Suppression Of CD8~+T Cell Proliferation And Activation By Regulatory Dendritic Cells And The Underlying Mechanisms

Dendritic cells (DCs) are the most potent professional antigen-presenting cell(APC) that play pivotal roles in the initiation of primary immune responses and induction of immunological tolerance. In recent years, more and more studies have shown that DCs are heterogeneous cell population, with many kinds of DC subsets exhibiting different phenotypes and functions, identification and characterization of phenotype and cytokine profile of DCs with regulatory properties (so-called regulatory DCs), and, investigation of their roles in the immune regulation and the pathogenesis of immune disorders attract much attention. Our previous studies have shown that splenic stromal cells, mimicking the secondary lymph organ microenvironment, can drive mature DCs (maDCs), which were previously thought to be terminally differentiated, to proliferate and differentiate into a novel subtype of regulatory DCs (Differentiated dentritic cells, diffDCs). diffDCs could strongly inhibit maDCs-induced CD4+ T cell proliferation via production of nitric oxide (NO), however, they could not induce the generation of regulatory T cells.T cells are the most important effector cells in the immune system. T cells recognize peptide fragments, which have been processed and become bound to major histocompatibility complex classâ… orâ…¡molecules and the co-stimulatory molecules on APCs, and then are activated to exhibit their variety of functions in the immunity anti-pathogens. CD8+ T cells, most of which are cytotoxic T lymphocytes(CTLs), recognize antigen presented on MHC classâ… . The most important role of CTLs is to eliminate the target cells infected with virus. However, the effector T cells-mediated immune response should be tightly regulated, so that T cell homeostasis is maintained, thus preventing the pathogenesis of immune disorders such as autoimmune diseases. So it is important to understand how to regulate adaptive T cell immune response to ensure there is no overactivation of T cell response and subsequent tissue damage. We have already found that diffDCs have powerful inhibitory effect on maDC-induced CD4+ T cell proliferation, and the inhibitory effect was partially mediated by NO. Do they have the similar suppressive effect on CD8+ T cells? Therefore, in this study, we have investigated the effects of diffDCs on the proliferation and activation of CD8+T cells and then further investigated the underlying mechanisms for the regulation of CD8+ T cells by diffDCs.Partâ… . Regulatory diffDCs inhibit antigen-specific CD8+T cell proliferation and activation.First we cocultured OVA257-264-specific CD8+T cells with OVA257-264peptide-pulsed maDCs in the presence or absence of graded numbers of diffDCs for 2 days and collected the CD8+ T cells and counted the viable cell number. The results showed that CD8+ T cells could not proliferate robustly in the presence of diffDCs.We then detected the activate status of CD8+ T cells on day 2. In the presence of diffDCs, the T cells expressed lower level of the activation-related molecules CD25. IL-2 production was also lower in the co-culture system in the presence of diffDCs. By detecting CFSE division and cell cycle, we found that CD8+ T cells had lower active cell division in the presence of diffDCs.. The results showed that diffDC could inhibit CD8+ T cell proliferation, but also inhibit T cell activation.In conclusion, we demonstrate that diffDC have powerful inhibitory effect on maDC-induced CD8+ T cell proliferation and activation, but this kind of inhibitory effect is possibly a relative tolerance status in the body, to keep maintenance of immune homeostasis.Partâ…¡. The mechanisms for the inhibition of antigen-specific CD8+ T cell proliferation and activation by regulatory diffDCs.In this part, we went further to investigate the underlying mechanisms of diffDC's inhibitory function.It has been shown that regulatory DCs can induce the generation of regulatory T cells (Treg). To find if diffDCs have the similar function, we analyzed the surface marker CD122 of 7AAD-CD8+ living OVA257-264 specific CD8 T cells in each cocultured group, and found that diffDCs could not induce activated OVA-specific CD8+ T cells to differentiate into CD122+ CD8+Tregs.We observed that the T cells had the tendency to apoptosis in the diffDC/maDC/CD8T co-culture system, so we detected CD8+ T cell apoptosis by 7-AAD staining. The percentage of 7-AAD+ apoptotic cells was similar in all groups. So we supposed that diffDC could not promote apoptosis of the activated OVA-specific CD8+ T cells.It has also been shown that regulatory DCs can induce T cell anergy and generation of suppressive or regulatory T cells, which are the main mechanisms by which regulatory DCs exert their inhibitory function. We cocultured OVA257-264 specific CD8+ T cells with peptide-pulsed maDCs or diffDCs and then purified T cells 2 days later. The T cells were rested in the presence of IL-2, and then restimulated with OVA257-264 peptide-pulsed maDCs. We found that the proliferation of CD8+ T cells remained unchanged even in the presence of diffDCs, indicating that regulatory diffDCs could not induce anergy or hyporesponsiveness of CD8+ T cells.We determined whether soluble factors from diffDCs or cell-cell contact between diffDCs and activated CD8+ T cells is required for the inhibition of the CD8+ T cells by diffDCs. The Transwell assays demonstrated that cell-cell contact between diffDCs and CD8+ T cells was indispensable for the inhibition of CD8+ T cell activation by diffDCs, and the negative control of CD8+ T cell proliferation by diffDCs was dependent on both cell-cell contact and soluble factors.As cell-cell contact between diffDCs and CD8+ T cells was required for diffDCs-induced inhibition of CD8+ T cell proliferation and activation, we wondered what membrane molecule(s) on diffDCs mediated the suppressive effect. It has been demonstrated that diffDC had a CD11bhi surface marker, and expressed FasL highly. We wondered if CD11b or FasL could be the key factors. Addition of in the neutralizing anti-CD11b or anti-FasL Ab into the diffDC/maDC/CD8+ T co-culture system could not reverse the inhibition of CD8+ T proliferation by diffDCs, indicating that CD11b and FasL are not involved in the suppressive effect of diffDCs. The significance of high level expression of CD11b and FasL on diffDC needs to be further investigated.Nitric oxide (NO) has been shown to be able to inhibit T cell proliferation and induce T cell apoptosis by blocking downstream signaling of IL-2, such as JAK3 and JAK1. We found that large amount of NO was produced in diffDC/maDC/CD8T co-culture system but not in maDC/CD8T co-culture system. And, when we used Transwell system to separate diffDC from maDC/CD8, there was lower NO detected, suggesting that NO may be one factor which mediates inhibitory effect of diffDCs on CD8+ T cell proliferation. So we added an arginine analog L-NAME to inhibit NO secretion, and found that the suppression of CD8+ T cell proliferation by diffDC could be significantly reversed. So, NO is the main effector molecule which mediates the inhibition of maDC-induced antigen-specific CD8+ T cell proliferation by diffDC.All together, the results demonstrate that regulatory dendritic cells (diffDCs) can suppress antigen-specific CD8+ T cell proliferation through NO in a cell-cell contact dependent manner. Our results will contribute to the better understanding of the mechanisms for the immune regulation and the pathogenesis of the immunological disorders, and also may be helpful to the design of immunotherapy for the immunological diseases.