No abstract

We have recently shown that effector T cells (TE) lacking either perforin or IFN-γ are highly effective mediators of tumor regression. To rule out compensation by either mechanism, TE deficient in both perforin and IFN-γ (perforin knockout (PKO)/IFN-γ knockout (GKO)) were generated. The adoptive transfer of PKO/GKO TE mediated complete tumor regression and cured wild-type animals with established pulmonary metastases of the B16BL6-D5 (D5) melanoma cell line. PKO/GKO TE also mediated tumor regression in D5 tumor-bearing PKO, GKO, or PKO/GKO recipients, although in PKO/GKO recipients efficacy was reduced. PKO/GKO TE exhibited tumor-specific TNF-α production and cytotoxicity in a 24-h assay, which was blocked by the soluble TNFRII-human IgG fusion protein (TNFRII:Fc). Blocking TNF in vivo by administering soluble TNFR II fusion protein (TNFRII:Fc) significantly reduced the therapeutic efficacy of PKO/GKO, but not wild-type TE. This study identifies perforin, IFN-γ, and TNF as a critical triad of effector molecules that characterize therapeutic antitumor T cells. These insights could be used to monitor and potentially tune the immune response to cancer vaccines.

Since multiple lines of experimental and clinical data clearly identified regulatory T cells as an integral part of the immune response, these cells have become a major focus of investigation in tumor immunology. Regulatory T cells are in place to dampen ongoing immune responses and to prevent autoimmunity, but they also have profound effects in blocking therapeutic anti-tumor activity. Therefore regulatory T cells are seen as a major hurdle that must be overcome in order for cancer immunotherapy to reach its therapeutic potential. Regulatory T cells are heterogeneous with sub-populations that exhibit distinct functional features. Here we will review the individual sub-populations in regards to their mode of action and their potential impact on blocking anti-tumor immunity. Approaches to measure function and frequency of regulatory T cells in model systems and clinical trails will be discussed. Finally, we will describe possible ways to interfere with regulatory T cell-mediated immune suppression with the focus on recent pre-clinical and clinical findings.

To investigate the mechanisms involved in Helicobacter pylori-mediated inducible nitric oxide synthase (iNOS) upregulation in mononuclear cells we cocultivated human THP-1 acute monocytic leukemia cells and murine J774A.1 professional macrophages with different H. pylori wild-type strains and mutants. We have shown that H. pylori-mediated iNOS induction in J774A.1 is independent of established virulence factors but dependent on direct interaction between bacteria and cells. In J774A.1, iNOS was equally upregulated by the wild-type strains J99, 26695, P12, and P1 as well as by mutants lacking the cag pathogenicity island, vacA, katA, alpAB genes and the hp0043 gene taking part in lipopolysaccharide biosynthesis when direct cell contact was allowed but not when bacteria and cells were separated by protein-permeable filter membranes. In contrast, iNOS was not induced in THP-1. This indicates that H. pylori-mediated iNOS induction in J774A.1 is independent of important virulence factors whereas cell contact is crucial which suggests a role of adhesion or phagocytosis.

Paraffin-embedded material from 69 patients with epithelial ovarian cancer FIGO stages I and II/A (including 21 patients with borderline carcinoma) was studied with automatic DNA image cytometry. Univariate analysis indicated a significant difference in survival based on the presence of nuclei with high DNA content (higher than 5 C). A group of patients with less than 0.2% cells with high DNA content had a 6-year survival of 87%, whereas in a group of patients with more than 0.2% of such cells, 6-year survival was 49%. This parameter remained significant when used in a group of stage I/a and I/b patients. Statistical analysis of diploid vs. non-diploid tumors also showed significant difference in survival. Separate analysis of 48 invasive ovarian cancers indicated that ploidy, the percentage of cells with high DNA content and tumor stage (stage I/a + b vs. stages I/c + II/a) reached significance for survival, whereas grading did not. In addition, comparison of clinical stage, grading, ploidy and the percentage of cells exceeding 5 C with a threshold at 0.2% by means of a multivariate analysis (Cox regression model) showed that only the percentage of cells exceeding 5 C remained statistically significant.

To investigate the mechanisms involved in Helicobacter pylori-mediated inducible nitric oxide synthase (iNOS) upregulation in mononuclear cells we cocultivated human THP-1 acute monocytic leukemia cells and murine J774A.1 professional macrophages with different H. pylori wild-type strains and mutants. We have shown that H. pylori-mediated iNOS induction in J774A.1 is independent of established virulence factors but dependent on direct interaction between bacteria and cells. In J774A.1, iNOS was equally upregulated by the wild-type strains J99, 26695, P12, and P1 as well as by mutants lacking the cag pathogenicity island, vacA, katA, alpAB genes and the hp0043 gene taking part in lipopolysaccharide biosynthesis when direct cell contact was allowed but not when bacteria and cells were separated by protein-permeable filter membranes. In contrast, iNOS was not induced in THP-1. This indicates that H. pylori-mediated iNOS induction in J774A.1 is independent of important virulence factors whereas cell contact is crucial which suggests a role of adhesion or phagocytosis.

In clinical trials therapeutic tumor vaccines have failed to provide evidence of efficacy. Based on preclinical tumor models showing that vaccination during homeostasis-driven proliferation improved therapeutic efficacy, we performed a translational phase I/II clinical trial in men with metastatic hormone-refractory adenocarcinoma of the prostate. This strategy has the theoretical advantage that it eliminates regulatory T cells (Treg). Patients were randomized to cohort A, B or C. Cohort A patients (n = 5) received prostate GVAX TM (two prostate cancer cell lines that secrete GM-CSF, Cell Genesys Inc.) vaccination only. Cohort B (n = 5) received cyclophosphamide followed by reinfusion of autologous PBMC and vaccine (GVAX). Cohort C patients (n=5) received same treatment as Cohort B with the addition of fludarabine. We hypothesized that the most lymphopenic patients would have the fewest Treg. While all cohorts showed an increase in the total number and frequency of peripheral Treg, the most lymphopenic patients (cohort C) had the greatest fold increase in peripheral Treg at weeks 2-6 (2.3,1.2,0.7 / p<0.05). Current studies are examining whether adoptive transfer of CD25 depleted PBMC will reduce Treg recovery and improve clinical response. Supported by CA119123, Robert W. Franz, Chiles foundation, Prostate Cancer Foundation and Providence Medical Foundation

No abstract