Mechanistic Insight Into T-cell Response By Mesenchymal Stem Cells

Background and objectives Ischemic heart diseases present a dramatic threat to the health of humans.More than one billion cardiomyocytes underwent dying after myocardial infarction.Necrotic heart tissues are always replaced by scar and lack of normal systolic and dilatory function,contributing to the bad life quality of patients.In recent years,stem-cell therapy is a promising candidate for healing myocardial infarction.Mesenchymal stem cells(MSCs)themselves are capable of differentiating towards cardiac myocytes and replace necrotic cardiomyocytes.Stem-cell therapy is effective on cardioprotection,while most of these transplanted stem cells fail to survive in vivo and few of them is supposed to differentiate into cardiomyocytes,indicating that the effects of stem cells are indirect somehow.MSCs mainly express CD73,CD90 and CD105.CD73 is regarded as ecto-5-nucleodiatse(NT5E),which catalyzes extracellular adenosine monophosphate(AMP)to adenosine(Ado).The ado is able to bind to ado receptors(ARs)that are classified into A1 R,A2AR,A2 BR and A3 R.A great number of current evidence indicates the role of immunomodulation of stem cells,by which cardiomyocytes can be protected.Activated T cells highly express A2 AR and A2 BR,and lots of study provides the evidence that the activation of A2 AR on T cells can direct themselves differentiating towards T regulatory cells(Tregs),which is beneficial to tissue repair and regeneration,whereas the effect of A2 BR on T cells during the process of myocardial infarction and stem-cell therapy remains unclear.The main hypothesis(CD73 on MSC?Ado?A2BRs on T cells ? Immunosuppression ? Cardioprotection)is established to verify if MSC-derived Ado mediates A2 BR on activated T cells in the setting of ischemia reperfusion.We aim to investigate the significance of CD73,Ado and A2 BRs on activated T cells in cardiac regeneration.By shedding light on the mechanism of MSC-induced T-cell response,it fulfills the basics for clinical practice.Materials and methods 1.The role of CD73 on MSC Wistar rats were selected and underwent sham-operation,left anterior artery ligation and ligation with MSCs injection.After 5 days,the rat hearts were isolated and perfused with Krebs-Henseleit Buffer under Langendorff apparatus.Cardiac interstitial transudate was collected in “Reverse Heart Model” and the quantity of adenosine was measured by high-pressure/performance liquid chromatography(HPLC).The adipose-derived mesenchymal stem cells(ADMSCs)were isolated from wild-type and CD73-/-C57BL/6 mice.After expansion of the ADMSCs in vitro,the cells were digested by trypsin to perform flow cytometery(FCM)and quantitative PCR(q PCR)for analysis of adenosine-related molecules(CD39 and CD73).In addition,the rest of cells were kept continually and used for co-culture experiments afterwards.2.The effect of CD73+MSC on CD4+ T cells The lymphocytes were isolated from the lymph nodes and spleens from wild-type C57BL/6 mice.Anti-CD3/CD28 antibody was applied to activate the T cells and magnetic sorting was performed after 3 days of cell culture.Sorted CD4+ T cells were co-cultured with ADMSCs from wild-type and CD73-/-mice for 24 hours.All CD4+ T cells were harvested after the 24 hours of co-cultivation for q PCR to analyze T-cell related genes.Due to the shortage of CD73 on ADMSC,we switched our stem-cell type to human umbilical vein mensenchymal stem cells(h UVMSCs,or MSC below)in the further experiments.Likewise,the CD4+ T cells were sorted as mentioned above from wild-type mice,stained with CFSE(analysis of proliferation)and co-cultured with CD73+ MSC for 24 hours.All CD4+ T cells or their supernatant were harvested after 24 hours of cocultivation for q PCR to analyze T-cell related genes,FCM to analyze anti-inflammatory mediators(CD39 and CD73)and proliferation status,and bioplex assay to detect interferon gamma(IFN-?)level.Naive CD4+ T cells were sorted,activated and co-cultivated with CD73+MSC for 3 days.FOXP3 staining were performed to measure the percentage of Tregs in culture.3.The effect of apoptotic CD73+MSC on CD4+ T cells Stem cells underwent apoptosis after the transplantation into the infarcted hearts,and therefore staurosporine-induced apoptosis of MSCs was established.The Annexin V/PI staining was applied to quantify the apoptosis rate of MSCs.The surface CD73 of normaland apoptotic MSCs was detected by flow cytometry and q PCR to conjecture if apoptosis of stem cells generated more extracellular adenosine.The CD4+ T cells were isolated from wild-type C57BL/6 mice,stained with CFSE and co-cultivated with apoptotic CD73+ MSCs for 24 hours.Q-PCR was performed to analyze T-cell related genes after 24 hours of co-cultivation.In addition,anti-inflammatory mediators(CD39 and CD73)and proliferation status were identified by FCM.Likewise,naive CD4+ T cells were activated and co-cultivated with apoptotic CD73+MSC for 3 days and FOXP3 measurement was acquired by FCM.4.A2 B receptor as a downstream mediator of CD73 in T-cell response The CD4+ T cells were isolated from wild-type and A2BR-/-C57BL/6 mice and cocultivated with normal and apoptotic CD73+ MSCs for 24 hours.Q-PCR was performed to analyze T-cell related genes and CD39 on cell surface was measured by FCM.A2 BR antagonist PSB603 was also applied to confirm the data In addition,naive CD4+ T cells were activated,applied with A2 BR inhibitor PSB603 and co-cultivated with normal and apoptotic CD73+MSC for 3 days.The subset of Tregs was identified by FOXP3 staining in FCM.Results 1.The role of CD73 on MSC The injection of CD73+MSCs into the infarcted hearts increased adenosine level of cardiac transudate in the model of Langendorff-perfused hearts.The CD73+ ADMSCs might generate more adenosine by expressing more adenosine-generating enzymes(CD39 and CD73)in comparison with the CD73-/-ADMSCs.2.The effect of CD73+MSC on CD4+ T cells CD73+MSC-induced immunosuppression of T cells was blocked by the knockout of CD73.In addition,more Tregs were identified as the long exposure to MSC.3.The effect of apoptotic CD73+MSC on CD4+ T cells Up-regulation of CD73 on the apoptotic MSC was identified by q RT-PCR and flow cytometry,by which extracellular adenosine level might be elevated.Apoptotic MSC manipulated CD4+ T cells expressing more anti-inflammatory factors and less proinflammatory factors,with a stronger suppression of T-cell response and proliferation.However,less Tregs were identified as the long exposure of CD4+T cells to apoptotic MSC,probably due to the overall inhibition of apoptotic MSC on T cells.4.A2 B receptor as a downstream mediator of CD73 in T-cell response A2 BR,as an inducer of Tregs,was involved in immunosuppression of both normal and apoptotic MSC on T cells.Conclusions Intramyocardial transplantation of CD73+MSC into the infarcted hearts leads to more extracellular adenosine generation,which is identified by techniques of Langendorff retroperfusion and reverse heart model.The extracellular adenosine takes beneficial effects on cardioprotection,by which CD73 on MSCs is essential to keep an anti-inflammatory milieu.Adenosine plays a role in the immunosuppression of MSC on T cells,and long exposure of MSC is able to promote the differentiation of Tregs.Apoptotic MSCs might generate more extracellular adenosine and exert a stronger anti-inflammatory property.However,the inhibitory effect of apoptotic MSC is mediated by the suppression of total T cells.The immunosuppression on T cells by normal and apoptotic MSC is partly mediated by A2 B receptors which are involved in the induction of Tregs.