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

Zinc Transporter ZIP14 Functions in Hepatic Zinc, Iron and Glucose Homeostasis during the Innate Immune Response (Endotoxemia)

<div><p>ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn²⁺ and non-transferrin-bound Fe²⁺ in vitro. Using a <em>Zip14^−/−</em> mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using <em>ZIP14^−/−</em> mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, <em>Zip14^−/−</em> mice absorbed more iron. The <em>Zip14</em> KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of <em>Zip14−/−</em> mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The <em>Zip14^−/−</em> phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The <em>Zip14</em> KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the <em>Zip14^−/−</em> mice and their phenotype shows defects in glucose homeostasis.</p> </div

LPS differentially regulates ZIP14 expression in mice.

<p>Young adult mice received LPS (2 mg/kg, i.p.) or the same volume (0.5 mL) of saline (control), 1–18 hr before being killed. (A) Total RNA was isolated and <i>Zip14</i> mRNA was measured by qPCR and expressed relative to TBP mRNA as the normalizer. ZIP14 protein abundance was measured by western analysis of liver homogenates. Representative western blots from multiple mice (n = 3−4) were measured for ZIP14 abundance by densitometry. (B, C) Zinc concentrations in serum and liver, in µg/mL and µg/g respectively, were measured by AAS. (D, E) Comparison of Zip14 mRNA and ZIP14 protein in WT and <i>Zip14</i> KO mice 18 hr after LPS, as measured by qPCR and western analysis. Values are mean ± SD, n = 3−5. (E) ZIP14 protein is increased at the plasma membrane of hepatocytes of WT mice but not Zip14^−/− mice following LPS. Localization was by confocal microscopy using ZIP14 antibody and Alexa fluor594 secondary antibody and DAPI as the nuclear marker. (F) Serum IL-6 as measured by ELISA was used as an indicator of efficiency of LPS administration. The IL6 response from LPS was attenuated in the <i>Zip14^−/−</i> mice. (* = P<0.05, ** = P<0.01, *** = P<0.001, **** = P<0.0001).</p

Deletion of <i>Zip14</i> in mice produces altered glucose homeostasis and IR functions.

<p>(A) Body composition of the WT and Zip14^−/− female mice was measured using a NMR Lean/Fat analyzer. (B) Serum and liver glucose from fed-mice were measured by OneTouch UltraMini and colorimetrically, respectively. (C, D) Serum insulin and liver cAMP were measured by ELISA. (E, F) Western analysis results from liver of three mice are shown for each treatment group. (G) Total RNA was isolated from livers and relative transcript abundance for GLUT2, PEPCK, SREBP-1c, FASN and SOCS-3 were measured by qPCR and expressed relative to TBP mRNA as the normalizer. Values are mean ± SE, n = 3−5.</p

<i>Zip14</i> null mice do not have depressed serum zinc after LPS administration.

<p>WT and <i>Zip14^−/−</i> mice were given LPS or saline administration (2 mg/kg; 0.5 mL; i.p.) 18 hr before being killed. (A, B) Zn concentrations in serum and liver were measured by AAS. (C, D) Global analysis of liver Zip and ZnT transcripts in KO and after LPS. Total RNA was isolated from the liver and <i>Zip14</i> mRNA was measured by qPCR and normalized to TBP mRNA. Values are mean ± SD, n = 3−5. (E) ZIP4 protein abundance in liver was shown by western analysis.</p

<i>Zip14^−/−</i> mice exhibit normal iron absorption but altered iron homeostasis.

<p>(A) WT and Zip14^−/− mice were administered LPS (2 mg/kg or saline 0.5 mL; ip), 18 hr before being killed. (A) Serum and liver iron concentrations were measured by AAS. Liver non-heme-iron was measured colorimetrically. (B) Fasted WT and <i>Zip14^−/−</i> mice received 2 µCi of ⁵⁹Fe by gavage and were killed 24 hr later. Percent absorption was calculated from the radioactivity administered. Serum and liver iron uptake was calculated from the specific activity of the ⁵⁹Fe. (C) Transcript abundance for liver hepcidin, TfR-1, DMT1 and ferritin was measured by qPCR and expressed relative to TBP mRNA as the normalizer. Values are mean ± SE, n = 5−10.</p