The ability of cancer cells to evade apoptosis may permit survival of a recombinant vaccinia lacking antiapoptotic genes in cancer cells compared with normal cells. We have explored the deletion of two vaccinia virus host range/antiapoptosis genes, SPI-1 and SPI-2, for their effects on the viral replication and their ability to induce cell death in infected normal and transformed cells in vitro. Indeed, in three paired normal and transformed cell types, the SPI-1 and SPI-2 gene-deleted virus (vSP) preferentially replicates in transformed cells or p53-null cells when compared with their normal counterparts. This selectivity may be derived from the fact that vSP-infected normal cells died faster than infected cancer cells. A fraction of infected cells died with evidence of necrosis as shown by both flow cytometry and detection of high-mobility group B1 protein released from necrotic cells into the culture supernatant. When administered to animals, vSP retains full ability to replicate in tumor tissues, whereas replication in normal tissues is greatly diminished. In a model of viral pathogenesis, mice treated with vSP survived substantially longer when compared with mice treated with the wild-type virus. The mutant virus vSP displayed significant antitumoral effects in an MC38 s.c. tumor model in both nude (P < 0.001) and immunocompetent mice (P < 0.05). We conclude that this recombinant vaccinia vSP shows promise for oncolytic virus therapy. Given its enhanced tumor selectivity, improved safety profile, and substantial oncolytic effects following systemic delivery in murine models, it should also serve as a useful vector for tumor-directed gene therapy.

We have recently demonstrated that cytolysis of human melanoma cells by immune effectors (both NK and T cells) is associated with release of the nuclear chromatin protein, high mobility group box I (HMGB1). Extracellular HMGB1 mediates a number of important functions including endothelial cell activation, stromagenesis, recruitment and activation of innate immune cells, and also dendritic cell maturation that, in the setting of cancer, lead to a chronic inflammatory response. This reparative inflammatory response promotes tumor cell survival, expansion, and metastases. Release of HMGB1 after chemotherapy-induced cytotoxicity has not been well characterized. We measured the release of HMGB1 after chemotherapy or immune cytolysis and demonstrated that this did not correlate with conventional markers of apoptosis and necrosis in several human colorectal, pancreatic, and melanoma tumor cell lines. Rather, we observed that tumor cells incubated with the platinating agent oxaliplatin, retained HMGB1 within the nucleus for significantly longer periods than other agents used at comparable cytotoxic concentrations or even with potent cytolytic cells. Thus, release of HMGB1 from dying tumor cells treated with chemotherapy or cells with lymphokine activated killer cell activity is not dependent solely on the mode of cell death. Sequestration of the damage associated molecular pattern molecule, HMGB1, may play a role in the clinical efficacy of platinating agents and suggests this as a superior agent for coupling with immunotherapeutic strategies, possibly enhancing their effectiveness.

Dendritic cells (DC) and natural killer (NK) cells are essential components of the innate immune system, which rapidly sense and eliminate invading pathogens and transformed cells, mediate inflammation, and initiate adaptive immune responses. During the early immune events, DC and NK cells interact and regulate each other. The cellular “cross talk” and its molecular mediators are believed to be critical to the quality and magnitude of innate and adaptive immune responses. The goal of the present manuscript is to identify and initially assess major molecular mediators of DC-NK cell interaction. We have previously shown that DC and NK cells constitutively express several tumor necrosis factor family ligands (TNFfLs) and corresponding TNF family receptors (TNFfRs). Therefore, DC and NK cells might be able to interact via cognate interplays of TNFfLs and TNFfRs. Here, we provide initial experimental evidence supporting this possibility. We found that combined but not individual ligation of several TNFfRs induced substantial increases in secretion of interleukin-12 and inteferon-γ by DC and NK cells, respectively. In contrast, specific, individual disruptions of the engagements of the corresponding TNfL-TNFfR pairs greatly impaired DC and NK cell abilities to reciprocally mediate the increases in cytokine secretion. These findings indicate that multiple TNFfLs mediate DC-NK cell interaction.

Several laboratories have developed culture systems that allow the generation of large numbers of human dendritic cells (DC) from monocytes using granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-4 (IL-4). In this work we provided evidence that GM-CSF (100 ng/ml) in combination with a low concentration of IL-4 (5 ng/ml) was efficient in the generation of immature, non-adherent, monocyte-derived DC as the same concentration of GM-CSF, and ten times higher concentration of IL-4 (50 ng/ml). This conclusion was based on the similar phenotype profile of DC such as the expression of CD1a, CD80, CD86, and HLA-DR, down-regulation of CD14, and the absence of CD83, as well as on their similar allostimulatory activity for T cells. A higher number of cells remained adherent in cultures with lower concentrations of IL-4 than in cultures with higher concentrations of the cytokine. However, most of these adherent cells down-regulated CD14 and stimulated the proliferation of alloreactive T cells. In contrast adherent cells cultivated with GM-CSF alone were predominantly macrophages as judged by the expression of CD14 and the inefficiency to stimulate alloreactive T cells. DC generated in the presence of lower concentrations of IL-4 had higher proapoptotic potential for the Jurkat cell line than DC differentiated with higher concentrations of IL-4, suggesting their stronger cytotoxic, anti-tumor effect.