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

Effects of cytokines on preadipocyte differentiation and Wnt signalling

The relationship between amount of adipose tissue, the Metabolic Syndrome and Type 2 diabetes has been recognized for several years. However, the mechanisms for this remain to be clarified. Not all obese individuals develop insulin resistance or Type 2 diabetes and these perturbations are also seen in non-obese individuals. Adipose cell enlargement is a marker of obesity, but adipocytes have a limited capacity for lipid storage. Adipose cell growth and differentiation are important steps for normal lipid storage. Interestingly, obesity and insulin resistance are associated with an inflamed adipose tissue and infiltration of macrophages. Thus, the cytokine levels are expected to be increased in the adipose tissue under those conditions. This thesis is focused on elucidating the consequences of increased adipose tissue cytokine levels on preadipocyte development and function.We first measured interstitial IL-6 concentrations (Paper I) and found that local IL-6 concentrations in the adipose tissue were markedly higher than in plasma. Interestingly, the interstitial IL-6 concentrations were positively correlated with adipose cell size. These high concentrations suggest that IL-6 might act as an autocrine/paracrine regulator in the adipose tissue. Adipose tissue biopsies were also incubated in vitro to examine the effects of IL-6. Several important adipose tissue differentiation markers such as adiponectin, aP2 and PPARgamma2 were decreased. Thus, high local IL-6 concentrations, as seen in obesity, can impair the differentiation of the adipose cells.In Paper II, we characterized the effects of PPARgamma and C/EBPalpha of adiponectin and aP2 expression. We differentiated C/EBPalpha-/- fibroblasts and found that PPARgamma2 was necessary for adiponectin expression but C/EBPalpha was required for full gene activation. We also found that the PPARgamma ligand, pioglitazone, increased adiponectin expression in the absence of C/EBPalpha suggesting that the adiponectin promoter contains functional PPRE elements. Both IL-6 and TNFalpha reduced the expression of the differentiation markers adiponectin and aP2. However, over-expressing C/EBPalpha prevented this effect of IL-6.In Paper III, we found that IL-6 impaired the terminal differentiation of preadipocytes to adipose cells and reduced the lipid accumulation. However, TNFalpha completely prevented differentiation. IL-6, like TNFalpha, reduced the expression of most genes related to normal adipocyte function, insulin signalling and action. Remarkably, the canonical Wnt signalling pathway remained activated in the presence of either IL-6 or TNFalpha. This was associated with low axin and high beta-catenin, thereby keeping the cells in a proliferative mode and preventing the terminal differentiation. Instead, both IL-6 and TNFalpha promoted an inflammatory phenotype of the (pre)adipocytes.CONCLUSIONS: Interstitial IL-6 levels in the adipose tissue were markedly higher than circulating IL-6 concentrations and correlated positively with adipose cell size. The inhibitory effect of IL-6 on adiponectin mRNA levels was prevented by over-expressing C/EBPalpha. Both IL-6 and TNFalpha impaired, or prevented, the normal preadipocyte differentiation to mature adipose cells and, instead, promoted an inflammatory phenotype. This was associated with a maintained Wnt signalling, thus preventing the normal differentiation. Together, these findings provide an explanation to why obesity is associated with insulin resistance, inflammation and ectopic lipid accumulation in other tissues

Type 2 Diabetes, Independent of Obesity and Age, is Characterized by Senescent and Dysfunctional Mature Human Adipose Cells

Obesity with dysfunctional adipose cells is the major cause of the current epidemic of T2D. We examined senescence in human adipose tissue cells from age- and BMI-matched lean, obese and obese T2D individuals and found mature and fully differentiated adipose cells from obese and, more pronounced, from T2D individuals to exhibit increased senescence similar to what we previously have shown in the progenitor cells. Degree of adipose cell senescence was positively correlated with whole-body insulin resistance and adipose cell size. Adipose cell protein analysis revealed dysfunctional cells in T2D with increased senescence markers, reduced PPARγ, GLUT4 and pS473AKT. Consistent with a recent study, we found the cell cycle regulator cyclin D1 to be increased in obese cells but also further elevated in T2D cells, closely correlating with senescence markers, ambient donor glucose and, more inconsistently, with plasma insulin levels. Furthermore, fully differentiated adipose cells were susceptible to experimentally induced senescence, to conditioned medium increasing cyclin D1 and also responsive to senolytic agents. Thus, fully mature human adipose cells from obese and, more pronounced, T2D subjects become senescent and SASP secretion by senescent progenitor cells can play an important role in addition to donor hyperinsulinemia

Toxicitetstest för pesticider med protozoer

I Sverige har man levt i förvissningen om att kemiska bekämpningsmedel bryts ner så effektivt i jorden att de inte kunde nå vattendrag och än mindre grundvatten. Uppståndelsen blev därför stor när vi fann halter i skånska åar som kunde skada såväl växt- och djur-livet i åarna som en del känsliga grönsakskulturer på land. Detta var i och för sig ingen ny kunskap, men att det var så pass utbrett viste vi inte, även om vi kunde räka oss till det på grund av våra studier av bekämpningsmedlens rörlighet i åkermark och på grund av utländska erfarenheter. (Se Ekohydrologi nr 20.) En annan viktig fråga är hur de marklevande små krypen mår när de duschas med sprutvätskor. I den två uppsatserna Toxicitetstest för pesticider med protozoer och Bekämpningsmedel - Utlakning från åkermark (tidigare publicerad i Konsulentavdelningens rapporter. Allmänt 84) behandlas dessa livsviktiga frågor. I motsats till bekämpningsmedlens sidoeffekter har växtnäringsämnenas betydelse för vattendrag, sjöar, hav och grundvatten diskuterats i årtionden mot bakgrunden av en omfattande forskning på alla fronter. Våra bidrag gäller jordbrukets andel och omfattar dels en registrerande, dels en orsakanalyserande del. Det förstnämnda löper i Naturvårdsverkets Program för övervakning av miljökvalitet (PMK)med försök i stora åkerskiften och det det sistnämnda i särskilt anlagda storruteförsök där olika styrande faktorer kan bemästras. Uppsatsen Odlingsårgärders inverkan på kvalitet hos yt- och grundvatten är vår senaste årsrapport till PMK och Lakning av fosfor ur jordar är en specialundersökning som knutits till PMK-nätet. Ekonomiska styrmedel är troligen det mest effektiva för att dämpa kväveläckaget. Konstgödselpris och spannmålspris inbegripet kvalitetsbetalning för protein är faktorer som påverkar gödslingen och därmed läckaget. Den frågan har också vållat livlig pressdebatt och behandlas i uppsatsen Vådan av proteingödsling