| Object:Regulatory T cells (Tregs) have been described as the most potent immunosuppressive cells and play a critical role in maintaining peripheral immunologic tolerance and controlling alloimmune disorders, infections, allergy, and transplantation tolerance. Among the Tregs, the characteristics of CD4+CD25+ regulatory T cells are well illuminated. CD4+CD25+ Tregs have two major subtypes, naturally occurring Tregs (nTregs) and induced Tregs (iTregs). The nTregs arise from the thymus and represent 5-10% of CD4+ T cells in mice and in human blood. The iTregs can differentiate from the CD4+CD25-Foxp3- precursors in the periphery outside the thymus under some conditions, such as TCR stimulation plus IL-10, or TGF-β.The iTregs had the same suppressive function as nTregs, both expressed CTLA-4, GITR and Foxp3. The transcript factor Foxp3, as the specific marker of Tregs, plays a critical role in the differentiation, development and function of Tregs. The utilization of Tregs to prevent or cure diseases by controlling inappropriate immune response has been a new strategy for immunotherapy. The adoptive transfer of Tregs was found to control experimental alloimmune diseases, and prevent allograft rejection in rodents. But the limited number of Tregs was the obstacle for their clinical application. So it's necessary to explore new methods to expand or induce CD4+CD25+ Tregs to facilitate the clinical application.Dendritic cells (DCs) are professional antigen presenting cells (APCs). The accumulating data showed that, DCs play the important role not only in the activation and proliferation of effector T cells, but also in both expansion of nTregs and induction of iTregs. On one hand, the antigen-loaded DCs together with IL-2 are able to expand nTregs. On the other hand, DCs with TGF-P, IL-10 or Vitamin D can induce naive CD4 CD25-T into Foxp3+Tregs. However, in previous protocols for the expansion or induction of Tregs, CD4+CD25-T or CD4+CD25+T cells needed to be purified from total CD4+T cell population and exogenous cytokines were required, which were costly and relatively complicated. Since total CD4+T cells contain both nTregs and CD4+CD25- precursors of iTregs, so whether is it feasible to generate Tregs directly from total CD4+ population stimulated by DCs without separation from each other and exogenous cytokines? Therefore, the first purpose of this study is to explore whether it is feasible or not to generate Tregs directly from total CD4+ population stimulated by DCs, which type of DCs (autogeneic or allogeneic, immature or mature DCs) work well, what is the mechanism? It is important to set up simpler and less-costly methods of generating Tregs to facilitate their clinical application.When DCs happen to differentiate, mature, and execute their responsibility, they are often in different microenvironment, in which oxygen tension is the important factor. While, under most physiological or pathological conditions, tissues and cells are always in different oxygenic microenvironment. For example, in the process of arterial embolism or organ transplantation, hypoxia was always found during the early phage. But after removing of the external factor or operation, the following return of blood flow and oxygen delivery in the hypoxic tissues lead to rapid change of oxygen tension. This reoxygenation (re-exposure to saturated oxygen) is a very important patho-physiological process, and it often exists ischemia-reperfusion injury (IRI). However, it is unclear whether the oxygenic microenvironment affects the differentiation and function of DCs, whether the DCs differentiated under different oxygenic microenvironment affect the T cell differentiation including the generation of Tregs. In the previous study, we found that hypoxia can inhibit the phenotypic maturation and function of DCs to stimulate T cell proliferation. Therefore, the second purpose of this study is to further explore the effect of hypoxia-reoxygenation (from hypoxia to normoxia) on the phenotype and function of mouse DCs and to analyze the effect of reoxygenated DCs on the T cell differentiation including the generation of Tregs on the basis of previous study. Also the internal molecular mechanism is researched. It is much helpful for explaining the pathogenesis of IRI, graft rejection in organ transplantation, and the immunotherapy for tumor.In a word, this project contains two parts, the first part is "The effect of allogeneic DCs on the generation of regulatory T cells and its mechanism"; the second part is "The effect of reoxygenated DCs on the generation of regulatory T cells and its mechanism".PART I THE EFFECT OF ALLOGENEIC DCS ON THE GENERATION OF REGULATORY T CELLS AND ITS MECHANISMMethods:1. To induce murine bone marrow derived DCs1) To separate murine bone marrow cells from C57BL/6 or BALB/c mice and stimulate with GM-CSF and IL-4.2) To stimulate BM-DCs maturation by 1μg/ml LPS or not at day 6 for 24h.2. To detect the ability of murine DCs to generate CD4+CD25+Tregs in different mixed lymphocyte reaction1) To purify CD4+T cells from the splenocytes of BALB/c mice, and cocultured with autogeneic (auto-DCs) or allogeneic DCs (allo-DCs).2) To detect the proportion of CD4+CD25+Tregs in the different cultural systems at the indicated time by Flow Cytometry (FCM).3. To detect the ability of human DCs to generate CD4+CD25+Tregs in allogeneic MLR1) To separate peripheral blood monouclear cells (PBMC) from different two human blood.2) To separate CD4+T cells by human CD4 immunomagnetic beads.3) To induce DCs by stimulating the adherent cells by recombinant human GM-CSF and IL-4.4) To coculture the induced human DCs with autogeneic or allogeneic human CD4+T cells for days.5) To detect the proportion of CD4+CD25+Foxp3+Treg in the cultures by FCM.4. To detect the suppressive function of the obtained CD4+CD25+Treg in allogeneic DCs stimulated system1) To separate the generated CD4+CD25+T cells from the above allogeneic MLR for using as suppressors. Freshly separated CD4+CD25-T cells were used as responders after labeling CFSE. Allogeneic DCs were stimulators.2) To detect the cell proliferation by FCM, freshly isolated CD4+CD25+T cells were used as positive suppressive control.5. To explore the mechanism of CD4+CD25+Tregs increasing5.1 To explore the effect of IL-2 on the generation of CD4+CD25+Tregs by allogeneic DCs1) To purify the CD4+CD25+T cells and label CFSE, to detect the effect of allo-DCs to stimulate the proliferation of Tregs in the presence of exogenous IL-2.2) To purify the CD4+CD25+T cells and label CFSE, remix with the CD4+CD25-T cells at the intrinsic ratio as the whole CD4+T cells, and to detect the effect of allo-DCs on the IL-2 production of the whole CD4+T cells.3) IL-2 blocking assay:to detect the inhibitive effect of anti-IL-2 neutralizing antibody on the proliferation of CFSE-labeled CD4+CD25+T cells abovementioned.5.2 To explore the effect of TGF-βon the generation of CD4+CD25+Tregs by allogeneic DCs1) To purify the CD4+CD25-T cells and label CFSE, to detect the ability of allo-DCs to induce Tregs without of exogenous cytokines.2) To detect the expression of cytokine TGF-P in the supernatant of above MLR using ELISA.3) TGF-βblocking assay: to detect the effect of different concentration of TGF-βRI Kinase InhibitorⅡ(ALK-5 inhibitorⅡ) on the expression of Foxp3. Results1. The murine allogeneic DCs effectively generated CD4+CD25+Foxp3+Tregs from total CD4+T cells population without exogenous cytokines in vitro, but the autogeneic DCs did notIn freshly purified total CD4+T cells (purity>95%), the percentage of CD4+CD25+Foxp3+Tregs was less than 10%. In cocultured CD4+T cells with auto-DCs, CD4+CD25+T cells were increased significantly (more than 90%), but only a small population (less than 10%) were Foxp3+Tregs among them. However, in cocultured CD4+T cells with allo-DCs, the percentage of Foxp3+Tregs in these CD4+CD25+T cells was more than 80%, which was closed to the freshly purified CD4+ T cells. As the cultural time extended, the proportion of CD4+CD25+Foxp3+T cells in total CD4+T cells was increased by 5-7 folds. Notably, the maturation of DCs did not significantly affect the generation of CD4+CD25+Foxp3+T cells, as both immature and mature allo-DCs were capable of increasing the percentage of CD4+CD25+Foxp3+ T cells.2. The human allogeneic DCs could generate CD4+CD25+Foxp3+ Tregs from total CD4+T cells population without exogenous cytokines in vitro as well as murine allogeneic DCsTo further demonstrate the ability of allo-DCs to generate Tregs in vitro, we repeated the abovementioned experiments in human systems. The results showed that, in freshly purified CD4+T cells (purity>95%), the percentage of CD4+CD25+ Foxp3+ T cells was about 5-6%. In auto-DCs stimulated system, although the CD4+CD25+T cells increased significantly, but only a small population (about 10%) were Foxp3+Tregs among them. However, in allo-DCs stimulated system, the percentage of Foxp3+ Tregs in these CD4+CD25+T cells was about 40%, which was much higher than that in the autogeneic DCs stimulated system. These data suggested that, human allo-DCs, as well as mouse DCs, can effectively generate CD4+CD25+Foxp3+Tregs from total CD4+T cells population without exogenous cytokines in vitro.3. Allogeneic DCs-generated Tregs possess the suppressive function in vitro To examine whether the allo-DCs generated Tregs possess suppressive function in vitro, we purified CD4+CD25+T cells (suppressor) from the abovementioned allo-DCs stimulated system, and added these cells to CFSE labeled CD4+ CD25-T cells (responder) at gradually increasing ratio of suppressor:responder, and stimulated with allo-DCs. The FCM result showed that the proliferation of CSFE-labeled CD4+CD25-T cells was effectively suppressed by the allo-DCs generated Tregs as well as the freshly purified nTregs.4. The endogenous IL-2 and TGF-βplay an important role in the generation of Tregs in allo-DCs stimulated systemsIn order to investigate the mechanism of generation of increased Tregs by allo-DCs, we further detect the role of endogenous IL-2 and TGF-P in the generation of Tregs in allo-DCs stimulated systems.4.1 IL-2 plays an important role in the expansion of CD4+CD25+Tregs in the allogeneic DCs stimulated system1) Tregs could proliferate when stimulated by allo-DCs in presence of exogenous IL-2CD4+CD25+ nTregs were purified and labeled with CFSE, and then co-cultured with allo-DCs plus IL-2 for indicated days. The FCM results showed that allo-DCs effectively expanded CD4+CD25+T cells in presence of exogenous IL-2.2) The endogenous IL-2 plays an important role in the generation of Tregs in allo-DCs stimulated systemCSFE labeled CD4+CD25+T cells were remixed with purified CD4+CD25-T cells according to their ratio in total CD4+ population and then coculured them with allo-DCs without cytokines. The ELISA result showed that the level of IL-2 in supernatant of coculture of CD4+T cells with allo-DCs was significant increased after 5 days of allostimulation compared with that in control group. To further clarify the role of IL-2 in proliferation of Tregs in this system, we administrated anti-IL-2 monoclonal antibody to block the role of IL-2 in MLR. The result showed that, blockage of IL-2 was able to markedly inhibit the proliferation of nTregs in CD4+ population. These results indicate that significant amounts of IL-2 in coculture plays an important role in the expansion of CD4+CD25+Tregs in CD4+ population by allo-DCs.4.2. TGF-βplays an important role in the induction of CD4+CD25+Tregs in the allogeneic DCs stimulated system1) Tregs could be induced by allo-DCsIn order to clarify whether the conversion of CD4+CD25+Tregs from CD4+CD25T cells was involved in allo-DCs generated Tregs in our system, the CFSE labeled CD4+CD25-T cells were cocultured with allo-DCs for different times and a dynamic analysis of Foxp3 expression was performed by FCM. The results showed that Foxp3+T cells increased significantly after 5 days coculture compared with freshly purified CD4+CD25-T cells. After 13 days of allostimulation, the expression of Foxp3 increased nearly two folds compared with that at day 5 of allostimulation. This indicates that CD4+CD25+Foxp3+ T cells can be induced by allo-imDCs without exogenous cytokines in vitro.2) Endogenous TGF-βexisted in allo-DCs stimulated systemThen the level of TGF-βin the supernatant of coculture was detected by ELISA. The results indicated that the total TGF-βsignificantly increased at day 5 and then elevated gradually along with the time of allostimulation. At day 13, the concentration of TGF-βwas about two times more than that at day 5 culture.3) The endogenous TGF-βplays an important role in the generation of Tregs in allo-DCs stimulated systemTo further confirm the role of endogenous TGF-βin the differentiation of Tregs by allo-DCs, we used the TGF-βRI Kinase InhibitorⅡ(ALK-5 inhibitorⅡ) to block the TGF-βsignal and then measured the change of percentage and total number of Foxp3+Tregs. We found that the percentage and absolute number of CD4+Foxp3high cells were gradually decreased when the concentration of ALK-5 inhibitorⅡincreased. Additionally, in this culture, CD4+Foxp3dim cells markedly decreased after administration of TGF-βRI Kinase InhibitorⅡin a dose-dependent manner. These results suggested that CD4+Foxp3dim cells may be the main population derived from the conversion of Foxp3+ Tregs, and TGF-β, to some extent, contributes to the conversion of CD4+CD25+T cells.Conclusions 1. The murine allogeneic DCs effectively generated CD4+CD25+Foxp3+Tregs from total CD4+T cells population without exogenous cytokines, and the generated Tregs also possessed the suppressive function in vitro.2. The human allogeneic DCs from PBMC could also generate CD4+CD25+Foxp3+ Tregs from total CD4+T cells population effectively.3. The mechanism of the increased CD4+CD25+Foxp3+Tregs:The endogenous IL-2 and TGF-β. play the important role in the generation of CD4+CD25+Foxp3+Tregs stimulated by allogeneic DCs.Originality1. It's the first time to provide evidence that the murine allogeneic but not autogeneic DCs effectively generated CD4+CD25+Foxp3+Tregs from total CD4+T cells population without exogenous cytokines in vitro and to explore the mechanism.2. It's the first time to look the CD4+T cells as a whole population to investigate the expansion and induction of Tregs. It is important to provide theoretical foundation for optimizing continuous empirical methods and application of Treg in the clinical therapy.Limitation of this study1. The detailed molecular mechanism for the allogeneic DCs to increase the proportion of Tregs in CD4+T population need to be further explored.2. The empirical methods need to be further optimized before the application of generated Treg in the clinical therapy. Methods1. Effect of reoxygenation on the phenotype of DCs1) To induce murine BM-DCs using GM-CSF and IL-4 under normoxic or hypoxic conditions, and stimulate maturation by LPS.2) To transform DCs differentiated under hypoxic condition to normoxia for various periods of continuous culture.3) To detect the phenotype (CD80, CD86, and MHC classⅡmolecules) of reoxygenated DCs by FCM.2. Effect of reoxygenation on the function of DCs to drive Th cells to proliferate and differentiate1) To separate CD4+T cells and label with CFSE and then coculture them with reoxygenated allogeneic or autogeneic DCs.2) To assay the CD4+T cells proliferation by FCM.3) To assay the CD4+T cells differentiation stimulated by reoxygenated DCs via FCM. 3. To explore the mechanism of the effect of reoxygenation on the phenotype and function of DCs1) To detect the expression of four adenosine receptors on reoxygenated DCs by real time PCR.2) To detect the effect of four adenosine receptor agonists on the phenotype of reoxygenated DCs by FCM.3) To detect the effect of different concentration of A2AR agonist on the phenotype of reoxygenated DCs by FCM.4) To detect the effect of A2AR agonist on the cytokines secreted by reoxygenated DCs by ELISA.Results 1. Reoxygenation reversed the effect of hypoxia on the phenotypic maturation of DCsTo determine whether oxygen delivery could influence the phenotypic maturation of hypoxic DCs, reoxygenation was performed on hypoxic DCs for various periods of time, related surface markers were examined by FCM. The results showed that reoxygenation obviously upregulated the expression of CD80, CD86 and MHC classⅡmolecules on DCs which were inhibited by hypoxia. And the percentage and mean fluorecence intensity (MFI) was gradually increased with the time of reoxygenation, and peaked after 24-48h of reoxygenation. This indicated that the reoxygenated DCs displayed a full mature phenotype.2. Reoxygenation enhanced the ability of hypoxic DCs to stimulate CD4+T cell proliferationSince reoxygenation promoted the phenotypic maturation of hypoxic DCs, we further analyzed the effects of reoxygenated DCs on the proliferation of CD4+T cells. The results showed that hypoxic DCs could not effectively drive T cells proliferation, but DCs reoxygenated for 6 h stimulated CD4+T cells to proliferate vigorously, not only in allogeneic DCs but also in anti-CD3 stimulated system. These data suggested that reoxygenation markedly enhances the ability of hypoxic DCs to stimulate CD4+T cells proliferation.3. Reoxygenated DCs decreased Foxp3+Treg cells, and induced Th17, Thl cell immune responsesIn order to investigate the CD4+T cells differentiation driven by reoxygenated DCs in the allogeneic MLR, the intracellular staining was performed. The FCM results showed that, the proportion of CD4+CD25+Foxp3+Tregs in the reoxygenated DCs stimulated system was much lower than that in the normoxic mature and immature DCs stimulated systems. However, reoxygenated DCs induced significantly higher number of IL-17+ CD4+T (Th17) cells than DCs differentiated under hypoxic or normoxic conditions did. And also the proportion of IFN-y+Thl cells in the allogeneic MLR and the level of IFN-y in the supernatant were also higher. In addition, we analyzed the cytokines profile of reoxygenated DCs and found that, hypoxic DCs exposed to oxygen for 24h produced significantly higher levels of IL-6 and slightly lower levels of TGF-β, .compared with that in hypoxia-differentiated DCs, suggesting the elevated IL-6 secretion by reoxygenated DCs along with significant amounts of TGF-β. abrogates Treg generation but drives CD4+T cells toward Th17 cells. Taken together, these results suggested that reoxygenated DCs had strong capacity to drive immune response toward a proinflammatory direction.4. The adenosine-A2AR pathway plays an important role in the process of hypoxia-reoxygenation of DCs4.1 Reoxygenation upregulated the mRNA level of A2AR on the DCs, but had no effect on the expression of A1R, A2BR and A3RTo investigate that did the adenosine receptor pathway play the important role in the process of reoxygenation, the expression of A1R, A2AR, A2BR and A3R on the mRNA level were detected by real time PCR. The results demonstrated that the A1, A2B and A3 receptors expression levels remained unaltered in response to reoxygenation compared with their levels in response to normoxia and hypoxia. In contrast, the expression of A2AR was predominantly increased in reoxygenated DCs.4.2 The effect of adenosine receptor agonists on the phenotype of reoxygenated DCsTo further clarify the effect of adenosine receptor pathway on the phenotype of reoxygenated DCs, different agonists for A1R, A2AR, A2BR and A3R were administrated when reoxygenation was performed on the hypoxic DCs. Twenty four hours later, the phenotype was detected by FCM. The results showed that, A2AR specific agonist CGS21680 could downregulate the expression of MHC-II, CD80 and CD86 of reoxygenated DCs, while the other agonists couldn't. It's that CGS21680 could restrain the upregulated function of reoxygenation on the expression of MHC-II, CD80 and CD86 which were inhibited by hypoxia.4.3 The A2AR specific agonist affects the phenotype of reoxygenated DCs in a concentration-dependent mannerFurthermore, we also studied the effect of different concentration of A2AR specific agonist CGS21680 on the phenotype of reoxygenated DCs, and the data demonstrated that MFI of MHC-II, CD80 and CD86 on the reoxygenated DCs were gradually decreased with the increased concentration of CGS21680, which was coincident with that in normoxic DCs. This indicated that A2AR signaling pathway did play an important role in the reoxygenated DCs.4.4 The A2AR agonist can obviously inhibit the proinflammatory cytokines secreted by reoxygenated DCs, but promote the anti-inflammatory cytokines' secretionWe further detected the effect of A2AR agonist on the cytokines secreted by reoxygenated DCs by ELISA. The results showed that A2AR specific agonist CGS21680 could inhibit the proinflammatory cytokines IL-1β, IL-6 and TNF-α, which were enhanced by reoxygenation. Well, the level of TGF-βwas significantly increased after the CGS21680 treatment. All these data suggested that A2AR signaling pathway may be a mediator for reoxygenation up-regulating the phenotype and function of hypoxic DCs.Conclusions1. Reoxygenation reversed the inhibition of maturation and function of DCs by hypoxia1) Reoxygenation upregulated the mature phenotype of DCs.2) Reoxygenation enhanced the ability of DCs to stimulate CD4+T cell proliferation, and induced Th1 and Th17 cell immune responses with decreased Foxp3+Treg cells.2. Adenosine receptor pathway may be a mediator for reoxygenation to upregulate the phenotype and function of DCs1) Reoxygenated DCs predominantly expressed higher level of A2AR, compared to that in normoxic and hypoxic DCs.2) A2AR specific agonist CGS21680 downregulated the phenotype of reoxygenated DCs in a concentration dependent manner.3) A2AR specific agonist CGS21680 inhibited the secretion of proinflammatory cytokines of reoxygenated DCs.Originality1. It's the first time to provide evidence that reoxygenation can reverse the maturation and function of hypoxic DCs. It is helpful for explaining the pathogenesis mechanism of IRI, and it also demonstrated that hypoxia-reoxygenation maybe one of internal mechanisms of IRI.2. It is the first time to provide evidence that, A2AR signaling pathway can inhibit the ability of reoxygenation to reverse the maturation and function of DCs. This is important for exploring new strategies to prevent or treat IRI at the immunologic point of view.Limitation of this study1. A2AR signaling pathway inhibits the ability of reoxygenation to reverse the maturation and function DCs, but the internal detailed molecular mechanism need to be further studied.2. Experiments of adenosine receptor agonist to treat IRI in animal model need to be further completed.
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