技术资料

Interactions with Ag-specific T cells drive B cell activation and fate choices that ultimately determine the quality of high-affinity Ab responses. As such,these interactions,and especially the long-lived interactions that occur before germinal center formation,may be important checkpoints to regulate undesirable responses. Using mouse model Ag systems,we directly observed interactions between T and B cells responding to the self-antigen myelin oligodendrocyte glycoprotein (MOG) and found that they are of lower quality compared with interactions between cells responding to the model foreign Ag nitrophenyl-haptenated OVA. This was associated with reduced expression of molecules that facilitate these interactions on the B cells,but not on T cells. B cell expression of these molecules was not dictated by the T cell partner,nor could the relative lack of expression on MOG-specific (MOG-sp.) B cells be reversed by a multivalent Ag. Instead,MOG-sp. B cells were inherently less responsive to BCR stimulation than MOG-non-sp. cells. However,the phenotype of MOG-sp. B cells was not consistent with previous descriptions of autoimmune B cells that had been tolerized via regular exposure to systemically expressed self-antigen. This suggests that alternate anergy pathways may exist to limit B cell responses to tissue-restricted self-antigens. View Publication

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders,hence they make suitable targets in proteomic studies. However,extraction of membrane proteins is challenging due to their low abundance,stability,and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction,the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols,internally identified as Protocol 1 and 2,were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation,while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer's recommendation. Using Protocol 2,protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150-200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships,particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states,such as ME/CFS. View Publication

Chimeric antigen receptor (CAR)-T cells are engineered to identify and eliminate cells expressing a target antigen. Current manufacturing protocols vary between commercial CAR-T cell products warranting an assessment of these methods to determine which approach optimally balances successful manufacturing capacity and product efficacy. One difference between commercial product manufacturing methods is whether T cell engineering begins with fresh (unfrozen) patient cells or cells that have been cryopreserved prior to manufacture. Starting with frozen PBMC material allows for greater manufacturing flexibility,and the possibility of collecting and storing blood from patients prior to multiple lines of therapy. We prospectively analyzed if second generation anti-CD19 CAR-T cells with either CD28 or 4-1BB co-stimulatory domains have different phenotype or function when prepared side-by-side using fresh or cryopreserved PBMCs. We found that cryopreserved PBMC starting material is associated with slower CAR-T cell expansion during manufacture but does not affect phenotype. We also demonstrate that CAR-T cell activation,cytokine production and in vitro anti-tumor cytotoxicity were not different when CAR-T cells were manufactured from fresh or cryopreserved PBMC. As CAR-T cell therapy expands globally,the need for greater flexibility around the timing of manufacture will continue to grow. This study helps support the concept that cryopreservation of PBMCs could be the solution to these issues without compromising the quality of the final CAR-T product. View Publication

The role of NK cells against HIV-1 infections remains to be elucidated in vivo. While humanized mouse models potentially could be used to directly evaluate human NK cell responses during HIV-1 infection,improved functional development of human NK cells in these hosts is needed. Here,we report the humanized MISTRG-6-15 mouse model,in which NK cells were quick to expand and exhibit degranulation,cytotoxicity,and proinflammatory cytokine production in nonlymphoid organs upon HIV-1 infection but had reduced functionality in lymphoid organs. Although HIV-1 infection induced functional impairment of NK cells,antiretroviral therapy reinvigorated NK cells in response to HIV-1 rebound after analytic treatment interruption. Moreover,a broadly neutralizing antibody,PGT121,enhanced NK cell function in vivo,consistent with antibody-dependent cellular cytotoxicity. Monoclonal antibody depletion of NK cells resulted in higher viral loads in multiple nonlymphoid organs. Overall,our results in humanized MISTRG-6-15 mice demonstrated that NK cells provided direct anti-HIV-1 responses in vivo but were limited in their responses in lymphoid organs. View Publication

Capsular polysaccharides are common virulence factors of extracellular,but not intracellular bacterial pathogens,due to the antiphagocytic properties of these surface structures. It is therefore paradoxical that Salmonella enterica subspecies enterica serovar Typhi,an intracellular pathogen,synthesizes a virulence-associated (Vi) capsule,which exhibits antiphagocytic properties. Here,we show that the Vi capsular polysaccharide has different functions when S. Typhi interacts with distinct subsets of host phagocytes. The Vi capsular polysaccharide allowed S. Typhi to selectively evade phagocytosis by human neutrophils while promoting human macrophage phagocytosis. A screen of C-type lectin receptors identified human DC-SIGN as the receptor involved in macrophage binding and phagocytosis of capsulated S. Typhi. Consistent with the anti-inflammatory activity of DC-SIGN,purified Vi capsular polysaccharide reduced inflammatory responses in macrophages. These data suggest that binding of the human C-type lectin receptor DC-SIGN by the Vi capsular polysaccharide contributes to the pathogenesis of typhoid fever. IMPORTANCE Salmonella enterica subspecies enterica serovar Typhi is the causative agent of typhoid fever. The recent emergence of S. Typhi strains which are resistant to antibiotic therapy highlights the importance of vaccination in managing typhoid fever. The virulence-associated (Vi) capsular polysaccharide is an effective vaccine against typhoid fever,but the role the capsule plays during pathogenesis remains incompletely understood. Here,we identify the human C-type lectin receptor DC-SIGN as the receptor for the Vi capsular polysaccharide. Binding of capsulated S. Typhi to DC-SIGN resulted in phagocytosis of the pathogen by macrophages and induction of an anti-inflammatory cytokine response. Thus,the interaction of the Vi capsular polysaccharide with human DC-SIGN contributes to the pathogenesis of typhoid fever and should be further investigated in the context of vaccine development. View Publication

BACKGROUND Systemic sclerosis (SSc) is a multiple-organ disease characterized by vascular damage,autoimmunity,and tissue fibrosis. Organ injuries such as interstitial lung diseases (ILD),resulting from inflammatory and fibrosis processes,lead to poor prognosis. Although autoantibodies are detected in the serum of patients with SSc,the mechanisms by which immune cells are involved in tissue inflammation and fibrosis is not fully understood. Recent studies have revealed carcinoembryonic antigen related cell adhesion molecule (CEACAM)-positive monocytes are involved in murine bleomycin-induced lung fibrosis. We investigated CEACAM-positive monocytes in patients with SSc to clarify the role of monocytes in the pathogenesis of SSc. METHODS The proportion of of CEACAM-positive classical monocytes in healthy controls (HCs) and patients with rheumatoid arthritis (RA) and SSc was evaluated using flow cytometry. The correlation between the proportion of CEACAM-positive monocytes and clinical parameters was analyzed in patients with SSc. Gene expression microarrays were performed in CEACAM-positive and negative monocytes in patients with SSc. Infiltration of CEACAM-positive monocytes into scleroderma skin was evaluated by immunohistochemical staining. RESULTS The proportion of CEACAM-positive classical monocytes was increased in patients with early SSc within 2 years after diagnosis,which positively correlated with ESR,serum IgG,and serum KL-6 and negatively correlated with %forced vital capacity. The percentage of CEACAM-positive monocytes decreased after immunosuppressive therapy. CEACAM6-positive cells among classical monocytes were significantly increased in patients with SSc compared with HCs and patients with rheumatoid arthritis. SSc serum induced CEACAM6 expression on monocytes from HCs. Functionally,CEACAM-positive monocytes produced higher levels of TNF-$\alpha$ and IL-1$\beta$ compared to CEACAM-negative cells and showed activation of the NF-$\kappa$B pathway. Furthermore,CEACAM6-positive monocytes infiltrated the dermis of SSc. CONCLUSIONS CEACAM-positive monocytes showed inflammatory phenotypes and may be involved in the tissue inflammation and fibrosis in early SSc. CEACAM-positive monocytes may be one of biomarkers to detect patients with progressive ILD,requiring therapeutic intervention. View Publication

A breakdown in cellular homeostasis is thought to drive na{\{i}}ve T cell aging however the link between na{\"{i}}ve T cell homeostasis and aging in humans is poorly understood. To better address this we developed a lymphoid organoid system that maintains resting na{\"{i}}ve T cells for more than 2 weeks in conjunction with high CD45RA expression. Deep phenotypic characterization of na{\"{i}}ve T cells across age identified reduced CD45RA density as a hallmark of aging. A conversion from CD45RAhigh naive cells to a CD45RAlow phenotype was reproduced within our organoid system by structural breakdown but not by stromal cell aging or reduced lymphocyte density and mediated by alternative CD45 splicing. Together these data suggest that external influences within the lymph node microenvironment may cause phenotypic conversion of na{\"{i}}ve T cells in older adults." View Publication

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

Regulatory T cells (Tregs) are critically involved in neovascularization,an important compensatory mechanism in peripheral artery disease. The contribution of G protein coupled receptor 174 (GPR174),which is a regulator of Treg function and development,in neovascularization remains elusive. Here,we show that genetic deletion of GPR174 in Tregs potentiated blood flow recovery in mice after hindlimb ischemia. GPR174 deficiency upregulates amphiregulin (AREG) expression in Tregs,thereby enhancing endothelial cell functions and reducing pro-inflammatory macrophage polarization and endothelial cell apoptosis. Mechanically,GPR174 regulates AREG expression by inhibiting the nuclear accumulation of early growth response protein 1 (EGR1) via G$\alpha$s/cAMP/PKA signal pathway activation. Collectively,these findings demonstrate that GPR174 negatively regulates angiogenesis and vascular remodeling in response to ischemic injury and that GPR174 may be a potential molecular target for therapeutic interventions of ischemic vascular diseases. View Publication

Neutrophils are central players in the innate immune system. To protect against invading pathogens,neutrophils can externalize chromatin to create neutrophil extracellular traps (NETs). While NETs are critical to host defense,they also have deleterious effects,and dysregulation of NETs formation has been implicated in autoimmune diseases,atherosclerosis and thrombotic conditions,cancer progression and dissemination,and acute respiratory distress syndrome. Here,we report that selinexor,a first-in-class selective inhibitor of nuclear export approved for the treatment of multiple myeloma and diffuse large B-cell lymphoma,markedly suppressed the release of NETs in vitro. Furthermore,we demonstrate a significant inhibitory effect of selinexor on NETs formation,but not on oxidative burst or enzymatic activities central to NETs release such as neutrophil elastase,myeloperoxidase or peptidyl arginine deiminase type IV. The inhibitory effect of selinexor was demonstrated in neutrophils activated by a variety of NETs-inducers,including PMA,TGF-$\beta$,TNF-$\alpha$ and IL-8. Maximal inhibition of NETs formation was observed using TGF-$\beta$,for which selinexor inhibited NETs release by 61.6%. These findings pave the way to the potential use of selinexor in an effort to reduce disease burden by inhibition of NETs. View Publication

BACKGROUND There is considerable evidence that epigenetic mechanisms and DNA methylation are critical drivers of immune cell lineage differentiation and activation. However,there has been limited coordinated investigation of common epigenetic pathways among cell lineages. Further,it remains unclear if long-lived memory cell subtypes differentiate distinctly by cell lineages. RESULTS We used the Illumina EPIC array to investigate the consistency of DNA methylation in B cell,CD4 T,and CD8 T na{\{i}}ve and memory cells states. In the process of na{\"{i}}ve to memory activation across the three lineages we identify considerable shared epigenetic regulation at the DNA level for immune memory generation. Further in central to effector memory differentiation our analyses revealed specific CpG dinucleotides and genes in CD4 T and CD8 T cells with DNA methylation changes. Finally we identified unique DNA methylation patterns in terminally differentiated effector memory (TEMRA) CD8 T cells compared to other CD8 T memory cell subtypes. CONCLUSIONS Our data suggest that epigenetic alterations are widespread and essential in generating human lymphocyte memory. Unique profiles are involved in methylation changes that accompany memory genesis in the three subtypes of lymphocytes." View Publication

BACKGROUND Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by recurrent airway infection and inflammation. There is no cure for PCD and to date there are no specific treatments available. Neutrophils are a crucial part of the immune system and are known to be dysfunctional in many inflammatory diseases. So far,the role of the neutrophils in PCD airways is largely unknown. The purpose of this study was to investigate the phenotype and function of airway neutrophils in PCD,and compare them to blood neutrophils. METHODS Paired peripheral blood and spontaneously expectorated sputum samples from patients with PCD (n??=??32) and a control group of patients with non-PCD,non-cystic fibrosis bronchiectasis (n??=??5) were collected. The expression of neutrophil-specific surface receptors was determined by flow cytometry. Neutrophil function was assessed by measuring the extent of actin polymerization,production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) in response to activating stimuli. RESULTS Sputum neutrophils displayed a highly activated phenotype and were unresponsive to stimuli that would normally induce ROS production,actin polymerization and the expulsion of NETs. In addition,PCD sputum displayed high activity of neutrophil elastase,and impaired the efferocytosis by healthy donor macrophages. CONCLUSIONS Sputum neutrophils in PCD are dysfunctional and likely contribute to ongoing inflammation in PCD airways. Further research should focus on anti-inflammatory therapies and stimulation of efferocytosis as a strategy to treat PCD. View Publication