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

Developmental Heterogeneity of Cardiac Fibroblasts Does Not Predict Pathological Proliferation and Activation

RationaleFibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown.ObjectiveWe sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury.Methods and resultsWe showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles.ConclusionsThe cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response

Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation.

RationaleFibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown.ObjectiveWe sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury.Methods and resultsWe showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles.ConclusionsThe cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response

Association of MAO-A Variant with Complicated Grief in Major Depression

Background/aimsIt has been suggested that monoamine oxidase A (MAO-A) activity is involved in the pathogenesis of major depression. Bereavement-related complicated grief significantly increases the risk of major depression and has been shown to be influenced by serotonergic tonus, possibly conferred by MAO-A activity. Complicated grief--whose inclusion in DSM-V as a separate mental disorder is under discussion--has been shown to be a distinct syndrome with symptoms not seen in depression. Therefore, in the present study, genetic variation in the MAO-A gene was investigated for its influence on complicated grief in major depression.MethodsSixty-six unrelated Caucasian patients (41 female, 25 male) with major depression and a history of bereavement were evaluated for complicated grief using the Inventory of Complicated Grief (ICG), the posttraumatic stress reaction after the loss by means of the Impact of Event Scale (IES-R) and further psychopathological measures. Patients were additionally genotyped for the functional variable number tandem repeat (VNTR) in the promoter region of the MAO-A gene.ResultsThe more active longer allele of the MAO-A VNTR was significantly associated with complicated grief in the female subgroup of patients (chi(2) = 9.471, p = 0.002, OR = 9.208, 95% CI 2.129-38.899, Bonferroni-corrected p = 0.012), whereas there was no such effect in male patients. Higher posttraumatic stress reaction was only nominally associated with the more active longer allele of the MAO-A VNTR in the female subgroup of patients (genotypes: chi(2) = 5.939, p = 0.015, OR = 5.333, 95% CI 1.366-20.557, Bonferroni-corrected p = 0.087). No significant associations of MAO-A VNTR with the severity of depressive symptoms (Beck Depression Inventory), anxiety symptoms (Spielberger State-Trait Anxiety Inventory), general mental health (Brief Symptom Inventory), or perceived social support (F-SozU) were found (all p > 0.10).ConclusionThe present pilot study for the first time suggests a gender-specific contribution of the more active MAO-A VNTR variant to an increased vulnerability for complicated grief as a potential intermediate phenotype of major depression.Anette Kersting, Kristin Kroker, Julia Horstmann, Bernhard T. Baune, Christa Hohoff, Lena Sünke Mortensen, Lisa C. Neumann, Volker Arolt, Katharina Domschk