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

Microbiota of the alimentary tract of children - implications for allergy and inflammatory bowel disease

Allergy, which is the most common chronic disease in Swedish children and adolescents, is associated with a high standard of living and Western lifestyle. According to the hygiene hypothesis, allergy is due to inadequate stimulation of the immune system by microbes during early childhood, leading to failed maturation of the immune system. The incidences of inflammatory bowel diseases, i.e., ulcerative colitis and Crohn’s disease, have also increased dramatically in Western countries over the last few decades, and currently, these diseases are often diagnosed already in childhood. Epidemiological evidence suggests links between alterations to the intestinal microbiota and these diseases. Thus, studies of the composition of the bacterial microbiota in infants and young children are of relevance for the pathogenesis of both allergic diseases and inflammatory bowel diseases. In this thesis, quantitative culture and DNA-based methods are compared for their abilities to characterize the gut microbiota in infants. Terminal-Restriction Fragment Length Polymorphism (T-RFLP) is based on differences in the 16S rRNA gene sequences between bacteria, as revealed by differences in fragment sizes after restriction enzyme digestion. A database was constructed to identify bacteria based on their fragment sizes. Multi-parallel sequencing of the 16S rRNA gene by pyrosequencing and T-RFLP were compared for sensitivity with quantitative culture of infant fecal samples. Bacterial genera that were present at >106 colony forming units/g feces, as determined by culture, were generally readily detected by DNA-based methods, with the exception of bifidobacteria, which generated only one sequence read per 108 viable bacteria. Clinically and immunologically relevant facultative bacteria, e.g., staphylococci, were often missed by the DNA-based methods due to having low counts in the fecal samples. The studies presented in this thesis indicate that cultivation and molecular-based assays are complementary in generating an overall picture of the complex gut microbiota. In the ALLERGYFLORA cohort study, T-RFLP was used to analyze the salivary microbiota in 4-month-old infants whose parents had the habit of “cleaning” their pacifier by sucking on it, and of control children whose parents did not have this habit. Sharing of the pacifier between parent and infant was associated with reduced risk of the child developing an allergy and altered salivary microbiota in the child. We hypothesize that the oral bacteria transmitted from the parents stimulate the child's immune system in such a way that allergy development is avoided. Samples of the duodenal fluids of children with newly diagnosed and untreated inflammatory bowel diseases (ulcerative colitis and Crohn's disease) and controls (having functional bowel disorders without signs of intestinal inflammation) were analyzed by culture and pyrosequencing. The microbiota of children with ulcerative colitis displayed lower bacterial diversity than that of the control children, and certain bacterial groups were less abundant in the former group. Taken together, the studies presented in this thesis suggest that the compositions of the commensal microbiota in the oral cavity and small intestine affect the risk of developing immunoregulatory diseases, such as allergies and inflammatory bowel diseases

Dietary oat bran reduces systemic inflammation in mice subjected to pelvic irradiation

Patients undergoing radiotherapy to treat pelvic-organ cancer are commonly advised to follow a restricted fiber diet. However, reducing dietary fiber may promote gastrointestinal inflammation, eventually leading to deteriorated intestinal health. The goal of this study was to evaluate the influence of dietary fiber on radiation-induced inflammation. C57BL/6J male mice were fed a High-oat bran diet (15% fiber) or a No-fiber diet (0% fiber) and were either irradiated (32 Gy delivered in four fractions) to the colorectal region or only sedated (controls). The dietary intervention started at 2 weeks before irradiation and lasted for 1, 6, and 18 weeks after irradiation, at which time points mice were sacrificed and their serum samples were assayed for 23 cytokines and chemokines. Our analyses show that irradiation increased the serum cytokine levels at all the time points analyzed. The No-fiber irradiated mice had significantly higher levels of pro-inflammatory cytokines than the High-oat irradiated mice at all time points. The results indicate that a fiber-rich oat bran diet reduces the intensity of radiation-induced inflammation, both at an early and late stage. Based on the results, it seems that the advice to follow a low-fiber diet during radiotherapy may increase the risk of decreased intestinal health in cancer survivors

Irradiation Induces Tuft Cell Hyperplasia and Myenteric Neuronal Loss in the Absence of Dietary Fiber in a Mouse Model of Pelvic Radiotherapy

Pelvic radiotherapy is associated with chronic intestinal dysfunction. Dietary approaches, such as fiber enrichment during and after pelvic radiotherapy, have been suggested to prevent or reduce dysfunctions. In the present paper, we aimed to investigate whether a diet rich in fermentable fiber could have a positive effect on radiation-induced intestinal damage, especially focusing on tuft cells and enteric neurons. Male C57BL/6 mice were fed either a purified non-fiber diet or the same purified diet with 5% or 15% oat fiber added, starting two weeks prior to sham-irradiation or irradiation with four fractions of 8 Gray. The animals continued on the diets for 1, 6 or 18 weeks, after which the gross morphology of the colorectum was assessed together with the numbers of enteric neurons, tuft cells and crypt-surface units. The results showed that dietary fiber significantly affected the intestinal morphometrics, both in the short and long-term. The presence of dietary fiber stimulated the re-emergence of crypt-surface unit structures after irradiation. At 18 weeks, the animals fed with the non-fiber diet displayed more myenteric neurons than the animals fed with the dietary fibers, but irradiation resulted in a loss of neurons in the non-fiber fed animals. Irradiation, but not diet, affected the tuft cell numbers, and a significant increase in tuft cells was found 6 and 18 weeks after irradiation. In conclusion, dietary fiber intake has the potential to modify neuronal pathogenesis in the colorectum after irradiation. The long-lasting increase in tuft cells induced by irradiation may reflect an as yet unknown role in the mucosal pathophysiology after pelvic irradiation

Low-complexity microbiota in the duodenum of children with newly diagnosed ulcerative colitis

<div><p>Background</p><p>Inflammatory bowel disease (IBD) is characterized by gut dysbiosis. To date, the large bowel microbiota has been in focus. However, the microbiota of the small intestine may also be of importance, as the small bowel is a site for the induction and control of mucosal immune responses, which can be modulated by constituents of the local microbiota.</p><p>Methods</p><p>Duodenal fluids were collected during diagnostic work-up of treatment-naïve children who were suspected of having IBD. The duodenal fluids were analyzed by pyrosequencing (average of 32,000 reads/sample, read length of 500 nucleotides). After diagnosis, the duodenal microbiota of subjects with ulcerative colitis (N = 8) or Crohn’s disease (N = 5), and non-IBD controls (N = 8) were compared.</p><p>Results</p><p>Pyrosequencing revealed that the duodenal microbiota of children with ulcerative colitis contained fewer Operational Taxonomic Units (OTUs) per individual than the duodenal microbiota of the controls (<i>P</i> = 0.005). This reduction in richness of the duodenal microbiota was seen for three major phyla: <i>Firmicutes</i>, <i>Actinobacteria</i>, and <i>Bacteroidetes</i>. Several bacterial genera were detected less frequently in the children with ulcerative colitis than in the non-IBD controls, including <i>Collinsella</i> (<i>P</i> = 0.001), <i>Lactobacillus</i> (<i>P</i> = 0.007), and <i>Bacillus</i> (<i>P</i> = 0.007), as well as a non-identified member of the order <i>Sphingobacteriales</i> (<i>P</i> = 0.007).</p><p>Conclusions</p><p>In this pilot study, we show that the duodenal microbiota of children with ulcerative colitis exhibits reduced overall richness, despite the fact that the inflammation is primarily localized to the colon. These results should be corroborated in a larger study.</p></div