Neonatal Infection with Species C Adenoviruses Confirmed in Viable Cord Blood Lymphocytes

Credible but conflicting reports address the frequency of prenatal infection by species C adenovirus. This question is important because these viruses persist in lymphoid cells and suppress double-stranded DNA-break repair. Consequently, prenatal adenovirus infections may generate the aberrant clones of lymphocytes that precede development of childhood acute lymphoblastic leukemia (ALL). The present study was designed to overcome technical limitations of prior work by processing cord blood lymphocytes within a day of collection, and by analyzing sufficient numbers of lymphocytes to detect adenovirus-containing cells at the lower limits determined by our previous studies of tonsil lymphocytes. By this approach, adenoviral DNA was identified in 19 of 517 (3.7%) samples, providing definitive evidence for the occurrence of prenatal infection with species C adenoviruses in a significant fraction of neonates predominantly of African American and Hispanic ancestry. Cord blood samples were also tested for the presence of the ETV6-RUNX1 translocation, the most common genetic abnormality in childhood ALL. Using a nested PCR assay, the ETV6-RUNX1 transcript was detected in four of 196 adenovirus-negative samples and one of 14 adenovirus-positive cord blood samples. These findings indicate that this method will be suitable for determining concordance between adenovirus infection and the leukemia-associated translocations in newborns

Turning T cells On: Epigenetically Enhanced Expression of Effector T-cell Costimulatory Molecules on Irradiated Human Tumor Cells

Background: Sub-lethal doses of radiation can alter the phenotype of target tissue by modulating gene expression and making tumor cells more susceptible to T-cell-mediated immune attack. We have previously shown that sublethal tumor cell irradiation enhances killing of colorectal carcinoma cells by tumor-specific cytotoxic T cells by unknown mechanisms. Recent data from our lab indicates that irradiation of tumor cells results in the upregulation of OX40L and 41BBL, and that T cells incubated with irradiated tumor cells displayed improved CTL survival, activation and effector activity. The objective of this current study was to determine the mechanism of enhanced OX40L and 41BBL expression in human colorectal tumor cells. Methods: Two colorectal carcinoma cell lines, HCT116 and SW620, were examined for changes in the expression of 41BBL and OX40L in response to inhibition of histone deacetylases (using TSA) and DNA methyltransferases (using 5-Aza-2′-deoxycytidine) to evaluate if epigenetic mechanisms of gene expression can modulate these genes. Tumor cells were treated with radiation, TSA, or 5-Aza-dC, and subsequently evaluated for changes in gene expression using RT-qPCR and flow cytometry. Moreover, we assessed levels of histone acetylation at the 41BBL promoter using chromatin immunoprecipitation assays in irradiated HCT116 cells. Results: Our data indicate that expression of 41BBL and OX40L can indeed be epigenetically regulated, as inhibition of histone deacetylases and of DNA methyltransferases results in increased OX40L and 41BBL mRNA and protein expression. Treatment of tumor cells with TSA enhanced the expression of these genes more than treatment with 5-Aza-dC, and co-incubation of T cells with TSA-treated tumor cells enhanced T-cell survival and activation, similar to radiation. Furthermore, chromatin immunoprecipitation experiments revealed significantly increased histone H3 acetylation of 41BBL promoters specifically following irradiation. Conclusions: Full understanding of specific mechanisms of immunogenic modulation (altered expression of immune relevant genes) of irradiated tumor cells will be required to determine how to best utilize radiation as a tool to enhance cancer immunotherapy approaches. Overall, our results suggest that radiation can be used to make human tumors more immunogenic through epigenetic modulation of genes stimulatory to effector T-cells. Keywords: External beam radiation, Immunogenic modulation, CTLs, Epigenetic, Effector co-stimulation

Combination Treatment with Sublethal Ionizing Radiation and the Proteasome Inhibitor, Bortezomib, Enhances Death-Receptor Mediated Apoptosis and Anti-Tumor Immune Attack

Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity

Sub-Lethal Irradiation of Human Colorectal Tumor Cells Imparts Enhanced and Sustained Susceptibility to Multiple Death Receptor Signaling Pathways

Background: Death receptors (DR) of the TNF family function as anti-tumor immune effector molecules. Tumor cells, however, often exhibit DR-signaling resistance. Previous studies indicate that radiation can modify gene expression within tumor cells and increase tumor cell sensitivity to immune attack. The aim of this study is to investigate the synergistic effect of sub-lethal doses of ionizing radiation in sensitizing colorectal carcinoma cells to death receptor-mediated apoptosis. Methodology/Principal Findings: The ability of radiation to modulate the expression of multiple death receptors (Fas/ CD95, TRAILR1/DR4, TRAILR2/DR5, TNF-R1 and LTbR) was examined in colorectal tumor cells. The functional significance of sub-lethal doses of radiation in enhancing tumor cell susceptibility to DR-induced apoptosis was determined by in vitro functional sensitivity assays. The longevity of these changes and the underlying molecular mechanism of irradiation in sensitizing diverse colorectal carcinoma cells to death receptor-mediated apoptosis were also examined. We found that radiation increased surface expression of Fas, DR4 and DR5 but not LTbR or TNF-R1 in these cells. Increased expression of DRs was observed 2 days post-irradiation and remained elevated 7-days post irradiation. Sub-lethal tumor cell irradiation alone exhibited minimal cell death, but effectively sensitized three of three colorectal carcinoma cells to both TRAIL and Fasinduced apoptosis, but not LTbR-induced death. Furthermore, radiation-enhanced Fas and TRAIL-induced cell death lasted as long as 5-days post-irradiation. Specific analysis of intracellular sensitizers to apoptosis indicated that while radiation di

Functional enhancement of Fas receptor pathway in sub-lethally irradiated colorectal tumor cells.

<p>Human tumor cells were mock-irradiated (0 Gy) or irradiated with 2.5, 5 or 10 Gy and re -cultured for 72 h (3-days). Cells were harvested and incubated with the indicated concentrations of Fas crosslinking antibody CH11 or IgM isotype control antibody for 3 h. A. Non-functional Fas receptor signaling in SW620 cells 72 h post-IR. B. Functional Fas receptor signaling in WiDr cells 72 h post-IR. C. Functional Fas receptor signaling in HCT116 cells 72 h post-IR. Experiment was repeated 3 times with similar results. D. Surface Fas levels, as determined by flow cytometry of stained cells 72 h post-IR.</p

Functional enhancement of TRAIL receptor pathway in sub-lethally irradiated colorectal tumor cells is sustained.

<p>Human tumor cells were mock-irradiated (0 Gy) or irradiated with 2.5, 5 or 10 Gy and re-cultured for the indicated times. Cells were harvested and incubated with the indicated concentrations of recombinant TRAIL protein or media for 3 h. Functional TRAIL receptor signaling in (A) SW620, (B) WiDr and (C) HCT116 cells 72 h post-IR (3-days). SW620 and WiDr cells were incubated with 100 ng/mL of recombinant protein and HCT116 cells were incubated with 25 ng/mL of protein. Experiments were repeated 3 times with similar results. Functional TRAIL receptor signaling in (D) SW620, (E) WiDr, and (F) HCT116 cells 120 h (5-days) post-IR. Experiments were repeated 2 times with similar results.</p

Radiation can modulate surface expression of some but not all TNF family death receptors in colorectal tumor cell lines.

<p>SW620, WiDr and HCT116 cells received 0 (black bar), 2.5 (dark grey bar), 5 (light gray bar) and 10 Gy (white bar) of radiation. Cells were re-cultured for 72 h and then analyzed by flow cytometry for surface (A) LTβR, (B) TNF-R1, (C) DR4, and (D) DR5 surface expression (inset; MFI of DR5 expression is shown for SW620 and HCT116 cells). Percent of cells expressing each death receptor is graphed. Cells stained with a fluorescently-labeled isotype control antibodies were negative (not shown). Staining was repeated 3 times with similar results.</p

Functional enhancement of Fas receptor pathway in sub-lethally irradiated colorectal tumor cells is sustained.

<p>Human tumor cells were mock-irradiated (0 Gy) or irradiated with 2.5, 5 or 10 Gy and re -cultured for 120 h (5-days). Cells were harvested and incubated with the indicated concentrations of Fas crosslinking antibody CH11 or IgM isotype control antibody for 3 h. A. Functional Fas receptor signaling in SW620 cells 120 h post-IR. B. Functional Fas receptor signaling in WiDr cells 120 h post-IR. C. Functional Fas receptor in HCT116 cells 120 h post-IR. Experiment was repeated 3 times with same results.</p

Changes in the expression of anti-apoptotic molecules following tumor cell irradiation.

<p>A–C. Expression of Bcl-X_L and c-FLIP was detected by western blot 72 h post-irradiation in SW620, WiDr, and HCT116 cells. β-actin was used as a loading control. D–E. Comparison of susceptibility to Fas (1 µg/mL) and TRAIL (100 ng/ml) death receptor induced apoptosis. Susceptibility to apoptosis is represented as: (0) denotes no apoptosis as <3%, (+) denotes 3–10%; (++) denotes 11–20%, (+++) denotes 21–30%, and (++++) denotes greater than 31% of cells positive for active caspase-3 in the functional death receptor assays. G–H. Expression of pro-apoptotic proteins Bid, Puma and Bax was detected by western blot 72 h post-irradiation in SW620, WiDr and HCT116 cells. β-actin was used as a loading control. Membranes were probed simultaneously and exposed to film for the same amount of time.</p

Enhanced sensitivity to receptor-mediated cell death by sub-lethal irradiation is apoptotic.

<p>Kinetics of dead and apoptotic cells were analyzed by annexin V–PE/7AAD staining of cells and flow cytometry. A–F. WiDr tumor cells were mock-irradiated (0 Gy) or irradiated with 5 or 10 Gy and re -cultured for 72 h. Cells were harvested and incubated with the indicated concentrations of Fas crosslinking antibody CH11 (A–C) or IgM isotype control antibody (D–F) for 5 h. Percent of apoptotic cells are represented in the upper left quadrant as Annexin-V⁺ and 7AAD⁻. B. Induction of apoptosis (Annexin-V⁺ and 7AAD⁻) following Fas receptor signaling in SW620 cells 120 h post-IR. Cells were treated with 1 µg/ml of CH11 mAb for 5 h. C. Induction of apoptosis (Annexin-V⁺ and 7AAD⁻) following TRAIL receptor signaling in HCT116 cells 72 h post-IR. Cells were incubated with 100 ng/ml of recombinant TRAIL protein for 5 h. Experiment was repeated 3 times with similar results.</p