Bone-derived MSCs encapsulated in alginate hydrogel prevent collagen-induced arthritis in mice through the activation of adenosine A2A/2B receptors in tolerogenic dendritic cells

Tolerogenic dendritic cells (tolDCs) facilitate the suppression of autoimmune responses by differentiating regulatory T cells (Treg). The dysfunction of immunotolerance results in the development of autoimmune diseases, such as rheumatoid arthritis (RA). As multipotent progenitor cells, mesenchymal stem cells (MSCs), can regulate dendritic cells (DCs) to restore their immunosuppressive function and prevent disease development. However, the underlying mechanisms of MSCs in regulating DCs still need to be better defined. Simultaneously, the delivery system for MSCs also influences their function. Herein, MSCs are encapsulated in alginate hydrogel to improve cell survival and retention in situ, maximizing efficacy in vivo. The three-dimensional co-culture of encapsulated MSCs with DCs demonstrates that MSCs can inhibit the maturation of DCs and the secretion of pro-inflammatory cytokines. In the collagen-induced arthritis (CIA) mice model, alginate hydrogel encapsulated MSCs induce a significantly higher expression of CD39+CD73+ on MSCs. These enzymes hydrolyze ATP to adenosine and activate A2A/2B receptors on immature DCs, further promoting the phenotypic transformation of DCs to tolDCs and regulating naïve T cells to Tregs. Therefore, encapsulated MSCs obviously alleviate the inflammatory response and prevent CIA progression. This finding clarifies the mechanism of MSCs-DCs crosstalk in eliciting the immunosuppression effect and provides insights into hydrogel-promoted stem cell therapy for autoimmune diseases.

Preparation of CD73/PD-L1 bispecific antibody and evaluation of its antitumor function in vitro / 中国生物制品学杂志

@#Objective To prepare bispecific antibody targeting cluster of differentiation 73(CD73) and programmed cell death-ligand 1(PD-L1),and evaluate its binding ability and killing ability in vitro.Methods Using genetic engineering method,PD-L1 single-chain fragment variable(scFv) was inserted into the hinge region of CD73 monoclonal antibody to construct anti-CD73/PD-L1 bispecific antibody(BS-21),which was screened by CHO GS expression system to obtain highly expressed cell line.After purified by Protein A and molecular sieve,the purity of antibody was detected by size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),the binding ability of antibody in vitro was detected by flow cytometry,and the killing ability in vitro was detected by using peripheral blood mononuclear cell(PBMC) to kill Calu 1 lung cancer cells in vitro.Results High-yield cell lines were obtained by pressure screening.A bispecific antibody BS-21 with a purity of 99.6% was obtained by purification,which bound to CD73 and PD-L1 molecules simultaneously.Compared with anti CD73 and anti PD-L1 groups,BS-21 group significantly increased the killing rate of immune cells to Calu 1 tumor cells(F=30.36,each P<0.001).Conclusion Bispecific antibody BS-21 reduced the immunosuppressive effect of CD73 and PD-Ll on immune cells simultaneously,and showed good anti-tumor function.

IL-6 regulates the migration, adhesion and proliferation of human placenta-derived mesenchymal stem cells by promoting CD73 expression / 中华微生物学和免疫学杂志

Objective:To investigate the mechanism of IL-6 affecting the expression of CD73 on human placenta-derived mesenchymal stem cells (hPMSCs) and regulating their migration, adhesion and proliferation.Methods:Flow cytometry (FCM) and Western blot were used to analyze the effects of exogenous IL-6 or IL-6 secreted by hPMSCs on the expression of CD73 on hPMSCs. The influence of IL-6 on the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) in hPMSCs was detected by monoclonal antibody blocking test and Western blot. Real-time cellular analysis (RTCA) was used to analyze the changes in the migration, adhesion and proliferation of hPMSCs after knockdown of CD73 expression or APCP pretreatment.Results:FCM and Western blot showed that both exogenous and autocrine IL-6 from hPMSCs promoted the expression of CD73 on hPMSCs ( P<0.001, P<0.01). Moreover, CD73 expression decreased significantly with the presence of IL-6R inhibitor ( P<0.01). IL-6 could up-regulate the levels of both p-STAT3 and CD73 in hPMSCs ( P<0.05, P<0.01), while CD73 expression decreased after adding STAT3 inhibitor ( P<0.01). RTCA showed that knockdown of CD73 expression on hPMSCs significantly inhibited the adhesion and proliferation ability of hPMSCs( P<0.01, P<0.05), but promoted the migration ability of hPMSCs ( P<0.05). Similarly, inhibiting the hydrolase activity of CD73 on hPMSCs by APCP also resulted in a significant decrease in the adhesion and proliferation capacities of hPMSCs, and an increase in the migration capacity of hPMSCs ( P<0.05). Conclusions:IL-6 enhanced the expression of CD73 on hPMSCs via the JAK/STAT3 pathway, thus affecting the migration, adhesion and proliferation of hPMSCs.

Protective effect of CD73 inhibitor α, ß-methylene ADP against amyloid-ß-induced cognitive impairment by inhibiting adenosine production in hippocampus

BACKGROUND: Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disease. Recent studies have reported the close association between cognitive function in AD and purinergic receptors in the central nervous system. In the current study, we investigated the effect of CD73 inhibitor α, ß-methylene ADP (APCP) on cognitive impairment of AD in mice, and to explore the potential underlying mechanisms. RESULTS: We found that acute administration of Aß1­42 (i.c.v.) resulted in a significant increase in adenosine release by using microdialysis study. Chronic administration of APCP (10, 30 mg/kg) for 20 d obviously mitigated the spatial working memory impairment of Aß1­42-treated mice in both Morris water maze (MWM) test and Y-maze test. In addition, the extracellular adenosine production in the hippocampus was inhibited by APCP in Aß-treated mice. Further analyses indicated expression of acetyltransferase (ChAT) in hippocampus of mice of was significantly reduced, while acetylcholinesterase (AChE) expression increased, which compared to model group. We observed that APCP did not significantly alter the NLRP3 inflammasome activity in hippocampus, indicating that anti-central inflammation seems not to be involved in APCP effect. CONCLUSIONS: In conclusion, we report for the first time that inhibition of CD73 by APCP was able to protect against memory loss induced by Aß1­42 in mice, which may be due to the decrease of CD73-driven adenosine production in hippocampus. Enhancement of central cholinergic function of the central nervous system may also be involved in the effects of APCP.

Adenosine Blockage in Tumor Microenvironment and Improvement of Cancer Immunotherapy

Immunotherapy has been introduced into cancer treatment methods, but different problems have restricted the efficacy of these protocols in clinical trials such as the presence of various immunomodulatory factors in the tumor microenvironment. Adenosine is an immunosuppressive metabolite produced by the tumor to promote growth, invasion, metastasis, and immune evasion. Many studies about adenosine and its metabolism in cancer have heightened interest in pursuing this treatment approach. It seems that targeting the adenosine pathway in combination with immunotherapy may lead to efficient antitumor response. In this review, we provide information on the roles of both adenosine and CD73 in the immune system and tumor development. We also describe recent studies about combination therapy with both purinergic inhibitors and other immunotherapeutic methods.

Correlation between CD39+ and CD73+ regulatory T cells and liver injury of primary biliary cholangitis / 临床检验杂志

Objective To evaluate the changes of adenosine metabolism pathway related molecules and their contribution to inflammatory injury in primary biliary cholangitis (PBC).Methods The consecutive samples of 49 subjects with PBC from The First People's Hospital of Taicang and The Second People's Hospital of Changshu were recruited from October 2016 to October 2017,and 36 healthy controls were involved in this study.The expression of CD39 and CD73 on CD4+T cells and Foxp3 + regulatory T cells were assayed by flow cytometry and the concentration of adenosine triphosphate (ATP) in serum was analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS).The correlations between Tregs,ATP and liver function were analyzed,i.e.,alanine aminotransferase (ALT),aspartate aminotransferase (AST),gamma-glutamyltransferase (GGT),alkaline phosphatase (ALP) and Mayo scores.Results In the patients with PBC,low proportions of CD4+CD39+T cells were noted compared with healthy controls [(5.28 ± 1.92) ％ vs (11.0l ± 3.19) ％,t =10.25,P ＜ 0.01].The patients with PBC also had significantly low proportion of CD4+CD25 + Foxp3+ CD39+ T cells compared with healthy controls [(23.75 ± 9.48) ％ vs (54.68 ± 5.18) ％,t =13.79,P ＜0.01].No significant difference of the proportion of CD4+CD73+T or CD4+CD25+ Foxp3+CD39+T cells was found between PBC and control groups (t values were 2.235 and 1.083,P ＞ 0.05).The level of serum ATP was higher in the patients with PBC than that of healthy controls [(200.28 ± 79.41) μg/L vs (89.20 ± 33.76) μg/L,t =8.367,P ＜ 0.01].A significant correlation was demonstrated between the proportion of CD39 + Treg in total Treg cells and the levels of ATP (r =-0.413,P =0.003),GGT (r=-0.378,P=0.007) and Mayo score (r=-0.382,P=0.007).Conclusion The low proportion of CD39+ Treg cells may contribute to the down-regulation of ATP hydrolysis in the patients with PBC.No significant change of CD73 + Treg cells was found in PBC patients.

Clinical significance of CD39 CD73 subsets and both of them positive subgroups of regulatory T cells in the microenvironment of colorectal cancer / 实用肿瘤学杂志

Objective The objectives of this study were to investigate the clinical significance of CD39,CD73,double positive subgroups for CD39 and CD73,and other lymphocytes with clinicopathological parameters in the microenvironment of colorectal cancer.Methods Tumor infiltrating lymphocyte(TIL)was collected from 24 patients with colorectal cancer after radical resection.The expression of CD39+,CD73+ or CD39+ with CD73+ in T cells were measured by flow cytometry.The association between these subgroups and clinicopathologic parameters was analyzed.Results The CD73+ and CD39+ with CD73+ subgroups were associated with lymph node metastasis and poor degree of differentiation,and this mechanism was closely related to tumor-associated inflammation.Conclusion CD39+ with CD73+ colorectal tumor infiltration Treg has a more unique biological activity than other Treg group.This study provides a new idea and theoretical basis for predicting the prognosis of colorectal cancer.

Overexpression of CD73 in epithelial ovarian carcinoma is associated with better prognosis, lower stage, better differentiation and lower regulatory T cell infiltration / 부인종양

OBJECTIVE: The purpose of the current study was to evaluate survival outcome according to the expression status of CD73 in patients with epithelial ovarian cancer. METHODS: A total of 167 patients with epithelial ovarian cancer were enrolled in the current study. For each patient, a retrospective review of medical records was conducted. Immunohistochemical staining for CD73, CD8, FoxP3, and CD68 was performed using tissue microarray made with paraffin embedded tissue block. RESULTS: Among the enrolled patients, 29.9% of patients (n=50) showed negative expression for CD73, whereas 70.1% of patients (n=117) showed positive expression for CD73. The CD73 positive group showed better prognosis compared to the CD73 negative group (5-year overall survival of CD73 positive group, 73.0%; that of CD73 negative group, 50.1%; p=0.023). CD73 was more frequently expressed in mucinous adenocarcinoma and clear cell carcinoma compared to serous or endometrioid adenocarcinoma. In addition, CD73 overexpressions were more frequently detected in patients with known good prognostic factors, i.e., low stage, well/moderate differentiation, negative peritoneal cytology, no lymphovascular involvement, and no macroscopic residual tumor after debulking surgery. There was significantly more infiltration of regulatory T cells in the CD73 negative group compared to the CD73 positive group. CONCLUSION: Good prognosis in patients with overexpression of CD73 may be due to that overexpression of CD73 was more frequently observed in epithelial ovarian cancer patients with known good prognostic factors. Therefore, this result means that favorable differentiation and stage have more influence on survival outcome than adverse effect of CD73 per se.

Overexpression of CD73 in epithelial ovarian carcinoma is associated with better prognosis, lower stage, better differentiation and lower regulatory T cell infiltration / 부인종양

OBJECTIVE: The purpose of the current study was to evaluate survival outcome according to the expression status of CD73 in patients with epithelial ovarian cancer. METHODS: A total of 167 patients with epithelial ovarian cancer were enrolled in the current study. For each patient, a retrospective review of medical records was conducted. Immunohistochemical staining for CD73, CD8, FoxP3, and CD68 was performed using tissue microarray made with paraffin embedded tissue block. RESULTS: Among the enrolled patients, 29.9% of patients (n=50) showed negative expression for CD73, whereas 70.1% of patients (n=117) showed positive expression for CD73. The CD73 positive group showed better prognosis compared to the CD73 negative group (5-year overall survival of CD73 positive group, 73.0%; that of CD73 negative group, 50.1%; p=0.023). CD73 was more frequently expressed in mucinous adenocarcinoma and clear cell carcinoma compared to serous or endometrioid adenocarcinoma. In addition, CD73 overexpressions were more frequently detected in patients with known good prognostic factors, i.e., low stage, well/moderate differentiation, negative peritoneal cytology, no lymphovascular involvement, and no macroscopic residual tumor after debulking surgery. There was significantly more infiltration of regulatory T cells in the CD73 negative group compared to the CD73 positive group. CONCLUSION: Good prognosis in patients with overexpression of CD73 may be due to that overexpression of CD73 was more frequently observed in epithelial ovarian cancer patients with known good prognostic factors. Therefore, this result means that favorable differentiation and stage have more influence on survival outcome than adverse effect of CD73 per se.

CD73 may impact the invasion and replication of Toxoplasma gondii in the host cells / 临床儿科杂志

Objective To explore the role of CD73 in Toxoplasma gondii (T. Gondii) infection; To verify the host cells absent with CD73 would have less infection and replication of T. Gondii. Methods CD73 knockout and wild-type mice were infected with 20 cysts of ME 49 by oral gavage. The survival rate, clinic symptoms, pathology in the gut, and cytokine production were investigated in infected mice. Macrophages and dendritic cells with CD73-/- and CD73+/+ were infected with RH-YFP. The infective rates, the number of intracellular RH-YFP in the host cells, and the replication of intracellular RH-YFP were detected in these two type cells. Results CD73 knockout mice had a higher survival rate and milder clinic presentation with less morphologies in the gut in the acute T. Gondii infection compared with wild-type mice. CD73-/- macrophages and dentritic cells had less T. Gondii infection, and less intracellular parasite number and replication compared with CD73+/+ macrophages and dentritic cells in vitro. Conclusions CD73 as a GPI-anchored surface protein of host cell might be involved in forming the parasite vacuole, and promote the parasite's attachment and invasion of host eeUs. Host cells absence with CD73 would be less infection and replication of T. Gondii, and relative resistant parasites infection.