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

Application of machine vision to determine the density of dingo teeth

Wild dogs are regarded as a pest by the Australian livestock industry. Determination of the age of free-ranging animals would be useful in developing and assessing the effectiveness of management strategies for such fauna. We have been examining techniques for determining the age of wild dogs and dingoes, using canine teeth removed from the skulls of known age animals. The skulls were obtained from a collection of wild dog skulls held at CSIRO Sustainable Ecosystems, Canberra

Bat fat: body composition analysis by total body electrical conductivity in northern long-eared bats

The ability to assess the 'condition' of small mammals in a non-invasive and non-stressful manner is important for understanding their natural history. More specifically, body condition is measured to determine if an animal is in positive or negative energy balance. Stored energy is a component of a total energy budget. One recent technology for assessing body condition is Total Body Electrical Conductivity (TOBEC). A TOBEC index should correlate with components of more traditional body composition analyses, such as body fat and lean mass. As part of our efforts to understand bat hibernation energetics, we made TOBEC measurements (EM-SCAN Model 3044) of northern long-eared bats (Myotis septentrionalis) to determine its accuracy and usefulness on animals of this size (6-8 g). The bats were then euthanized, dried to a constant mass, subjected to a Soxhlet fat extraction process, and re-dried to constant mass. Standard body composition parameters were calculated. A preliminary analysis of these data indicate no significant correlations between the raw TOBEC measurements and carcass lean wet mass, fat mass, or percent body fat. These animals may be too small for use of TOBEC in assessing their energy storage, although additional calculations and a larger sample size may prove useful

Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, antechinus flavipes

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC

Heterogeneity of alpha-cardiac myosin heavy chains in a small marsupial, Antechinus flavipes, and the effect of hypothyroidism on its ventricular myosins

The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin components were found: V1a, V1b, V1c, V2 and V3, in order of decreasing mobility. In western blots, V1a, V1b, and V1c reacted with anti-a-MyHC antibody, but not with anti-B-MyHC, whereas V2 and V3 reacted with anti-B-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which bound anti-a-MyHC antibody while the third bound anti-B-MyHC antibody. We conclude that V1a, V1b, V1c are triplets arising from the dimerization of two distinct a-MyHC isoforms. Hypothyroidism, veriWed by metabolic studies, decreased a-MyHC content significantly (t-test, P < 0.001) from 91.6 5.9% (SEM, n = 4) in control animals to 67.2 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in B-MyHC content. We conclude that in adult marsupials, ventricular myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians