PD-L1) leads to T cell exhaustion, we stimulated patient PBMC with QYDPVAALF peptide for 14 days in the presence or absence of anti PD-L1 antibody which blocks PD-1/PD-L1 inhibitory pathway. Blockade of PD-1/PD-L1 pathway resulted in 3.9-fold increase in patient CMV specific T cells. These findings demonstrate that PD-1 is associated with the exhaustion of CMV specific CD8+ T cells during persistent CMV infection in this patient. To examine the effect of patient serum on CMV specific CD8+ T cells, we stimulated patient PBMC with QYDPVAALF peptide for 14 days in culture media with patient or control serum. CMV specific CD8+ T cells increased 4-fold and 55-fold in the presence of patient and control serum, respectively. Patient serum led to higher PD-1 expression on CMV specific CD8+ T cells, compared with control serum (Fig). These findings suggest that patient serum may contain what regulates PD-1 expression level of exhausted T cells. Further investigations to identify factors regulating PD-1 expression in patient serum are in progress. The identification of the factors may provide new strategies to improve exhausted T cell function. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Lower expression levels of the programmed death 1 receptor on CD4+CD25+ T cells and correlation with the PD‐1.3A genotype in patients with systemic lupus erythematosus

A genetic polymorphism in the programmed death 1 (PD-1) gene encoding the coinhibitory PD-1 immunoreceptor, PD-1.3A, is associated with systemic lupus erythematosus (SLE). The aim of this study was to assess PD-1 receptor expression in patients with SLE, in comparison with relatives and unrelated healthy controls, and to identify correlations of lower expression levels of PD-1 receptor with the PD-1.3A genotype. Patients with SLE, patients' relatives, and unrelated healthy control subjects from Iceland and Sweden were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3/anti-CD28, and PD-1 expression was analyzed by flow cytometry. PD-1.3A/G genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. PD-1 expression on PBMCs was induced after antibody stimulation, showing increases of 2.1-fold in SLE patients, 3.1-fold in relatives, and 5.1-fold in healthy controls. The frequency of PD-1+ cells was significantly lower in SLE patients compared with relatives and healthy controls. PD-1 expression on PD-1+ cells and on CD4+CD25+ T cells was significantly lower in SLE patients and relatives compared with healthy controls. PD-1 expression was significantly elevated on CD25(high) cells. Levels of PD-1 expression on CD25(high) and CD25(intermediate) cells were significantly lower in SLE patients compared with healthy controls. PD-1 was expressed on both FoxP3- and FoxP3+ cells. Lower expression of PD-1 was significantly correlated with the PD-1.3A/G genotype. The results demonstrate significantly lower PD-1 receptor expression in SLE patients and their relatives and reveal a significant correlation of lower PD-1 expression with the PD-1.3A allele. Thus, PD-1.3A may contribute to abnormalities in PD-1 receptor expression on CD4+CD25+ T cells in patients with SLE, providing support for an important role of the PD-1 pathway in SLE and, possibly, in other autoimmune diseases.

Cloning and identification of porcine programmed death 1

Programmed death 1 (PD-1) is a member of the immunoglobulin (Ig) superfamily, which is expressed on activated T cells, B cells and monocytes. Many researches have demonstrated that a high level of PD-1 expression is closely related to persistent infection and immune evasion in some human infections. In order to study the relationship between PD-1 expression and persistent infections caused by some porcine viruses, we first cloned the porcine PD-1 from porcine PBMCs based on the blast result in the EST database using the human PD-1 sequence. Sequence analysis showed that the cloned PD-1 molecule shares 63 and 54% amino acid sequence identity with human and murine PD-1, respectively. Its molecular structure is also similar to that of human and murine PD-1, containing an IgV-like domain in the extracellular region and two immune regulatory motifs in its cytoplasmic tail. The in vitro T cell proliferation assay showed that the cloned PD-1 could inhibit porcine T cell proliferation by 71% and secretion of IFN-γ and IL-2 by 64 and 53%, respectively. These data suggest that porcine PD-1 negatively regulates the porcine immune response in a similar manner to that of its counterpart in the human and mouse immune system.

Inhibitory TCR Coreceptor PD-1 Is a Sensitive Indicator of Low-Level Replication of SIV and HIV-1

Ongoing antigenic stimulation appears to be an important prerequisite for the persistent expression of programmed death 1 (PD-1), an inhibitory TCR coreceptor of the CD28 family. Although recent publications have emphasized the utility of PD-1 as a marker for dysfunctional T cells in chronic viral infections, its dependence on antigenic stimulation potentially renders it a sensitive indicator of low-level viral replication. To explore the antigenic threshold for the maintenance of PD-1 expression on virus-specific T cells, we compared PD-1 expression on virus-specific and memory T cell populations in controlled and uncontrolled SIV and HIV-1 infection. In both controlled live attenuated SIV infection in rhesus macaques and HIV-1 infection in elite controllers, elevated levels of PD-1 expression were observed on SIV- and HIV-1-specific CD8(+) T cells. However, in contrast to chronic wild-type SIV infection and uncontrolled HIV-1 infection, controlled SIV/HIV-1 infection did not result in increased expression of PD-1 on total memory T cells. PD-1 expression on SIV-specific CD8(+) T cells rapidly decreased after the emergence of CTL escape in cognate epitopes, but was maintained in the setting of low or undetectable levels of plasma viremia in live attenuated SIV-infected macaques. After inoculation of naive macaques with a single-cycle SIV, PD-1 expression on SIV-specific CD8(+) T cells initially increased, but was rapidly downregulated. These results demonstrate that PD-1 can serve as a sensitive indicator of persistent, low-level virus replication and that generalized PD-1 expression on T lymphocytes is a distinguishing characteristic of uncontrolled lentiviral infections.