Study Of Effects And Mechanisms Of Notch Signaling Deficiency On Mouse Dendritic Cell-dependent Anti-tumor Immunity

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that initiate specific immune responses against pathogens and tumor cells. Immature DCs which locate in the tissues and the peripheral lymphoid organs persistently surveillance the environment and recognize the exogenous and endogenous antigen, and capture antigens by phagocytosis, micropinocytosis, and endocytosis. After antigen recognition and uptake, the immature DCs will undergo a series of maturation events, including the up-regulation of MHC II and the co-stimulatory molecules, the secretion of cytokines, the outgrowth of dendrites, and the modulation of chemokine receptor expression profile accompanied by the migration into the peripheral lymphoid organs. The antigen-loaded mature DCs can activate T-cells through the interaction between MHC II-peptide and T-cell receptor (TCR) complex, and can activate B-cells and NK-cells through specific ligands expressed on DCs and cytokines secreted by DCs. DCs play a critical role in the initiation, programming and regulation of the anti-tumor immunity. DCs have been considered as a promising agent to generate effective anti-tumor immune responses, because DCs can be generated in large numbers, and the cultured immature DCs could be converted into mature DCs through antigen loading. These DCs can be delivered to the tumor site or lymph nodes to activate T-cell responses against tumor. However, although the use of mature DCs as cellular vaccines showed promising anti-tumor effect in many mouse tumor models, the application of mature DCs in phase III clinical trails in human cancer patients with prostate cancer or melanoma have largely failed. Therefore, the fully understanding of the molecular mechanisms regulating DC maturation and activation is a prerequisite for the DC-based anti-tumor therapies.The Notch signaling pathway is an evolutionarily conserved pathway that regulates development by participating in cell fate determinations and cell proliferation, differentiation and apoptosis during embryonic and postnatal stages. There are four Notch receptors (Notch1-4) and five ligands (Jagged1, Jagged2, and Delta-like ligands (Dll) 1, 3, and 4) in mammals. After the triggering of the Notch receptors by the binding of the Notch ligands, the Notch intracellular domain (NIC) translocates into the nucleus, where it interacts with RBP-J. This protein complex will recruit other transcription co-activators, and transactivate the transcription of the target genes.The Notch signaling plays an important role in the immune system, including the development of T cell, B cell, NK cell, DC and myeloid cell, formation of immunological synapse and polarization of Th1/Th2, etc. During the DC development, the effects of Notch signaling are different according to different ligand-receptor binding. Delta-like-1 could induce DC differentiation, whereas Jagged-1 could inhibit DC differentiation, promote the accumulation of immature myeloid cells and induce the maturation of the monocyte-derived human DCs. The differentiation of DCs was significantly impaired in Notch-1 down-regulated mice. Although many studies reported the effects of Notch signaling on DC development, the detail mechanisms remain to be further investigated.Wnt signaling is a complex pathway that is highly conserved among species. Wnt signaling was originally identified as aβ-catenin dependent signaling pathway. However, within the past 15 years, at least three Wnt-mediated pathways have been proposed that function independent ofβ-catenin. Calcium has been implicated as an important second messenger in all of these pathways. Wnt proteins are secreted glycoproteins, such as Wnt1, Wnt3a, Wnt8, etc. Wnt receptors include seven transmembrane Frizzled (Fzd) family, as well as LRP-5 or LRP-6. After activation, Wnt signaling will directs cell proliferation, survival and alters cell fates. The interaction between Notch and Wnt signaling has been reported by series of studies in the past, especially in DCs. The Wnt signaling is downstream of Notch pathway and they cooperatively play an important role in the differentiation of DCs.We have recently shown that the RBP-J-mediated Notch signaling plays a critical role in the maturation of the LPS-induced DCs. In order to investigate whether the Notch signaling participates in the DC-mediated anti-tumor immunity, we established tumor-bearing mouse models by using several mouse tumor cell lines and RBP-J-deleted DCs. We observed the growth of tumor, and analysed the immunity levels. We have identified the effects of Notch signaling deficiency on the DC-dependent anti-tumor immunity and the potential mechanisms, and also provided experimental foundation and theoretical support for DC-based anti-tumor therapy. The main results are as follows:1. Notch signaling changes were detected in tumor infiltration DCs.B16 cells (5×106) were injected into WT C57BL/6 mice s.c. at the abdomen, PBS was considered as a control. The mice were sacrificed 10 days later, and CD11c+ DCs were sorted by magnetic beads from mouse spleens. Compared with control, the relative mRNA level of Notch receptors and downstream genes which were detected by qPCR were significantly changed in tumor bearing mouse spleen DCs. This indicated that Notch signaling was involved in immune system inhibition in tumor bearing mouse.2. Conditional RBP-J knockout transgenic mice were obtained, proper inoculation tumor cells were selected, proper amount of tumor cells injected subcutaneously and the time point for tumor measurement were identified.Lots of Mx-Cre-RBP-Jflox/WT (RBP-J-/+) and Mx-Cre-RBP-Jflox/flox (RBP-J-/-) mice were obtained by mating RBP-Jflox/WT and Mx-Cre mice. The RBP-J gene was conditionally knocked out by 12 times of poly I:C injection. Nine tumor cell lines were injected s.c. including B16 melanoma, H22 hepatoma, S180 osteosarcoma, LLC lewis lung cancer, L1210 leukemia, EL4 lymphoma, CT26 colon cancer, MFC gastric cancer, EMT6 breast cancer, only the first four tumor cell lines could survive successfully. Tumor volume was monitored every 2 days from the 5th day after inoculation and the mice were sacrificed on the 17th day.3. RBP-J deficient DCs are unable to repress tumor growth when co-inoculated with tumor cells in mice.The DCs were sorted by magnetic beads from RBP-J-/+ and RBP-J-/- mice and incubated with tumor cell antigens for 12 h. Then the mature DCs (1×106) were mixed with tumor cells (5×106) and injected s.c. into WT C57BL/6 mice. Compared with control, the tumors containing RBP-J-/- DCs grew significantly faster. Further the amount of infiltrated T cells, B cells and NK cells were significantly lower in tumors containing RBP-J-/- DCs compared with control. This indicated that the anti-tumor immunity of DCs was significantly impaired after Notch signaling knockout and the infiltration of immune cells to tumor tissues was also decreased.4. Notch signaling deficiency inhibited DC maturation, decreased migration, weakened IS formation and attenuated T cell activation.The ability of antigen uptake by DCs appeared comparable between the RBP-J-/- DCs and control. Expression of MHC II, CD80 and CD86 were decreased in RBP-J-/- DCs compared with control after tumor antigen stimulation. The expression of CCR7 was significantly down-regulated in RBP-J-/- DCs. The migration ability to draining lymph nodes was significantly decreased. The T cell activation ability was significantly impaired according to in vivo and in vitro experiments, this may resulted from the malformation of IS between DCs and T cells. We found that the expressions of CTx and MHC II were significantly lower in IS in RBP-J-/- DC-T cell conjugates. The polarization of F-actin,α-tubulin and vinculin were decreased in RBP-J-/- DCs after contacted with T cells. These results indicated that the impaired anti-tumor immunity of RBP-J-/- DC may result from maturation disorder, decreased migration and T cell activation.5. Notch signaling deficiency down-regulated the expression of Wnt receptors of DCs and resulted in decreased free calcium levels in DCs.The relative mRNA expressions of Wnt receptors of DCs were detected by qPCR. We found that most of the Wnt receptors mRNA expressions were down-regulated in RBP-J-/- DCs compared with control. The free calcium level of RBP-J-/- DCs was significantly lower than control after LPS stimulation. These indicated that Notch signaling deficiency may impair DC function through down-regulating Wnt/calcium signaling pathway.6. ConclusionsThe current study showed that Notch signaling may be involved in the immune system inhibition in tumor bearing mouse. Notch deficiency could significantly impair anti-tumor immunity of mouse DCs, decrease the infiltration of T cells, B cells and NK cells to tumor tissues, finally resulted in faster tumor growth.Further investigation demonstrated that Notch signaling deficiency impeded DC maturation after tumor antigen stimulation, inhibited the up-regulation of MHC II, CD80 and CD86, decreased the expression of CCR7, attenuated the migration ability to draining lymph nodes, weakened IS formation and decreased the T cell activation.Mechanism exploration indicated that Notch signaling may down-regulate the level of Wnt receptor, decrease the activation of Wnt/calcium signaling and the free calcium level, influence cytoskeleton polarization in DCs after contacted with T cells and finally weaken the formation of IS between DCs and T cells.These results implied that Notch signaling plays a critical role in DC-dependent anti-tumor immunity, provided further comprehension of DC vaccine based anti-tumor therapy. Conversely, it indicated that the anti-tumor immunity may be strengthened through Notch signaling up-regulation in DCs, and also provided new ideas for DC vaccine application in clinic.