The Role Of Notch Signaling In Potential Of Antigen Presentation And Th1/Th2 Polarization Of Mouse Bone Marrow-derived Dendritic Cells

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. Exploiting the immune-regulatory capacitiy of dendritic cells holds great promise for the treatment of cancer. A DC vaccine is defined as DCs loaded with tumor associated antigen. Upon administration into patients, the vaccine is thought to induce an antigen-specific T-cell response against the tumor. Several groups reported that Notch ligand Jagged-1 is an inhibitor of DC differentiation. We co-cultured the OP9-DLL1 cells which have higher level of Delta like-1 expression with cells of mouse bone marrow to generate DCs. We shed light on the critical role of Delta-like 1 in differentiation and the antigen presentation capacity of dendritic cells. We hope to acquire more effective DC vaccine by the regulation of Notch signaling.DCs are the most potent antigen presenting cells. Recognition of pathogens by DC through interaction with pattern recognition receptors including Toll like receptors (TLR) is crucial for directing Th cell differentiation toward the Th1 or Th2 type. It is known that intracellular organisms are primarily capable of instructing DCs to induce Th1 responses, whereas extracts of parasitic helminthes have been demonstrated to drive Th2 skewed responses. Th1 cells produce interferon-γ(IFN-γ) and Th2 cells produce interleukin-4 (IL-4), IL-5 and IL-13. Th1 and Th2 polarization is promoted by IL-12 and IL-10, respectively. The corresponding functional DC subsets are called dendritic cell type 1 (DC1), dendritic cell type 2 (DC2). A key TLR signaling event is activation of the NF-κB family of transcription factors. Numerous reports have described regulation of NF-κB by Notch. The RBP-J-deficient DCs possess significantly less dendrites, lower level of surface MHCⅡmolecules, and reduced capacity of migration and antigen presentation both in vitro and in vivo. It was known that TLR ligands induce expression of the Notch ligands on DCs. We used LPS or Poly I:C to stimulate RBP-J+/-DCs and RBP-J-/-DCs. By detecting the amounts of cytokines and levels of Notch ligands expression, we observed the effect of RBP-J-deficient DCs on Th cell differentiation. Notch ligands transcription is mediated by nuclear factor-κB (NF-κB). Upon engagement of TLR, signaling cascades are initiated that involve activation of NF-κB and Notch, and induction of expressed genes of Notch ligands and cytokines involved in DC maturation and the ability to prime and skew T cell responses.The main finds are as follows:1. In the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4, mouse bone marrow cells were co-cultured with OP9-DLL1 and OP9-GFP cell lines respectively. Compared with OP9-GFP, the bone marrow cells co-cultured with OP9-DLL1 produced significantly more CD11c+ DCs .2. The surface molecules of the activated DCs were analyzed by flow cytometry. We found that the DCs generated from bone marrow cells co-cultured with OP9-DLL1 possessed higher levels of surface molecules expression including MHCⅡ, CD80 and CD86 after tumor antigen stimulation.3. The culture supernatants were detected by ELISA. The DCs generated from bone marrow cells co-cultured with OP9-DLL1 secreted higher amounts of IL-12 and less IL-10.4. We analyzed the proliferation of T-cells co-cultured with DCs by FACS. The DCs generated from bone marrow cells co-cultured with OP9-DLL1 also resulted in significantly stronger T-cell proliferation response.5. We cultured BM cells from the Poly I:C-induced RBP-J knockout and control mice for 8 days, in the presence of GM-CSF and IL-4. Notch ligands mRNA levels of the DCs treated with LPS or poly I:C for 16 h were assessed by Real-time PCR. LPS can upregulate expression of the ligands (Jagged-1, Delta-like 1 and 4) in DCs via TLR4. In addition, RBP-J-/- DCs have much weaker response than RBP-J+/- DCs. Compared to RBP-J-/+ DCs, the levels of mRNA expression of the three ligands in RBP-J-/- DCs were lower. The expression of the other ligands (Jagged-2, Delta-like 3) had not significant difference. The same effect was detected when DCs were stimulated by poly I:C.6. After stimulated by LPS or poly I:C, the RBP-J-/- DC secreted higher amounts of IL-10 and less IL-12 than RBP-J-/+ DC. Although, when we detected IL-6 and TNF-αby ELISA, there were no significant differences.7. Na?ve T cells through interaction with RBP-J+/- DCs could produce higher level of IFN-γ. RBP-J+/- DCs could be polarized to the DC1 after LPS or poly I:C stimulation. In contrast, na?ve T cells through interaction with RBP-J-/- DCs could produce higher level of IL-4. So RBP-J-/- DCs could be polarized to the DC2.8. When T cells were co-cultured with DCs at 3d and 5d, the proliferation was analyzed by FACS respectively. The results showed that T cells through interaction with RBP-J-/- had significantly weaker proliferation response.It was concluded that DLL1 could promote the differentiation of DCs from mouse bone marrow cells and enhance their antigen presentation capacity. We provided the evidence that flexibility of mouse bone marrow-derived DCs in directing T Helper Type 1 and 2 cells was regulated by Notch/RBP-J signaling. NF-κB might involve in the regulation process. Th1 differentiation was affected by the Notch ligands (Jagged-1, Delta-like 1 and 4) expression on dendritic cells. Deletion of RBP-J in dendritic cells compromises TLR triggering-induced Notch ligand up-regulation and Th1 activation.