Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis

Francisella tularensis is a highly infectious gram-negative coccobacillus that causes the zoonosis tularemia. This bacterial pathogen causes a plague-like disease in humans after exposure to as few as 10 cells. Many of the mechanisms by which the innate immune system fights Francisella are unknown. Here we show that wild-type Francisella, which reach the cytosol, but not Francisella mutants that remain localized to the vacuole, induced a host defense response in macrophages, which is dependent on caspase-1 and the death-fold containing adaptor protein ASC. Caspase-1 and ASC signaling resulted in host cell death and the release of the proinflammatory cytokines interleukin (IL)-1β and IL-18. F. tularensis–infected caspase-1– and ASC-deficient mice showed markedly increased bacterial burdens and mortality as compared with wild-type mice, demonstrating a key role for caspase-1 and ASC in innate defense against infection by this pathogen

Molecular residual disease detection in resected, muscle-invasive urothelial cancer with a tissue-based comprehensive genomic profiling–informed personalized monitoring assay

IntroductionCirculating tumor DNA (ctDNA) detection postoperatively may identify patients with urothelial cancer at a high risk of relapse. Pragmatic tools building off clinical tumor next-generation sequencing (NGS) platforms could have the potential to increase assay accessibility.MethodsWe evaluated the widely available Foundation Medicine comprehensive genomic profiling (CGP) platform as a source of variants for tracking of ctDNA when analyzing residual samples from IMvigor010 (ClinicalTrials.gov identifier NCT02450331), a randomized adjuvant study comparing atezolizumab with observation after bladder cancer surgery. Current methods often involve germline sampling, which is not always feasible or practical. Rather than performing white blood cell sequencing to filter germline and clonal hematopoiesis (CH) variants, we applied a bioinformatic approach to select tumor (non-germline/CH) variants for molecular residual disease detection. Tissue-informed personalized multiplex polymerase chain reaction–NGS assay was used to detect ctDNA postsurgically (Natera).ResultsAcross 396 analyzed patients, prevalence of potentially actionable alterations was comparable with the expected prevalence in advanced disease (13% FGFR2/3, 20% PIK3CA, 13% ERBB2, and 37% with elevated tumor mutational burden ≥10 mutations/megabase). In the observation arm, 66 of the 184 (36%) ctDNA-positive patients had shorter disease-free survival [DFS; hazard ratio (HR) = 5.77; 95% confidence interval (CI), 3.84–8.67; P < 0.0001] and overall survival (OS; HR = 5.81; 95% CI, 3.41–9.91; P < 0.0001) compared with ctDNA-negative patients. ctDNA-positive patients had improved DFS and OS with atezolizumab compared with those in observation (DFS HR = 0.56; 95% CI, 0.38–0.83; P = 0.003; OS HR = 0.66; 95% CI, 0.42–1.05). Clinical sensitivity and specificity for detection of postsurgical recurrence were 58% (60/103) and 93% (75/81), respectively.ConclusionWe present a personalized ctDNA monitoring assay utilizing tissue-based FoundationOne® CDx CGP, which is a pragmatic and potentially clinically scalable method that can detect low levels of residual ctDNA in patients with resected, muscle-invasive bladder cancer without germline sampling

TCR-mediated signaling in thymocyte selection

grantor: University of TorontoTCR-mediated signals have been shown to induce both positive and negative selection of thymocytes. How the same TCR can induce both forms of selection introduces a conceptual paradox. A generally accepted model of thymocyte selection is the "strength of signal" or "affinity/avidity" hypothesis. This model predicts that the absence of any signal from the TCR results in death by neglect, a "strong" signal results in negative selection and a "weak" signal results in positive selection. My thesis examines how these TCR-peptide/MHC interactions are translated into intracellular signals that discriminate between positive and negative selection. The thesis investigates the relationship between the extent of TCR internalization in response to various peptide/MHC stimuli and thymocyte selection outcomes. Our results demonstrate a direct correlation: peptides that induce strong TCR down-regulation are most efficient at mediating negative selection, whereas ligand interactions that induce suboptimal TCR internalization are more efficient at triggering positive selection. These data suggest that T cell selection is mediated by differing intensities of the same TCR-mediated signal, rather than by distinct signals. My thesis also assesses the role of the mitogen-activated protein kinase (MAPK) signaling cascade, specifically the extracellular signal-regulated kinase (ERK) module in regulating both positive and negative selection. We find that negatively selecting conditions induce stronger ERK activation in thymocytes than do positively selecting conditions. We further demonstrate that attenuating the ERK activity by pharmacological inhibition can shift the activation threshold for negative selection to that for positive selection. Moreover, in order to study the kinetics of ERK activation upon stimulation with positive and negative selection stimuli, we devised an 'in vitro ' assay system for thymocyte selection. Data from these studies suggest that weak, sustained ERK activation is characteristic of positive selection, whereas strong but transient ERK activation leads to positive selection. Therefore, both timing and extent of ERK activation appear to be important for thymocyte selection. Since positively selecting stimuli do not induce efficient TCR internalization, the large proportion of TCR complexes that remain on the cell surface may relay continuous signals required for survival and differentiation. Together, these results add a novel viewpoint to thymocyte selection.Ph.D

POPing the fire into the pyrin?

Initiation of an inflammatory response requires the co-ordinated participation of proteins within a scaffold in the cytosol of responding cells. The scaffold proteins contain members of a newly discovered family of pyrin-domain adaptor proteins that regulate complex assembly for initiation of nuclear factor kappaB and interleukin-1beta signalling. A paper in this issue of the Biochemical Journal by Stehlik et al. identifies a new member of the pyrin family that may control signalling by sequestering pro-inflammatory components, shedding light on the origin of human inflammatory disorders

COMMENTARY POPing the fire into the pyrin?

Initiation of an inflammatory response requires the co-ordinated participation of proteins within a scaffold in the cytosol of responding cells. The scaffold proteins contain members of a newly discovered family of pyrin-domain adaptor proteins that regulate complex assembly for initiation of nuclear factor κB and interleukin-1β signalling. A paper in this issue of the Biochemical Journal by Stehlik et al. identifies a new member of the pyrin family that may control signalling by sequestering pro-inflammatory components, shedding light on the origin of human inflammatory disorders. Key words: cryopyrin, inhibitory κB kinase (IKK), innate immunity, interleukin-1β (IL-1β), pyrin. Pyrin domains (PyD; also known as PAAD and DAPIN) are protein–protein interaction modules found at the N-termini of proteins thought to be involved in inflammatory signalling pathway

ctDNA guiding adjuvant immunotherapy in urothelial carcinoma

Minimally invasive approaches to detect residual disease after surgery are needed to identify patients with cancer who are at risk for metastatic relapse. Circulating tumour DNA (ctDNA) holds promise as a biomarker for molecular residual disease and relapse1. We evaluated outcomes in 581 patients who had undergone surgery and were evaluable for ctDNA from a randomized phase III trial of adjuvant atezolizumab versus observation in operable urothelial cancer. This trial did not reach its efficacy end point in the intention-to-treat population. Here we show that ctDNA testing at the start of therapy (cycle 1 day 1) identified 214 (37%) patients who were positive for ctDNA and who had poor prognosis (observation arm hazard ratio = 6.3 (95% confidence interval: 4.45-8.92); P < 0.0001). Notably, patients who were positive for ctDNA had improved disease-free survival and overall survival in the atezolizumab arm versus the observation arm (disease-free survival hazard ratio = 0.58 (95% confidence interval: 0.43-0.79); P = 0.0024, overall survival hazard ratio = 0.59 (95% confidence interval: 0.41-0.86)). No difference in disease-free survival or overall survival between treatment arms was noted for patients who were negative for ctDNA. The rate of ctDNA clearance at week 6 was higher in the atezolizumab arm (18%) than in the observation arm (4%) (P = 0.0204). Transcriptomic analysis of tumours from patients who were positive for ctDNA revealed higher expression levels of cell-cycle and keratin genes. For patients who were positive for ctDNA and who were treated with atezolizumab, non-relapse was associated with immune response signatures and basal-squamous gene features, whereas relapse was associated with angiogenesis and fibroblast TGFβ signatures. These data suggest that adjuvant atezolizumab may be associated with improved outcomes compared with observation in patients who are positive for ctDNA and who are at a high risk of relapse. These findings, if validated in other settings, would shift approaches to postoperative cancer care

Duration and strength of extracellular signal-regulated kinase signals are altered during positive versus negative thymocyte selection

During thymocyte development, high-affinity/avidity TCR engagement leads to the induction of negative selection and apoptosis, while lower TCR affinity-avidity interactions lead to positive selection and survival. To elucidate how these extracellular interactions are translated into intracellular signals that distinguish between positive and negative selection, we developed a culture system in which naive double-positive thymocytes were either induced to differentiate along the CD8+ lineage pathway or were triggered for clonal deletion. Using this system, we show that sustained low level activation of extracellular signal-regulated kinases (ERKs) promotes positive selection, whereas strong but transient ERK activation is coupled with negatively selecting stimuli. Importantly, similar ERK activation profiles were demonstrated during positive selection for strong agonist ligands presented at low concentrations or weak agonist ligands. This is consistent with the affinity/avidity model and a role for strong or weak agonists during positive selection. Surprisingly, the addition of a pharmacological inhibitor which blocks ERK activation prevented the induction of negative selection. These data suggest that the duration and strength of the TCR signal is involved in discriminating between positive and negative selection