31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Noninvasive whole-body imaging of phosphatidylethanolamine as a cell death marker using 99mTc-duramycin during TNF-induced SIRS

Systemic inflammatory response syndrome (SIRS) is an inflammatory state affecting the whole body. It is associated with the presence of pro- and antiinflammatory cytokines in serum, including tumor necrosis factor (TNF). TNF has multiple effects and leads to cytokine production, leukocyte infiltration, and blood pressure reduction and coagulation, thereby contributing to tissue damage and organ failure. A sterile mouse model of sepsis, TNF-induced SIRS, was used to visualize the temporal and spatial distribution of damage in susceptible tissues during SIRS. For this, a radiopharmaceutical agent, Tc-99m-duramycin, that binds to exposed phosphatidylethanolamine on dying cells was longitudinally visualized using SPECT/CT imaging. Methods: C57BL/6J mice were challenged with intravenous injections of murine TNF or vehicle, and necrostatin-1 was used to interfere with cell death. Two hours after vehicle or TNF treatment, mice received Tc-99m-duramycin intravenously (35.44 +/- 3.80 MBq). Static whole-body Tc-99m-duramycin SPECT/CT imaging was performed 2, 4, and 6 h after tracer injection. Tracer uptake in different organs was quantified by volume-of-interest analysis using PMOD software and expressed as SUVmean. After the last scan, ex vivo biodistribution was performed to validate the SPECT imaging data. Lastly, terminal deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed to correlate the obtained results to cell death. Results: An increased Tc-99m-duramycin uptake was detected in mice injected with TNF, when compared with control mice, in lungs (0.55 +/- 0.1 vs. 0.34 +/- 0.05), intestine (0.75 +/- 0.13 vs. 0.56 +/- 0.1), and liver (1.03 +/- 0.14 vs. 0.64 +/- 0.04) 4 h after TNF and remained significantly elevated until 8 h after TNF. The imaging results were consistent with ex vivo gamma-counting results. Significantly increased levels of tissue damage were detected via TUNEL staining in the lungs and intestine of mice injected with TNF. Interestingly, necrostatin-1 pretreatment conferred protection against lethal SIRS and reduced the Tc-99m-duramycin uptake in the lungs 8 h after TNF (SUV, 0.32 +/- 0.1 vs. 0.51 +/- 0.15). Conclusion: This study demonstrated that noninvasive Tc-99m-duramycin SPECT imaging can be used to characterize temporal and spatial kinetics of injury and cell death in susceptible tissues during TNF-induced SIRS, making it useful for global, whole-body assessment of tissue damage during diseases associated with inflammation and injury

Acidification of forested podzols in North Belgium during the period 1950-2000

Acidification of forest soils in Europe and North America has been an important concern over the last decades. The last area-covering survey of forest soil acidification in Flanders (North Belgium) goes back to 1985 [Ronse A, De Temmerman L, Guns M, De Borger R. Evolution of acidity, organic matter content, and CEC in uncultivated soils of North Belgium during the past 25 years. Soil Sci; 146, (1988), 453-460] and highlighted a significant acidification of the upper layer (0.3-0.4 m) of forested podzols during the period 1950-1985. The present study aimed to assess (1) to what extent further acidification of forested podzols occurred during the period 1985-2000 at different depths and (2) whether the average annual acidification rate accelerated or slowed down between 1985 and 2000 compared to the period 1950-1985. Average soil pH-KCl values of podzols in northern Belgium dropped during the period 1985-2000. This decline extends to a depth of about 50 cm but was most pronounced and significant in the A horizon. In the A0, A1 and A2 horizons, average pH dropped with 0.2, 0.3 and 0.1 units, and in the Bir and C horizons with 0.1 units. No change in average pH value occurred in the Bh horizon. Average annual acidification rate of the A1 horizon was significantly higher in the period 1985-2000 than in the period 1950-1985. Changes in pH occurred in the entire soil profile during the period 1950/67-1985 likely because sulphate was the major form of acid deposition before 1985. After 1985, acid sulphur deposition decreased with more than 50% in North Belgium. In contrast, ammonium deposition almost doubled between 1950 and 1980, which may explain why soil acidification between 1985 and 2000 has been restricted to the upper soil horizons

Recovery of post-agricultural forest: tree species determines soil properties and possibilities for ecosystem restoration

Reforestation of agricultural land in many European countries has progressed more slowly than anticipated. Many years’ fertilization has caused an accumulation of nutrients in the top soil layer. Phosphorus (P) has proven to be particularly persistent and elevated concentrations in soil and vegetation are found up to thousands of years after agricultural abandonment. This persistently elevated bioavailable P is a paradox for ecosystem recovery in post-agricultural forests: on one hand, higher P bioavailability stimulates stand productivity and tree growth but, on the other hand, it limits the development of typical forest plant communities (Baeten et al. in press) and associated organisms. Limitation of bioavailable P seems to be a necessity for the recovery of species-rich target communities and for the conservation of endangered species (Wassen et al. 2005). Reforestation of agricultural land results in a decrease in soil pH with increasing forest age, but the extent and rate of soil acidification differs highly between tree species. Gymnosperm tree species are often contrasted to angiosperms because of their acidifying impact on soils. We will present data showing that rapid (within 2 decades) changes in soil properties can also occur beneath different broadleaved tree species. Ca concentra¬tions in leaf litter seem to be the driving factor for differences between species: tree species with higher litter Ca concentrations such as Populus and Fraxinus support a significantly higher soil pH, base saturation degree and lower quantities of bioavailable aluminum compared to the Ca-poor Fagus and Quercus species. Tilia and Prunus trees are intermediate between these ‘soil preservers’ and ‘soil acidifiers’. Furthermore, tree species have a major influence on the short-term and long-term bioavailability of P. Below ‘soil acidifiers’ we found significantly higher bioavailable and slowly cycling P fractions compared to ‘soil preservers’ which mainly retained P in less soluble pools. To date, there are no integrated studies on the long-term effect of different broadleaved tree species on soil properties in general and the bioavailability of P through soil acidification in particular. Most studies evaluating the bioavailability of P use extraction methods that only provide short-term dose-response insights into the readily available P pool for plants and soil microbes. However, for studying the role of P in recovering (semi)natural ecosystems, it is much more relevant to gain insight into time-scales extending a single growing season (Richter et al. 2006) and to include more slowly cycling P fractions. We discuss six common garden experiments in which broadleaved tree species have been planted on loamy agricultural soil for between 5 and 35 years. We argue that vegetation choice can be an important driver of regional biogeochemistry and biodiversity