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Dendritic cells (Dendritic cell, DC) is considered to be the most powerful known professional antigen-presenting cells (antigen presenting cells, APC), capable of sensing, integrating a range of exotic pathogens signal and passes it to lymphocytes, moderate immune response starts to clear the body of invading pathogens. Immature DC in microbial infection or post-transplant identification, phagocytosis exogenous antigens, fast maturity. DC maturation after phagocytosis weakened, but the cell costimulatory molecules and upregulation of adhesion molecules and secretion of proinflammatory cytokines, starting the initial T-cell-mediated immune response. In recent years, more and more studies show that DC is a heterogeneous population of cells, located in different anatomic sites, with different subpopulations of cells, at different stages of maturity, to express different phenotypes and cytokines, its function is also diversity. Which has a negative role in immune regulation of DC subsets found its features and mechanism of action is important progress in this field in recent years, one of these has a negative role in immune regulation DC subsets are known to regulate of DC (Regulatroy DC). now that, by selective adjustment of the DC induced mainly Th2 type immune response or induce naive CD4 and CD8 T cells to secrete IL-10 in regulatory T cells in order to negatively regulate immune responses. In the previous study, in mice as experimental subjects, we found that in the past was considered terminally differentiated cells mature DC, and spleen were cultured stromal cells to further proliferate and differentiate into a class through the secretion of NO and inhibition of T proliferation of new regulatory dendritic cells, which we named \the antigen-presenting task, in secondary lymphoid organs microenvironment, still able to further proliferation and differentiation, and thus was given a new life and functionality. In different physiological or pathological conditions, diffDC around a lot with the presence of immune cells interact with each other precision adjustment, for example, through the secretion of IL-10 activation of NK cells and promote their killing capacity, and through the secretion of IP-10 selectively chemotactic Thl cells and inhibit their proliferation, in order to maintain the body's homeostasis. However, these views are derived from the experimental results for the mouse, in the human body mature dendritic cells and splenic stromal microenvironment is how to effect it? Whether it will produce such as mice diffDC has a negative regulatory function human dendritic cell subsets do? If there were such a person DC subsets, the biological significance and its related mechanism of action and what is it? Given the nature of the problem into two cell interactions, we are in the present study is divided into three parts, namely around the splenic stromal cell lines, novel human regulatory dendritic cell subsets as well as the features and functions of the human body cell subsets verify three aspects were discussed. A human splenic stromal cell line establishment and identification HESSCs First, we isolated from April to June abortion fetus fresh spleen, using tissue culture method splenic stromal cells. About a month or so, the growth of adherent stromal cell culture plate covered, digestion and passage formed after the initial splenic stromal cell lines. At this point the splenic stromal cell lines were observed in visible form by several kinds and sizes of cells, purity is poor. With further cultivation, mainly from the purification step of endothelial-like cells of the stromal cell lines, cell growth rate stabilized, so that after about 2-3 months of culture, the formation of a stable splenic stromal cell lines. Next, we have built the human splenic stromal cell lines were preliminarily identified by flow cytometry analysis showed that the cell line is mainly composed of CD105 endothelial cells did not express CD11c, CDllb other myeloid cell markers. mRNA analysis showed that stromal cell lines to endothelial cells mainly detected by ELISA detection of this cell line secreting high TGF-p, suggesting that the cell lines may have some ability to induce differentiation, we constructed splenic stromal cell lines is a group composed mainly of endothelial cells have the potential ability to induce differentiation stromal cell line, which we call HESSCs (Human Endothelial-like Spleen Stromal Cells) two, HESSCs induced regulation of novel human dendritic cell subsets in the and function in this part of the experiment, we focus on research by human splenic stromal cell lines HESSCs induced from human peripheral blood monocyte-derived dendritic cells maturation comes CD11bhiCTLA-4 novel human regulatory dendritic cell subsets features and functions. First, we were isolated from normal human peripheral blood samples of human peripheral blood mononuclear cells (PBMC), further purification of CD14 monocytes with GM-CSF and IL-4 induction of differentiation combined typical immature dendritic cells (imDC), then the TLR ligand LPS stimulated their differentiation into mature dendritic cells (maDC), the previously established with the maDC splenic stromal cell lines HESSC mixed culture, observe the interaction between the two. It was found that removal of cytokines induced by GM-CSF and IL-4, the control medium in the most maDC apoptosis or death in 1-2 weeks, and most of the HESSCs mixed culture of dendritic cells still alive , indicating HESSCs support monocyte derived maDC survival. Based on the above phenomenon, we selected co-cultured with HESSC 1-2 weeks after maDC as research subjects, phenotypic and functional aspects of testing. By flow cytometric analysis found that these cells relative to maDC mainly as CD11clow, CD11bhi, HLA-DRlow, CD80low, CD83low, CD86low and CTLA-4, also confirmed that the group of cytokines cell hypersecretion PGE2, TGF-p, however, is not yet found that the group of cells that secrete high IL-10, NO and other negative regulatory factor. It is interesting that the group of cells express inhibitory receptor CTLA-4, together with its high level of secretion can PGE2, TGF-p, etc. cytokine negative, suggesting that the novel group of dendritic cell subsets may play a certain amount of immune suppression. By maDC and the novel dendritic cell subsets mRNA analysis and comparison proves that the group of novel dendritic cell subsets TGF-β, CTLA-4 mRNA levels are significantly higher than maDC, while not yet found the dendritic cell subsets in IL-10, PD-1, PDL-1, PDL-2 mRNA levels and other important molecules have significantly increased. Dendritic cells as the most important antigen-presenting cells in the development and progression of inflammation plays an important regulatory role, then our group of novel dendritic cell subsets in the inflammatory response to state how about it? Experiments show that the novel dendritic cell subsets in comparison with maDC TLR4 ligand LPS stimulation, can secrete high levels of inflammatory cytokines, including IL-10, TNF-α, PGE2, TGF-β and so on. The results suggest that our novel dendritic cell subsets in the inflammatory state may play a bigger role in immune regulation. HESSCs induced by a group of new regulatory dendritic cells express CTLA-4, and CTLA-4 as an important negative regulator of molecules in the inhibition of T-cell immune responses play an important role in the process, so we speculated that the novel dendritic cell subsets may have immunosuppressive function. Mixed lymphocyte reaction in vitro experiments, we found that the new DC subsets can indeed significantly inhibit maDC stimulated proliferation of allogeneic CD4 T cells, the specific mechanism needs further study. As we all know, DC in an immature state with strong phagocytosis, when it encounters the antigen stimulation or maturation after phagocytosis phagocytosis basically lost. The differentiation comes from the mature DC The new DC subsets but greatly enhanced phagocytic ability, this phenomenon for our in-depth study was to investigate its function provides a great imagination. In summary of the above experiment, the spleen HESSCs stromal cells can be induced in peripheral blood mononuclear cells as a source of differentiation maDC hypersecretion PGE2, TGF-β and other regulatory factors unique phenotype CD1 1bhiCTLA-4 of the novel human regulatory dendritic cell subsets. We will initially named CD11bhiCTLA-4 DCreg. Third, the human body, the new regulatory dendritic cell subsets identified in this part of the research work, we focus on the human spleen finding and preliminary validation of such new regulatory dendritic cells exist. In order to verify that the new DC subsets is really present in the body, we find that the human spleen DC subsets. We first used the new DC subsets specific surface markers (CD11c CD11bhiCTLA-4) will be the same phenotype cell sorting out, and then by detecting its inhibition of cytokine secretion and its function in the human body to determine whether there is such a group has not been found to DC subsets. We used flow sorter will CD11c spleen cells of the human body elected CD11chiHLA-DRhiCD11blow, CD11chiHLA-DRhiCD11bhi and CD11cintHLA-DRlowCD11bhi three cell subsets to study, through to cell phenotype, mixed lymphocyte reaction, cytokine secretion and phagocytosis compare various aspects of the ability to detect, confirm that the memory in a group of human spleen cells CD11chiHLA-DRhiCD11bhi express CTLA-4, and secrete large amounts of PGE2, TGF-β, IL-10 and other cytokines, has a strong phagocytosis, in the accounted for a larger proportion of cases can be effectively suppressed in vitro maDC induced allogeneic CD4 T lymphocyte proliferation. Therefore, we determine the initial human spleen cells within CD11chiHLA-DRhiCD11bhi splenic stromal cell lines in vitro induced differentiation HESSCs CD11bhiCTLA-4 DCreg have similar features and functionality. In summary, we found through experiments, human splenic stromal cell lines HESSCs can induce human peripheral blood monocyte-derived DC differentiation into mature group of people with negative immunomodulatory effects of novel human dendritic cell subsets. The cell subsets have unique cellular phenotype CD11bhiCTLA-4, high secretion of PGE2, TGF-β and other negative regulatory factor, can effectively inhibit the proliferation of CD4 T lymphocytes, and initially validated in humans (spleen) within exists. These results enrich the immune microenvironment negative regulation of the immune response understanding of multi-cellular networks, for the further study of dendritic cells involved in the immune mechanism of negative regulation provides a new direction and some experimental base for exploring the tumor, self-tolerance and autoimmune diseases such as pathogenesis and immunotherapy applications provide new avenues of research.
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