技术资料

The integrated stress response (ISR) facilitates cellular adaptation to unfavorable conditions by reprogramming the cellular response. ISR activation was reported in neurological disorders and solid tumors; however,the function of ISR and its role as a possible therapeutic target in hematological malignancies still remain largely unexplored. Previously,we showed that the ISR is activated in chronic myeloid leukemia (CML) cells and correlates with blastic transformation and tyrosine kinase inhibitor (TKI) resistance. Moreover,the ISR was additionally activated in response to imatinib as a type of protective internal signaling. Here,we show that ISR inhibition combined with imatinib treatment sensitized and more effectively eradicated leukemic cells both in vitro and in vivo compared to treatment with single agents. The combined treatment specifically inhibited the STAT5 and RAS/RAF/MEK/ERK pathways,which are recognized as drivers of resistance. Mechanistically,this drug combination attenuated both interacting signaling networks,leading to BCR-ABL1- and ISR-dependent STAT5 activation. Consequently,leukemia engraftment in patient-derived xenograft mice bearing CD34+ TKI-resistant CML blasts carrying PTPN11 mutation responsible for hyperactivation of the RAS/RAF/MAPK and JAK/STAT5 pathways was decreased upon double treatment. This correlated with the downregulation of genes related to the RAS/RAF/MAPK,JAK/STAT5 and stress response pathways and was associated with lower expression of STAT5-target genes regulating proliferation,viability and the stress response. Collectively,these findings highlight the effect of imatinib plus ISRIB in the eradication of leukemic cells resistant to TKIs and suggest potential clinical benefits for leukemia patients with TKI resistance related to RAS/RAF/MAPK or STAT5 signaling. We propose that personalized treatment based on the genetic selection of patients carrying mutations that cause overactivation of the targeted pathways and therefore make their sensitivity to such treatment probable should be considered as a possible future direction in leukemia treatment. View Publication

Although studies have identified the presence of gut-associated cells in the enthesis of joints affected by spondylarthritis (SpA),a direct link through cellular transit between the gut and joint has yet to be formally demonstrated. Using KikGR transgenic mice to label in situ and track cellular trafficking from the distal colon to the joint under inflammatory conditions of both the gut and joint,we demonstrate bona-fide gut-joint trafficking of T cells from the colon epithelium,also called intraepithelial lymphocytes (IELs),to distal sites including joint enthesis,the pathogenic site of SpA. Similar to patients with SpA,colon IELs from the TNF$\Delta$ARE/+ mouse model of inflammatory bowel disease and SpA display heightened TNF production upon stimulation. Using ex vivo stimulation of photo-labeled gut-joint trafficked T cells from the popliteal lymph nodes of KikGR and KikGR TNF$\Delta$ARE/+ we saw that the CD4+ photo-labeled population was highly enriched for IL-17 competence in healthy as well as arthritic mice,however in the TNF$\Delta$ARE/+ mice these cells were additionally enriched for TNF. Using transfer of magnetically isolated IELs from TNF+/+ and TNF$\Delta$ARE/+ donors into Rag1 -/- hosts,we confirmed that IELs can exacerbate inflammatory processes in the joint. Finally,we blocked IEL recruitment to the colon epithelium using broad spectrum antibiotics in TNF$\Delta$ARE/+ mice. Antibiotic-treated mice had reduced gut-joint IEL migration,contained fewer Il-17A and TNF competent CD4+ T cells,and lessened joint pathology compared to untreated littermate controls. Together these results demonstrate that pro-inflammatory colon-derived IELs can exacerbate inflammatory responses in the joint through systemic trafficking,and that interference with this process through gut-targeted approaches has therapeutic potential in SpA. View Publication

BACKGROUND Beh{\c{c}}et's disease (BD) is a multisystem autoinflammatory disease characterised by mucosal ulceration,ocular,neural,joint and skin inflammation. The cause of BD is not known but there is a strong genetic association with HLA-B*51,IL10 and IL23R. Neutrophils are a first line of defence against invading pathogens and have been described as activated in patients with BD. Neutrophils can now be separated into different subsets,such as low density (LDN) and normal density (NDN) that have diverse functional roles. We wished to address neutrophil heterogeneity in patients with BD. METHODS Peripheral blood neutrophils were obtained from 32 BD patients and 37 healthy aged-matched controls. Percoll isolation was used to isolate all neutrophils,while Ficol-Hypaque was used to obtain LDN and NDN. Phagocytic capacity and production of reactive oxygen species (ROS),and neutrophil extracellular traps (NET) stimulated with phorbol 12-myristate 13-acetate (PMA) and Escherichia coli (E.coli) were assessed in both groups. RESULTS We have demonstrated reduced phagocytic capacity and ROS production but greater NET production by total neutrophils stimulated with PMA or E.coli from BD patients in comparison with healthy controls. Patients with BD had elevated numbers of LDN and lower number of NDN compared with healthy controls. However,both neutrophil subsets showed the same reduced ROS production and phagocytic function as total neutrophils in both groups. CONCLUSION Our novel findings indicate that the neutrophil population in BD is heterogeneous and the increased number of LDN in combination with greater NET production may contribute to the inflammatory response and pathogenesis. View Publication

CD8+ memory T (TM) cells play a critical role in immune defense against infection. Two common $\gamma$-chain family cytokines,IL-2 and IL-7,although triggering the same mTORC1-S6K pathway,distinctly induce effector T (TE) cells and TM cells,respectively,but the underlying mechanism(s) remains elusive. In this study,we generated IL-7R-/and AMPK$\alpha$1-knockout (KO)/OTI mice. By using genetic and pharmaceutical tools,we demonstrate that IL-7 deficiency represses expression of FOXO1,TCF1,p-AMPK$\alpha$1 (T172),and p-ULK1 (S555) and abolishes T cell memory differentiation in IL-7R KO T cells after Listeria monocytogenesis rLmOVA infection. IL-2- and IL-7-stimulated strong and weak S6K (IL-2/S6Kstrong and IL-7/S6Kweak) signals control short-lived IL-7R-CD62L-KLRG1+ TE and long-term IL-7R+CD62L+KLRG1- TM cell formations,respectively. To assess underlying molecular pathway(s),we performed flow cytometry,Western blotting,confocal microscopy,and Seahorse assay analyses by using the IL-7/S6Kweak-stimulated TM (IL-7/TM) and the control IL-2/S6Kstrong-stimulated TE (IL-2/TE) cells. We determine that the IL-7/S6Kweak signal activates transcriptional FOXO1,TCF1,and Id3 and metabolic p-AMPK$\alpha$1,p-ULK1,and ATG7 molecules in IL-7/TM cells. IL-7/TM cells upregulate IL-7R and CD62L,promote mitochondria biogenesis and fatty acid oxidation metabolism,and show long-term cell survival and functional recall responses. Interestingly,AMPK$\alpha$1 deficiency abolishes the AMPK$\alpha$1 but maintains the FOXO1 pathway and induces a metabolic switch from fatty acid oxidation to glycolysis in AMPK$\alpha$1 KO IL-7/TM cells,leading to loss of cell survival and recall responses. Taken together,our data demonstrate that IL-7-stimulated weak strength of mTORC1-S6K signaling controls T cell memory via activation of transcriptional FOXO1-TCF1-Id3 and metabolic AMPK$\alpha$1-ULK1-ATG7 pathways. This (to our knowledge) novel finding provides a new mechanism for a distinct IL-2/IL-7 stimulation model in T cell memory and greatly impacts vaccine development. View Publication

We present a real-time,high-throughput,and cost-effective method of detecting apoptosis in vitro using a previously developed reagent that detects caspase activation by fluorescence. Current methods of assessing apoptosis fail to account for the dimension of time,and thus are limited in data yielded per sample. This reagent allows real-time detection of apoptosis,but until now has been restricted to a costly automated detection system. Here,we describe apoptosis detection with the Essen Bioscience IncuCyte Caspase-3/7 Reagent using a multimode microplate reader,a common instrument in biological laboratories,which may be used prior to or in lieu of the automated system. This modified microplate reader apoptosis assay was validated against the established automated system,and was shown to detect a strong dose-response relationship (automated system r2 = 0.9968,microplate reader r2 = 0.9924). We also propose a quick and reliable method of quantifying cell density by Hoechst 33342 nuclear staining in microplates (r2 = 0.8812 between Hoechst signal and cell density). We assert that the dimension of time should not be overlooked,and that the method presented here is an accessible strategy for many researchers due to low startup cost and precise detection of apoptosis in real time. View Publication

Dengue virus (DENV) infection can result in severe complications. However,the understanding of the molecular correlates of severity is limited,partly due to difficulties in defining the peripheral blood mononuclear cells (PBMCs) that contain DENV RNA in vivo. Accordingly,there are currently no biomarkers predictive of progression to severe dengue (SD). Bulk transcriptomics data are difficult to interpret because blood consists of multiple cell types that may react differently to infection. Here,we applied virus-inclusive single-cell RNA-seq approach (viscRNA-Seq) to profile transcriptomes of thousands of single PBMCs derived early in the course of disease from six dengue patients and four healthy controls and to characterize distinct leukocyte subtypes that harbor viral RNA (vRNA). Multiple IFN response genes,particularly MX2 in naive B cells and CD163 in CD14+ CD16+ monocytes,were up-regulated in a cell-specific manner before progression to SD. The majority of vRNA-containing cells in the blood of two patients who progressed to SD were naive IgM B cells expressing the CD69 and CXCR4 receptors and various antiviral genes,followed by monocytes. Bystander,non-vRNA-containing B cells also demonstrated immune activation,and IgG1 plasmablasts from two patients exhibited clonal expansions. Lastly,assembly of the DENV genome sequence revealed diversity at unexpected sites. This study presents a multifaceted molecular elucidation of natural dengue infection in humans with implications for any tissue and viral infection and proposes candidate biomarkers for prediction of SD. View Publication

Though lymphotoxin (LT) is highly expressed by type I helper T (Th1) cells,its contribution to CD4+ T cell differentiation during infections and diseases remains a mystery. In HSV-1 infection,we observed that LTbetaR signaling is required to limit the Th1 response. Using bone marrow chimeric mice,mixed-T-cell chimeric mice,and LTbetaR in vivo blockades,we unexpectedly observed that LT,especially T cell-derived LT,played an indispensable role in limiting the Th1 response. The LTbetaR-Ig blockade promoted the Th1 response by increasing infiltration of monocytes and monocyte-derived DCs and up-regulating IL-12 secretion in the lymphoid environment. Our findings identified a novel role for T cell-derived LT in manipulating Th1 differentiation. View Publication

Accurate characterization of hematopoietic stem cells (HSC) products is needed to better anticipate the hematopoietic reconstitution and the outcome in patients. Although CD34+ viable cells enumeration is a key predictor of time to correction of aplasia,it does not fully inform about functionality of cells contained in the graft. CFU assay is the gold standard in vitro potency assay to assess clonogenicity of HSC and consists on the count and identification of colonies several days after culture in a semi solid media. Manual count of colonies with optic microscope is the most commonly used method but its important variability and subjectivity hinders the universal implementation of this potency assay. The aim of this study is to validate a standardized method using the STEMvision™ system,the first semi-automated instrument for imaging and scoring hematopoietic colonies,according to French and European recommendations. Results obtained highlight better performance criteria with STEMvision™ system than the manual method. This semi-automatic device tends to reduce the coefficients of variation of repeatability,inter-operator variability and intermediate precision. This newly available platform could represent an interesting option,significantly improving performances of CFU assays used for the characterization of hematopoietic progenitors. View Publication

T helper 17 (Th17) cells are pathogenic in many inflammatory diseases,but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here,we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines,were characterized by a metabolism typical of quiescent or memory T cells,and did not participate in inflammatory processes. In contrast,infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines,disseminated widely into the periphery,and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population. View Publication

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral replication and the emergence of the virus from the latent state,therefore,inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However,discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here,we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat,which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties,the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181-TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors. View Publication

Interleukin (IL)-23 is thought to be critical in the pathogenesis of psoriasis,and anti-IL-23 monoclonal antibodies (mAbs) have been approved for the treatment of psoriasis. We speculated that an anti-IL-23 receptor mAb might have greater efficacy than an anti-IL-23 mAb in the treatment of local inflamed lesions with high IL-23 levels. We previously generated an anti-human IL-23 receptor mAb,AS2762900-00,which potently blocked IL-23-induced cell proliferation,regardless of the concentration of IL-23. Here,we evaluated the therapeutic potential of AS2762900-00 in the treatment of psoriasis. Compared with untreated control,AS2762900-00 significantly reduced the epidermal thickness of lesions in a clinically relevant psoriatic human skin xenograft model. The expression of inflammatory genes including genes downstream of IL-23 signaling in the lesion tended to be lower in the AS2762900-00 group than the untreated group,suggesting that the inhibitory effects of AS2762900-00 in the psoriatic human skin xenograft model might occur via blockade of IL-23 signaling pathways. Further,AS2762900-00 showed an inhibitory effect on signal transducer and activator of transcription 3 (STAT3) phosphorylation as a downstream signal of IL-23 receptor activation in whole blood from patients with psoriasis. We also confirmed that AS2762900-00 inhibited IL-23-induced STAT3 phosphorylation in a concentration-dependent manner using whole blood from healthy donors. These data suggest that AS2762900-00 is a promising drug candidate for the treatment of psoriasis. In addition,STAT3 phosphorylation in whole blood may be a useful biomarker for the evaluation of the pharmacodynamic effects of AS2762900-00 in healthy volunteers in clinical development. View Publication

Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However,CTC investigation is hampered by their low number,making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy,and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype,stem-cell like characteristics,angiogenesis potential,an osteomimetic signature and the capacity to escape from the immune system. Moreover,they showed changes in mRNA and protein expression (e.g.,DEFA6,ABCB1 and GAL),whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits,treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time. View Publication