Human Splenic Stromal Cells Induce A Novel Subset Of Regulatory Dendritic Cells And The Underlying Mechanisms

Dendritic cells (DCs), the most potent professional antigen-presenting cells (APCs), are effective in the integration of a wide array of incoming signals and convey of them to lymphocytes, initiating the appropriate immune responses. It is well known that immature DCs (imDCs) undergo rapid maturation following microbial infection or allograft transplantation. Mature DCs lose endocytic activity, upregulate the surface expression of adhesion and costimulatory molecules, secrete proinflammatory cytokines and initiate primary T cell-mediated immune responses. In recent years, more and more studies have shown that DCs are heterogeneous cell population, with many kinds of DC subsets exhibiting different phenotype and functions. Among the DCs subsets, one population of DCs with ability to negatively regulate the immune response, so-called regulatory DCs, attracts much attention, and more evidences show that the regulatory DCs negatively regulate immune response by promoting naive CD4+and CD8+T cells to differentiate into IL-10-producing T regulatory/suppressor cells or inducing a preferential Th2 response or T cell anergy. However, the detailed machanisms by which regulatory DCs negatively regulate immune response remain to be fully understood to date.Our previous studies have shown that mouse splenic stromal cells, mimicking the secondary lymph organ microenvironment, can drive mouse mature DCs to proliferate and differentiate into a novel subtype of regulatory DCs (diffDCs), which express a higher level of IL-10 but minimal IL-12p70 and inhibit antigen-specific T-cell proliferation. However, all of these are the experimental results from mouse model. How is about the effect of human splenic stromal cells on the proliferation and differentiation of human DCs? Are there diffDCs in human? If there are diffDC-like cells in human, we wonder how they exert their regulatory function and what is the underlying mechanism?In three parts of our current study, we have established the human splenic stromal cell line (HESSCs), and identified the characteristics and regulatory function of the new subset of human dendritic cells induced by HESSCs and also went further to identify the natural counterpart of the new subset of human DCs in the human spleen. 1. The establishment and identification of the human splenic stromal cell lineFirst, we got the spleens from dead fetus 4-6 months old by induced abortion. We cultured the stromal cells by tissue culture methods from these spleens. Stromal cells would develop to cover the plate in one month so that we got the primary human spleen stromal cells after the primary passage. However, the stromal cell line at this moment was composed of many kinds of cells, such as epithelial-like cells, endothelial-like cells, fibroblast-like cells and even macrophage-like cells. Following the continual cultures and passages, the splenic stromal cells line mostly seemed like endothelial-like cells and fibroblast-like cells after 2-3 months when they began to proliferate steadily, and then the human splenic stromal cells line was preliminary established.To further identify the splenic stromal cell line we established, we analyzed the phenotype of the splenic stromal cells line by flow cytometry, and we found these cells expressed high level of CD 105 which was regarded as a marker of endothelial cells and but not CD11c, CD11b, CD14. In addition, we found the stromal cell line secreted high level of transforming growth factor-β(TGF-β). Accorrding to these results, we claimed that we had established the human splenic stromal cell line which was mainly composed of endotheial-like cells and expressed high level of TGF-β.We termed the cell line as human endotheial-like splenic stromal cells (HESSCs).2. The characteristics and function of the new subset of human regulatory dendritic cells induced by HESSCs.In this part of the study, we investigated proliferation and differentiation of human mature peripheral blood monocyte-derived dendritic cells (MoDC) induced by co-coculture with HESSCs. We found that the mature MoDC (maDCs) could differentiate to a new subset of regulatory dendritic cells after cocultured with HESSCs for a certain period.First, we isolated human peripheral blood mononuclear cells (PBMC) from peripheral blood of healthy donors and then got the CD14+monocytes by magnetic bead sorting. After cultured with rhGM-CSF and rhIL-4 for more than 6 days, the immature dendritic cells (imDC) which were differentiated from monocytes were stimulated with LPS for another 2 days, and then became the mature dendritic cells (maDC). These maDCs had been cocultured with HESSCs for 1-2 weeks to investigate the interaction between them. By assessing the proliferation and survival of the maDCs, we found most maDCs cultured without HESSCs had become apoptosis and death in 2 weeks, while the maDCs cocultured with HESSCs had been survival. The data suggested that HESSCs could support the survival of mature monocyte-derived dendritic cells, but we found HESSCs could not induce further proliferation of maDCs.Based on the above results, we analyzed the phenotype and cytokine profiles of the DCs cocultured with HESSCs. The flow cytometry revealed that the DCs had a phenotype of CD11bhi CD11clow HLA-DRlow CD80low CD83low CD86low CTLA-4+, which was ifferent from that of maDCs. Meanwhile, the DCs after co-cocultured with HESSCs secreted higher level of PGE2, TGF-P than maDCs. It was interesting that the DCs expressed high level of inhibitory receptor CTLA-4. The expression of PGE2, TGF-βand CTLA-4 suggested that the DCs, after cococulture with HESSC, may play roles in the immune suppression. Assay of mRNA expession of TGF-βand CTLA-4 in the DCs also confirmed higher expression of TGF-βand CTLA-4 in the DCs than maDC.Next, we investigated the function of the DCs with unique pahenotype and cytokine profiles after co-cocultured with HESSCs. We found that the DCs could secrete more inflammatory cytokines, including IL-10, TNF-a, PGE2, TGF-βin response to LPS stimulation. In the mixed lymphocyte reaction (MLR) in vitro, we found that the DCs could potently inhibit the proliferation of allogeneic CD4+T lymphocytes but could not affect the activation of CD4+T lymphocytes triggered by maDCs.It was well known that DCs in the immature state had a potent phagocytic capacity which maDCs had very weak phagocytic capacity. As compared to the maDCs, the DCs exerted the increased phagocytic capacity.In a conclusion, we found that humann splenic stromal cells (HESSCs) could induce human monocyte-derived mature DCs to differentiate into a new subet of DCs with the unique phenotype (CD11bhiCTLA-4+regulatory function), high secretion of PGE2 and TGF-β, and more importantly, the regulatory function. So, we designated this new subset of human DCs with regulatory function as the regulatory DCs (DCreg).3. Identification of the natural counterpart in vivo of the new subset of human DCreg.The primary aim of this part of our study is to verify whether the new type of DC subsets really exists in human body. We wanted to identify the counterpart in the human spleen derived from the trauma patients. Above all, we used those specific surface markers(CD11c+, HLA-DR+,CD11bhi, CTLA-4+, etc.) to sort the splenic cells with the similar phenotype. Afterwards, we detected their secretion of several relevant cytokines and possible function for inhibiting the proliferation of allogeneic CD4+T lymphocytes so as to confirm whether there were the natural counterpart of DCreg with similar phenotype and function. The results showed that there were a group of CD11chiHLA-DRhiCD11bhi cells expressing CTLA-4 in human spleens. Furthermore, these cells secreted large amounts of PGE2, TGF-β, IL-10, especially after stimulation with LPS. Besides, they could effectively inhibit the proliferation of allogeneic CD4+T lymphocytes in MLR. Therefore, we successfully identified one natural counterpart existing in human spleen of human DCreg which we identified in vitro through cococulture system with HESSCs.In summary, we have demonstrate that human splenic stromal cells can induce human monocyte-derived mature dendritic cells to differentiate into a new subset of human regulatory DCs which have unique phenotype CD11c+CD11bhiCTLA-4+and cytokine profiles of high PGE2 and TGF-β.More importantly, we show this new subset of human regulatory DCs can effectively inhibit the proliferation of allogeneic CD4+T lymphocytes in vitro. We also successfully identified one natural counterpart of human DCreg, existing in human spleen and exhibiting similar phenotype and cytokine profiles. Our results provide the experimental evindence that the immune microenvironment in human secondary lymph organs can control the immune response by inducing generation of rwgulatory immune cells such as DCreg. The disorder of the regulatory network may be one of the mechanisms for the pathiogenesis of immunological disorders such as autoimmune diseases.