For survival studies, mice were treated with using a 2-dose (7 and 19 d), 3-dose (7, 19, 31 d), or 6-dose (7, 8, 11, 12, 24, and 36 d) schedule. and increased IFN production by T-cell populations. treatment provided a significant therapeutic benefit in pancreatic tumor-bearing mice. This therapeutic benefit depended on IL12 and IFN production, MyD88 signaling, and CD8+ T-cell populations. Although CD4+ T cells exhibited activated effector phenotypes and produced IFN, CD4+ T cells as well as NK cells were not required for the therapeutic benefit. In addition, CD8+ T cells isolated from treatment and demonstrates the significance of targeting tumor-associated myeloid cells as a mechanism to stimulate more effective immunity to pancreatic cancer. pyrimidine synthesis pathway to establish a uracil auxotroph (14, 15). invades and replicates in cells exogenously supplemented with uracil and during infection in normal or immune deficient mice (14C19), actively invades cells but fails to replicate making this vaccine strain avirulent and safe. vaccination elicits strong induction of interleukin-12 (IL12) and local interferon-gamma (IFN) that drives development of a potent CD8+ T-cell immunity and memory against infection (14, 15, 17, 19C22). Immunotherapeutic treatment of mice bearing established aggressive ovarian cancer or B16 melanoma recently was shown to stimulate potent antitumor responses and tumor-free survival (16, 23C25). In this study, we investigated immunotherapy using a highly aggressive, non-immunogenic disseminated peritoneal PDA model. We demonstrated treatment prolonged survival of mice bearing disseminated pancreatic tumors and examined the mechanisms underlying this effective immunotherapeutic treatment. treatment rapidly increased expression of co-stimulatory molecules and IL12 production by tumor-associated macrophages and dendritic cells (DC), particularly in myeloid cells actively invaded by treatment relied on invasive parasites, IL12 and IFN production, MyD88 signaling, and CD8+ T cells. Our findings demonstrate immunotherapy with the attenuated vaccine CP-724714 strain neutralized suppressive myeloid-cell mechanisms in PDA and stimulated effective antitumor T-cell responses. These results highlight the significance of targeting suppressive myeloid-cell populations as an effective immunotherapeutic mechanism to combat pancreatic cancer. Materials and Methods Mice and cell lines 6C8 week CP-724714 old female C57BL/6 (000664), IL12p35?/? (002692), IFN?/? (002287), MyD88?/? (009088) and CD8a?/? (002665) were purchased HOX1H from Jackson Laboratory. All animal work was performed at the Dartmouth Hitchcock Medical Center animal facility with Dartmouth IACUC approval. The murine pancreatic adenocarcinoma Pan02 cell line, also known as Panc02 (26), was acquired from the Division of Cancer Treatment Tumor Repository (NCI). Pan02 cells were maintained in high glucose Roswell Park Memorial Institute (RPMI) 1640 media. ID8-GFP cells (27) were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM). Human foreskin fibroblasts (HFF) (28) cultures were maintained in Eagle’s Minimum Essential Medium (EMEM). All cell culture media was supplemented with 10% FBS, L-glutamine, and penicillin/streptomycin. Parasites Tachyzoites of the vaccine strain were grown in HFF cells supplemented with 300 M of uracil (14, 15). Tachyzoites were purified through a 3.0 m nuclepore membrane and washed twice with phosphate buffer saline (PBS) prior to treatment of tumor-bearing mice. For experiments tracking cell types invaded by treatments used 2.0 106 tachyzoites injected i.p. For survival studies, mice were treated with using a 2-dose (7 and 19 d), 3-dose (7, 19, 31 d), or 6-dose (7, 8, 11, 12, 24, and 36 d) schedule. For cytokine analysis, mice were treated once at 7 d. For all cellular analysis studies, mice were treated with at 14 d. Tissue and cell isolation For spleen and mesenteric lymph node isolations, tissues were homogenized with DMEM in 10% FBS and single-cell suspensions were obtained by disrupting the organs using a cell strainer (40 m). Peritoneal cells were harvested by lavage at the time of sacrifice. Red blood cells were lysed in cell suspensions using red blood cell lysis buffer (eBioscience). Serum and peritoneal fluid was stored at ?80C. Cellular analysis and flow cytometry For intracellular staining CP-724714 studies, cells were incubated with Brefeldin A for 5 h at 37C. Antibody reagents were obtained from Biolegend: AF647-conjugated anti-mouse CD45 (30-F11), PE-conjugated and AF647-conjugated anti-mouse CD11b (M1/70), Brilliant Violet 421-conjugated anti-mouse CD11c (N418), PE-Cy7-conjugated anti-mouse CD4 and PE-conjugated anti-mouse CD4 (GK1.5),.