Immature Dendritic Cells Generated From Human PBMC Are Maturation Resistant

In the treatment of major burns,early and effective coverage of the wound plays a critical role. Allogeneic skin transplantation is a good method, but the rejection after transplantation limits its application.Prolongation of skin allografts survival by immunosuppressive drugs increases the potential risk for life-threatening infections.This is why clinical skin allografting practically does not exist. Therefore, it is crucial to induce host alloantigen-specific tolerance to donor. Dendritic cells(DCs) are the most important professional antigen-presenting cells(APC),which play critical roles in innate and acquired immunity.They display a heterogeneous group of cells that represent distinct origin,developmental stages and subsets.Immature DCs with deficient costimulatory molecules of CD40,CD86 can induce T anergy and promote alloantigen-specific tolerance. Isolation procedures of immature DCs from tissues directly are time consuming and cell yields are low, because they are rare in all body tissues. Furthermore, maturation of immature DCs could be induced by LPS or TNF-, which are high levels in major burn patients. IL-10 is defined as an anti-inflammatory cytokine,which was found to inhibit the proliferation of T cells in an allogeneic mixed lymphocyte reaction(MLR). Recent studies have reported that IL-10 treatment of recipient animals prior to grafting enhanced graft survival, but its inhibitory actions may attribute to its actions on APC. In this study,DCs(IL-10-imDC) were cultured from human peripheral blood progenitors with rhGM-CSF and rhIL-4 for 9 days, rhIL-10 was added on the 7th culture day. The cells in suspension were analysed on morphology, phenotype and functional tests. Cultured cells were also stimulated with LPS and TNF-αfor 2 days respectively, and their functional changes were observed by MLR. The study was divided into two parts:1.Generation of immature dendritic cells from human peripheral blood.2.Maturation resistance of immature dendritic cells. Results 1. The suspending cells displayed typical morphological features of DC, anomalous shape, bigger body, numerous dendrites and cascading plica were observed by fluorescent microscope and scanning electronic microscope. 2. The immunological phenotype of IL-10-imDC by flow cytemeter was CD1a++ HLA-DR+ CD83-CD40-CD86+/-, and costimulatory molecules of CD40, CD86 were down-regulated.Futhermore,a marked inhibition of the mean fluorescence intensity of HLA-DR molecules in IL-10-imDC. 3. IL-10-imDC inhibited the proliferation of non-sensitized T lymphocyte proliferation in MLR. 4. Their stimulating capacity of IL-10-imDC was not increased after exposure to LPS or TNF-and induced alloantigen-specific T cell unresponsiveness in MLR. Conclusion 1. IL-10-imDC exhibited typical morphological characteristics of DC and had immunological phenotype and function of immature DC. The results suggested the method was feasible. 2. IL-10-imDC were resistant to maturation by lipopolysaccharide(LPS) and TNF-α,which may be owed to the down-regulation of the costimulatory molecules and of class II MHC molecules of immature dendirtic cells with IL-10.