Cholinergic signaling influences the expression of immune checkpoint inhibitors, PD-L1 and PD-L2, and tumor hallmarks in human colorectal cancer tissues and cell lines.

BACKGROUND: Cancer cells express immunosuppressive molecules, such as programmed death ligands (PD-L)1 and PD-L2, enabling evasion from the host's immune system. Cancer cells synthesize and secrete acetylcholine (ACh), acting as an autocrine or paracrine hormone to promote their proliferation, differentiation, and migration. METHODS: We correlated the expression of PD-L1, PD-L2, cholinergic muscarinic receptor 3 (M3R), alpha 7 nicotinic receptor (α7nAChR), and choline acetyltransferase (ChAT) in colorectal cancer (CRC) tissues with the stage of disease, gender, age, risk, and patient survival. The effects of a muscarinic receptor blocker, atropine, and a selective M3R blocker, 4-DAMP, on the expression of immunosuppressive and cholinergic markers were evaluated in human CRC (LIM-2405, HT-29) cells. RESULTS: Increased expression of PD-L1, M3R, and ChAT at stages III-IV was associated with a high risk of CRC and poor survival outcomes independent of patients' gender and age. α7nAChR and PD-L2 were not changed at any CRC stages. Atropine and 4-DAMP suppressed the proliferation and migration of human CRC cells, induced apoptosis, and decreased PD-L1, PD-L2, and M3R expression in CRC cells via inhibition of EGFR and phosphorylation of ERK. CONCLUSIONS: The expression of immunosuppressive and cholinergic markers may increase the risk of recurrence of CRC. These markers might be used in determining prognosis and treatment regimens for CRC patients. Blocking cholinergic signaling may be a potential therapeutic for CRC through anti-proliferation and anti-migration via inhibition of EGFR and phosphorylation of ERK. These effects allow the immune system to recognize and eliminate cancer cells.

Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors.

Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.

Investigation of pd-l1 (cd274), pd-l2 (pdcd1lg2), and ctla-4 expressions in malignant pleural mesothelioma by immunohistochemistry and real-time polymerase chain reaction methods.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an aggressive malignant disease with a poor prognosis, which affects the surface mesothelium of the pleural cavity. Immune checkpoints are responsible for controlling the immune system to avoid autoimmunity and prevent tissue damage. In this study, we aimed to investigate the expression of cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2) immuno-control receptors in MPM patients and the relationship of the expression with tumour types and prognostic parameters. MATERIAL AND METHODS: In this study, we evaluated 50 MPM cases. Immunohistochemically CTLA-4, PD-L1, and PD-L2 were detected by using monoclonal anti-CTLA-4, anti-PD-L1, and anti-PD-L2. Real-time polymerase chain reaction (RT-PCR) analysis was performed with the primers CTLA-4, PD-L1, and PD-L2. RESULTS: Statistically, no significant relation was determined between the PD-L1, PD-L2, and CTLA-4 expressions (immunohistochemical and RT-PCR methods) and the MPM histological type. Interestingly significant correlation was observed between the mean survival time and immunohistochemical PD-L2 expression; thus, long-term survival was observed in cases with PD-L2 expression. CONCLUSIONS: Programmed death ligand 1, PD-L2, and CTLA-4 expression were observed in some MPM cases, suggesting that treatments targeting immune checkpoints may be effective. Because immunohistochemical expression of PD-L2 is associated with better prognosis, it may provide useful clues in the follow-up of patients.

An Extended PD-L2 Cytoplasmic Domain Results From Alternative Splicing in NSCLC Cells.

Antibody-based immunotherapy targeting the interaction between programmed cell death 1 (PD-1) and its ligand PD-L1 has shown impressive clinical outcomes in various cancer types, including nonsmall cell lung cancer (NSCLC). However, regulatory mechanisms in this immune checkpoint pathway still needs clarification. PD-L2 is structurally homologous to PD-L1 and is a second PD-1 ligand. Alternative mRNA splicing from the CD274 and PDCD1LG2 genes holds the potential to generate PD-L1 and PD-L2 isoforms, respectively, with novel functionality in regulation of the PD-1 immune checkpoint pathway. Here, we describe alternative splicing in NSCLC cells potentially generating eight different PD-L2 isoforms from the PDCD1LG2 gene. Extension of exon 6 by four nucleotides is the most prominent alternative splicing event and results in PD-L2 isoform V with a cytoplasmic domain containing a 10 amino acid extension. On average 13% of the PDCD1LG2 transcripts in NSCLC cell lines and 22% of the transcripts in NSCLC tumor biopsies encode PD-L2 isoform V. PD-L2 isoform V localizes to the cell surface membrane but less efficiently than the canonical PD-L2 isoform I. The cytoplasmic domains of PD-1 ligands can affect immune checkpoint pathways by conferring membrane localization and protein stability and thereby represent alternative targets for immunotherapy. In addition, cytoplasmic domains are involved in intracellular signalling cascades in cancer cells. The presented observations of different cytoplasmic domains of PD-L2 will be important in the future delineation of the PD-1 immune checkpoint pathway.

Exome sequencing identifies PD-L2 as a potential predisposition gene for lymphoma.

To investigate germline predisposition in lymphoma, we performed whole-exome sequencing and discovered a novel variant (c.817-1G>T) in programmed cell death 1 ligand 2 (PD-L2) in a family with early-onset lymphomas and other cancers. The variant was present in the proband with follicular lymphoma and his son with Hodgkin's lymphoma. It was in the terminal splice acceptor site of PD-L2 and embedded in a putative enhancer of Janus kinase 2 (JAK2) and programmed cell death 1 ligand (PD-L1). We also found that gene expression of PD-L2, PD-L1, and JAK2 was significantly increased. Using 3' rapid amplification of cDNA ends (3' RACE), we detected an abnormal PD-L2 transcript in the son. Thus, the c.817-1G>T variant may result in the elevated PD-L2 expression due to the abnormal PD-L2 transcript and the elevated PD-L1 and JAK2 expression due to increased enhancer activity of PD-L1 and JAK2. The PD-L2 novel variant likely underlies the genetic etiology of the lymphomas in the family. As PD-L2 plays critical roles in tumor immunity, identification of PD-L2 as a germline predisposition gene may inform personalized immunotherapy in lymphoma patients.

Anti-Tumor Potency of Short-Term Interleukin-15 Dendritic Cells Is Potentiated by In Situ Silencing of Programmed-Death Ligands.

Dendritic cell (DC) vaccines have proven to be a valuable tool in cancer immune therapy. With several DC vaccines being currently tested in clinical trials, knowledge about their therapeutic value has been significantly increased in the past decade. Despite their established safety, it has become clear that objective clinical responses are not yet robust enough, requiring further optimization. Improvements of this advanced therapy medicinal product encompass, among others, regulating their immune stimulating capacity by in situ gene engineering, in addition to their implementation in combination therapy regimens. Previously, we have reported on a superior monocyte-derived DC preparation, including interleukin-15, pro-inflammatory cytokines and immunological danger signals in the culture process. These so-called IL-15 DCs have already proven to exhibit several favorable properties as cancer vaccine. Evolving research into mechanisms that could further modulate the immune response towards cancer, points to programmed death-1 as an important player that dampens anti-tumor immunity. Aiming at leveraging the immunogenicity of DC vaccines, we hypothesized that additional implementation of the inhibitory immune checkpoint molecules programmed death-ligand (PD-L)1 and PD-L2 in IL-15 DC vaccines would exhibit superior stimulatory potential. In this paper, we successfully implemented PD-L silencing at the monocyte stage in the 3-day IL-15 DC culture protocol resulting in substantial downregulation of both PD-L1 and PD-L2 to levels below 30%. Additionally, we validated that these DCs retain their specific characteristics, both at the level of phenotype and interferon gamma secretion. Evaluating their functional characteristics, we demonstrate that PD-L silencing does not affect the capacity to induce allogeneic proliferation. Ultimately designed to induce a durable tumor antigen-specific immune response, PD-L silenced IL-15 DCs were capable of surpassing PD-1-mediated inhibition by antigen-specific T cells. Further corroborating the superior potency of short-term IL-15 DCs, the combination of immune stimulatory components during DC differentiation and maturation with in situ checkpoint inhibition supports further clinical translation.

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.

Nicotine treatment regulates PD-L1 and PD-L2 expression via inhibition of Akt pathway in HER2-type breast cancer cells.

The immune checkpoint molecules such as PD-L1 and PD-L2 have a substantial contribution to cancer immunotherapy including breast cancer. Microarray expression profiling identified several molecular subtypes, namely luminal-type (with a good-prognosis), HER2-type (with an intermediate-prognosis), and triple-negative breast cancer (TNBC)-type (with a poor-prognosis). We found that PD-L1 and PD-L2 mRNA expressions were highly expressed in TNBC-type cell lines (HCC1937, MDA-MB-231), moderately expressed in HER2-type cell line (SK-BR-3), and poorly expressed in luminal-type cell lines (MDA-MB-361, MCF7). The PD-L1 and PD-L2 expression in SK-BR-3 cells, but not those in HCC1937 and MDA-MB-231 cells, decreased by nicotine stimulation in a dose-dependent manner. In addition, nicotine treatment decreased the phosphorylation of Akt in SK-BR-3 cells, but not in other cell lines. These results show that nicotine regulates the expression of immune checkpoint molecules, PD-L1 and PD-L2, via inhibition of Akt phosphorylation. This findings may provide the new therapeutic strategies for the treatment of breast cancer.

PD-L1 and PD-L2 Mutations in Pediatric Hodgkin Lymphoma: Do They Have Any Prognostic Significance?

Background: Reed-Sternberg cells can escape from the immune system by enhancement of the expression of PD-L1 and PD-L2. Objectives: The aim of the present study was to investigate the significance PD-L1 and PD-L2 gene mutations in childhood Hodgkin Lymphoma's (HL). Methods: The study included 39 pediatric classical HL cases. PD-L1 and PD-L2 mutations were determined by Sanger sequencing. Clinicopathological parameters were obtained from patients' records. Results: Eight cases (20.5%) showed p.R260C mutations, and three (7.7%) p.R234L in the exome 5 of PD-L1 gene. None of the cases had PD-L2 mutations. p.R260C mutation exhibited a significant relationship with older age and nodular sclerosing (NS) histology and was associated with longer event free survival. Conclusions: Although PD-L1 mutational status did not show statistically significance with well-established prognostic factors, our preliminary data indicate that p.R260C mutation of PD-L1 gene may be associated with longer event free survival in older patients and NS histology in pediatric HL.

Differential Involvement of Programmed Cell Death Ligands in Skin Immune Responses.

PD-1 is an immunoregulatory receptor that can bind PD-L1 or PD-L2 expressed on stimulated antigen-presenting cells. In this study, isolated antigen-presenting cells (macrophages and dendritic cells) were cultured with IFN-γ, IL-4, or IL-17A, and the expression of PD-L1 and PD-L2 was compared by flow cytometry. Strong upregulation of PD-L1 expression was observed on IFN-γ stimulation of both antigen-presenting cells as well as in response to IL-17A stimulation of macrophages compared with the expression in unstimulated controls. In contrast, only stimulation with IL-4 could upregulate PD-L2 expression on both antigen-presenting cells. Therefore, experiments were performed in murine models, including DNFB-induced contact hypersensitivity, calcipotriol-induced atopic dermatitis-like skin inflammation, and imiquimod-induced psoriasis-like dermatitis models, to trigger IFN-γ‒mediated T helper type (Th)1-, IL-4‒mediated Th2-, and IL-17A‒mediated Th17-type responses, respectively. In both Th1- and Th17-type immunity models, changes in ear thickness were more severe in Pd-l1‒deficient mice than in wild-type or Pd-l2‒deficient mice. In the Th2-type immunity model, changes in thickness in Pd-l2‒deficient mice were more severe than that in wild-type or Pd-l1‒deficient mice. Collectively, PD-L1 has predominant roles in Th1 and Th17 type immunity, whereas PD-L2 is involved in Th2-type immunity.

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.

m6A Regulator-Mediated Methylation Modification Patterns and Tumor Microenvironment Infiltration Characterization in Acute Myeloid Leukemia.

Recent studies have demonstrated epigenetic regulation of immune responses. Nevertheless, the underlying effect of RNA N6-methyladenosine (m6A) modifications on tumor microenvironment cell infiltration remains elusive. In this study, we thoroughly assessed m6A modification patterns of 255 myeloid leukemia specimens based on 23 m6A regulators. Consensus clustering of the 23 m6A regulators was performed to determine three distinct m6A modification patterns that were remarkably consistent with three immunophenotypes of tumors: immunorejection, immune activation, and immune inertness. Further evaluation and prognostic analysis of the m6A modification patterns of individual tumors revealed that low m6A score was characterized by increased mutational burden, immune activation, and survival rates, whereas high m6A score was characterized by poorer survival rates and the absence of effective immune infiltration. In addition, this study investigated the association between m6A regulators and antitumor immune responses and discovered higher expression of the immune regulators PD-L1, PD-L2, MRP1, and MRP2 in low m6A scores. Generally, the expression pattern of m6A regulators was remarkably associated with prognostic results and antitumor immune responses in acute myeloid leukemia and may be an underlying target and biological marker for immune therapies.

Programmed Cell Death Protein Ligand 2 Is a Potential Biomarker That Predicts the Efficacy of Immunotherapy.

Immune checkpoint inhibitor (ICI) responses vary, and biomarkers for predicting responders are urgently needed. Growing evidence points to the association between programmed cell death protein ligand 2 (PDL2) and ICI benefits, while clinical evidences were lacking. Thus, we consolidated five public ICI-treated cohorts to investigate the association between PDL2 expression and ICI treatment prognosis. Immune cell signatures and IFN-γ signatures are investigated in The Cancer Genome Atlas (TCGA) dataset and later in ICI-treated cohorts to explore the association between PDL2 and antitumor immunity in the tumor microenvironment (TME). We found that immune cell signatures and IFN-γ signatures were enriched in the PDL2-high group in TCGA pooled cohorts and most cancers. Consistently, in ICI-treated cohorts, patients with high PDL2 expression experienced longer overall survival time (OS) and were more likely responsive to ICIs than patients with low PDL2 expression. Immune cell scores of the high PDL2 expression patients were significantly higher (P < 0.05) than those of the low PDL2 expression patients in ICI-treated cohorts. In conclusion, our findings suggest that PDL2 is a potential predictive biomarker for ICIs.

Regulation of Immunity in Clear Cell Renal Carcinoma: Role of PD-1, PD-L1, and PD-L2.

Regulation of immunity is a unique oncogenic mechanism that differs in different cancers. VHL deficient clear cell renal cell carcinomas (ccRCC) trigger the immune response resulting in cancer progression. This study aimed to investigate PD-1, PD-L1, and PD-L2 expression in ccRCC primary cancers and metastatic tissues associated with the p-VHL content, transcriptional, and growth factors expression. METHODS: A total of 62 patients with RCC were enrolled in the study. Investigation of mRNA level was performed by PCR in real-time. Western blotting analysis was used for detecting the p-VHL protein content in tissues. RESULTS: The PD-L2 prevalence in metastatic cancers is crucial in tumor progression. The VHL expression and p-VHL content determined the aggressive cancer behavior and elevated in disseminated tumors. The cancer dissemination was accompanied by an increase in both mRNA and VHL content. CONCLUSION: We present a new instrument targeting pathologies with p-VHL/HIF altered function that impact the PD-L2 expression through the change in transcriptional, growth factors, and AKT/mTOR modulation.

Molecular and Immune Correlates of PDCD1 (PD-1), PD-L1 (CD274), and PD-L2 (PDCD1LG2) DNA Methylation in Triple Negative Breast Cancer.

Immune checkpoints are important targets in oncological therapy. Recent studies have proven efficacy of immune checkpoint inhibition (ICI) in treatment of triple negative breast cancer (TNBC). However, only a proportion of TNBC-patients benefit from ICI. Thus, current scientific efforts in this context are focused on the identification of a robust biomarker that enables patient stratification. In the present study, we investigated the epigenetic regulation of PD-1 (PDCD1), PD-L1 (CD274), and PD-L2 (PDCD1LG2). Methylation data of PD-1, PD-L1, and PD-L2, and complex immunogenomic data were obtained from The Cancer Genome Atlas (TCGA). Methylation were systematically analyzed with regard to the transcriptional activity of the studied immune checkpoint genes and the tumor microenvironment. We found differential methylation of PD-1, PD-L1, and PD-L2 in normal adjacent tissue and TNBC tumor tissue. In the TNBC-TCGA cohort, methylation status of PD-1, PD-L1, and PD-L2 were significantly correlated with mRNA levels indicating a strong epigenetic regulation of the transcriptional activity. Moreover, PD-1, PD-L1, and PD-L2 methylation status was strongly associated with a distinct immune cell infiltration pattern. Our results indicate an epigenetic regulation of immune checkpoint genes through DNA methylation in TNBC. In addition, the methylation status was associated with a distinct composition of the tumor microenvironment. Overall, this provides a strong rationale for assessing the value of PD-1, PD-L1, and PD-L2 DNA methylation to predict response to ICI and immunogenicity in TNBC.

DNA Methylation Patterns in the HLA-DPB1 and PDCD1LG2 Gene Regions in Patients with Autoimmune Thyroiditis from Different Water Iodine Areas.

Background: Epigenetic disorders play an important role in the pathogenesis of autoimmune thyroiditis (AIT). Therefore, the study of the possible role of DNA methylation in AIT is of great significance to explore the pathogenesis of AIT. Methods: From May 2019 to June 2019, whole blood samples were collected from 176 AIT patients and 176 controls from different water iodine levels in Shandong Province, China. We used the Illumina Methylation 850K BeadChip to determine significant differences in methylation status of genes and used the MethylTarget™ assay to verify the methylation level in 176 cases and 176 controls. The relative mRNA levels of genes were detected by quantitative real-time-polymerase chain reaction. Results: There were multiple differential methylation sites in the HLA-DPB1 and PDCD1LG2 genes between the case and control population with different water iodine levels. Some target regions of HLA-DPB1 and PDCD1LG2 genes were negatively correlated with relative mRNA expression in the case and control populations and with different water iodine levels. Conclusions: There is differential methylation status in genomic DNA in patients with AIT. The methylation patterns of HLA-DPB1 and PDCD1LG2 genes related to cell adhesion molecule pathway may be different based on different water iodine levels. HLA-DPB1 and PDCD1LG2 genes related to the cell adhesion molecules pathway may play a role in the development of AIT. This study is registered with Chinese Clinical Trial Registry, www.chictr.org.cn, number ChiCTR2000039105.

Characterization of human FDCs reveals regulation of T cells and antigen presentation to B cells.

Stromal-derived follicular dendritic cells (FDCs) are essential for germinal centers (GCs), the site where B cells maturate their antibodies. FDCs present native antigen to B cells and maintain a CXCL13 gradient to form the B cell follicle. Yet despite their essential role, the transcriptome of human FDCs remains undefined. Using single-cell RNA sequencing and microarray, we provided the transcriptome of these enigmatic cells as a comprehensive resource. Key genes were validated by flow cytometry and microscopy. Surprisingly, marginal reticular cells (MRCs) rather than FDCs expressed B cell activating factor (BAFF). Furthermore, we found that human FDCs expressed TLR4 and can alter antigen availability in response to pathogen-associated molecular patterns (PAMPs). High expression of PD-L1 and PD-L2 on FDCs activated PD1 on T cells. In addition, we found expression of genes related to T cell regulation, such as HLA-DRA, CD40, and others. These data suggest intimate contact between human FDCs and T cells.

Mifepristone inhibited the expression of B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis.

BACKGROUND: The immune mechanism was shown to be involved in the development of adenomyosis. The aim of the current study was to evaluate the expression of the immune checkpoints B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and to explore the effect of mifepristone on the expression of these immune checkpoints. METHODS: The expression of B7-H2, B7-H3, B7-H4 and PD-L2 in normal endometria and adenomyosis patient samples treated with or without mifepristone was determined by immunohistochemistry analysis. RESULTS: In adenomyosis patient samples, the expression of B7-H2, B7-H3 and B7-H4 was increased in the eutopic and ectopic endometria compared with normal endometria, both in the proliferative and secretory phases. Moreover, the expression of B7-H2 and B7-H3 was higher in adenomyotic lesions than in the corresponding eutopic endometria, both in the proliferative and secretory phases. The expression of PD-L2 was higher in adenomyotic lesions than in normal endometria in both the proliferative and secretory phases. In the secretory phase but not the proliferative phase, the expression of B7-H4 and PD-L2 in adenomyotic lesions was significantly higher than that in the corresponding eutopic endometria. In normal endometria and eutopic endometria, the expression of B7-H4 was elevated in the proliferative phase compared with that in the secretory phase, while in the ectopic endometria, B7-H4 expression was decreased in the proliferative phase compared with the secretory phase. In addition, the expression of B7-H2, B7-H3, B7-H4 and PD-L2 was significantly decreased in adenomyosis tissues after treatment with mifepristone. CONCLUSIONS: The expression of the immune checkpoint proteins B7-H2, B7-H3, B7-H4 and PD-L2 is upregulated in adenomyosis tissues and is downregulated with mifepristone treatment. The data suggest that B7 immunomodulatory molecules are involved in the pathophysiology of adenomyosis.

New insights into the important roles of tumor cell-intrinsic PD-1.

PD-1 (Programmed cell death protein-1) is mainly expressed in various immune cells, while its ligands PD-L1/PD-L2 (Programmed death ligand-1/Programmed death ligand-2) are mostly expressed in tumor cells. Generally, the binding of PD-L1/PD-L2 and PD-1 could lead to the tumor immune evasion. However, some recent studies showed that PD-1 could also be expressed in tumor cells and could activate mTOR (Mammalian Target of Rapamycin) or Hippo signaling pathway, therefore facilitating tumor proliferation independent of the immune system. While there was evidence that tumor cell-intrinsic PD-1 inhibited the activation of AKT and ERK1/2 pathways, thereby inhibiting tumor cell growth. Based on TCGA and CCLE database, we found that PD-1 was expressed in a variety of tumors and was associated with patient's prognosis. Besides, we found that PD-1 may be involved in many carcinogenic signaling pathway on the basis of PD-1 gene enrichment analysis of cancer tissues and cancer cells. Our understanding of the tumor cell-intrinsic PD-1 function is still limited. This review is aimed at elaborating the potential effects of tumor cell-intrinsic PD-1 on carcinogenesis, providing a novel insight into the effects of anti-PD-1/PD-L1 immunotherapy, and helping to open a major epoch of combination therapy.

Current progress and future perspectives of research on intravascular large B-cell lymphoma.

Intravascular large B-cell lymphoma is a rare disease of the large B cells characterized by selective growth in the lumina of small vessels in systemic organs. Since first reported in 1959, the difficulty of obtaining sufficient tumor cells from biopsy specimens has hampered the elucidation of its underlying biology. Recent progress using xenograft models and plasma cell-free DNA has uncovered genetic features that are similar to those of activated B-cell type diffuse large B-cell lymphoma, including MYD88 and CD79B mutations and frequent alterations in immune check point-related genes such as PD-L1 and PD-L2. Given the improvement in clinical outcomes and a higher risk of secondary central nervous system (CNS) involvement in the rituximab era, a phase 2 trial of R-CHOP combined with high-dose methotrexate and intrathecal chemotherapy as a CNS-oriented therapy has been conducted. This trial, the PRIMEUR-IVL study, has displayed good progression-free survival and a low cumulative incidence of secondary CNS involvement. Long-term follow-up within this trial is still ongoing. Further understanding of the pathophysiology of the disease and improvements in clinical outcomes are still needed.