The Role of the Transcription Factor SIM2 in Prostate Cancer

Background: Recent reports have suggested a possible involvement of Single-minded homolog 2 (SIM2) in human solid cancers, including prostate cancer. However, the exact role of SIM2 in cancer in general, and in prostate cancer in particular, remains largely unknown. This study was designed to elucidate the role of SIM2 in prostate cancer using a shRNA-based approach in the PC3 prostate cancer cell line. Methods: Lentiviral shRNAs were used to inhibit SIM2 gene and protein levels in PC3 cells. Quantitative RT-PCR and branched DNA were performed to evaluate transcript expression. SIM2 protein expression level was measured by western blot. Profiling of gene expression spanning the whole genome, as well as polar metabolomics of several major metabolic pathways was performed to identify major pathway dysregulations. Results: SIM2 gene and protein products were significantly downregulated by lenti-shRNA in PC3 cell line. This low expression of SIM2 affected gene expression profile, revealing significant changes in major signaling pathways, networks and functions. In addition, major metabolic pathways were affected. Conclusion: Taken together, our results suggest an involvement of SIM2 in key traits of prostate tumor cell biology an

Immunization with a Peptide Containing MHC Class I and II Epitopes Derived from the Tumor Antigen SIM2 Induces an Effective CD4 and CD8 T-Cell Response

Here, we sought to determine whether peptide vaccines designed harbor both class I as well as class II restricted antigenic motifs could concurrently induce CD4 and CD8 T cell activation against autologous tumor antigens. Based on our prior genome-wide interrogation of human prostate cancer tissues to identify genes over-expressed in cancer and absent in the periphery, we targeted SIM2 as a prototype autologous tumor antigen for these studies. Using humanized transgenic mice we found that the 9aa HLA-A*0201 epitope, SIM2237–245, was effective at inducing an antigen specific response against SIM2-expressing prostate cancer cell line, PC3. Immunization with a multi-epitope peptide harboring both MHC-I and MHC-II restricted epitopes induced an IFN-γ response in CD8 T cells to the HLA-A*0201-restricted SIM2237–245 epitope, and an IL-2 response by CD4 T cells to the SIM2240–254 epitope. This peptide was also effective at inducing CD8+ T-cells that responded specifically to SIM2-expressing tumor cells. Collectively, the data presented in this study suggest that a single peptide containing multiple SIM2 epitopes can be used to induce both a CD4 and CD8 T cell response, providing a peptide-based vaccine formulation for potential use in immunotherapy of various cancers

The Scavenger Receptor MARCO Modulates TLR-Induced Responses in Dendritic Cells

The scavenger receptor MARCO mediates macrophage recognition and clearance of pathogens and their polyanionic ligands. However, recent studies demonstrate MARCO expression and function in dendritic cells, suggesting MARCO might serve to bridge innate and adaptive immunity. To gain additional insight into the role of MARCO in dendritic cell activation and function, we profiled transcriptomes of mouse splenic dendritic cells obtained from MARCO deficient mice and their wild type counterparts under resting and activating conditions. In silico analysis uncovered major alterations in gene expression in MARCO deficient dendritic cells resulting in dramatic alterations in key dendritic cell-specific pathways and functions. Specifically, changes in CD209, FCGR4 and Complement factors can have major consequences on DC-mediated innate responses. Notably, these perturbations were magnified following activation with the TLR-4 agonist lipopolysaccharide. To validate our in silico data, we challenged DC‘s with various agonists that recognize all mouse TLRs and assessed expression of a set of immune and inflammatory marker genes. This approach identified a differential contribution of MARCO to TLR activation and validated a major role for MARCO in mounting an inflammatory response. Together, our data demonstrate that MARCO differentially affects TLR-induced DC activation and suggest targeting of MARCO could lead to different outcomes that depend on the inflammatory context encountered by DC

A safety-modified SV40 Tag developed for human cancer immunotherapy

Simian virus 40 (SV40)-like DNA sequences have been found in a variety of human tumors, raising the possibility that strategies targeting SV40 may provide a potential avenue for immunotherapy directed against SV40 large T Antigen (Tag)-expressing tumors. We generated a recombinant vaccinia (vac-mTag) expressing mTag and herein assessed the ability of mTag to transform cells and to interact with anti-oncoproteins, as well as screened for the presence of potential HLA-A2.1-restricted epitopes within mTag. We found that transfection of cells with mTag did not lead to their transformation. Also, we demonstrated that mTag protein is degraded rapidly in cells. In addition, our work revealed that mTag did not physically interact with certain anti-oncoproteins. Finally, two potential HLA-A2.1-restricted functional epitopes within mTag sequence were identified. Our results show that mTag lacks the oncogenecity of full-length Tag and harbors potential HLA-A2.1-restricted immunogenic epitopes, hence suggesting the safety of vac-mTag for use in cancer immunotherapy

NAD+ protects against EAE by regulating CD4+ T-cell differentiation

CD4+ T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD+) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4+IFNγ+IL-10+ T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD+ regulates CD4+ T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD+, the frequency of T-bet−/− CD4+IFNγ+ T cells was twofold higher than wild-type CD4+ T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4+ T-cell differentiation and demonstrate that NAD+ may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases

Loss of Tff1 Promotes Pro-Inflammatory Phenotype with Increase in the Levels of RORγt+ T Lymphocytes and Il-17 in Mouse Gastric Neoplasia

Background: TFF1 deficiency induces a mucosal pro-inflammatory phenotype that contributes to gastric tumorigenesis in mouse and human. Methods: We utilized the Tff1-KO mouse model to assess the impact of TFF1 loss on immune cells infiltration in the stomach. We used single cell suspension, flow cytometry, immunohistochemistry, and quantitative PCR (qPCR) assays. Results: The Tff1-KO gastric mucosa demonstrated high chronic inflammatory scores (score: 3-4) at age 2 months, which exacerbated at age 8 months (score: 4-6). We next used single-cell suspensions for flow cytometry analysis of total leukocytes (CD45+ cells), total T lymphocytes (CD45+CD3+cells), T cell subsets (CD4+, CD8+, and CD3+CD4-CD8-cells), and monocytes/macrophages (CD45+F4/80+cells). The results demonstrated an age-dependent (2 → 8 month age) significant increase of leukocytes (p<0.05), T cells (p<0.05), and monocytes/macrophages (p<0.001) in the gastric mucosa of the Tff1-KO mice, as compared to Tff1-WT. A similar increase was observed in blood samples (p<0.05). Using ionomycin to activate CD4+ splenocytes, the results indicated that Tff1-KO CD4+ splenocytes secreted higher levels of IL-17A (p<0.05 at 2 and p<0.001 at 8 months) and IL-17F (p<0.05 at 2 and 8 months) than Tff1-WT splenocytes. Conversely, Tff1-KO CD8+-cells secreted less IL-17F, but comparable levels of IL-17A. In addition, we detected a significant upregulation of Il-17 mRNA expression in gastric tissues in the Tff1-KO, as compared to Tff1-WT (p<0.001). Conclusions: The results identify TFF1 loss as a major pro-inflammatory step that modulates the tumor microenvironment and immune cell infiltration in the stomach. Furthermore, the data suggest that the increase of IL-17A and IL-17F in Th17 cells, derived from CD4+ T cells, reflects the chronic inflammation in gastric mucosa, whereas the absence of change of IL-17A and decrease of IL-17F in CD8+Tc17 cells suggest loss of cytotoxic function of CD8+Tc17 cells during gastric tumorigenesis of the Tff1-KO mice

The Macrophage Scavenger Receptor SR-AI/II and Lung Defense against Pneumococci and Particles

The class A macrophage scavenger receptor SR-AI/II is implicated as a pattern recognition receptor for innate immunity, but its functional role in lung defense has not been studied. We used mice genetically deficient in SR-AI/II and their wild-type C57BL/6 counterparts to investigate the contribution of this receptor to defense against pneumococcal infection and inhaled particles. SR-AI/II deficiency caused impaired phagocytosis of fluorescent bacteria in vivo, diminished clearance of live bacteria from the lungs, and substantially increased pneumonic inflammation. Survival studies also showed increased mortality in SR-AI/II–deficient mice with pneumococcal lung infection. Similarly, after challenge of the airways with TiO2 particles, SR-AI/II–deficient mice showed increased proinflammatory cytokine levels in lung lavage fluid and a more pronounced neutrophilic inflammation. The data indicate that the lung macrophage class A scavenger receptor SR-AI/II contributes to innate defense against bacteria and inhaled particles