With a 5-year survival rate of <5%, pancreatic cancer is one of the most rapidly fatal malignancies. Current protocols for the treatment of pancreas cancer are not as effective as we desire. In this study, we show that a novel Mucin-1 (MUC1)-based vaccine in combination with a cyclooxygenase-2 inhibitor (celecoxib), and low-dose chemotherapy (gemcitabine) was effective in preventing the progression of preneoplastic intraepithelial lesions to invasive pancreatic ductal adenocarcinomas. The study was conducted in an appropriate triple transgenic model of spontaneous pancreatic cancer induced by the KRASG12D mutation and that expresses human MUC1 as a self molecule. The combination treatment elicited robust antitumor cellular and humoral immune responses and was associated with increased apoptosis in the tumor. The mechanism for the increased immune response was attributed to the down-regulation of circulating prostaglandin E2 and indoleamine 2, 3,-dioxygenase enzymatic activity, as well as decreased levels of T regulatory and myeloid suppressor cells within the tumor microenvironment. The preclinical data provide the rationale to design clinical trials with a combination of MUC1-based vaccine, celecoxib, and gemcitabine for the treatment of pancreatic cancer.

Recent data have indicated that an important instructive class of signals regulating the immune response is Notch ligand–mediated activation. Using quantitative polymerase chain reaction, we observed that only Delta-like 4 (dll4) was up-regulated on bone marrow–derived dendritic cells after respiratory syncytial virus (RSV) infection, and that it was dependent on MyD88-mediated pathways. Using a polyclonal antibody specific for dll4, the development of RSV-induced disease was examined. Animals treated with anti-dll4 had substantially increased airway hyperresponsiveness compared with control antibody-treated animals. When the lymphocytic lung infiltrate was examined, a significant increase in total CD4+ T cells and activated (perforin+) CD8+ T cells was observed. Isolated lung CD4+ T cells demonstrated significant increases in Th2-type cytokines and a decrease in interferon γ, demonstrating an association with increased disease pathogenesis. Parellel in vitro studies examining the integrated role of dll4 with interleukin-12 demonstrated that, together, both of these instructive signals direct the immune response toward a more competent, less pathogenic antiviral response. These data demonstrate that dll4-mediated Notch activation is one regulator of antiviral immunity.

Respiratory syncytial virus (RSV) is the leading cause of respiratory disease in infants worldwide. The induction of innate immunity and the establishment of adaptive immune responses are influenced by the recognition of pathogen-associated molecular patterns by TLRs. One of the primary pathways for TLR activation is by MyD88 adapter protein signaling. The present studies indicate that MyD88 deficiency profoundly impacts the pulmonary environment in RSV-infected mice characterized by the accumulation of eosinophils and augmented mucus production. Although there was little difference in CD4 T cell accumulation, there was also a significant decrease in conventional dendritic cells recruitment to the lungs of MyD88−/− mice. The exacerbation of RSV pathophysiology in MyD88−/− mice was associated with an enhanced Th2 cytokine profile that contributed to an inappropriate immune response. Furthermore, bone marrow-derived dendritic cells (BMDC) isolated from MyD88−/− mice were incapable of producing two important Th1 instructive signals, IL-12 and delta-like4, upon RSV infection. Although MyD88−/− BMDCs infected with RSV did up-regulate costimulatory molecules, they did not up-regulate class II as efficiently and stimulated less IFN-γ from CD4+ T cells in vitro compared with wild-type BMDCs. Finally, adoptive transfer of C57BL/6 BMDCs into MyD88−/− mice reconstituted Th1 immune responses in vivo, whereas transfer of MyD88−/− BMDCs into wild-type mice skewed the RSV responses toward a Th2 phenotype. Taken together, our data indicate that MyD88-mediated pathways are essential for the least pathogenic responses to this viral pathogen through the regulation of important Th1-associated instructive signals.

CD4 and CD8 T cells produce different cytokines during immune response. Using a model of respiratory syncytial virus (RSV) infection, isolated CD4 T cells were found to produce predominantly Th2, while CD8 produced predominantly Th1 cytokines. To better understand the contribution of CD4 and CD8 T cells in immune pathology during RSV infection, CD4 and CD8 T cells were depleted using specific depleting antibodies and immune response was compared with control mice during RSV infection. Depletion of CD8 T cells showed increased airway hyperresponsiveness (AHR), production of IL-13, as well as increased mucus-associated gene, Gob5, expression. Specific depletion of CD4 induced lower AHR compared to control mice, lower Th2 cytokines and reduced Gob5 production. PAS staining showed both control and CD8 depleted mice with increase mucus production and increased goblet cell hyperplasia whereas mice depleted of CD4 T cell did not produce mucus. These results indicate that CD8 T cells are protective whereas CD4 T cells are responsible for increased pathology observed during RSV infection. This work was supported in part by NIH RO1AI036302.

Notch has recently been implicated in skewing T cell phenotype during immune responses. It has previously been reported that DCs from MyD88 deficient mice do not express the notch ligand delta-like 4. Our recent data has shown that CD4+ T cells from MyD88 deficient mice are skewed toward a Th2 phenotype during RSV infection. Here we demonstrate that the addition of recombinant dll4 to a dendritic cell/T cell co-culture reduced the amount of Th2 cytokines produced upon restimulation of MyD88 deficient T cells with RSV. In this system there was no change in the amount of IFNγ produced. To further investigate the role of dll4 in viral infection, we developed a polyclonal antibody to dll4 and blocked the protein in vivo during the RSV response. We found that mice receiving anti-dll4 had significantly higher airway hyperreactivity, an increase in the number of CD69+ CD4+ T cells in the lung, and a lower number of CD69+CD8+ T cells. CD4+ T cells isolated from lung draining lymph nodes during RSV infection and restimulated with RSV pulsed DCs produced significantly more IL-4, IL-5 and IL-13, however no change was observed in IFNγ. To investigate the role of dll4 during primary T cell responses, recombinant dll4 was used in co-cultures of DCs and DO11 ova-specific transgenic T cells. The presence of dll4 during a primary ova response decreased IL-4 and IL-13 production and increased IFNγ.