技术资料

Despite the effectiveness of tyrosine kinase inhibitors (TKIs) against chronic myeloid leukemia (CML),they are not usually curative as some patients develop drug-resistance or are at risk of disease relapse when treatment is discontinued. Studies have demonstrated that primitive CML cells display unique miRNA profiles in response to TKI treatment. However,the utility of miRNAs in predicting treatment response is not yet conclusive. Here,we analyzed differentially expressed miRNAs in CD34+ CML cells pre- and post-nilotinib (NL) therapy from 58 patients enrolled in the Canadian sub-analysis of the ENESTxtnd phase IIIb clinical trial which correlated with sensitivity of CD34+ cells to NL treatment in in vitro colony-forming cell (CFC) assays. We performed Cox Proportional Hazard (CoxPH) analysis and applied machine learning algorithms to generate multivariate miRNA panels which can predict NL response at treatment-na{\{i}}ve or post-treatment time points. We demonstrated that a combination of miR-145 and miR-708 are effective predictors of NL response in treatment-na{\"{i}}ve patients whereas miR-150 and miR-185 were significant classifiers at 1-month and 3-month post-NL therapy. Interestingly incorporation of NL-CFC output in these panels enhanced predictive performance. Thus this novel predictive model may be developed into a prognostic tool for use in the clinic." View Publication

New strategies that augment T cell responses are required to broaden the therapeutic arsenal against cancer. CD96,TIGIT,and CD226 are receptors that bind to a communal ligand,CD155,and transduce either inhibitory or activating signals. The function of TIGIT and CD226 is established,whereas the role of CD96 remains ambiguous. Using a panel of engineered antibodies,we discovered that the T cell stimulatory activity of anti-CD96 antibodies requires antibody cross-linking and is potentiated by Fc$\gamma$ receptors. Thus,soluble Fc silent" anti-CD96 antibodies failed to stimulate human T cells whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype and it was dependent on antibody trans-cross-linking by Fc$\gamma$RI. In contrast neither human IgG2 nor variants with increased Fc$\gamma$ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T cell activation leading to proliferation cytokine secretion and resistance to Treg suppression. Furthermore CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma and its cross-linking activated tumor-infiltrating T cells thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy." View Publication

BACKGROUND In kidney transplantation,early allograft inflammation impairs long-term allograft function. However,precise mediators of early kidney allograft inflammation are unclear,making it challenging to design therapeutic interventions. METHODS We used an allogeneic murine kidney transplant model in which CD45.2 BALB/c kidneys were transplanted to CD45.1 C57BL/6 recipients. RESULTS Donor kidney resident macrophages within the allograft expanded rapidly in the first 3 days. During this period,they were also induced to express a high level of Ccl8,which,in turn,promoted recipient monocyte graft infiltration,their differentiation to resident macrophages,and subsequent expression of Ccl8. Enhanced graft infiltration of recipient CCR8+ T cells followed,including CD4,CD8,and ?? T cells. Consequently,blocking CCL8-CCR8 or depleting donor kidney resident macrophages significantly inhibits early allograft immune cell infiltration and promotes superior short-term allograft function. CONCLUSIONS Targeting the CCL8-CCR8 axis is a promising measure to reduce early kidney allograft inflammation. View Publication

Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset,which express na{\{i}}ve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of na{\"{i}}ve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy." View Publication

Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed tissues. Therapeutic induction of neutrophil apoptosis,an immunologically silent form of cell death,may be beneficial in these diseases,provided that the apoptotic neutrophils are efficiently cleared from the tissue. Previous research in our group identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both in vitro and in vivo. Here,we extend that work using a clinical ErbB inhibitor,neratinib,which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration o an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute,but not chronic,lung injury,suggesting that the drug may have therapeutic benefits in acute inflammatory settings. Phosphoproteomic analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis,migration,and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and,through examination of the neutrophil phosphoproteome,provides important insights into the mechanisms by which this may be occurring. View Publication

As a critical immune checkpoint molecule,PD-L1 is expressed at significantly higher levels in multiple neoplastic tissues compared to normal ones. PD-L1/PD-1 axis is a critical target for tumor immunotherapy,blocking the PD-L1/PD-1 axis is recognized and has achieved unprecedented success in clinical applications. However,the clinical efficacy of therapies targeting the PD-1/PD-L1 pathway remains limited,emphasizing the need for the mechanistic elucidation of PD-1/PD-L1 expression. In this study,we found that RNF125 interacted with PD-L1 and regulated PD-L1 protein expression. Mechanistically,RNF125 promoted K48-linked polyubiquitination of PD-L1 and mediated its degradation. Notably,MC-38 and H22 cell lines with RNF125 knockout,transplanted in C57BL/6 mice,exhibited a higher PD-L1 level and faster tumor growth than their parental cell lines. In contrast,overexpression of RNF125 in MC-38 and H22 cells had the opposite effect,resulting in lower PD-L1 levels and delayed tumor growth compared with parental cell lines. In addition,immunohistochemical analysis of MC-38 tumors with RNF125 overexpression showed significantly increased infiltration of CD4+,CD8+ T cells and macrophages. Consistent with these findings,analyses using The Cancer Genome Atlas (TCGA) public database revealed a positive correlation of RNF125 expression with CD4+,CD8+ T cell and macrophage tumor infiltration. Moreover,RNF125 expression was significantly downregulated in several human cancer tissues,and was negatively correlated with the clinical stage of these tumors,and patients with higher RNF125 expression had better clinical outcomes. Our findings identify a novel mechanism for regulating PD-L1 expression and may provide a new strategy to increase the efficacy of immunotherapy. View Publication

Lymphopenia is a common observation in patients with COVID-19. To explore the cause of T cell lymphopenia in the disease,laboratory results of 64 hospitalized COVID-19 patients were retrospectively analyzed and six patients were randomly selected to trace their changes of T lymphocytes and plasma concentration of IL-6 for the course of disease. Results confirmed that the T-cell lymphopenia,especially CD4+ T cell reduction in COVID-19 patients,was a reliable indicator of severity and hospitalization in infected patients. And CD4+ T cell count below 200 cells/$\mu$L predicts critical illness in COVID-19 patients. In vitro assay supported that exposure to key contributors (IL-1$\beta$,IL-6,TNF-$\alpha$ and IFN-$\gamma$) of COVID-19 cytokine storm caused substantial death of activated T cells. Among these contributors,IL-6 level was found to probably reversely correlate with T cell counts in patients. And IL-6 alone was potent to induce T cell reduction by gasderminE-mediated pyroptosis,inferring IL-6 took a part in affecting the function and status of T cells in COVID-19 patients. Intervention of IL-6 mediated T cell pryprotosis may effectively delay disease progression,maintain normal immune status at an early stage of infection. View Publication

Oncolytic viruses have been emerging as a promising therapeutic option for cancer patients,including lung cancer. Orf virus (ORFV),a DNA parapoxvirus,can infect its natural ungulate hosts and transmit into humans. Moreover,the ORFV has advantages of low toxicity,high targeted,self-amplification and can induce potent Th1-like immunity. This study explored the therapeutic potential of ORFV infection for human lung cancer therapy and investigated the molecular mechanisms. We used a previously described ORFV NA1/11 strain and tested the oncolysis of ORFV NA1/11 in two lines of lung cancer cells in vitro and in vivo. Treatment of both cell lines with ORFV NA1/11 resulted in a decrease in cell viability by inducing cell cycle arrest in G2/M phase,suppressing cyclin B1 expression and increasing their apoptosis in a caspase-dependent manner. The ORFV NA1/11-infected lung cancer cells were highly immunogenic. Evidently,ORFV NA1/11 infection of lung cancer cells induced oncolysis of tumor cells to release danger-associated molecular patterns,and promoted dendritic cell maturation,and CD8 T cell infiltration in the tumors by enhancing CXCL16 secretion. These findings may help to understand the molecular mechanisms of ORFV oncolysis and aid in the development of novel therapies for lung cancer. View Publication

Cardiovascular calcification is the lead predictor of cardiovascular events and the top cause of morbidity and mortality worldwide. To date,only invasive surgical options are available to treat cardiovascular calcification despite the growing understanding of underlying pathological mechanisms. Key players in vascular calcification are vascular smooth muscle cells (SMCs),which transform into calcifying SMCs and secrete mineralizing extracellular vesicles that form microcalcifications,subsequently increasing plaque instability and consequential plaque rupture. There is an increasing,practical need for a large scale and inexhaustible source of functional SMCs. Here we describe an induced pluripotent stem cell (iPSC)-derived model of SMCs by differentiating iPSCs toward SMCs to study the pathogenesis of vascular calcification. Specifically,we characterize the proteome during iPSC differentiation to better understand the cellular dynamics during this process. First,we differentiated human iPSCs toward an induced-SMC (iSMC) phenotype in a 10-day protocol. The success of iSMC differentiation was demonstrated through morphological analysis,immunofluorescent staining,flow cytometry,and proteomics characterization. Proteomics was performed throughout the entire differentiation time course to provide a robust,well-defined starting and ending cell population. Proteomics data verified iPSC differentiation to iSMCs,and functional enrichment of proteins on different days showed the key pathways changing during iSMC development. Proteomics comparison with primary human SMCs showed a high correlation with iSMCs. After iSMC differentiation,we initiated calcification in the iSMCs by culturing the cells in osteogenic media for 17 days. Calcification was verified using Alizarin Red S staining and proteomics data analysis. This study presents an inexhaustible source of functional vascular SMCs and calcifying vascular SMCs to create an in vitro model of vascular calcification in osteogenic conditions,with high potential for future applications in cardiovascular calcification research. View Publication

The past two decades have witnessed significant strides in leukemia therapies through approval of therapeutic inhibitors targeting oncogene-driving dysregulated tyrosine kinase activities and key epigenetic and apoptosis regulators. Although these drugs have brought about complete remission in the majority of patients,many patients face relapse or have refractory disease. The main factor contributing to relapse is the presence of a small subpopulation of dormant drug-resistant leukemia cells that possess stem cell features (termed as leukemia stem cells or LSCs). Thus,overcoming drug resistance and targeting LSCs remain major challenges for curative treatment of human leukemia. Chronic myeloid leukemia (CML) is a good example,with rare,propagating LSCs and drug-resistant cells that cannot be eradicated by BCR-ABL-directed tyrosine kinase inhibitor (TKI) monotherapy and that are responsible for disease relapse/progression. Therefore,it is imperative to identify key players in regulating BCR-ABL1-dependent and independent drug-resistance mechanisms,and their key pathways,so that CML LSCs can be selectively targeted or sensitized to TKIs. Here,we describe several easily adaptable gene knockdown approaches in CD34+ CML stem/progenitor cells that can be used to investigate the biological properties of LSCs and molecular effects of genes of interest (GOI),which can be further explored as therapeutic modalities against LSCs in the context of human leukemia. View Publication

In response to an intracellular infectious agent,the immune system produces a specific cellular response as well as a T cell-dependent Ab response. Precursor T cells differentiate into effector T cells,including Th1 cells,and T follicular helper (TFH) cells. The latter cooperate with B cells to form germinal centers and induce the formation of Ab-forming plasmacytes. One major focal point for control of T cell differentiation is the transcription factor BCL6. In this study,we demonstrated that the Bcl6 gene is regulated by FOXO1-binding,cis-acting sequences located in a highly conserved region of the first Bcl6 intron. In both mouse and human T cells,deletion of the tandem FOXO1 binding sites increased the expression of BCL6 and enhanced the proportion of TFH cells. These results reveal a fundamental control point for cellular versus humoral immunity. View Publication

BACKGROUND Neutrophils are the most abundant innate immune cells in the circulating blood,and they act as the first responder against bacterial and fungal infection. However,accumulation of activated neutrophils can cause severe inflammation and tissue damage. Recently,neutrophil trogocytosis or membrane transfer with neighboring cells was reported to modulate immune responses. Extracellular cold-inducible RNA binding protein (eCIRP) is a newly identified damage-associated molecular pattern (DAMP). eCIRP can activate neutrophils to be more pro-inflammatory. This study aimed to identify the role of eCIRP in neutrophil trogocytosis during their trans-endothelial migration. METHODS A trans-endothelial migration (TEM) assay using bone marrow neutrophils and mouse primary lung vascular endothelial cells was conducted using transwell chambers and neutrophil trogocytosis was assessed in vitro. In an in vivo mouse model of acute lung injury,neutrophil trogocytosis was assessed from bronchoalveolar lavage fluid. RESULTS In TEM assay,the trogocytosis of neutrophils occurred during trans-endothelial migration and eCIRP significantly increased the percentage of these neutrophils. The trogocytosed neutrophils acquired the endothelial membrane containing junctional adhesion molecule-C (JAM-C) and VE-cadherin,and these membrane patches were polarized by Mac-1 binding. Furthermore,eCIRP-induced JAM-C positive trogocytosed neutrophils are more pro-inflammatory than the JAM-C negative counterpart. JAM-C positive trogocytosed neutrophils were also observed in the bronchoalveolar lavage fluid of a mouse model of acute lung injury. CONCLUSION These data suggest that during the paracellular trans-endothelial migration of neutrophils in response to inflammation,eCIRP induces trogocytosis of neutrophils,and the trogocytosed neutrophils exhibit an exaggerated pro-inflammatory phenotype promoting acute lung injury. View Publication