技术资料

OBJECTIVE Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However,evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS Using complementary cell-based techniques,metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS Although TKI suppressed glycolysis,compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However,clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION Targeting glutamine metabolism with the FDA-approved drug,Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells. View Publication

Rheumatoid arthritis (RA) is characterized by an impaired articular bone immune microenvironment,which is associated with regulatory T cells (Tregs) hypofunction and osteoclasts (OCs) hyperfunction and leads to articular bone erosion and systemic bone loss. Studies have shown that Tregs slow bone loss in RA by regulating the bone resorption function of OCs and the JAK/STAT signaling pathway can regulate the immunosuppressive function of Tregs and reduce the bone erosion function of OCs. Yi Shen Juan Bi Pill (YSJB) is a classic Chinese herbal compound for the treatment of RA. However,whether YSJB regulates bone immune microenvironment homeostasis through JAK/STAT signaling pathway remains unclear. Based on in vitro OC single culture,Treg single culture and OC-Treg coculture systems,treatments were performed using drug-containing serum,AG490 and JAK2 siRNA to explore whether YSJB-containing serum regulates the homeostasis of the bone immune microenvironment through the JAK/STAT signaling pathway. In vitro,YSJB treatment decreased the number of TRAP+ cells and the areas of bone resorption and inhibited the expression of RANK,NFATc1,c-fos,JAK2,and STAT3 in both the OC single culture system and the OC-Treg coculture system. Tregs further reduced the number of TRAP+ cells and the areas of bone resorption in the coculture system. YSJB promoted the secretion of IL-10 while inhibiting the expression of JAK2 and STAT3 in Tregs. Moreover,inhibiting the expression of JAK2 with the JAK2 inhibitor AG490 and JAK2 siRNA improved the immunosuppressive functions of Treg,inhibited OC differentiation and bone resorption. Our study demonstrates that YSJB can regulate OC-mediated bone resorption and Treg-mediated bone immunity through the JAK2/STAT3 signaling pathway. This study provides a new strategy for regulating the bone immune microenvironment in RA with traditional Chinese medicine. View Publication

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here,we describe a method to produce human memory-like T cells from naive human T cells in culture. Using commercially available human T-cell differentiation kits,both purified naive CD8+ T cells and purified naive CD4+ T cells were activated via T-cell receptor signaling and appropriate cytokines for several days in culture. All the T-cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O2 atmosphere) for several more days; during this period,most of the cells died,but some survived in a quiescent state for a month. The survivors had small round cell bodies,expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T-cell activators were added back. We could also induce memory-like T cells from naive human T cells without hypoxia,if we froze the activated T cells or prepared the naive T cells from chilled filter buffy coats. View Publication

Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multiorgan inflammation that affects tens of thousands of people worldwide. The causative gene,cystic fibrosis transmembrane conductance regulator (CFTR),is mutated in CF patients. CFTR functions in epithelial cells have traditionally been thought to cause the disease symptoms. Recent work has shown an additional defect: monocytes from CF patients show a deficiency in integrin activation and adhesion. Because monocytes play critical roles in controlling infections,defective monocyte function may contribute to CF progression. In this study,we demonstrate that monocytes from CFTR$\Delta$F508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type (WT) bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with WT monocytes,significantly improved survival,and reduced inflammation. WT/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. WT mice reconstituted with CF bone marrow also show lethality,suggesting that the CF defect in monocytes is not only necessary but also sufficient to cause disease. We also show that monocyte-specific knockout of CFTR retards weight gains and exacerbates dextran sulfate sodium-induced colitis. Our findings show that providing WT monocytes by bone marrow transfer rescues mortality in CF mice,suggesting that similar approaches may mitigate disease in CF patients. View Publication

Our previous studies have shown that ethanol intoxication combined with burn injury increases intestinal bacterial growth,disrupts the intestinal barrier,and enhances bacterial translocation. Additionally,studies show that Th17 effector cytokines IL-17 and IL-22,which are dependent on IL-23,play important roles in maintaining intestine mucosal barrier integrity. Recent findings suggest neutrophils are a significant source of IL-17 and IL-22. We determined the effect of ethanol and burn injury on neutrophil IL-17 and IL-22 production,as well as their ability to phagocytose and in bacterial clearance,and whether these effects are modulated by IL-23. Mice were given ethanol 4 h prior to receiving ˆ¼12.5% total body surface area burn and were euthanized day 1 after injury. We observed that intoxication combined with burn injury significantly decreases blood neutrophil phagocytosis and bacteria killing,as well as their ability to produce IL-17 and IL-22,compared with sham vehicle mice. The treatment of neutrophils with rIL-23 significantly increases IL-22 and IL-17 release and promotes expression of IL-23R,retinoic acid-related orphan receptor $\gamma$t,Lipocalin2,and Nod-like receptor 2 following ethanol and burn injury. Furthermore,IL-22- and IL-17-producing neutrophils have enhanced neutrophil extracellular trap formation and bacterial killing ability,which are dependent on IL-23. Finally,although we observed that peritoneal neutrophils harvested after casein treatment are functionally different from blood neutrophils,both blood and peritoneal neutrophils exhibited the same response to rIL-23 treatment. Together these findings suggest that IL-23 promotes neutrophil IL-22 and IL-17 production and their ability to kill bacteria following ethanol and burn injury. View Publication

Sialic acids are negatively charged carbohydrates that cap the glycans of glycoproteins and glycolipids. Sialic acids are involved in various biological processes including cell-cell adhesion and immune recognition. In dendritic cells (DCs),the major antigen-presenting cells of the immune system,sialic acids emerge as important regulators of maturation and interaction with other lymphocytes including T cells. Many aspects of how sialic acids regulate DC functions are not well understood and tools and model systems to address these are limited. Here,we have established cultures of murine bone marrow-derived DCs (BMDCs) that lack sialic acid expression using a sialic acid-blocking mimetic Ac53FaxNeu5Ac. Ac53FaxNeu5Ac treatment potentiated BMDC activation via toll-like receptor (TLR) stimulation without affecting differentiation and viability. Sialic acid blockade further increased the capacity of BMDCs to induce antigen-specific CD8+ T cell proliferation. Transcriptome-wide gene expression analysis revealed that sialic acid mimetic treatment of BMDCs induces differential expression of genes involved in T cell activation,cell-adhesion,and cell-cell interactions. Subsequent cell clustering assays and single cell avidity measurements demonstrated that BMDCs with reduced sialylation form higher avidity interactions with CD8+ T cells. This increased avidity was detectable in the absence of antigens,but was especially pronounced in antigen-dependent interactions. Together,our data show that sialic acid blockade in BMDCs ameliorates maturation and enhances both cognate T cell receptor-MHC-dependent and independent T cell interactions that allow for more robust CD8+ T cell responses. View Publication

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity,immunodeficiencies,and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies,which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-$\gamma$ (IFN-$\gamma$) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization,revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits,revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions,which could inform the design of immunotherapies. View Publication

Stress hormones are believed to skew the CD4 T-cell differentiation towards a Th2 response via a T-cell-extrinsic mechanism. Using isolated primary human na{\{i}}ve and memory CD4 T cells here we show that both adrenergic- and glucocorticoid-mediated stress signalling pathways play a CD4 na{\"{i}}ve T-cell-intrinsic role in regulating the Th1/Th2 differentiation balance. Both stress hormones reduced the Th1 programme and cytokine production by inhibiting mTORC1 signalling via two parallel mechanisms. Stress hormone signalling inhibited mTORC1 in na{\"{i}}ve CD4 T cells (1) by affecting the PI3K/AKT pathway and (2) by regulating the expression of the circadian rhythm gene period circadian regulator 1 (PER1). Both stress hormones induced the expression of PER1 which inhibited mTORC1 signalling thus reducing Th1 differentiation. This previously unrecognized cell-autonomous mechanism connects stress hormone signalling with CD4 T-cell differentiation via mTORC1 and a specific circadian clock gene namely PER1." View Publication

CD8+ T cell longevity regulated by metabolic activity plays important roles in cancer immunotherapy. Although in vitro-polarized,transferred IL-9-secreting CD8+ Tc9 (cytotoxic T lymphocyte subset 9) cells exert greater persistence and antitumor efficacy than Tc1 cells,the underlying mechanism remains unclear. Here,we show that tumor-infiltrating Tc9 cells display significantly lower lipid peroxidation than Tc1 cells in several mouse models,which is strongly correlated with their persistence. Using RNA-sequence and functional validation,we found that Tc9 cells exhibited unique lipid metabolic programs. Tc9 cell-derived IL-9 activated STAT3,upregulated fatty acid oxidation and mitochondrial activity,and rendered Tc9 cells with reduced lipid peroxidation and resistance to tumor- or ROS-induced ferroptosis in the tumor microenvironment. IL-9 signaling deficiency,inhibiting STAT3,or fatty acid oxidation increased lipid peroxidation and ferroptosis of Tc9 cells,resulting in impaired longevity and antitumor ability. Similarly,human Tc9 cells also exhibited lower lipid peroxidation than Tc1 cells and tumor-infiltrating CD8+ T cells expressed lower IL9 and higher lipid peroxidation- and ferroptosis-related genes than circulating CD8+ T cells in patients with melanoma. This study indicates that lipid peroxidation regulates Tc9 cell longevity and antitumor effects via the IL-9/STAT3/fatty acid oxidation pathway and regulating T cell lipid peroxidation can be used to enhance T cell-based immunotherapy in human cancer. View Publication

BACKGROUND B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers,leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet,the mechanism underlying this regulation remains unexplored. METHODS Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. RESULTS Through RNA sequencing,P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-$\kappa$B (nuclear factor kappa B),leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings,P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. CONCLUSIONS This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover,analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects,suggesting P62 as a new immunomodulatory target for treating atherosclerosis. View Publication

Immunological non-responders (InRs) are HIV-infected individuals in whom the administration of combination antiretroviral therapy (cART),although successful in suppressing viral replication,cannot properly reconstitute patient circulating CD4+ T-cell number to immunocompetent levels. The causes for this immunological failure remain elusive,and no therapeutic strategy is available to restore a proper CD4+ T-cell immune response in these individuals. We have recently demonstrated that platelets harboring infectious HIV are a hallmark of InR,and we now report on a causal connection between HIV-containing platelets and T-cell dysfunctions. We show here that in vivo,platelet-T-cell conjugates are more frequent among CD4+ T cells in InRs displaying HIV-containing platelets (<350 CD4+ T cells/$\mu$l blood for >1 year) as compared with healthy donors or immunological responders (IRs; >350 CD4+ T cells/$\mu$l). This contact between platelet containing HIV and T cell in the conjugates is not infectious for CD4+ T cells,as coculture of platelets from InRs containing HIV with healthy donor CD4+ T cells fails to propagate infection to CD4+ T cells. In contrast,when macrophages are the target of platelets containing HIV from InRs,macrophages become infected. Differential transcriptomic analyses comparing InR and IR CD4+ T cells reveal an upregulation of genes involved in both aerobic and anaerobic glycolysis in CD4+ T cells from InR vs. IR individuals. Accordingly,InR platelets containing HIV induce a dysfunctional increase in glycolysis-mediated energy production in CD4+ T cells as compared with T cells cocultured with IR platelets devoid of virus. In contrast,macrophage metabolism is not affected by platelet contact. Altogether,this brief report demonstrates a direct causal link between presence of HIV in platelets and T-cell dysfunctions typical of InR,contributing to devise a platelet-targeted therapy for improving immune reconstitution in these individuals. View Publication

Allergic airway inflammation is a universal airway disease that is driven by hyperresponsiveness to inhaled allergens. Group 2 innate lymphoid cells (ILC2s) produce copious amounts of type 2 cytokines,which lead to allergic airway inflammation. Here,we discovered that both peripheral blood of human and mouse lung ILC2s express the endothelin-A receptor (ETAR),and the expression level of ETAR was dramatically induced upon interleukin-33 (IL-33) treatment. Subsequently,both preventive and therapeutic effects of BQ123,an ETAR antagonist,on allergic airway inflammation were observed,which were associated with decreased proliferation and type 2 cytokine productions by ILC2s. Furthermore,ILC2s from BQ123 treatment were found to be functionally impaired in response to an interleukin IL-33 challenged. And BQ123 treatment also affected the phosphorylation level of the extracellular signal-regulated kinase (ERK),as well as the level of GATA binding protein 3 (GATA3) in activated ILC2s. Interestingly,after BQ123 treatment,both mouse and human ILC2s in vitro exhibited decreased function and downregulation of ERK signaling and GATA3 stability. These observations imply that ETAR is an important regulator of ILC2 function and may be involved in ILC2-driven pulmonary inflammation. Therefore,blocking ETAR may be a promising therapeutic strategy for allergic airway inflammation. View Publication