G1 arrest and differentiation can occur independently of Rb family function

Repression of E2F target genes is required for cell cycle arrest in Rb family (Rb, p107, and p130)-deficient cells

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

The influence of therapeutic radiation on the patterns of bone marrow in ovary-intact and ovariectomized mice.

The functional components of bone marrow (i.e., the hematopoietic and stromal populations) and the adjacent bone have traditionally been evaluated incompletely as distinct entities rather than the integrated system. We perturbed this system in vivo using a medically relevant radiation model in the presence or absence of ovarian function to understand integrated tissue interaction.Ovary-intact and ovariectomized mice underwent either no radiation or single fractional 16 Gy radiation to the caudal skeleton (I ± R, OVX ± R). Marrow fat, hematopoietic cellularity, and cancellous bone volume fraction (BV/TV %) were assessed. Ovariectomy alone did not significantly reduce marrow cellularity in non-irradiated mice (OVX-R vs. I-R, p = 0.8445) after 30 days; however it impaired the hematopoietic recovery of marrow following radiation exposure (OVX+R vs. I+R, p = 0.0092). The combination of radiation and OVX dramatically increases marrow fat compared to either factor alone (p = 0.0062). The synergistic effect was also apparent in the reduction of hematopoietic marrow cellularity (p = 0.0661); however it was absent in BV/TV% changes (p = 0.2520). The expected inverse relationship between marrow adiposity vs. hematopoietic cellularity and bone volume was observed. Interestingly compared with OVX mice, intact mice demonstrated double the reduction in hematopoietic cellularity and a tenfold greater degree of bone loss for a given unit of expansion in marrow fat.Ovariectomy prior to delivery of a clinically-relevant focal radiation exposure in mice, exacerbated post-radiation adipose accumulation in the marrow space but blunted bone loss and hematopoietic suppression. In the normally coupled homeostatic relationship between the bone and marrow domains, OVX appears to alter feedback mechanisms. Confirmation of this non-linear phenomenon (presumably due to differential radiosensitivity) and demonstration of the mechanism of action is needed to provide strategies to diminish the effect of radiation on exposed tissues

Semi-quantitative assessment of vascular abnormalities in intact control (I−R) and irradiated (I+R) mice estimated by a board certified veterinary clinical pathologist (LS) according to standard protocols.

<p>Numerical scale: 0 not present, +1 mild, +2 moderate, +3 marked, +4 severe. Radiation caused moderate to marked sinusoidal dilation and venous congestion on days 3 and 8 that was mostly resolved by Day 30, and transient edema on Day 8. Wilcoxon rank-sum test:</p>*<p>Irradiated (marginally) significantly greater sinusoidal dilation compared with control of the same day, Day 3, p = 0.0507; Day 8, p = 0.0182.</p>†<p>Irradiated significantly greater vascular congestion compared with control of the same day, Day3, p = 0.0247; Day8, p = 0.0217.</p>‡<p>Irradiated marginally significantly greater edema compared with control of the same day, Day 8, p = 0.0507.</p

Megakaryocyte number and myeloid: erythroid ratio in intact control (I−R) and irradiated (I+R) mice estimated by a board certified veterinary clinical pathologist (LS) according to standard protocols.

<p>Cells contain the data for individual mice (n = 5/group); N = normal, NA = too few cells to reliably estimate. Numerical scale: −4 severely decreased, −3 markedly decreased, −2 moderately decreased, −1 mildly decreased, +1 mildly increased, +2 moderately increased, +3 markedly increased, +4 severely increased, normal assigned a value of 0 for statistical calculations. Megakaryocyte numbers were significantly depressed by radiation exposure on Days 3 and 8, with a trend towards ongoing suppression on Day 30. Too few hematopoietic precursors were available for interpretation in the markedly hypocellular Day 3 post-irradiation marrow, but the recovery phase was accompanied by significantly increased myeloid to erythroid ratios characterized by persistent erythroid hypoplasia. Wilcoxon rank-sum test:</p>*<p>Megakaryocytes, Irradiated significantly decreased compared with Control of the same day, Day 3, p = 0.0182; Day 8, p = 0.0236; Day 30, p = 0.0450.</p>†<p>Myeloid:Erythroid ratio, Irradiation significantly increased when compared with Control, Day8, p = 0.0182; Day 30, p = 0.0236.</p

Experimental Plan Schematic.

<p>Sixteen week old BALB/c mice were ovariectomized (OVX) and maintained in the vivarium for 57 days in order to attain skeletal hemostasis. Both intact (I) and OVX mice were then irradiated with 16Gy delivered to the caudal skeleton or maintained as controls. Groups of animals were euthanized at 3, 8, and 30 days post irradiation in order to perform histological evaluations of the distal femur; microCT measurements were conducted at the 30 day time point only.</p