PD-L1 and PD-L2 expression in pancreatic ductal adenocarcinoma and their correlation with immune infiltrates and DNA damage response molecules.

Immunotherapy targeting programmed cell death-1 (PD-1) has considerably improved the prognosis of patients with advanced cancers; however, its efficacy in the treatment of pancreatic ductal adenocarcinoma (PDAC) is unfavourable. To address the issue of PDAC immunotherapy, we investigated the expression of two PD-1 ligands, PD-L1 and PD-L2, in PDAC, analysed their role in survival, and explored their correlation with clinicopathological features, immune infiltration, and DNA damage response molecules. Immunohistochemistry was performed on 291 surgically resected PDAC samples. In tumour cells (TCs) and immune cells (ICs), the positivity of PD-L1 expression was 30 and 20% and that of PD-L2 expression was 40 and 20%, respectively. Moreover, PD-L1 expression on TCs correlated with its expression on ICs (p < 0.0001); a similar result was observed for PD-L2 (p < 0.0001). Nonetheless, no correlation was observed between PD-L1 and PD-L2 expression. Positive PD-L1 expression on TCs was related to N1 stage (p = 0.011) and AJCC II stage (p = 0.002), whereas positive PD-L2 expression on TCs was associated with high FOXP3+ cell infiltration (p = 0.001) and high BRCA2 expression (p < 0.0001). Survival analysis revealed that positive PD-L1 (p = 0.046) and PD-L2 (p = 0.028) expression on TCs was an independent risk factor for unfavourable disease-specific survival (DSS). Furthermore, positive PD-L2 expression on TCs was an independent risk factor for lower DSS in the pN0 (p = 0.023), moderate and well tumour differentiation (p = 0.004), low BRCA1 (p = 0.017), wild-type p53 (p = 0.034), and proficient mismatch repair (p = 0.004) subgroups. Moreover, post-operative adjuvant chemotherapy could significantly affect DSS, regardless of PD-L1/PD-L2 expression status (positive or negative) on TCs, while it only prolonged DSS in PDL1-ICs(-) (p < 0.0001) and PDL2-ICs(-) (p < 0.0001) subgroups. This study provides a comprehensive understanding of the roles of PD-L1 and PD-L2 in PDAC, supporting anti-PD-1 axis immunotherapy for PDAC.

Inhibition of PI3Kδ Differentially Regulates Poly I:C- and Human Metapneumovirus-Induced PD-L1 and PD-L2 Expression in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are front sentinels eliciting innate and adaptive immunity to respiratory viral pathogens. Recognition of viral double-stranded RNA induces antiviral interferon (IFN) responses in bronchial epithelial cells. Co-inhibitory molecules programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) were also induced on bronchial epithelial cells, which bind programmed cell death 1 on T cell and inhibit the function of virus-specific cytotoxic T lymphocyte. A previous study showed that antiviral type I IFN increased PD-L1 and PD-L2 expression in cultured melanoma cells. However, it remains unknown whether antiviral IFNs affect PD-L1 and PD-L2 expression in bronchial epithelial cells. In addition, we previously reported that inhibition of PI3Kδ signaling enhanced antiviral IFN responses in human primary bronchial epithelial cells (PBECs). Here we assessed the effect of exogenous IFNs or a selective PI3Kδ inhibitor IC87114 on PD-L1 and PD-L2 in PBECs stimulated with a synthetic double-stranded RNA poly I:C or human metapneumovirus. Treatment with IFNß or IFNλ increased PD-L1 and PD-L2, and IFNß or IFNλ treatment plus poly I:C further increased both expressions. Treatment with IC87114 or transfection with siRNA targeting PI3K p110δ enhanced poly I:C-induced gene and protein expression of PD-L2, whereas IC87114 suppressed poly I:C-induced PD-L1. IC87114 enhanced poly I:C-induced gene expression of IFNß, IFNλ, and IFN-regulated genes via increased TBK1 and IRF3 phosphorylation. Transfection with siIRF3 counteracted the enhancement of poly I:C-induced PD-L2 by IC87114, whereas IC87114 suppressed poly I:C-induced PD-L1 regardless of transfection with siNC or siIRF3. Similar effects of IC87114 on PD-L1 and PD-L2 expression were observed in human metapneumovirus-infected PBECs. We showed for the first time that type I and type III IFNs induced the expression of PD-L1 and PD-L2 in PBECs. Our findings suggest that during viral infections, inhibition of PI3Kδ differentially regulates PD-L1 and PD-L2 expression in bronchial epithelial cells.

New Insights into the Role of PD-1 and Its Ligands in Allergic Disease.

Programmed cell death 1 (PD-1) and its ligands PD-L1 and PD-L2 are receptors that act in co-stimulatory and coinhibitory immune responses. Signaling the PD-1/PD-L1 or PD-L2 pathway is essential to regulate the inflammatory responses to infections, autoimmunity, and allergies, and it has been extensively studied in cancer. Allergic diseases include asthma, rhinoconjunctivitis, atopic dermatitis, drug allergy, and anaphylaxis. These overactive immune responses involve IgE-dependent activation and increased CD4+ T helper type 2 (Th2) lymphocytes. Recent studies have shown that PD-L1 and PD-L2 act to regulate T-cell activation and function. However, the main role of PD-1 and its ligands is to balance the immune response; however, the inflammatory process of allergic diseases is poorly understood. These immune checkpoint molecules can function as a brake or a kick-start to regulate the adaptive immune response. These findings suggest that PD-1 and its ligands may be a key factor in studying the exaggerated response in hypersensitivity reactions in allergies. This review summarizes the current understanding of the role of PD-1 and PD-L1 and PD-L2 pathway regulation in allergic diseases and how this immunomodulatory pathway is currently being targeted to develop novel therapeutic immunotherapy.

PD-L2 glycosylation promotes immune evasion and predicts anti-EGFR efficacy.

BACKGROUND: Combination therapy has been explored for advanced head and neck squamous cell carcinoma (HNSCC) owing to the limited efficacy of anti-epidermal growth factor receptor (EGFR) therapy. Increased expression and glycosylation of immune checkpoint molecules in tumors are responsible for cetuximab therapy refractoriness. The role of programmed death ligand 2 (PD-L2), a ligand of PD-1, in the immune function is unclear. Here, we examined the regulatory mechanism of PD-L2 glycosylation and its role in antitumor immunity and cetuximab therapy. METHODS: Single-cell RNA sequencing and immunohistochemical staining were used to investigate PD-L2 expression in cetuximab-resistant/sensitive HNSCC tissues. The mechanism of PD-L2 glycosylation regulation was explored in vitro. The effects of PD-L2 glycosylation on immune evasion and cetuximab efficacy were verified in vitro and using mice bearing orthotopic SCC7 tumors. RESULTS: The PD-L2 levels were elevated and N-glycosylated in patients with cetuximab-resistant HNSCC. Glycosylated PD-L2 formed a complex with EGFR, which resulted in the activation of EGFR/signal transducer and activator of transcription 3 (STAT3) signaling and decreased the cetuximab binding affinity to EGFR. The N-glycosyltransferase fucosyltransferase (FUT8), a transcriptional target of STAT3, was required for PD-L2 glycosylation. Moreover, glycosylation modification stabilized PD-L2 by blocking ubiquitin-dependent lysosomal degradation, which consequently promoted its binding to PD-1 and immune evasion. Inhibition of PD-L2 glycosylation using Stattic, a specific STAT3 inhibitor, or PD-L2 mutation blocking its binding to FUT8, increased cytotoxic T lymphocyte activity and augmented response to cetuximab. CONCLUSIONS: Increased expression and glycosylation of PD-L2 in tumors are an important mechanism for cetuximab therapy refractoriness. Thus, the combination of PD-L2 glycosylation inhibition and cetuximab is a potential therapeutic strategy for cancer.

Adipogenesis in triple-negative breast cancer is associated with unfavorable tumor immune microenvironment and with worse survival.

Cancer-associated adipocytes are known to cause inflammation; however, the role of adipogenesis, the formation of adipocytes, in breast cancer is unclear. We hypothesized that intra-tumoral adipogenesis reflects a different cancer biology than abundance of intra-tumoral adipocytes. The Molecular Signatures Database Hallmark adipogenesis gene set of gene set variant analysis was used to quantify adipogenesis. Total of 5,098 breast cancer patients in multiple cohorts (training; GSE96058 (n = 3273), validation; TCGA (n = 1069), treatment response; GSE25066 (n = 508) and GSE20194 (n = 248)) were analyzed. Adipogenesis did not correlate with abundance of adipocytes. Adipogenesis was significantly lower in triple negative breast cancer (TNBC). Elevated adipogenesis was significantly associated with worse survival in TNBC, but not in the other subtypes. High adipogenesis TNBC was significantly associated with low homologous recombination deficiency, but not with mutation load. High adipogenesis TNBC enriched metabolism-related gene sets, but neither of cell proliferation- nor inflammation-related gene sets, which were enriched to adipocytes. High adipogenesis TNBC was infiltrated with low CD8+ T cells and high M2 macrophages. Although adipogenesis was not associated with neoadjuvant chemotherapy response, high adipogenesis TNBC was significantly associated with low expression of PD-L1 and PD-L2 genes, and immune checkpoint molecules index. In conclusion, adipogenesis in TNBC was associated with cancer metabolism and unfavorable tumor immune microenvironment, which is different from abundance of adipocytes.

Downregulation of the PD-1/PD-Ls pathway in peripheral cells correlates with asbestosis severity.

BACKGROUND: Asbestosis and silicosis are characterized by diffuse or nodular interstitial lung fibrosis resulting from exposure to asbestos or silica dust, respectively. This study was designed to detect programmed cell death protein (PD-1)/programmed death ligands (PD-Ls) expression in patients with asbestosis and silicosis and to explore the possible clinical significance of PD-1/PD-Ls expression in patients with the two diseases. METHODS: Thirty patients with asbestosis, 23 patients with silicosis and 25 healthy controls were consecutively recruited and provided informed consent to participate in the study. Clinical data were collected from patients' clinical charts. PD-1/PD-Ls expression in peripheral blood (PB) was detected using flow cytometry. RESULTS: PD-1 was expressed at significantly lower levels on CD4+ or CD8+ peripheral T cells from patients with asbestosis and silicosis than on cells from healthy controls. Similarly, significantly lower PD-L1 and PD-L2 expression was detected on CD14+ monocytes from patients with asbestosis and silicosis than on cells from healthy controls. In addition, no significant differences in PD-1, PD-L1 and PD-L2 expression were observed between the asbestosis and silicosis groups. Moreover, the proportions of PD-1+ CD4+ T cells and PD-1+ CD8+ T cells in patients with asbestosis were positively correlated with the percentage of forced vital capacity predicted. CONCLUSIONS: Decreased PD-1 expression on CD4+ T or CD8+ T cells in PB was positively correlated with the asbestosis severity, implying that pulmonary fibrosis development in patients with asbestosis was positively correlated with the downregulation of the PD-1/PD-Ls pathway.

PD-1 signaling pathway in sepsis: Does it have a future?

Sepsis is characterized by high mortality and poor prognosis and is one of the leading causes of death among patients in the intensive care unit (ICU). In the past, drugs that block early inflammatory responses have done little to reverse the progression of sepsis. Programmed cell death receptor 1 (PD-1) and its two ligands, programmed cell death receptor ligand 1(PD-L1) and programmed cell death receptor ligand 2 (PD-L2), are negative regulatory factors of the immune response of the body. Recently, the role of the PD-1 signaling pathway in sepsis has been widely studied. Studies showed that the PD-1 signaling pathways are closely related to the mortality and prognosis of sepsis patients. In the immunotherapy of sepsis, whether in animal experiments or clinical trials, anti-PD-1/PD-L1 antibodies have shown good promise. In this review, firstly, we focus on the immunosuppressive mechanism of sepsis and the structure and function of the PD-1 signaling pathway. The variety of the PD-1 signaling pathways in sepsis is introduced. Then, the relationship between the PD-1 signaling pathway and immune cells and organ dysfunction and the regulatory factors of the PD-1 signaling pathway in sepsis is discussed. Finally, the application of the PD-1 signaling pathway in sepsis is specifically emphasized.

Programmed Death-Ligand 2 Deficiency Exacerbates Experimental Autoimmune Myocarditis in Mice.

Programmed death ligand 2 (PD-L2) is the second ligand of programmed death 1 (PD-1) protein. In autoimmune myocarditis, the protective roles of PD-1 and its first ligand programmed death ligand 1 (PD-L1) have been well documented; however, the role of PD-L2 remains unknown. In this study, we report that PD-L2 deficiency exacerbates myocardial inflammation in mice with experimental autoimmune myocarditis (EAM). EAM was established in wild-type (WT) and PD-L2-deficient mice by immunization with murine cardiac myosin peptide. We found that PD-L2-deficient mice had more serious inflammatory infiltration in the heart and a significantly higher myocarditis severity score than WT mice. PD-L2-deficient dendritic cells (DCs) enhanced CD4+ T cell proliferation in the presence of T cell receptor and CD28 signaling. These data suggest that PD-L2 on DCs protects against autoreactive CD4+ T cell expansion and severe inflammation in mice with EAM.

Frequent genetic alterations in immune checkpoint-related genes in intravascular large B-cell lymphoma.

Intravascular large B-cell lymphoma (IVLBCL) is a unique type of extranodal lymphoma characterized by selective growth of tumor cells in small vessels without lymphadenopathy. Greater understanding of the molecular pathogenesis of IVLBCL is hampered by the paucity of lymphoma cells in biopsy specimens, creating a limitation in obtaining sufficient tumor materials. To uncover the genetic landscape of IVLBCL, we performed whole-exome sequencing (WES) of 21 patients with IVLBCL using plasma-derived cell-free DNA (cfDNA) (n = 18), patient-derived xenograft tumors (n = 4), and tumor DNA from bone marrow (BM) mononuclear cells (n = 2). The concentration of cfDNA in IVLBCL was significantly higher than that in diffuse large B-cell lymphoma (DLBCL) (P < .0001) and healthy donors (P = .0053), allowing us to perform WES; most mutations detected in BM tumor DNA were successfully captured in cfDNA and xenograft. IVLBCL showed a high frequency of genetic lesions characteristic of activated B-cell-type DLBCL, with the former showing conspicuously higher frequencies (compared with nodal DLBCL) of mutations in MYD88 (57%), CD79B (67%), SETD1B (57%), and HLA-B (57%). We also found that 8 IVLBCL (38%) harbored rearrangements of programmed cell death 1 ligand 1 and 2 (PD-L1/PD-L2) involving the 3' untranslated region; such rearrangements are implicated in immune evasion via PD-L1/PD-L2 overexpression. Our data demonstrate the utility of cfDNA and imply important roles for immune evasion in IVLBCL pathogenesis and PD-1/PD-L1/PD-L2 blockade in therapeutics for IVLBCL.

Prognostic and clinicopathological utility of PD-L2 expression in patients with digestive system cancers: A meta-analysis.

OBJECTIVE: Programmed death ligand-2 (PD-L2)has been detected in various cancers. However, its prognostic value in digestive system cancers (DSCs) remains unclear. Accordingly, this meta-analysis investigated the prognostic and clinicopathological utility of PD-L2 in patients with DSCs. METHODS: We systematically searched PubMed, EMBASE, Web of Science, ClinicalTrials.gov., Scopus, and Cochrane Library databases for eligible studies up to April 30, 2020. The hazard ratio (HR), odds ratio (OR), and corresponding 95% confidence interval (CI) of the outcomes were calculated. RESULTS: Twenty two studies with 4886 patients were included in this meta-analysis. The pooled results showed that PD-L2 overexpression was significantly associated with poor overall survival (OS) (HR 1.470, 95% CI: 1.252-1.728, p < 0.001) and worse disease-free survival (DFS) (HR1.598, 95% CI: 1.398-1.826, p < 0.001). Subgroup analysis revealed that elevated PD-L2 was a significant prognostic indicator of worse OS in hepatocellular carcinoma (HR 1.703, 95% CI: 1.456-1.991, p < 0.001) and colorectal cancer (HR 3.811, 95% CI: 1.718-8.454, p = 0.001). Concerning clinicopathologic factors, PD-L2 overexpression was associated with lymphatic metastasis (OR 1.394., 95% CI: 1.101-1.764, p = 0.006), tumor metastasis (OR 1.599, 95% CI: 1.072-2.383, p = 0.021), and the histopathological stage (OR 0.704, 95% CI: 0.566-0.875, p = 0.002). CONCLUSION: PD-L2 overexpression in DSCs after surgery might predict a poor prognosis, especially in hepatocellular carcinoma and colorectal cancer. Larger patient cohorts are needed to validate its prognostic role.

Dual Roles of PU.1 in the Expression of PD-L2: Direct Transactivation with IRF4 and Indirect Epigenetic Regulation.

PD-L2, which has been identified as a PD-1 ligand, is specifically expressed in dendritic cells (DCs) and macrophages. The transcription factors that determine the cell type-specific expression of PD-L2 are largely unknown, although PD-1 and its ligands, which have been shown to play important roles in T cell suppression, have been vigorously analyzed in the field of cancer immunology. To reveal the mechanism by which Pdcd1lg2 gene expression is regulated, we focused on DCs, which play key roles in innate and acquired immunity. The knockdown of the hematopoietic cell-specific transcription factors PU.1 and IRF4 decreased PD-L2 expression in GM-CSF-induced mouse bone marrow-derived DCs. Chromatin immunoprecipitation assays, luciferase assays, and electrophoretic mobility shift assays demonstrated that PU.1 and IRF4 bound directly to the Pdcd1lg2 gene via an Ets-IRF composite element sequence and coordinately transactivated the Pdcd1lg2 gene. Furthermore, PU.1 knockdown reduced the histone acetylation of the Pdcd1lg2 gene. The knockdown of the typical histone acetyltransferase p300, which has been reported to interact with PU.1, decreased the expression and H3K27 acetylation of the Pdcd1lg2 gene. GM-CSF stimulation upregulated the Pdcd1lg2 gene expression, which was accompanied by an increase in PU.1 binding and histone acetylation in Flt3L-generated mouse bone marrow-derived DCs. The involvement of PU.1, IRF4, and p300 were also observed in mouse splenic DCs. Overall, these results indicate that PU.1 positively regulates Pdcd1lg2 gene expression as a transactivator and an epigenetic regulator in DCs.

Indoleamine 2,3-Dioxygenase-1 Expression in Adrenocortical Carcinoma.

BACKGROUND: Indoleamine 2,3-dioxygenase 1 (IDO-1) is overexpressed in many human carcinomas and a successful target for therapy in mouse models. Prognosis of patients with advanced adrenocortical carcinoma (ACC) is poor due to the lack of effective treatments, and new therapies are therefore needed. Herein, we investigate whether IDO-1 is expressed in human ACC tissues. METHODS: 53 tissue samples from patients with ACC, adrenal adenoma (AA), adrenocortical tumors (ACTs), and normal adrenal were identified. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded slides for IDO-1. Samples were scored for cytoplasmic staining as per intensity and the percent of positive cells and for stromal staining by percent of positive cells. Tumor characteristics, PD-L1, PDL-2, and CD-8+ T-lymphocyte expression were also determined. RESULTS: Samples from 32 ACC, 3 ACT, 15 AA, and 3 normal adrenal were analyzed. IDO-1 was expressed in tumor tissue in 22 of 32 ACC samples, compared with 8 of 15 AA sample (P = 0.344). IDO-1 expression was significantly increased in stromal tissue of ACC samples (16 of 33), compared with AA samples (0 of 15) (P = 0.001). IDO-1 expression in ACC and AA samples was associated with PD-L2 expression (P = 0.034). IDO-1 expression in ACC stromal tissue was associated with CD8+ T-lymphocyte infiltration (P = 0.028). CONCLUSIONS: IDO-1 is expressed in a majority of ACC samples. Its expression in tumor tissue is associated with PD-L2 expression, and expression in stroma is associated with CD8+ cell infiltration. IDO-1 inhibition, alone or in combination with PD-1 inhibition, could therefore be an interesting target in treatment of ACC.

PD-1, CTLA4, PD-L1 and PD-L2 DNA methylation in papillary thyroid carcinoma.

Aim: We investigated DNA methylation patterns of immune checkpoint genes PD-1, PD-L1, PD-L2, CTLA4 and an adjacent long noncoding RNA in papillary thyroid carcinoma (PTC). Materials & methods: DNA methylation and mRNA expression were examined in PTCs. DNA methylation was correlated with mRNA expression, BRAF and RAS mutational status, and immune cell infiltration. Results: Inverse correlations between DNA methylation and mRNA expression were observed. Immune checkpoint expression correlated positively, and DNA methylation negatively, with immune cell infiltration. Higher DNA methylation levels accompanied by lower immune checkpoint expression were observed in RAS-mutated tumors. Conclusion: We suggest epigenetic regulation of immune checkpoints in PTC. Methylation was associated with BRAF and RAS mutation status. DNA methylation might be a promising biomarker candidate in the context of immunotherapies in PTC.

Clinical relevance and prognostic significance of PD-1/PD-Ls in non-metastatic bladder cancer: A role for PD-L2.

BACKGROUND: Bladder cancer (BC) can be successfully treated by manipulating immune responses with intravesical Bacillus Calmette-Guerin instillation or targeting the PD-1/PD-L signaling pathway. In the present study we investigated the prognostic significance of the immune checkpoint inhibitor PD-1 and its ligands PD-L1 and PD-L2 on tumor cells and infiltrating lymphocytes, in the tumor microenvironment and draining lymph nodes in patients with non-metastatic BC. PATIENTS AND METHODS: Cells were mechanically isolated from tissues and draining lymph nodes from 58 patients, and surface-stained for CD45, PD-1, PD-L1 and PD-L2. The cells were then analyzed with a flow cytometric method. RESULTS: Approximately 2% of CD45-negative tumor and stromal cells expressed PD-L1. Expression was not associated with the main clinicopathological characteristics of the disease or with survival. However, as tumors progressed the frequency of PD-L1+CD45hi cells and the mean expression of PD-1 on CD45hi cells increased remarkably on immune cells in tumor tissues and draining lymph nodes. In addition, frequency analysis showed that cell percentages as well as mean expression of PD-L2 on total CD45+ lymphocytes and their CD45hi subpopulation in tumor-draining lymph nodes was significantly associated with cancer-related death (P < 0.05). Multiple Cox regression also revealed that while CD45+ (hazard ratio: 0.596, 95 % CI 0.439-0.809, P = 0.001) was associated with improved survival, CD45neg (HR: 0.615, 95 % CI 0.454-0.831, P = 0.002), and PD-L2+CD45+ cells (hazard ratio: 1.472, 95 % CI 1.023-2.120, P = 0.038) in draining lymph nodes were associated with lower survival. CONCLUSION: Our results suggest that in patients with BC, PD-1 and PD-L expression on immune cells, especially in draining lymph nodes, is valuable for predicting prognosis and survival, and possibly responsiveness to immunotherapy. However, expression of the inhibitor molecule or its ligands on tumor cells was not associated with prognosis. The results highlight the significance of PD-L2 as a second important suppressive molecule in tumors.

GATA2 Regulates Constitutive PD-L1 and PD-L2 Expression in Brain Tumors.

Encouraging clinical results using immune checkpoint therapies to target the PD-1 axis in a variety of cancer types have paved the way for new immune therapy trials in brain tumor patients. However, the molecular mechanisms that regulate expression of the PD-1 pathway ligands, PD-L1 and PD-L2, remain poorly understood. To address this, we explored the cell-intrinsic mechanisms of constitutive PD-L1 and PD-L2 expression in brain tumors. PD-L1 and PD-L2 expression was assessed by flow cytometry and qRT-PCR in brain tumor cell lines and patient tumor-derived brain tumor-initiating cells (BTICs). Immunologic effects of PD-L2 overexpression were evaluated by IFN-γ ELISPOT. CD274 and PDCD1LG2 cis-regulatory regions were cloned from genomic DNA and assessed in full or by mutating and/or deleting regulatory elements by luciferase assays. Correlations between clinical responses and PD-L1 and PD-L2 expression status were evaluated in TCGA datasets in LGG and GBM patients. We found that a subset of brain tumor cell lines and BTICs expressed high constitutive levels of PD-L1 and PD-L2 and that PD-L2 overexpression inhibited neoantigen specific T cell IFN-γ production. Characterization of novel cis-regulatory regions in CD274 and PDCD1LG2 lead us to identify that GATA2 is sufficient to drive PD-L1 and PD-L2 expression and is necessary for PD-L2 expression. Importantly, in TCGA datasets, PD-L2 correlated with worse clinical outcomes in glioma patients.. By perturbing GATA2 biology, targeted therapies may be useful to decrease inhibitory effects of PD-L2 in the microenvironment.

Expression of PD-L1, PD-L2, and IDO1 on tumor cells and density of CD8-positive tumor-infiltrating lymphocytes in early-stage lung adenocarcinoma according to histological subtype.

PURPOSE: This study examined the expression of programmed cell death-ligand 1 (PD-L1), programmed cell death-ligand 2 (PD-L2), and indoleamine 2,3-dioxygenase-1 (IDO1) in tumor cells and cluster of differentiation 8 (CD8)-positive tumor-infiltrating lymphocytes (TILs) in early-stage lung adenocarcinoma according to histological subtypes. METHODS: We evaluated PD-L1, PD-L2, and IDO1 expression in tumor cells and CD8-positive TILs in surgically resected specimens from 196 stage 0 or I lung adenocarcinoma patients by immunohistochemical staining. We also examined the relationships between the expression of PD-L1, PD-L2, and IDO1 in tumor cells and the density of CD8-positive TILs and clinical factors. Patients were divided into three groups: A, adenocarcinoma in situ and minimally invasive adenocarcinoma (N = 32); B, lepidic predominant invasive adenocarcinoma (IAD; LPA; N = 66); and C, IAD except for LPA (N = 98). RESULTS: PD-L1 was expressed only in Group C, but not in Groups A or B. The positive ratio of PD-L2 was significantly higher in Group C (63.3%), and that of IDO1 was also significantly higher in Group C (65.3%). The density of CD8-positive TILs was significantly higher in Group C (45 ± 2.4). There was no significant difference between the positive ratios of PD-L2 and IDO1 and the density of CD8-positive TILs in Group A (50.0%, 21.9%, and 36 ± 4.1, respectively) or Group B (60.6%, 25.8%, and 44 ± 3.0, respectively). CONCLUSIONS: No cases in Groups A and B expressed PD-L1. The expression of immune-related factors, especially PD-L1 and IDO1, was significantly associated with Group C. This is the first report of the detailed examination of PD-L1, PD-L2, IDO1, and CD8 expression in lung adenocarcinoma subtypes with lepidic predominant components. Our results could help identify patients who would benefit from perioperative immunotherapy.

Immune suppression caused by PD-L2 expression on tumor cells in gastric cancer.

BACKGROUND: Gastric cancer (GC) patients with PD-L1-negative tumor occasionally have a favorable response to anti-PD-1 mAb. The aim of the present study was to investigate the regulatory mechanism and immunosuppressive role of PD-L2 in GC. METHODS: We used immunohistochemistry to evaluate the expression of PD-L2 in primary tumors from 194 patients with GC. The mechanism of PD-L2 expression was assessed in TCGA stomach adenocarcinoma tissue dataset and in vitro assay using GC cell lines. The immunosuppressive role of PD-L2 was evaluated by cytotoxicity of CTL clone against PD-L2 expressing GC cells. RESULTS: PD-L2 was expressed on tumor cells (TCs) of 28.4% patients and PD-L2 expression on TCs was significantly associated with tumor progression. TCGA dataset revealed that IFN-γ and, to a lesser extent, IL-4 signature significantly correlated with PD-L2 expression. In vitro assay showed that IFN-γ and, also to a lesser extent, IL-4 can upregulate PD-L2 expression on GC cells. Anti-PD-L2 mAb significantly enhanced the cytotoxicity of CTL clone against GC cell lines expressing PD-L2. CONCLUSIONS: PD-L2 is expressed on GC cells and PD-1/PD-L2 interaction are functionally involved in anti-tumor CTL activities. PD-L2 expression should be considered when determining the optimal immunotherapy for GC.

[Programmed cell death protein 1 and its ligands regulate immune balance in allergic rhinitis].

Objective: To explore the expression and significance in regulating immune balance of programmed cell death protein 1 (PD-1) and its ligands PD-L1, PD-L2 in allergic rhinitis (AR). Methods: Eighty-two patients who received outpatient treatment due to high nasal reaction symptoms or were hospitalized due to nasal septum deviation and underwent nasal septum correction surgery in Department of Otorhinolaryngology Head and Neck Surgery of Renmin Hospital of Wuhan University from May 2018 to May 2019 were enrolled, including 42 males and 40 females, with the age ranging from 14 to 38 years old. Blood, inferior turbinate nasal mucosal tissue and relevant clinical data were collected. Patients were divided into AR group and control group due to clinical manifestation, skin prick test and detection of specific IgE (sIgE) in serum. Immunohistochemistry was used to detect the expression of PD-1 and its ligands in nasal mucosa of the two groups. Flow cytometry was used to detect the proportions of PD-1(+)CD4(+)T cells, PD-L1(+) myeloid dendritic cells (mDCs), PD-L2(+)mDCs and Th2 cells in peripheral blood of the two groups. The expression levels of total IgE, sPD-1, sPD-L1 and sPD-L2 in serum of the two groups were detected by ELISA. The measurement data of normal distribution or normal distribution after the logarithm conversion to Ln were compared by t test. Pearson correlation or Spearman correlation was used to analyze the correlation among the indicators. P<0.05 was considered statistically significant. Results: The expression of PD-1 and its ligands on the surface of immune cells in the nasal mucosa of the AR group was significantly higher than that of the control group. The ratio of PD-1(+)CD4(+)T cells, PD-L1(+)mDCs and Th2 cells in peripheral blood of AR group was significantly higher than that of the control group ((15.24±6.45)% vs (8.71±5.33)%, (8.79±2.01)% vs (5.74±2.90)%, (7.89±1.95)% vs (2.52±1.34)%, all P<0.05). There was no significant difference in the ratio of PD-L2(+)mDCs between the two groups. Correlation analysis found that the proportion of PD-1(+)CD4(+) T cells was positively correlated with the Visual Analogue Scale (VAS) score of AR, total IgE concentration and the serum sIgE concentration (r value was 0.501, 0.541, 0.608, respectively, all P<0.05). The proportion of PD-L1(+)mDCs was positively correlated with the VAS score of AR and the serum sIgE concentration (r value was 0.604, 0.563, respectively, all P<0.05). The proportion of Th2 cells in peripheral blood was positively correlated with the proportion of PD-L1(+)mDCs and PD-1(+)CD4(+)T cells (r value was 0.538, 0.623, respectively, all P<0.05). Serum total IgE, sPD-1 and sPD-L1 in the AR group were significantly higher than those in the control group ((6.34±1.38) ng/ml vs (4.89±1.10) ng/ml, (4.40±1.01) pg/ml vs (3.79±1.21) pg/ml, (3.88±0.25) pg/ml vs (3.57±0.23) pg/ml, all P<0.05), and there was no significant difference in sPD-L2 levels between the two groups. Correlation analysis showed that sPD-L1 was positively correlated with total IgE and sIgE concentration (r values was 0.32, 0.45, respectively, all P<0.05). Conclusions: PD-1 and PD-L1 are highly expressed on the surface of immune cells in peripheral blood and nasal mucosa of AR patients, and sPD-1 and sPD-L1 expression levels in peripheral blood of AR patients are increased. The PD-1/PD-L1 signaling pathway promote AR inflammatory response by inducing Th2 type immune response.

The polymorphism rs975484 in the protein arginine methyltransferase 1 gene modulates expression of immune checkpoint genes in hepatocellular carcinoma.

Protein arginine methyltransferase 1 (PRMT1) is a key regulator of hepatic immune responses. Recently, we reported that PRMT1 regulates the tumor immune response in hepatocellular carcinoma (HCC). Here we found that PRMT1 expression in human HCC correlates with that of programmed cell death 1 ligand 1 (PD-L1), PD-L2, and other checkpoint genes. PRMT1 deletion in mice reduced PD-L1 and PD-L2 expression in tumors and reduced the efficiency of PD-1 antibody treatment in a diethylnitrosamine-induced HCC mouse model, suggesting that PRMT1 regulates the hepatic immune checkpoint. Mice had reduced PD-L1 and PD-L2 expression when PRMT1 was specifically deleted in tumor cells or macrophages, but PRMT1 deletion in dendritic cells did not alter PD-L1 and PD-L2 expression. rs975484 is a common polymorphism in the human PRMT1 gene promoter, and we found that it alters PRMT1 expression in blood monocytes and tumor-associated macrophages in human HCC. PRMT1 expression was higher in individuals with a GG genotype than in individuals with a CC genotype, and heterozygous carriers had intermediate expression. Luciferase reporter assays indicated that this differential expression is due to an extra C/EBPß-binding site in the PRMT1 promoter of individuals carrying the minor G allele. The rs975484 genotype also correlated with PRMT1 target expression in HCC. Individuals with the GG genotype had significantly higher levels of the PRMT1 targets PD-L1, PD-L2, and VISTA than those with the CC genotype. We conclude that PRMT1 critically controls immune checkpoints in mice and humans and that the PRMT1 polymorphism rs975484 affects checkpoint gene expression in HCC.