Defective Induction of IL-27-Mediated Immunoregulation by Myeloid DCs in Multiple Sclerosis.

The purpose of this study was to examine whether myeloid dendritic cells (mDCs) from patients with multiple sclerosis (MS) and healthy controls (HCs) become similarly tolerogenic when exposed to IL-27 as this may represent a potential mechanism of autoimmune dysregulation. Our study focused on natural mDCs that were isolated from HCs and MS patient peripheral blood mononuclear cells (PBMCs). After a 24-h treatment with IL-27 ± lipopolysaccharide (LPS), the mDCs were either harvested to identify IL-27-regulated gene expression or co-cultured with naive T-cells to measure how the treated DC affected T-cell proliferation and cytokine secretion. mDCs isolated from HCs but not untreated MS patients became functionally tolerogenic after IL-27 treatment. Although IL-27 induced both HC and untreated MS mDCs to produce similar amounts of IL-10, the tolerogenic HC mDCs expressed PD-L2, IDO1, and SOCS1, while the non-tolerogenic untreated MS mDCs expressed IDO1 and IL-6R. Cytokine and RNA analyses identified two signature blocks: the first identified genes associated with mDC tolerizing responses to IL-27, while the second was associated with the presence of MS. In contrast to mDCs from untreated MS patients, mDCs from HCs and IFNb-treated MS patients became tolerogenic in response to IL-27. The genes differentially expressed in the different donor IL-27-treated mDCs may contain targets that regulate mDC tolerogenic responses.

Cellular and molecular regulation of the programmed death-1/programmed death ligand system and its role in multiple sclerosis and other autoimmune diseases.

Programmed Cell Death 1 (PD-1) receptor and its ligands (PD-Ls) are essential to maintain peripheral immune tolerance and to avoid tissue damage. Consequently, altered gene or protein expression of this system of co-inhibitory molecules has been involved in the development of cancer and autoimmunity. Substantial progress has been achieved in the study of the PD-1/PD-Ls system in terms of regulatory mechanisms and therapy. However, the role of the PD-1/PD-Ls pathway in neuroinflammation has been less explored despite being a potential target of treatment for neurodegenerative diseases. Multiple Sclerosis (MS) is the most prevalent, chronic, inflammatory, and autoimmune disease of the central nervous system that leads to demyelination and axonal damage in young adults. Recent studies have highlighted the key role of the PD-1/PD-Ls pathway in inducing a neuroprotective response and restraining T cell activation and neurodegeneration in MS. In this review, we outline the molecular and cellular mechanisms regulating gene expression, protein synthesis and traffic of PD-1/PD-Ls as well as relevant processes that control PD-1/PD-Ls engagement in the immunological synapse between antigen-presenting cells and T cells. Also, we highlight the most recent findings regarding the role of the PD-1/PD-Ls pathway in MS and its murine model, experimental autoimmune encephalomyelitis (EAE), including the contribution of PD-1 expressing follicular helper T (TFH) cells in the pathogenesis of these diseases. In addition, we compare and contrast results found in MS and EAE with evidence reported in other autoimmune diseases and their experimental models, and review PD-1/PD-Ls-targeting therapeutic approaches.

Increasing the expression of programmed death ligand 2 (PD-L2) but not 4-1BB ligand in colorectal cancer cells.

Immune checkpoint (ICP) molecules modulate the immune response by either inducing or preventing T cell activation. Over-expression of some ICPs on malignant cells has been shown to regulate anti-tumor immune responses. We aimed to investigate the expression levels of two immune checkpoint molecules which have not been studied extensively in patients with colorectal cancer (CRC). Programmed Death Ligand 2 (co-inhibitory) and 4-1BB ligand (co-stimulatory) were assessed in tumor tissues of CRC patients compared to the adjacent normal tissues. Following tissue excision during surgical operation from 21 CRC patients, RNA extraction, cDNA synthesis and semi-quantitative real-time PCR were done for measuring the expressions of PD-L2 and 4-1BBL genes. In protein level, indirect immunohistochemistery (IHC) was performed on tissue sections. We revealed that PD-L2 was expressed in about 81% CRCs and insignificantly correlated with the tumor differentiation grade. Although a 3.25-fold change in the gene expression of PD-L2 was found in tumor tissues compared to the adjacent normal tissues (P = 0.005), but decreased level of 4-1BBL in counterpart tissues was not significant. Our results were confirmed by IHC for PDL-2 (P = 0.02) and 4-1BBL, however it was not statistically significant for the latter one. Although not significant, we could find an association between the elevated expression of PD-L2 and the tumor differentiation grade. Increased expression of negative regulator of the anti-tumor immune responses like PD-L2, as a prominent way of tumor escape, can be considered for cancer immunotherapy approaches in CRC patients using blocking monoclonal antibodies.

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.

Molecular cloning, expression and characterization of Pekin duck programmed death-1.

Programmed death-1 (PD-1) has a pivotal role in the attenuation of adaptive immune responses and peripheral tolerance. Here we describe the identification of the Pekin duck programmed death-1 orthologue (duPD-1). The duPD-1 cDNA encodes a 283-amino acid polypeptide that has an amino acid identity of 70%, 32% and 31% with chicken, murine and human PD-1, respectively. The duck PD-1 gene shares five conserved exons with chicken, murine and human PD-1 genes. A cluster of putative regulatory elements within the conserved region B (CR-B) of the basal promotor is conserved. Homology modeling was most compatible with the two ß-sheet IgV domain structure of murine PD-1. Contact residues, shown to be critical for binding of the respective human and murine PD-1 ligands are mostly conserved between avian and mammalian species, whereas residues that define the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) are highly conserved across higher vertebrates and frog. Constitutive expression of duPD-1 transcripts was predominantly found in lymphocyte-rich tissues, and mitogen-stimulation of duck peripheral blood mononuclear cells transiently increased duPD-1 mRNA expression. A soluble duPD-1 protein was expressed and shown to engage the identified duck PD-1 ligands. Our observations show considerable evolutionary conservation between mammalian and avian PD-1 orthologues. This work will facilitate further investigation of the role of PD-1 signaling in adaptive immunity in the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.

Expression and prognostic value of PD-L1 and PD-L2 in ovarian cancer.

BACKGROUND: In the present study, we aimed to investigate the expression and prognostic value of co-stimulatory molecules, programmed death ligand-1 (PD-L1) and PD-L2, in ovarian cancer (OC). METHODS: Immunohistochemical (IHC) staining was used to assess the expressions of PD-L1 and PD-L2 in 77 cases of OC, and 10 cases of benign ovarian cyst were employed as negative controls. Moreover, χ2 test was used to analyze the correlation between the PD-L1/PD-L2 expression and clinicopathological parameters. Kaplan-Meier method was used to compare the effects of PD-L1/PD-L2 expression level on the overall survival (OS) of OC patients. RESULTS: PD-L1 and PD-L2 were mainly expressed on membrane and in cytoplasm of OC cells. The high-expression rate of PD-L1 and PD-L2 in OC tissues was 44.16% (34/77) and 22.08% (17/77), respectively. The expression of PD-L1 in OC cells was significantly correlated with FIGO stage (P=0.026), while its expression was not significantly correlated with other clinicopathological parameters. There was no significant correlation between PD-L2 and any clinicopathological parameters. Kaplan-Meier survival analysis showed that the OS of high PD-L1 expression group was significantly shorter compared with the low PD-L1 expression group (HR =2.689, 95% CI: 1.400-5.163). Patients with high PD-L2 expression also exhibited significantly shorter OS (HR =2.204, 95% CI: 1.037-4.682). Multivariable analysis displayed that high expression of PD-L1 (HR =2.275, 95% CI: 1.120-4.169), high expression of PD-L2 (HR =2.314, 95% CI: 1.136-4.714) and FIGO stage (HR =11.229, 95% CI: 1.373-91.865) were independent prognostic factors of OC. When negative expressions of both PD-L1 and PD-L2 were used as a combined prognostic factor, the OS was significantly prolonged (HR =3.396, 95% CI: 1.858-6.029). According to our previous studies, patients with negative PD-L1 expression and high T-bet+ TIL infiltration have higher OS than other patients. Patients with positive PD-L1 expression and low T-bet+ TIL infiltration exhibit the shortest OS. Collectively, our findings provided the basis for PD-1/PD-L1 or PD-1/PD-L2 blockade therapy for OC patients. CONCLUSIONS: Co-stimulatory molecules, PD-L1 and PD-L2, were highly expressed in OC tissues, and their expression levels were correlated with FIGO stage, age and prognosis. These results suggested that PD-L1 and PD-L2 were involved in the occurrence and development of malignant OC, indicating their potential value in clinical diagnosis and prognosis of OC.

Human placenta-derived mesenchymal stem cells ameliorate GVHD by modulating Th17/Tr1 balance via expression of PD-L2.

AIMS: To examine whether human placenta mesenchymal stem/stromal cells (hpMSCs) mitigate graft-versus-host-disease (GVHD) via regulation of Th17 and Tr1. MATERIALS AND METHODS: hpMSCs or phosphate buffered saline (PBS, as a control) were injected into humanized xeno-GVHD NOD/SCID mouse model. Effects on body weights and survival times were determined. In addition, various assays, including flow cytometry (FCM) and HE stain, were performed on tissues (liver, spleen, lung and intestine) from these hpMSCs versus PBS treated GVHD mice. Th17 cell number in vitro was analyzed by FCM. KEY FINDINGS: hpMSCs reduced weight loss, along with IL-6 and IL-17 production to prolong the survival of GVHD mice. Th17 cell number was down-regulated obviously in hpMSCs treated GVHD mice. Conversely, Tr1 cell number and TGF-ß production were enhanced by hpMSCs. Moreover, knockdown of programmed death ligand 2 (PD-L2) increased Th17 cell number from PMA activated T cells co-cultured with hpMSCs. SIGNIFICANCE: hpMSCs can modulate the balance between Th17 and Tr1 cells to alleviate GVHD. In addition, PD-L2 as expressed on hpMSCs inhibits the generation of Th17 subset from activated T cells. These data suggest that hpMSCs attenuate GVHD through inhibition of severe inflammatory responses resulting from T cell differentiation.

Poly(I:C) primes primary human glioblastoma cells for an immune response invigorated by PD-L1 blockade.

Prognosis of glioblastoma remains dismal, underscoring the need for novel therapies. Immunotherapy is generating promising results, but requires combination strategies to unlock its full potential. We investigated the immunomodulatory capacities of poly(I:C) on primary human glioblastoma cells and its combinatorial potential with programmed death ligand (PD-L) blockade. In our experiments, poly(I:C) stimulated expression of both PD-L1 and PD-L2 on glioblastoma cells, and a pro-inflammatory secretome, including type I interferons (IFN) and chemokines CXCL9, CXCL10, CCL4 and CCL5. IFN-ß was partially responsible for the elevated PD-1 ligand expression on these cells. Moreover, real-time PCR and chloroquine-mediated blocking experiments indicated that poly(I:C) triggered Toll-like receptor 3 to elicit its effect. Cocultures of poly(I:C)-treated glioblastoma cells with peripheral blood mononuclear cells enhanced lymphocytic activation (CD69, IFN-γ) and cytotoxic capacity (CD107a, granzyme B). Additional PD-L1 blockade further propagated immune activation. Besides activating immunity, poly(I:C)-treated glioblastoma cells also doubled the attraction of CD8+ T cells, and to a lesser extent CD4+ T cells, via a mechanism which included CXCR3 and CCR5 ligands. Our results indicate that by triggering glioblastoma cells, poly(I:C) primes the tumor microenvironment for an immune response. Secreted cytokines allow for immune activation while chemokines attract CD8+ T cells to the front, which are postulated as a prerequisite for effective PD-1/PD-L1 blockade. Accordingly, additional blockade of the concurrently elevated tumoral PD-L1 further reinforces the immune activation. In conclusion, our data proposes poly(I:C) treatment combined with PD-L1 blockade to invigorate the immune checkpoint inhibition response in glioblastoma.