The Role Of CD40 Signal In Up-regulating B7-H3 Expression In Mouse Dendritic Cell And Its Control Of Th Cell Polarization

Dendritic cells (DCs) are professional APC that are specialized for the initiation and regulation of T cell immunity. Critical determinants of T cell activation induced by DCs include the density of peptide-MHC ligand available for TCR engagement as well as the provision of costimulatory signals. DCs also play an important role in Th1 or Th2 polarization. In vitro, DCs can be derived from bone marrow precursor cells when the appropriate cytokine signals are provided. Previous studies have shown that apoptotic tumor cells could be efficiently phagocytosed by immature DCs via cell surface expression of αvβ3, αvβ5 and CD36. DCs process, cross-present tumor Ag and stimulate antigen-specific, class Ⅰ or Ⅱ-restricted CD8~+ T cells. The utilization of apoptotic tumor cells as tumor antigens raises the possibility that antitumor specific immune responses activated by DCs without identification of TAA or TSA. In order to induce DCs maturation and trigger their transitions from immature Ag-capturing cells to mature Ag-presenting cells, various cytokines or biological factors were applied, such as sCD40L, CD40L transfectants or agonist CD40mAb, tumor necrosis factor-α (TNF-α). Notably, studies demonstrated that CD40 triggering DCs were most potent to elicit antitumor immunity. Moreover, several reports suggested that CD40 activated mature DCs preferable induced Th1 immune response in vitro or in vivo. However, the rational mechanisms remain unknown.Although appreciated as being fundamental to T cell activation, costimulatory molecules have also been implicated in promoting Th1 and Th2 specificity. Among the most important costimulatory signals are those delivered by the B7 family. Like most other members of this family, B7-H3 was described as a type I membrane protein. Collectively, the as-yet-unidentified receptor is expressed on activated T cells,and binding to B7-H3 might participate in the regulation of cellular and humoral immune responses..In the present report, we generated mouse myeloid DCs from bone marrow precursor in vitro using GM-CSF and IL-4. Phagocytosis of apoptotic tumor cells by DCs was observed by Confocal Microscopy. Immature DCs loaded with apoptotic tumor cells were stimulated with CD40L transfectants or TNF-a to prepare mature DCs. 3H-thymidine incorporation test was used to detect the T cell proliferation stimulated by mature DCs. The concentration IFN-y in supernatants of MLR from dendritic cell-driven T cells activation was analyzed by ELISA. Intracellular staining and FCM were used to detect the percent of CD4+IFN-y+T cells and CD4+IL-4+T cells hi T cells activated by mature DCs. Our results demonstrated that allogeneic MLR responses induced by CD40 activated DCs generated more Thl effectors than the responses induced by TNF-a stimulated DCs in vitro. FCM showed that during the process of maturation, DCs up-regulated the expression of CD40, CD80, CD86, and no significant differences between CD40 activated and TNF-a stimulated DCs were found in these molecules(P>0.05). In further study, we examined the expression and function of mouse B7-H3 on dendritic cell. Expression of B7-H3 in DCs was detected by FCM, and mRNA of B7-H3 was detected by RT-PCR. 3H-TdR incorporation test was used to detect the T cell proliferation stimulated by DCs with or without blocking B7-H3 by mAb respectively. The concentrations of IFN-y in supernatants of MLR from distinct groups were analyzed by ELISA. The concentration of IFN-y in supernatant of DCs was determined by ELISA. The data showed that B7-H3 expression on dendritic cells appears to be up-regulated by CD40 ligation, and differential expression of B7-H3 was obvious on DCs stimulated by mCD40L-CHO cells or TNF-a. In the blockade assay, we found an antagonistic mAb to B7-H3 inhibited CD40 activated DCs mediated T cells proliferation and IFN-y production in vitro. CD40 ligation was a potent signal to enhance apoptotic tumor cells pulsing DCs to secrete IFN-y. We hypothesized that auto-secreting IFN-y contributed toup-regulating expression of B7-H3 in DCs ligated by CD40, which sequentially favored CD40 activated DCs to enhance Thl- potential and mediate Thl immune response.In conclusion, our studies substantially gave evidences for a precise balance of costimulatory molecules determining the outcome of a T cell response and expanded our knowledge of costimulatory molecules regulatory network. Furthermore, this study was performed to provide some guidance about the DCs preparation we might take into clinical studies. It might be anticipated that DCs triggered by CD40 signal in vitro will be entirely compatible with their preprogrammed function. Remarkable advances in our understanding of the role of CD40 activated tumor specific DCs in generation of effectors T lymphocyte had shed new light on the regulation of the immune responses. Once we understand what the ideal T lymphocyte responders require, this new knowledge will help develop better therapies for controlling the immune responses. Ultimately the clinical data and practical issues will decide which DCs preparation clinicians will select, and DCs activated by CD40 signal would have a promising tumor immunotherapeutic prospect.