The Study Of VEGF-based Cancer Vaccine And The Survival Mechanism Of Adoptive CD8~+ Effector T Cells

Vascular endothelial growth factor(VEGF)plays an important role in the progression of various cancers.The VEGF specific antibodies bevacizumab when combined with chemotherapy was shown to significantly improve progression-free survival in certain cancers.However,repeated administration is necessary for effective suppression of VEGF,thereby making the therapy expensive and cumbersome.Thus it is urgent to develop alternative reagents such as VEGF vaccines.Here we report a VEGF based antigen,consisting of the receptor binding domain of VEGF and diphtheria toxin T-domain(DTT),not only stimulated neutralizing antibody response,but also induced type1 immune response as well as anti-tumor cytotoxic T lymphocytes in mice when administered with aluminum hydroxide adjuvant.Antibodies triggered by DTT-VEGF immunization inhibited the binding of VEGF to VEGF receptor and prevented the increasing of VEGF level in tumor bearing mice.VEGF specific IgG2a and IgG2b antibodies as well as type 1 cytokines were stimulated by DTT-VEGF vaccination.Splenocytes from DTT-VEGF immunized mice showed cytotoxic activity against VEGF expressing B16-F10 cells.Extensive necrosis with severe hemorrhage and enhanced CD8~+T cells infiltration were observed in tumors from DTT-VEGF immunized mice.The percentage of CD31~+vascular areas in the tumor sections from DTT-VEGF immunized mice was significant lower than that from control mice.DTT-VEGF significantly inhibited tumor growth in preventive and therapeutic vaccination settings in mouse model.Our data suggest that DTT is an effective antigen carrier to break self-tolerance and our vaccine design has potential to be used for human cancer therapy.Adoptive T cell immunotherapy is an alternative approach to bypass vaccination,leading to directly kill the cancer cells.However,the adoptively transferred effector T cells are not able to persist in the host for a long time due to the activation-induced cell death(AICD),thereby limiting their long-term efficacy.Lymphopenia promotes adoptive effector T-cells survival and memory formation.The underlying molecular mechanism is not yet fully understood.Here,we established an irradiation-induced lymphopenic mouse model,and transferred in vitro-activated transgenic OT-I CD8~+effector T-cells into irradiated wild-type C57BL/6,IL-7 knockout(KO)and IL-15 KO mice.We demonstrate that transferred T-cells prolong their survival and enhance their memory in lymphopenic mice,in a manner that depends on IL-15 signaling,but not IL-7.We determine that in vitro stimulation of na?ve or effector T-cells with IL-7 and IL-15 reduces IL-7R?,and increases and/or maintains IL-15R?expression,respectively.Moreover,the expression of IL-7R?and IL-15R is down-and up-regulated,respectively,on transferred T-cells in the early phase post T-cell transfer in lymphopenia.We further show that in vitro IL-15restimulation-induced memory T-cells(compared to IL-2 restimulation-induced effector T-cells)and in vivo transferred T-cells in irradiated IL-15-sufficient C57BL/6 mice(compared to IL-15-deficient IL-15 KO mice)have increased mitochondrial content,but less NADH and lower mitochondrial potential,and demonstrate greater phosphorylation of signal transducers and activators of transcription-5(STAT5)and Unc-51-like kinase-1(ULK1),and higher expression of B-cell leukemia/lymphoma-2(Bcl-2)and memory-,autophagy-and mitochondrial biogenesis-related molecules.Taken together,our data demonstrate that irradiation-induced lymphopenia promotes effector T-cell survival via IL-15 signaling the STAT5/Bcl-2 pathway,enhances T-cell memory formation via IL-15activation of the forkhead box(Fox)/eomesodermin(Eomes)memory and ULK1/autophagy related 7(Atg7)autophagy pathways and the mitochondrial remodeling.Our results thus identify important targets to consider when designing potent adoptive T-cell immunotherapies of cancer.