Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common autosomal dominant inherited diseases. Mutations in the human

Pancreatic ductal adenocarcinoma is poorly immunogenic, and immune suppression prevents T cell activation. We developed a microbial-based immunotherapeutic concept for selective delivery of immunogenic tetanus toxoid (TT856-1313) antigen into tumor cells by attenuated Listeria monocytogenes, and reactivation of TT-specific memory T cells to kill infected tumor cells. Treatment of KPC mice with Listeria-TT resulted in TT accumulation inside tumor cells, and attraction of TT-specific CD4 and CD8 T cells.Lymph node-like structures were frequently observed in contact with pancreatic tumors of treated mice, and exhibited CD4 and CD8 T cells producing IFNg. Gemcitabine combined with Listeria-TT significantly improved migration of CD4 T cells into tumors, and enhanced perforin and granzyme B production. The combination treatment also significantly reduced pancreatic tumors and metastases in Panc-02 and KPC mouse models, with minimal side effects, turning "cold" tumors into "hot" tumors. This study provides insight into mechanisms for improving immunotherapy for pancreatic cancer.selectively attracts MDSC through the production of cytokines and factors 11,20 , where MDSC deliver Listeria to the TME as a Trojan horse 10,19 . Once at the tumor site, Listeria spreads from MDSC into tumor cells through a cell-to-cell mechanism unique to Listeria 21 . Listeria can also infect tumor cells directly 22 . Listeria would be rapidly killed in healthy tissues, but is protected from immune clearance in the TME through strong immune suppression. Because of this combination of selective attraction of MDSC by bacteria and cancer with the strong immune suppression occurring in the TME but not in normal tissues, Listeria can selectively enter, multiply, and survive in the TME but not in normal tissues 10,19,23,24 . Based on these results we now use Listeria as a platform for the selective delivery of anticancer agents to the TME 19,23,25 .In the current study, we tested the combination of Listeria-TT+GEM (Fig 1b) in two mouse models of pancreatic cancer, a syngeneic Panc-02 model 26 and a transgenic KPC model 27,28 . Listeria-TT was administered intraperitoneally, and the in vivo uptake by tumor cells was verified by intravital imaging. We demonstrate that the combination of Listeria-TT+GEM robustly reduced pancreatic cancer at early and advanced stages in both Panc-02 and KPC mouse models. Our results unveil new mechanisms of Listeria to improve immunotherapy for PDAC. RESULTS Development and characterization of Listeria-TTThe Listeria construct used to develop Listeria-TT includes pGG34 29 , a truncated non-cytolytic Listeriolysin O (LLO) fused to a non-toxic TT856-1313 fragment, and a myc sequence for detection as outlined in Fig 2a. Secretion of LLO-TT856-1313 protein into the culture medium by the bacteria was detected by western blotting (Fig 2b). Infection of Panc-02 tumor cells with Listeria-TT856-1313 resulted in the expression of TT protein in the tumor cells (Fig 2c).

The B7 family, and their receptors, the CD28 family, are major immune checkpoints that regulate T-cell activation and function. In the present study, we explore the role of two B7 immune-checkpoints: HERV-H LTR-Associating Protein 2 (HHLA2) and B7 Family Member, H4 (B7x), in the progression of gastrointestinal and pancreatic neuroendocrine tumors (GINETs and PNETs). We demonstrated that both HHLA2 and B7x were expressed to a high degree in human GINETs and PNETs. We determined that the expression of B7x and HHLA2 correlates with higher grade and higher incidence of nodal and distant spread. Furthermore, we confirmed that HIF-1 overexpression is associated with the upregulation of B7x both in our in vivo (animal model) and in vitro (cell culture) models. When grown in vitro, islet tumor β-cells lack B7x expression, unless cultured under hypoxic conditions, which results in both hypoxia inducible factor 1 subunit alpha (HIF-1α) and B7x upregulation. In vivo, we demonstrated that Men1/B7x double knockout (KO) mice (with loss of B7x expression) exhibited decreased islet β-cell proliferation and tumor transformation accompanied by increased T-cell infiltration compared with Men1 single knockout mice. We have also shown that systemic administration of a B7x mAb to our Men1 KO mice with PNETs promotes an antitumor response mediated by increased T-cell infiltration. These findings suggest that B7x may be a critical mediator of tumor immunity in the tumor microenvironment of NETs. Therefore, targeting B7x offers an attractive strategy for the immunotherapy of patients suffering from NETs.