Eradication of Metastatic Renal Cell Carcinoma after Adenovirus-Encoded TNF-Related Apoptosis-Inducing Ligand (TRAIL)/CpG Immunotherapy

Despite evidence that antitumor immunity can be protective against renal cell carcinoma (RCC), few patients respond objectively to immunotherapy and the disease is fatal once metastases develop. We asked to what extent combinatorial immunotherapy with Adenovirus-encoded murine TNF-related apoptosis-inducing ligand (Ad5mTRAIL) plus CpG oligonucleotide, given at the primary tumor site, would prove efficacious against metastatic murine RCC. To quantitate primary renal and metastatic tumor growth in mice, we developed a luciferase-expressing Renca cell line, and monitored tumor burdens via bioluminescent imaging. Orthotopic tumor challenge gave rise to aggressive primary tumors and lung metastases that were detectable by day 7. Intra-renal administration of Ad5mTRAIL+CpG on day 7 led to an influx of effector phenotype CD4 and CD8 T cells into the kidney by day 12 and regression of established primary renal tumors. Intra-renal immunotherapy also led to systemic immune responses characterized by splenomegaly, elevated serum IgG levels, increased CD4 and CD8 T cell infiltration into the lungs, and elimination of metastatic lung tumors. Tumor regression was primarily dependent upon CD8 T cells and resulted in prolonged survival of treated mice. Thus, local administration of Ad5mTRAIL+CpG at the primary tumor site can initiate CD8-dependent systemic immunity that is sufficient to cause regression of metastatic lung tumors. A similar approach may prove beneficial for patients with metastatic RCC

Activation of PPARγ in Myeloid Cells Promotes Lung Cancer Progression and Metastasis

Activation of peroxisome proliferator-activated receptor-γ (PPARγ) inhibits growth of cancer cells including non-small cell lung cancer (NSCLC). Clinically, use of thiazolidinediones, which are pharmacological activators of PPARγ is associated with a lower risk of developing lung cancer. However, the role of this pathway in lung cancer metastasis has not been examined well. The systemic effect of pioglitazone was examined in two models of lung cancer metastasis in immune-competent mice. In an orthotopic model, murine lung cancer cells implanted into the lungs of syngeneic mice metastasized to the liver and brain. As a second model, cancer cells injected subcutaneously metastasized to the lung. In both models systemic administration of pioglitazone increased the rate of metastasis. Examination of tissues from the orthotopic model demonstrated increased numbers of arginase I-positive macrophages in tumors from pioglitazone-treated animals. In co-culture experiments of cancer cells with bone marrow-derived macrophages, pioglitazone promoted arginase I expression in macrophages and this was dependent on the expression of PPARγ in the macrophages. To assess the contribution of PPARγ in macrophages to cancer progression, experiments were performed in bone marrow-transplanted animals receiving bone marrow from Lys-M-Cre+/PPARγflox/flox mice, in which PPARγ is deleted specifically in myeloid cells (PPARγ-Macneg), or control PPARγflox/flox mice. In both models, mice receiving PPARγ-Macneg bone marrow had a marked decrease in secondary tumors which was not significantly altered by treatment with pioglitazone. This was associated with decreased numbers of arginase I-positive cells in the lung. These data support a model in which activation of PPARγ may have opposing effects on tumor progression, with anti-tumorigenic effects on cancer cells, but pro-tumorigenic effects on cells of the microenvironment, specifically myeloid cells

Comprehensive Analysis Reveals Dynamic and Evolutionary Plasticity of Rab GTPases and Membrane Traffic in Tetrahymena thermophila

Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways

Emerging concepts in biomarker discovery; The US-Japan workshop on immunological molecular markers in oncology

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations

Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+) T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8(+) T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+) T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency

Rapid maturation of effector T cells in tumors, but not lymphoid organs, during tumor regression.

Increasing the efficacy of adoptively transferred, tumor antigen specific T cells is a major goal of immunotherapy. Clearly, a more thorough understanding of the effector phase of T cell responses, within the tumor site itself, would be beneficial. To examine this issue, we adoptively transferred tumor antigen-specific effector T cells into tumor-bearing mice, then performed kinetic evaluations of their phenotype, function, and survival in tumors, draining lymph nodes (dLNs), and spleens during regression of murine fibrosarcomas. Effector function in tumors was quantitated through the use of a novel intratumoral cytolytic assay. This approach revealed dynamic changes in the phenotype, cytolytic capacity, and viability of tumor infiltrating effector T cells during the course of tumor regression. Over a period of days, T cells within tumors rapidly transitioned from a CD25(hi)/CD27(hi) to a CD25(low)/CD27(low) phenotype and displayed an increase in cytolytic capacity, indicative of effector maturation. Simultaneously, however, the viability of maturing T cells within tumors diminished. In contrast, transferred T cells trafficking through lymphoid organs were much more static, as they maintained a stable phenotype, robust cytolytic activity, and high viability. Therefore, there exists a marked phenotypic and functional divergence between tumor-infiltrating effector T cells and their counterparts in lymphoid organs. Our results indicate that the population of tumor-infiltrating T cells is unique in experiencing rapid effector maturation post-transfer, and suggest that strategies aimed at prolonging the survival of CD25(low)/CD27(low) full effectors, which displayed the highest levels of intratumoral cytolytic activity, should enhance the efficacy of T cell based tumor immunotherapies

Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1

The mechanisms by which tumor microenvironments modulate nucleic acid-mediated innate immunity remain unknown. Here, we identified the receptor TIM-3 as key to circumventing the stimulatory effects of nucleic acids in tumor immunity. TIM-3 is highly expressed on tumor-associated dendritic cells (DC) in murine tumors and cancer patients. DC-derived TIM-3 suppresses innate immune responses through Toll-like receptor and cytosolic sensor recognition of nucleic acids via a galectin-9 independent mechanism. Instead, TIM-3 interacts with the HMGB1 to interfere with recruitment of nucleic acids into DC endosomes and attenuates the therapeutic efficacy of DNA vaccination and chemotherapy by reducing immunogenicity of nucleic acids released from dying tumor cells. Together, these findings define a novel mechanism by which tumor microenvironments suppress antitumor immunity mediated by nucleic acids