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

A low gastric pH mouse model to evaluate live attenuated bacterial vaccines.

The low pH of the stomach serves as a barrier to ingested microbes and must be overcome or bypassed when delivering live bacteria for vaccine or probiotic applications. Typically, the impact of stomach acidity on bacterial survival is evaluated in vitro, as there are no small animal models to evaluate these effects in vivo. To better understand the effect of this low pH barrier to live attenuated Salmonella vaccines, which are often very sensitive to low pH, we investigated the value of the histamine mouse model for this application. A low pH gastric compartment was transiently induced in mice by the injection of histamine. This resulted in a gastric compartment of approximately pH 1.5 that was capable of distinguishing between acid-sensitive and acid-resistant microbes. Survival of enteric microbes during gastric transit in this model directly correlated with their in vitro acid resistance. Because many Salmonella enterica serotype Typhi vaccine strains are sensitive to acid, we have been investigating systems to enhance the acid resistance of these bacteria. Using the histamine mouse model, we demonstrate that the in vivo survival of S. Typhi vaccine strains increased approximately 10-fold when they carried a sugar-inducible arginine decarboxylase system. We conclude that this model will be a useful for evaluating live bacterial preparations prior to clinical trials

<b>Bacterial strains and plasmids used in this study.</b>

a<p>In genotype descriptions, the subscripted number refers to a composite deletion and insertion of the indicated gene. P, promoter; TT, T4 ip III transcription terminator; <i>ori</i>, origin of replication; Kan^R, kanamycin resistance; Str/Spc^R, streptomycin/spectinomycin resistance; Amp^R, ampicillin resistance.</p

Gastric pH following histamine injection.

<p>Following a 6 h fast, mice were injected at time 0 with 10/kg histamine. The pH of the gastric contents was monitored for 4 h post histamine injection. Data shown are the mean and standard error of the mean of at least five mice per time point.</p

Survival of strains cultured under non-acid resistance inducing conditions.

<p>Wild-type enteric strains were grown in LB medium to late-log phase under aerobic conditions. <b>(A)</b> Cells were challenged in EG medium (pH 3.0) containing 0.1% casamino acids. Survival during EG medium challenge was assayed hourly for 4 h by plating onto LB agar. Data shown are the mean and SEM of three independent experiments. <b>(B)</b> Mice were either fasted for 6 h (fasted mouse model) or fasted and low gastric pH was induced by histamine injection (histamine mouse model) and then inoculated with 10⁹ CFU of each strain. Sixty min after inoculation, mice were euthanized and the entire small intestine removed and homogenized. Strain survival was assayed by plating onto LB agar containing kanamycin. Data are expressed as the percent of initial inoculum recovered (% survival). The geometric mean and 95% confidence interval of two independent experiments (8 mice total) is depicted.</p

Effect of arginine decarboxylase on the gastric survival of <i>S.</i> Typhi.

<p>Pairs of attenuated <i>S.</i> Typhi strains differing only in their arginine decarboxylase locus were grown to stationary phase in EGA medium under aerobic conditions. Cells were combined in a 1:1 ratio in PBS containing 1 mM arginine. Low gastric pH was induced by histamine injection in mice fasted for 6 h. Mice were inoculated with 10⁹ CFU of each strain. Sixty min after inoculation, mice were euthanized and the entire small intestine removed and homogenized. Strain survival was assayed by plating onto LB agar containing kanamycin or streptomycin. Data shown are the competitive index of the two strains in each mouse with the geometric mean of two independent experiments (10 mice total) indicated as a solid line.</p

Visualization of the bacterial inoculum in the gastrointestinal tract via light emission.

<p>Low gastric pH was induced in mice by histamine injection prior to inoculation with 1×10⁹ CFU of <i>S.</i> Typhi Ty2(pGEN-<i>luxCDABE</i>). At 30, 60 and 90 min post-inoculation, the gastrointestinal tract was removed and examined for the production of light by luciferase. A visible signal is equivalent to approximately 5×10⁵ CFU. Luminescence is reported as the number of photons detected per s per square cm. Images shown are representative results from groups of seven mice.</p

Correlation between in vitro and in vivo survival.

<p>The log₁₀ of the geometric mean number of CFU that survived in vitro challenge at pH 3.0 for 2 h was plotted against the log₁₀ of the geometric mean number of CFU recovered from the intestinal tissue in the <b>(A)</b> histamine mouse model or <b>(B)</b> fasted mouse model. Linear regression was performed on each data set and the r² value of the best-fit line is depicted for each model.</p

Acquisition of Dutch as a Second Language: The Explanative Power of Cognate and Genetic Linguistic Distance Measures for 11 West-European First Languages.

Contains fulltext : 86583.pdf (publisher's version ) (Open Access)32 p