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

Mono-(2-Ethylhexyl) Phthalate Promotes Pro-Labor Gene Expression in the Human Placenta.

Women exposed to phthalates during pregnancy are at increased risk for delivering preterm, but the mechanism behind this relationship is unknown. Placental corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2) are key mediators of parturition and are regulated by the non-canonical NF-kB (RelB/p52) signaling pathway. In this study, we demonstrate that one of the major phthalate metabolites, mono-(2-ethylhexyl)-phthalate (MEHP), increased CRH and COX-2 mRNA and protein abundance in a dose-dependent manner in primary cultures of cytotrophoblast. This was coupled with an increase in nuclear import of RelB/p52 and its association with the CRH and COX-2 promoters. Silencing of NF-kB inducing kinase, a central signaling component of the non-canonical NF-kB pathway, blocked MEHP-induced upregulation of CRH and COX-2. These results suggest a potential mechanism mediated by RelB/p52 by which phthalates could prematurely induce pro-labor gene activity and lead to preterm birth

MEHP upregulates expression of CRH and COX-2 in primary cultures of human cytotrophoblast.

<p>Primary cultures of term cytotrophoblast were exposed to different concentrations of MEHP for 24 hours with DMSO as the vehicle control. (<b>A</b>) Trypan blue exclusion test of cell viability for primary cytototrophoblast exposed to MEHP at concentrations as indicated (N = 3 independent experiments). (<b>B</b>) Representative gel patterns and analysis in whole cell lysates from three independent term placentas by Western blot assay with use of antibody as indicated. (<b>C</b>) Total RNAs were extracted and RT-qPCR was performed for assessment of CRH and COX-2 mRNA levels with normalization to GAPDH mRNA level. Bars represent the average of relative mRNA levels and error bars represent standard deviation from experiments performed in three independent term placentas. * p < 0.05; ** p < 0.01.</p

The effects of MEHP exposure on interaction of RelB/p52 with the <i>CRH/COX-2</i> gene promoters.

<p>Term cytotrophoblast were exposed to MEHP for 24 hrs at concentrations as indicated. ChIP assays were performed to determine occupancy of RelB/p52 at <i>CRH</i> (<b>A</b>) and <i>COX-2</i> (<b>B</b>), and also occupancy of RelA at the <i>CRH</i> (<b>C</b>) and <i>COX-2</i> (<b>D</b>) gene promoter. Fold enrichment was derived with normalization to a human DNA a satellite, a non-coding DNA sequence to which no transcriptional factors should bind. Rabbit IgG was used as a non-specific control. The bars indicate the average of fold enrichment with error bars representing the standard deviation from three independent experiments. * p < 0.01.</p

NIK knockdown attenuates MEHP-induced upregulation of <i>CRH</i> and <i>COX-2</i> in the human placenta.

<p>Primary term cytotrophoblast were incubated with siRNAs targeting NIK (siNIK) for 24 hrs, and then treated with MEHP for an additional 24 hrs. Scramble siRNA (Scr-siRNA) was used as the non-targeting control. Total RNAs were extracted and RT-qPCR was performed to determine mRNA levels of CRH (<b>A</b>), COX-2 (<b>B</b>), or NIK (<b>C</b>) (N = 3 independent experiments). * p < 0.05 (compared to DMSO). ** p< 0.01 (compared to DMSO). ## p < 0.01 (compared to Scr-siRNA). (<b>D</b>) Representative gel pattern of Western blot analysis on whole cell lysates from three independent experiments. (<b>E</b>) IF staining was performed to directly assess intracellular distribution of RelB. These images represent data obtained from three independent placentas. Original magnification, 200Χ.</p

<i>Bacillus anthracis</i> Inosine 5′-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion‑, Substrate‑, and Product-Bound Complexes

Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of <i>Bacillus anthracis</i> IMPDH, in a phosphate ion-bound (termed “apo”) form and in complex with its substrate, inosine 5′-monophosphate (IMP), and product, xanthosine 5′-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known <i>Cryptosporidium parvum</i> IMPDH inhibitors was examined for the <i>B. anthracis</i> enzyme and compared with those of three bacterial IMPDHs from <i>Campylobacter jejuni</i>, <i>Clostridium perfringens</i>, and <i>Vibrio cholerae</i>. The structures contribute to the characterization of the active site and design of inhibitors that specifically target <i>B. anthracis</i> and other microbial IMPDH enzymes