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Epithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy.We evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55).We uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3Our results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

BackgroundAberrant expression of CD70 in several malignancies is potentially associated with poor patient prognosis and could serve as a therapeutic target. However, the clinical relevance of CD70 expression in pancreatic cancer has not been thoroughly explored.MethodsWe evaluated CD70 expression in 166 surgical specimens obtained from human patients with pancreatic cancer. We analyzed the function of CD70 in proliferation and migration using pancreatic cancer cell lines with silenced CD70 expression.ResultsCD70 expression was positively stained in 42 patients (25%). In the whole cohort, high CD70 expression was not associated with overall survival (OS: 33.1 vs. 40.8 months, P = 0.256), although it was significantly associated with inferior OS in a population of patients that completed adjuvant chemotherapy (P = 0.027). Moreover, the incidence of hematogenous metastasis was significantly higher in patients with high CD70 expression than in those with low CD70 expression (P = 0.020). This finding was also statistically significant in multivariate analyses (P = 0.001). In vitro experiments demonstrated that CD70 expression contributed to cancer cell proliferation independently of gemcitabine treatment as well as cell migration. Furthermore, real-time polymerase chain reaction analysis of frozen surgical tissues showed a correlation between the expression of CD70 and mesenchymal markers.ConclusionsCD70 expression in pancreatic cancer might be involved in hematogenous metastasis. Furthermore, our results imply that CD70 overexpression can serve as a novel prognostic factor and a potential therapeutic target in patients who have completed adjuvant chemotherapy.

Regulatory B10 cells have been shown to exhibit impaired functions in autoimmune diseases. However, the underlying mechanism is still obscure. In the present study, we aimed to understand the regulatory characteristics of regulatory B10 cells and how these cells are involved in the development of rheumatoid arthritis (RA). Here, we chose CD19+CD24hiCD27+ as the phenotype of regulatory B10 cells. We found that the frequencies of CD19+CD24hiCD27+ regulatory B10 cells were decreased and that their IL-10-producing function was impaired in patients with RA compared with healthy controls (HCs). The impairment in CD19+CD24hiCD27+ B10 cells was partially attributed to the decreased expression of CD27 induced by the upregulated CD70 expression on CD19 + B cells and CD4 + T cells. The proportion of CD19+CD24hiCD27+ regulatory B10 cells could be restored by blocking the CD70-CD27 interaction with an anti-CD70 antibody. Furthermore, the CD70-CD27 interaction significantly elevated IL-10 expression and might compensate for the decreased number of CD19+CD24hiCD27+ B cells. Hence, the CD70-CD27 interaction might play a critical role in the numerical and functional impairments of regulatory B10 cells, thus contributing to RA pathogenesis. In conclusion, the change in CD19+CD24hiCD27+ regulatory B10 cells in RA was only a consequence, not the cause, of RA development, but the increased expression of CD70 might be the culprit.

The troponin complex regulates the Ca(2+) activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500–700 million years ago during early animal evolution. Troponin T (TnT) is the thin-filament–anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and they also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly-diverged structure consisting of a long glutamic acid–rich C-terminal extension of ∼70 residues with unknown function. We found here that C-terminally truncated Drosophila TnT (TpnT–CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnT(CD70) gene residing on the X chromosome resulted in lethality in male flies. We demonstrate that this X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific mAb indicated expression of TpnT–CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and EM examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT–CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca(2+) overloading induced by high-frequency electrical pacing. Our findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca(2+) buffer/reservoir and is potentially critical to the high-frequency asynchronous contraction of flight muscles.

Background Galectin 3 (LGALS3) gene expression is associated with poor survival in acute myeloid leukemia (AML) but the prognostic impact of LGALS3 protein expression in AML is unknown. LGALS3 supports diverse survival pathways including RAS mediated cascades, protein expression and stability of anti-apoptotic BCL2 family members, and activation of proliferative pathways including those mediated by beta Catenin. CD74 is a positive regulator of CD44 and CXCR4 signaling and this molecule may be critical for AML stem cell function. At present, the role of LGALS3 and CD74 in AML is unclear. In this study, we examine protein expression of LGALS3 and CD74 by reverse phase protein analysis (RPPA) and identify new protein networks associated with these molecules. In addition, we determine prognostic potential of LGALS3, CD74, and their protein networks for clinical correlates in AML patients. Methods RPPA was used to determine relative expression of LGALS3, CD74, and 229 other proteins in 231 fresh AML patient samples and 205 samples were from patients who were treated and evaluable for outcome. Pearson correlation analysis was performed to identify proteins associated with LGALS3 and CD74. Progeny clustering was performed to generate protein networks. String analysis was performed to determine protein:protein interactions in networks and to perform gene ontology analysis. Kaplan-Meir method was used to generate survival curves. Findings LGALS3 is highest in monocytic AML patients and those with elevated LGALS3 had significantly shorter remission duration compared to patients with lower LGALS3 levels (median 21.9 vs 51.3 weeks, p = 0.016). Pearson correlation of LGALS3 with 230 other proteins identifies a distinct set of 37 proteins positively correlated with LGALS3 expression levels with a high representation of proteins involved in AKT and ERK signaling pathways. Thirty-one proteins were negatively correlated with LGALS3 including an AKT phosphatase. Pearson correlation of proteins associated with CD74 identified 12 proteins negatively correlated with CD74 and 16 proteins that are positively correlated with CD74. CD74 network revealed strong association with CD44 signaling and a high representation of apoptosis regulators. Progeny clustering was used to build protein networks based on LGALS3 and CD74 associated proteins. A strong relationship of the LGALS3 network with the CD74 network was identified. For AML patients with both the LGALS3 and CD74 protein cluster active, median overall survival was only 24.3 weeks, median remission duration was 17.8 weeks, and no patient survived beyond one year. Interpretation The findings from this study identify for the first time protein networks associated with LGALS3 and CD74 in AML. Each network features unique pathway characteristics. The data also suggest that the LGALS3 network and the CD74 network each support AML cell survival and the two networks may cooperate in a novel high risk AML population. Fund Leukemia Lymphoma Society provided funds to SMK for RPPA study of AML patient population. Leukemia Texas provided funds to PPR and SMK to study CD74 and LGALS3 expression in AML patients using RPPA. No payment was involved in the production of this manuscript.

Single-stranded DNA aptamers recognizing CD70 molecule overexpressed in some tumor cell lines was isolated by means of systematic evolution of ligands by exponential enrichment (SELEX). Both purified CD70 protein and CD70-expressing cells were used to isolate target-specific aptamer. After certain rounds of protein-based SELEX and cell-SELEX (ten and seven rounds, respectively), Aptamer 928 (hereafter Apt928) with high affinity and specificity for CD70 was obtained. The specific binding of the aptamer to its target could block interaction of CD70 with CD27 (known as CD70 receptor). Dissociation constant of Apt928 was calculated to be 66 nmol L−1. Moreover, ATTO 674N-labeled Apt928 was applied as a fluorescent aptasensor for rapid and sensitive detection of SKOV-3 cells as CD70-positive cells. The detection limit of this aptasensor was 14 cells mL−1.

CD4A total of 43 control subjects, 42 unstable angina patients, and 90 AMI patients were enrolled in the present study. The serum levels of soluble CD27 (sCD27) in patients were measured, revealing a significant increase in serum sCD27 levels in AMI patients within 24 h of the cardiac event, after which they decreased. Correlation analyses revealed that serum sCD27 was positively correlated with cardiac troponin I (c-TnI) (r = 0.267, P = 0.011). When anti-CD70 antibody was used to block the CD27-CD70 pathway in MI model mice, we found that this treatment increased left ventricular end-diastolic dimension (LVEDD) (P 0.01) and left ventricular end-systolic dimension (LVESD) (P 0.01), and decreased ejection fraction (P 0.01). Flow cytometric analysis revealed that the percentage of regulatory T cells was lower in blocking antibody-treated mice (P 0.01), while neutrophils levels were higher (P 0.01). The number of CD31-positive endothelial cells (P = 0.026) and α-smooth muscle actin-positive arterioles (P 0.01) were significantly down-regulated in anti-CD70 treated-AMI mice. The formation of the extracellular matrix (ECM) was also impaired.Serum sCD27 may be a potential biomarker for AMI. Blockade of the CD27-CD70 pathway worsens cardiac dysfunction, aggravates left ventricular remodeling, and impairs scar healing after AMI, resulting in heart failure.

Chimeric antigen receptor (CAR) T cell therapy is effective in lymphoid malignancies, but there has been limited data in myeloid cancers. Here, we start with a CD27-based CAR to target CD70 (‘native’) in acute myeloid leukemia (AML), and we find modest efficacy in vivo, consistent with prior reports. We then use orthogonal approaches to increase binding on both the tumor and CAR-T cell sides of the immune synapse: a pharmacologic approach (azacitidine) to increase antigen density of CD70 in myeloid tumors, and an engineering approach to stabilize binding of the CAR to CD70. To accomplish the latter, we design a panel of hinge-modified regions to mitigate cleavage of the extracellular portion of CD27. Our CD8 hinge and transmembrane-modified CD70 CAR-T cells are less prone to cleavage, have enhanced binding avidity, and increased expansion, leading to more potent in vivo activity. This enhanced CD70-targeted CAR is a promising candidate for further clinical development.

Immune checkpoint proteins (ICPs) are regulators of immune system. The ICP dysregulation silences the host immune response to cancer-specific antigens, contributing to the occurrence and progress of various cancers. MiRNAs are regulatory molecules and function in mRNA silencing and post-transcriptional regulation of gene expression. MiRNAs that modulate the immunity via ICPs have received increasing attention. Many studies have shown that the expressions of ICPs are directly or indirectly repressed by miRNAs in multiple types of cancers. MiRNAs are also subject to regulation by ICPs. In this review, recent studies of the relationship between miRNAs and ICPs (including the PD-1, PD-L1, CTLA-4, ICOS, B7-1, B7-2, B7-H2, B7-H3, CD27, CD70, CD40, and CD40L) in cancer immune escape are comprehensively discussed, which provide critical detailed mechanistic insights into the functions of the miRNA-ICP axes and their effects on immune escape, and will be beneficial for the potential applications of immune checkpoint therapy and miRNA-based guidance for personalized medicine as well as for predicting the prognosis.

Objectives In accordance with the Precision Medicine Initiative, new treatment strategies for head and neck squamous cell carcinoma (HNSCC) are needed to yield better therapeutic outcomes. The purpose of this study was to establish and validate chimeric antigen receptor (CAR)-T cells targets in HNSCC. Methods Putative CAR-T antigens were identified in The Cancer Genome Atlas database. To validate antigen suitability, quantitative RT-PCR, flow cytometry, and immunofluorescent staining were performed. A retroviral human CD70 CAR construct, using truncated CD27 conjugated with 4-1BB and CD3-zeta costimulatory molecules, was used to transduce activated human T cells to generate CD70 CAR-T cells. Cell-based cytotoxicity and cytokine ELISAs were used to measure efficacy of killing. Results Nine potential CAR-T targets (CD276, EGFR, MICA, MICB, MAGE-A4, FAP, EPCAM, CD70, B4GALNT1) were identified based on their high expression in tumors compared to flanking control tissues. CD70 was selected for further proof-of-principle analysis based on its differential expression in several tumor subtypes, and showed substantial heterogeneity in individual tumors analyzed. Cell surface CD70 protein and CD70 mRNA were detected from low to high levels in established HNSCC cancer cell lines. CD70 was highly expressed in 4 of 21 tumor biopsies (19%), and 3 of 4 specimens showed strong CD70 expression on the tumor cell surface. CD70-specific CAR-T cells were generated and further demonstrated to recognize and kill CD70-positive HNSCC cells efficiently, but not CD70-negative cancer cells. Conclusion CD70-specific CAR-T cells specifically recognized and efficiently eliminated CD70-positive HNSCC cells. This study provides the basis for further investigation into CD70 and other CAR-T targets.

Objective Circulating lymphocyte subtypes are not fully explored parameters for monitoring chronic T cell activation during inflammatory bowel disease (IBD). Tumor necrosis factor α (TNFα), one of the main mediators of IBD related inflammation induces expression of CD70 on T cells. CD70 limits T cell expansion and controls CD27 receptor on activated B lymphocytes. Aim of this study was to assess the number and the frequency of CD70+ T cells and CD27+ B cells in IBD patients during inactive phase of the disease under or without anti-TNFα treatment. Design We studied 91 patients with inactive IBD, 31 untreated, 29 treated with infliximab (IFX), and 31 treated with adalimumab (ADA). Lymphocyte phenotypes were assessed by flow cytometry using anti-CD45, CD19, CD27, CD3, and CD70 monoclonal antibodies. IFX and ADA actual capacity of TNFα neutralization in serum was estimated by the recoveryELISA technique. Results Whereas CD3+ T cells were increased in treated compared to untreated patients, the percentage of the CD70+ T cells was significantly lower in treated patients indicating a ‘cooling’ effect of the biological therapy. This effect differs between samples according to the therapeutic range of the circulating drug. Although the CD19+ B-cell percentage tended to be lower in treated patients, CD19+27+ memory B cells did not show significant differences between groups. Conclusions Frequency of peripheral blood CD70+ T cells was significantly reduced by treatment with anti-TNFα antibodies. Monitoring of this parameter of T cells can give better insight to the disease progression and therapy application in IBD patients.

Background The expression of CD70 on T cells is greatly enhanced by antigen-presenting cell (APC)-associated signals, such as tumour necrosis factor(TNF)-α, which is constitutionally high in patients with inflammatory bowel disease (IBD). Experimentally, the chronic activation of CD27 as a result of the constitutive expression of CD70 leads to the demise of B cells in bone marrow (BM) and the secondary lymphoid organs. The aim of this study was to assess the number and phenotype of circulating B cell in untreated IBD patients and their counterparts treated with biological anti-TNF drugs. Methods The study involved 13 untreated IBD patients, 36 IBD patients treated with biological drugs, and 10 healthy controls. The B cell phenotypes were assessed by means of flow cytometry using monoclonal antibodies specific for CD20, CD19, CD3, CD27 and CD43. In order to evaluate B cell development in bone marrow and peripheral B cell activation, we identified four B cell subsets: hematogones (HBs: CD20+ 19+ 3− 27− 43+), memory B cells (MBs: CD20+ 19+ 3− 27+ 43−), pre-plasmablasts (PPBs: CD20+ 19+ 3− 27+ 43+), and plasmablasts (PBs: CD20− 19+ 3− 27+ 43+). Results The total number of B cells in the untreated patients was three times lower than that in the patients treated with biological drug (p

CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction.

Isoform 1 of uracil-DNA glycosylase (UNG1) is the major protein for initiating base-excision repair in mitochondria and is in close proximity to the respiratory chain that generates reactive oxygen species (ROS). Effects of ROS on the stability of UNG1 have not been well characterized. In the present study, we found that overexpression of UNG1 enhanced cells' resistance to oxidative stress and protected mitochondrial DNA (mtDNA) from oxidation. Proteomics analysis showed that UNG1 bound to eight proteins in the mitochondria, including PAPSS2, CD70 antigen, and AGR2 under normal growth conditions, whereas UNG1 mainly bound to Peroxiredoxin 3 (PRDX3) via a disulfide linkage under oxidative stress. We further demonstrated that the UNG1-PRDX3 interaction protected UNG1 from ROS-mediated degradation and prevented mtDNA oxidation. Moreover, our results show that ROS-mediated UNG1 degradation was Lon protease 1 (LonP1)-dependent and mitochondrial UNG1 degradation was aggravated by knockdown of PRDX3 expression. Taken together, these results reveal a novel function of UNG1 in the recruitment of PRDX3 to mtDNA under oxidative stress, enabling protection of UNG1 and UNG1-bound DNA from ROS damage and enhancing cell resistance to oxidative stress.

Objective To critically examine current evidence regarding the role of the CD27–CD70 pathway in the pathophysiology of autoimmune disease with a focus on understanding the contributions of this pathway as a potential new therapeutic target for systemic lupus erythematosus and rheumatoid arthritis. Methods A PubMed search for articles was conducted using the following key words: (“CD27” OR “CD70”) AND (“autoimmune disease” OR “systemic lupus erythematosus” OR “rheumatoid arthritis”). The search was limited to publications in English and included human and animal studies. The reference lists of identified articles were searched for further relevant citations. Publications on the list that was developed by this approach were assessed and those with relevance to CD27–CD70 pathway mediated pathophysiology in autoimmune disease were chosen for the detailed review. Results Data from human diseases and animal models document a major role for the CD27–CD70 receptor–ligand pair in providing signals that regulate T and B lymphocyte activation. The membrane receptor CD27 and its soluble form (sCD27) transmit co-stimulatory signals and induce activation and proliferation of T and B lymphocytes. CD70-expressing CD4 T lymphocytes are increased in autoimmune disease including systemic lupus erythematosus and rheumatoid arthritis and have been shown to produce pro-inflammatory cytokines. At the same time, preclinical evidence suggests that the outcome of CD27–CD70 signals may vary qualitatively between cell subsets and differentiation stages, especially for B lymphocytes. Blockade of the CD27–CD70 pathway has been shown to ameliorate disease manifestations in animal models including murine collagen-induced arthritis and experimental colitis. Conclusion Current evidence from animal models and human diseases suggests that CD27–CD70 pathway contributes to the pathophysiology of autoimmunity. Although a number of basic questions still remain open, the available findings suggest that targeting the components of this pathway could provide useful and novel therapeutic interventions.

DNA methylation is the covalent addition of a methyl group to a DNA base, typically the cytosine of cytosine-phosphate-guanosine (CpG) dinucleotides. It is catalysed by methyltransferase enzymes using an S-adenosyl methionine donor, which is a heritable, stable and reversible DNA modification. Aberrant DNA methylation can influence gene expression without changing nucleotide sequences, inducing occurrence and development in autoimmune diseases, such as systemic lupus erythematosus and immune thrombocytopenia. Immune thrombocytopenia is an autoimmune disease characterised by bleeding and thrombocytopenia of peripheral blood, a normal or increased number of megakaryocytes and a maturation disorder. Recently, it was proven that aberrant DNA methylation is associated with the aetiology of immune thrombocytopenia. The defective methylation induces overexpression of methylation-related genes, such as CD70 and FOXP3, which can take part in autoreactive immune responses, and ultimately accelerated the progression of immune thrombocytopenia. Targeting the DNA methylation can be used as a new treatment for immune thrombocytopenia. As a demethylated drug, decitabine promotes megakaryocyte maturation and platelet release under the action of tumour necrosis factor-related apoptosis inducing ligand (TRAIL) promoter. This review highlights recent evidence on the role of DNA methylation in immune thrombocytopenia by describing the relationship between DNA methylation and immune thrombocytopenia, and the DNA methylation-related genes. Identifying and regulating abnormal DNA methylation provides new ideas for the diagnosis and treatment of immune thrombocytopenia.

Systemic sclerosis (SSc) is a heterogeneous and life-threatening autoimmune disease characterized by damage to small blood vessels, interruption of immune homeostasis and ultimately, fibrosis. Currently, the mechanisms involved in SSc pathogenesis remain unknown. An increasing amount of data shows that, via certain signaling pathways, epigenetic mechanisms, including DNA methylation, histone modification, and miRNAs, are closely related to the three primary processes that characterize SSc: vascular abnormalities, activation of immune system, and excessive extracellular matrix deposition. In the clinical setting, identification of molecules and biomarkers for determining disease severity, predicting disease progression and assessing response to treatment remains challenging. In this review, we aim to summarize the key epigenetic mechanisms involved in the pathogenesis of SSc. Certain cytokines or molecules, such as CD40, CD70, and Fli-1, are expressed at varying rates in SSc due to epigenetic modification and play important roles in SSc. It is therefore likely that these molecules may be biomarkers for SSc. In addition, epigenetic changes of certain genes, including Fli-1, BMPRII, CD11a, Foxp3, and eNOS, influence the expression of these genes to ultimately result in an anti-fibrotic effect. The influence that epigenetics has on SSc pathogenesis suggests that epigenetics-targeting drugs may have potential therapeutic effects against SSc. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

Primary immunodeficiency (PID) comprises a heterogeneous group of over 330 genetic disorders, caused mainly by single-gene mutations, such as CD70. We generated human induced pluripotent stem cell lines, PHAi003-A and PHAi003-B, from a PID patient carrying the homozygous frameshift CD70 mutation c.250delT. The CD70 c.250delT genotype results in a complete loss of expression variant. This patient is one of the five CD70 deficient individuals described to date, and presented hypogammaglobulinemia, EBV associated Hodgkin's lymphoma and susceptibility to other viral infections. The PHAi003-A and PHAi003-B lines are a unique resource for PID modeling and studying CD70-mediated immunity in human cells.

Systemic lupus erythematosus (SLE), a chronic autoimmune disease characterized by overproduction of autoantibodies against a series of nuclear antigens, causes widespread tissue damage. Although multiple genes take part in determining the predisposition to SLE, environmental factors, largely reflected by epigenetic alterations, DNA methylation changes in particular, have been demonstrated to be crucial for the pathogenesis of SLE. DNA methylation is a most widely studied epigenetic mark that is stable, inheritable, and reversible. Gene transcription can be repressed through hypermethylation of active promoter regions associated with CpG-rich sequences. In contrast to gene hypermethylation, DNA hypomethylation facilitates transcription factor binding to promoter regions, thereby promoting gene expression.

We investigated regulation of human NK cell function by CD27 engagement using a recombinant soluble CD70 protein. CD27 was preferentially expressed on CD56(bright) NK cells, and soluble CD70 protein bound to CD27(+)CD56(bright) NK cells. While soluble CD70 protein enhanced IFN-γ secretion by CD56(bright) NK cells in the presence of IL-12, it augmented neither cytolytic activity nor proliferation of NK cells. Thus, we next asked if soluble CD70 protein could be used to induce non-cytolytic antiviral activity of NK cells using an in vitro hepatitis C virus (HCV) infection system. Soluble CD70 protein stimulated NK cells to suppress HCV replication by enhancing NK cell IFN-γ secretion without killing infected cells. Taken together, we demonstrate that CD27 engagement by a soluble CD70 protein enhances non-cytolytic antiviral activity of CD56(bright) NK cells by IFN-γ secretion. Thus, this soluble CD70 protein may be useful for the treatment of viral infections such as HCV infection.

DNA hypomethylation is a characteristic feature of systemic lupus erythematosus (SLE) immune cells. Numerous reports have implicated the involvement of the MEK/ERK pathway in the reduction of DNA methyltransferase (DNMT) expression, hence inducing the transcription of methylation-sensitive genes in SLE patients. However, the molecular mechanisms involved remain unclear. Here, we investigated whether the catalytic subunit of protein phosphatase 2A (PP2Ac), which is overexpressed in SLE T-cells, contributes to reduced DNA methylation. We show that both chemical suppression and siRNA silencing of PP2Ac in T-cells resulted in sustained phosphorylation of MEK and ERK following stimulation with phorbol 12-myristate 13-acetate and ionomycin. Furthermore, PP2Ac suppression resulted in increased DNMT enzyme activity, DNA hypermethylation, and decreased expression of methylation-sensitive genes. Similarly, in SLE T-cells, suppression of PP2Ac resulted in increased MEK/ERK phosphorylation, enhanced DNMT1 expression and suppressed expression of the methylation-sensitive CD70 gene. Our results demonstrate that PP2A regulates DNA methylation by influencing the phosphorylation of MEK/ERK. We propose that enhanced PP2Ac in SLE T-cells may dephosphorylate and activate the signaling pathway upstream of DNMT1, thus disturbing the tight control of methylation-sensitive genes, which are involved in SLE pathogenesis.

Interleukin-7 (IL-7) promotes the maintenance and activation of peripheral T cells, whereas it does not act directly on mature B cells due to the lack of IL-7Rα expression on these. We report here that, in spite of the insensitivity of B cells to IL-7, high concentration of IL-7 can lead to increased B cell survival and antibody production in the presence of T cells, without the use of any further B cell stimulatory signal. IL-7 promoted B cell activation through inducing CD70 expression on resting T cells, particularly on CD4+ memory cells. The interaction of CD70 molecules with the B cell costimulatory receptor CD27 led to B cell proliferation, the accumulation of CD38 + CD20- plasmablasts and antibody production. In addition, IL-7 treatment induced BAFF secretion from resting peripheral T cells thereby promoting B cell survival. IL-7 levels can increase in lymphopenic conditions, in autoimmune diseases or in patients receiving T cell regenerative IL-7 therapy. Based on our findings high IL-7 levels can lead to increased B cell activation by inducing the B cell regulatory proteins CD70 and BAFF in resting T cells. Such activity might be beneficial in short term immune-stimulatory IL-7 therapies; permanently increased IL-7 levels, on the other hand, can contribute to impaired B cell tolerance.

CD70 (TNFSF7), as a methylation susceptive immune gene, was hypomethylated in some autoimmune diseases. To investigate the status of CD70 methylation and the expressions of genes that regulated methylation in immune thrombocytopenia (ITP) patients, the expressions of CD70 mRNA and protein in CD4(+) T cells from ITP and controls were measured respectively by real-time PCR and flow cytometry. Methylation specific high resolution melting (MS-HRM) technology was applied to detect CD70 promoter methylation indices. Transcription levels of DNA methyltransferases (DNMTs), methylated CpG binding protein 2 (MBD2) were measured. The results showed that CD70, DNMTs and MBD2 was over expressed and methylation indices of CD70 promoter in CD4(+) T cells from ITP patients were lower than that from healthy controls. The transcription levels of CD70 showed significantly inverse correlation with methylation indices in ITP patients but significantly positive correlation with that of DNMTs. We concluded that DNMTs functioned as demethylases as MBD2, while increased DNMTs and MBD2 may cause demethylation and over expression of CD70 in CD4(+) T cells, potentially contributing to the pathogenesis of ITP.

Background: Alloreactive memory T cells are a major obstacle to transplantation acceptance due to their capacity for accelerated rejection. Methods: C57BL/6 mice that had rejected BALB/c skin grafts 4 weeks earlier were used as recipients. The recipient mice were treated with anti-CD154/LFA-1 with or without anti-CD70 during the primary skin transplantation and anti-CD154/LFA-1 or not during the secondary transplantation of BALB/c heart. We evaluated the impact of combinations of antibody-mediated blockade on the generation of memory T cells and graft survival after fully MHC-mismatched transplantations. Results: One month after the primary skin transplantation, the proportions of CD4(+) memory T cells/CD4(+) T cells and CD8(+)memory T cells/CD8(+) T cells in the anti-CD154/LFA-1 combination group were 47.32 +/- 428% and 23.18 +/- 2.77%, respectively. In the group that included anti-CD70 treatment, the proportions were reduced to 34.10 +/- 2.71% and 12.19 +/- 3.52% (P

DNA demethylation and histone hyperacetylation of CD11a and CD70 regulatory regions contribute to the development of autoreactivity and autoantibody overstimulation in CD4 + T cells of patients with systemic lupus erythematosus (SLE). However, the mechanisms causing these changes remain largely unknown. We report that the expression and activity of the transcription factor RFX1 are decreased in SLE CD4 + T cells. We demonstrate that RFX1 affects DNA methylation and histone acetylation in CD4 + T cells by recruiting the co-repressors DNMT1 and HDAC1 to the CD11a and CD70 promoters, and thereby represses their expression. Reducing RFX1 in CD4 + T cells is sufficient to cause lupus-like T and B cell hyperactivity, whereas overexpressing RFX1 suppresses T cell reactivity. These findings reveal a crucial role for RFX1 in regulating the epigenetic status of T cells, and demonstrate that autoimmune responses in SLE are due in part to RFX1 downregulation.

T cell DNA methylation levels decline with age, activating genes such as KIR and TNFSF7 (CD70), implicated in lupus-like autoimmunity and acute coronary syndromes. The mechanisms causing age-dependent DNA demethylation are unclear. Maintenance of DNA methylation depends on DNA methyltransferase 1 (Dnmt1) and intracellular S-adenosylmethionine (SAM) levels, and is inhibited by S-adenosylhomocysteine (SAH). SAM levels depend on dietary micronutrients including folate and methionine. SAH levels depend on serum homocysteine concentrations. T cell Dnmt1 levels also decline with age. We hypothesized that age-dependent Dnmt1 decreases synergize with low folate, low methionine or high homocysteine levels to demethylate and activate methylation-sensitive genes. T cells from healthy adults ages 22-81, stimulated and cultured with low folate, low methionine, or high homocysteine concentrations showed demethylation and overexpression of KIR and CD70 beginning at age approximately 50 and increased further with age. The effects were reproduced by Dnmt1 knockdowns in T cells from young subjects. These results indicate that maintenance of T cell DNA methylation patterns is more sensitive to low folate and methionine levels in older than younger individuals, due to low Dnmt1 levels, and that homocysteine further increases aberrant gene expression. Thus, attention to proper nutrition may be particularly important in the elderly.

Ligation of CD27, a member of the tumor necrosis factor (TNF) receptor family, by its ligand CD70 is thought to be important in T cell activation, expansion and survival, B cell activation, and NK cell activation. We examined the role of CD70 in Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) mice. Blocking of CD70 in effector phase by anti-CD70 monoclonal antibody (mAb) suppressed the development of TMEV-IDD. The number of IFN-gamma- or TNF-alpha-producing cells in the spleen and mRNA levels of IFN-gamma and TNF-alpha in spinal cord were decreased in mice treated with anti-CD70 mAb at the effector phase. In contrast, treatment with anti-CD70 mAb in induction phase failed to reduce these responses, compared to nonspecific IgG-treated control mice. These data suggest that CD70 is critically involved in the pathogenesis of TMEV-IDD and that antibodies against CD70 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.

Objective We have used mass-spectrometry (MS) based proteomics platform to identify cell surface proteins over-expressed on a cisplatin resistant derivative of an ovarian cancer cell line A2780. Methods Membrane associated glycoproteins from A2780 and its cisplatin resistant derivative cell line, A2780cis, were processed for liquid chromatography (LC)-MS based analysis. The expression of proteins found at elevated levels in A2780cis cell line was confirmed using flow cytometry and Taqman analysis. The expression of these proteins was further evaluated in unrelated ovarian cancer cell lines using MS analysis and flow cytometry. Immunohistochemical (IHC) analysis was performed using ovarian tumor tissues to evaluate the protein density on the cell surface. Monoclonal antibodies were used in an alamar blue proliferation assay to examine the cytotoxic effects on cell proliferation in resistant cell lines. Results Six proteins were identified by LC-MS as being over-expressed on cell surface of A2780cis cell line. Mass spectrometry and flow cytometry confirmed the over-expression of CD49f, CD70 and Her-2/neu in other cisplatin resistant ovarian cancer cell lines. Immunohistochemical analysis revealed that only CD70 was expressed at moderate levels in ovarian tumors. When cisplatin resistant ovarian cell lines A2780cis and SKOV-3 were treated with antibody against CD70, there was a significant decrease in cell proliferation. Conclusion Using a MS based proteomics approach we have shown that expression of CD70 is associated with cisplatin resistance in ovarian cancer cell lines. Follow-up examination of these tumor cell line findings in clinical tumor specimens with available pathology staging and cisplatin treatment history is warranted.

Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies (mAbs), forming antibody-drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The drugs were conjugated with a linker construct that is labile to lysosomal proteases and incorporates a valine-alanine-p-aminobenzyl (PAB)-amino linkage for direct attachment to the electron-deficient amine functional groups present in both drugs. The resulting ADCs release drug following internalization into antigen-positive cancer cells. The drug linkers were conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) via alkylation of reduced interchain disulfides to give ADCs loaded with 4 drugs/mAb. The streptonigrin ADCs were potent and immunologically specific on a panel of cancer cell lines in vitro and in a Hodgkin lymphoma xenograft model. We conclude that streptonigrin ADCs are candidates for further research, and that the novel linker system used to make them is well-suited for the conjugation of cytotoxic agents containing electron-deficient amine functional groups.

Targeting leukocyte differentiation antigens is a validated approach to develop therapeutic agents for the treatment of cancer, autoimmunity, and inflammatory diseases. A subset of activation antigens transiently induced on leukocytes is particularly interesting because many of them are absent from normal tissues, including those of most vital organs, and therapeutic agents' targeting of such antigens is expected to impart limited toxicity. One such antigen, CD70, has recently emerged as an attractive potential drug target for the treatment of cancers. Whereas CD70 is only transiently expressed on activation T and B cells and mature dendritic cells, it is found to be aberrantly expressed on a variety of tumor cells, including Waldenström's macroglobulinemia. In this report, we discuss potential antibody-based therapeutic approaches targeting CD70 for tumor elimination where various mechanisms such as antibody effector functions, immune enhancement, blockade of paracrine growth loop, and delivery of cytotoxic payloads can be exploited to achieve efficacy. Indeed, early clinical trials with therapeutic anti-CD70 antibodies are currently in progress, and those for anti-CD70 drug conjugates will soon follow.

Background Abnormally high serum uric acid levels have been associated with several disease conditions including gout and kidney stone disease. More recently, it was shown that uric acid crystals stimulate dendritic cell maturation, activate the NALP3 inflammasome, and enhance antigen-specific immune responses. We hypothesize that uric acid can also stimulate T cells directly and in the absence of antigen presentation. Methods Purified primary human T cells were incubated with and without uric acid at concentrations of 50, 100, 150, and 200 μ g/mL. The expression of T-cell activation markers CD25 and CD70 was assessed by flow cytometry. In other experiments, Jurkat T cells were used and the expression of the costimulatory molecule CD70 was determined at the mRNA level. Results Uric acid directly activates primary human T cells in the absence of antigen presentation. Furthermore, primary human T cells and Jurkat T cells treated with uric acid overexpress the costimulatory molecule CD70, which plays an important role in T cell-B cell interaction and antibody production. Conclusions The finding that uric acid directly promotes T-cell activation in an antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gouty arthritis and other immune-mediated diseases.

SummarySteady-state dendritic cells (DCs) maintain peripheral T cell tolerance, whereas mature DCs generate immunity. CD70 is a costimulatory ligand acquired upon DC maturation. To determine its impact on T cell fate, we have generated mice that constitutively express CD70 in conventional DCs (cDCs). In these mice, naive CD4+ and CD8+ T cells spontaneously convert into effector cells. Administration of peptide without adjuvant, which is ordinarily tolerogenic, elicited tumor-eradicating CD8+ T cell responses and robust CD4+ T cell-independent memory. CD70 was also constitutively expressed in cDCs that inducibly present viral epitopes. In this case, tolerance induction was prevented as well. The antigen-presenting DCs generated protective immunity to virus infection and broke a pre-existing state of CD8+ T cell tolerance. Thus, the sole expression of CD70 by otherwise immature cDCs sufficed to convert CD8+ T cell tolerance into immunity, defining the importance of CD27-CD70 interactions at the interface between T cell and DC.

Objective Chronic lymphocytic leukemia (CLL) B cells from most patients express both membrane-bound CD27 (mCD27) and soluble CD27 (sCD27). Expression of sCD27 inhibits CD27-dependent T-cell or CLL-cell activation mediated by its ligand, CD70. In this study, we evaluated whether protease inhibitors could inhibit the release of sCD27 from CLL cells and enhance T-cell activation mediated by CD27-CD70 interaction. Methods CLL cells exposed to hydroxamic acid-based matrix metalloprotease (MMP) inhibitors were evaluated for the release of sCD27 by sandwich enzyme-linked immunosorbent assay and immunoprecipitation. We examined for phenotypic changes in CLL cells treated with MMP inhibitors by flow cytometry and T-cell activation by CLL cells was assessed by [ 3 H] thymidine incorporation assay and the production of interferon-gamma. Results Treatment of CLL cells with MMP inhibitors blocked the release of sCD27 to the culture supernatant. In contrast, a non–hydroxamic acid control compound or inhibitors of other proteases, including serine, cysteine, and aspartyl proteases, were ineffective. Furthermore, CLL cells treated with MMP inhibitors expressed significantly higher levels of accessory molecules, such as CD54, CD80, and CD95. Consistent with such changes, we found that CLL cells treated with MMP inhibitors, but not control treated cells, could stimulate allogeneic and autologous T cells in mixed lymphocyte reactions. Conclusion These data reveal that metalloprotease inhibitors can block production of sCD27, which can interfere with mCD27-CD70 interactions that induce expression of immune costimulatory molecules on CLL B cells. Conceivably, treatment of CLL cells with metalloprotease inhibitors may enhance their potential for stimulating cellular immune recognition of leukemia-associated antigens.

The considerable advances made in the fields of molecular biology, genomics, proteomics and protein engineering have led to the identification of a vast range of potential vaccine antigens for a host of man's most serious diseases. However, experience informs us that vaccines based on recombinant proteins and synthetic peptides lack the immunogenicity of the whole, killed pathogens used in traditional vaccines and, as such, clinical use of these immunogens remains negligible. In order to fully realize the potential benefits of recombinant antigen-based vaccines there is a pressing need to identify powerful adjuvants which can safely enhance these weak responses with a minimum of undesirable side effects. Adjuvant research represents a vibrant and fast moving field and recent developments suggest the goal of generating effective, safe and affordable ways of enhancing immune responses appears to be almost within our grasp. The purpose of this article is to review recent advances in adjuvant development using approaches that directly exploit the immune system's own co-stimulatory pathways to exert their function; with a particular emphasis on CD40 and CD28 based therapies.

In vitro work has defined the TNF receptor family member CD27 as a T and B cell co-stimulatory molecule. Its activity is governed by the transient availability of its TNF-like ligand CD70 on lymphocytes and dendritic cells. Recent studies, enforcing or abrogating CD27 function by genetic or protein intervention in mouse models have revealed key contributions of the CD27-CD70 system to effector and memory T cell formation, which is probably based on improved cell survival. The stimulatory effects of CD27 on B cell function appear to oppose those of CD70, which also has a signaling role. Targeting CD27-CD70 for therapy is attractive but should take into account the fact that constitutive CD27 stimulation culminates in lethal immunodeficiency.

The NF-kappaB-inducing kinase (NIK) induces proteolytic processing of NF-kappaB2/p100 and, hence, the generation of NF-kappaB dimers such as p52:RelB but was suggested not to signal for the processing of IkappaB. Here, we show that although the induction of IkappaB degradation in lymphocytes by TNF is independent of NIK, its induction by CD70, CD40 ligand, and BLyS/BAFF, which all also induce NF-kappaB2/p100 processing, does depend on NIK function. Both CD70 and TNF induce recruitment of the IKK kinase complex to their receptors. In the case of CD70, but not TNF, this process is associated with NIK recruitment and is followed by prolonged receptor association of just IKK1 and NIK. Recruitment of the IKK complex to CD27, but not that of NIK, depends on NIK kinase function. Our findings indicate that NIK participates in a unique set of proximal signaling events initiated by specific inducers, which activate both canonical and noncanonical NF-kappaB dimers.

Members of both the CD28 and TNFR families can have costimulatory roles in T cell activation. Gene targeted mice as well as in vivo blocking experiments have established distinct roles for CD28/B7; ICOS/ICOSL; CD27/CD70; 4-1BB/4-1BBL and OX40/OX40L during viral infection. Many issues remain to be addressed, including the timing and location of the interactions, the possibility of partial redundancy between related family members and the molecular basis for the specific phenotypes observed in the different gene targeted mice.

Background: T-cell-mediated myocardial damage is known to be involved in acute myocarditis and dilated cardiomyopathy. Recently, we found that tumor necrosis factor (TNF) ligand superfamily costimulatory molecules, especially 4-1BBL, played an important role in the myocardial damage of murine acute viral myocarditis. Methods and results: To investigate the roles for CD27L, CD30L, OX40L and 4-1BBL, which belong to TNF ligand superfamily, in the development of acute myocarditis and dilated cardiomyopathy, we analyzed the expression of these antigens in the myocardial tissues of patients with acute myocarditis and dilated cardiomyopathy. We also examined expression of the receptors for these molecules, CD27, CD30, OX40 and 4-1BB, which belong to TNF receptor superfamily, on the infiltrating cells. Strong expression of CD27L, CD30L and 4-1BBL and weak to moderate expression of OX40L was found in the cardiac myocytes of patients with acute myocarditis. Moderate expression of CD27L, CD30L and 4-1BBL and weak expression of OX40L was found on the cardiac myocytes of patients with dilated cardiomyopathy. Most of the infiltrating cells expressed CD27, CD30 and 4-1BB and a part of the infiltrating cells expressed OX40. Conclusions: Our findings suggest that expression of TNF ligand superfamily costimulatory molecules on cardiac myocytes may play a role in the cell-mediated myocardial damage in patients with acute myocarditis and dilated cardiomyopathy as in murine viral myocarditis.

The mechanism of hypergammaglobulinemia in patients infected with HIV has remained unclear in spite of the identification of a reduction of CD4+ T cells. The amounts of CD27+ memory B cells were remarkably reduced in the peripheral blood and immunoglobulin (Ig) production was diminished in HIV-infected patients. Some of the freshly isolated patients' T cells expressed the CD70 (CD27 ligand) on the surface and the CD70 expression on both of the CD4+ and CD8+ T cells was greatly enhanced by various stimuli. It was also striking that plasmacytosis was observed in patients' bone marrow. Thus, our findings suggest that CD70 expressed spontaneously or by activation on T cells of HIV-infected patients stimulates memory B cells via CD27 and promotes their differentiation into plasma cells, resulting in the elevation of serum Ig levels and the elimination of circulating memory B cells in HIV-infected patients.

CD70/CD27 are cell surface molecules belonging to the TNF/TNF-receptor families. Ligation of CD27 by its ligand CD70 is thought to be important in T cell activation and T cell–B cell interaction. However, the in vivo function of these molecules during the establishment of cell-mediated immunity remains unclear. In this study, we examined the contribution of CD70–CD27 interactions to cell-mediated immunity by investigating the effect of anti-CD70 mAb on the development of experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with anti-CD70 mAb prevented EAE induced by immunization with PLP 139–151 . The preventive effect of anti-CD70 mAb was not due to the inhibition of T cell priming and antibody production from B cells, or immune deviation. However, TNF-α production was suppressed by treatment with anti-CD70 mAb, indicating that the ameliorating effect of anti-CD70 mAb appeared, at least in part, to be mediated by the inhibition of TNF-α production. These results indicate that the CD70–CD27 interaction plays a pivotal role in the development of cell-mediated autoimmune disease.