技术资料

Here,we describe the discovery of a naturally occurring human antibody (Ab),FluA-20,that recognizes a new site of vulnerability on the hemagglutinin (HA) head domain and reacts with most influenza A viruses. Structural characterization of FluA-20 with H1 and H3 head domains revealed a novel epitope in the HA trimer interface,suggesting previously unrecognized dynamic features of the trimeric HA protein. The critical HA residues recognized by FluA-20 remain conserved across most subtypes of influenza A viruses,which explains the Ab's extraordinary breadth. The Ab rapidly disrupted the integrity of HA protein trimers,inhibited cell-to-cell spread of virus in culture,and protected mice against challenge with viruses of H1N1,H3N2,H5N1,or H7N9 subtypes when used as prophylaxis or therapy. The FluA-20 Ab has uncovered an exceedingly conserved protective determinant in the influenza HA head domain trimer interface that is an unexpected new target for anti-influenza therapeutics and vaccines. View Publication

The importance of early cancer diagnosis and improved cancer therapy has been clear for years and has initiated worldwide research towards new possibilities in the care strategy of patients with cancer using technological innovations. One of the key research fields involves the separation and detection of circulating tumor cells (CTC) because of their suggested important role in early cancer diagnosis and prognosis,namely,providing easy access by a liquid biopsy from blood to identify metastatic cells before clinically detectable metastasis occurs and to study the molecular and genetic profile of these metastatic cells. Provided the opportunity to further progress the development of technology for treating cancer,several CTC technologies have been proposed in recent years by various research groups and companies. Despite their potential role in cancer healthcare,CTC methods are currently mainly used for research purposes,and only a few methods have been accepted for clinical application because of the difficulties caused by CTC heterogeneity,CTC separation from the blood,and a lack of thorough clinical validation. Therefore,the standardization and clinical application of various developed CTC technologies remain important subsequent necessary steps. Because of their suggested future clinical benefits,we focus on describing technologies using whole blood samples without any pretreatment and discuss their advantages,use,and significance. Technologies using whole blood samples utilize size-based,immunoaffinity-based,and density-based methods or combinations of these methods as well as positive and negative enrichment during separation. Although current CTC technologies have not been truly implemented yet,they possess high potential as future clinical diagnostic techniques for the individualized therapy of patients with cancer. Thus,a detailed discussion of the clinical suitability of these new advanced technologies could help prepare clinicians for the future and can be a foundation for technologies that would be used to eliminate CTCs in vivo. View Publication

Inflammation is a risk factor for cancer development. Individuals with preleukemic TET2 mutations manifest clonal hematopoiesis and are at a higher risk of developing leukemia. How inflammatory signals influence the survival of preleukemic hematopoietic stem and progenitor cells (HSPCs) is unclear. We show a rapid increase in the frequency and absolute number of Tet2-KO mature myeloid cells and HSPCs in response to inflammatory stress,which results in enhanced production of inflammatory cytokines,including interleukin-6 (IL-6),and resistance to apoptosis. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis,resulting in increased expression of a novel anti-apoptotic long non-coding RNA (lncRNAs),Morrbid,in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss with regard to hyperactivation of Stat3 and Morrbid. In vivo,pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2 mutant mice rescues inflammatory-stress-induced abnormalities in HSPCs and mature myeloid cells,including clonal hematopoiesis. View Publication

Aneurysms and dissections affecting thoracic aorta are associated with smooth muscle cell (SMC) dysfunction. NO/cGMP signaling pathway in smooth muscle cells has been shown to be affected in sporadic thoracic aortic aneurysms. We analyzed the mRNA levels of PDE5,a cGMP-hydrolyzing enzyme highly expressed in aortic SMCs,that regulates arterious vascular tone by lowering cGMP levels. We found that aortic tissue obtained from Marfan,tricuspid and bicuspid thoracic aneurysms expressed lower levels of PDE5 mRNA compared to control aortas. In particular,we found that affected aortas showed lower levels of all the PDE5A isoforms,compared to control aortas. Transfection of vascular SMCs (VSMCs) with NOTCH3 activated domain (NICD3) induced the expression of PDE5A1 and A3 protein isoforms,but not that of the corresponding mRNAs. VSMC stimulation with GSNO,a nitric oxide analogue or with 8-br-cGMP,but not with 8-br-cAMP,up-regulated PDE5 and NOTCH-3 protein levels,indicating a negative feedback loop to protect the arterial wall from excessive relaxation. Finally,we found that PDE5 is expressed early during human aorta development,suggesting that if loss of function mutations of PDE5 occur,they might potentially affect aortic wall development. View Publication

We investigated the contribution of host immune cells to bacterial killing in a whole-blood bactericidal activity (WBA) assay,an ex vivo model used to test efficacy of drugs against mycobacterium tuberculosis (Mtb). We performed WBA assays with immuno-magnetic depletion of specific cell types,in the presence or absence of rifampicin. Innate immune cells decreased Mtb growth in absence of drug,but appeared to diminish the cidal activity of rifampicin,possibly attributable to intracellular bacterial sequestration. Adaptive immune cells had no effect with or without drug. The WBA assay may have potential for testing adjunctive host-directed therapies acting on phagocytic cells. View Publication

Systemic lupus (SLE) is characterized by a break of B cell tolerance that plays a central role in disease pathophysiology. An early checkpoint defect occurs at the transitional stage leading to the survival of autoreactive B cells and consequently the production of pathogenic autoantibodies. The main purpose of our work was to determine whether transitional B cells,as the most immature na{\{i}}ve B cell subset upstream of pathogenic B cells display specific features compared to healthy non SLE subjects. Through extensive analysis of transitional B cells from untreated or low treated mostly Caucasian SLE patients we demonstrated that transitional (T1 and T2) B cell frequencies were increased in SLE and positively correlated with disease activity. SLE transitional B cells displayed defects in two closely inter-related molecules (i.e. TLR9 defective responses and CD19 downregulation). RNA sequencing of sorted transitional B cells from untreated patients revealed a predominant overexpression of interferon stimulated genes (ISGs) even out of flares. In addition early transitional B cells from the bone marrow displayed the highest interferon score reflecting a B cell interferon burden of central origin. Hence the IFN signature in transitional B cells is not confined to African American SLE patients and exists in quiescent disease since the medullary stage. These results suggest that in SLE these 3 factors (i.e. IFN imprintment CD19 downregulation and TLR9 responses impairment) could take part at the early transitional B cell stage in B cell tolerance by-pass ultimately leading in periphery to the expansion of autoantibodies-secreting cells." View Publication

Leukocyte adhesion requires beta2-integrin activation. Resting integrins exist in a bent-closed conformation-i.e.,not extended (E-) and not high affinity (H-)-unable to bind ligand. Fully activated E+H+ integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans. E-H- transitions to E+H+ through E+H- or through E-H+,which binds to ICAMs on the same cell in cis. Spatial patterning of activated integrins is thought to be required for effective arrest,but no high-resolution cell surface localization maps of activated integrins exist. Here,we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest,E-H+ integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern,we blocked integrin binding to ICAMs in cis,which significantly relieved the face-to-face orientation. View Publication

HIV persists in latently infected CD4+ T cells during antiretroviral therapy (ART). Immune checkpoint molecules,including PD-1,are preferentially expressed at the surface of persistently infected cells. However,whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4+ T cells from HIV-infected individuals,we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely,PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption. View Publication

Phospho flow is a powerful approach to detect cell signaling aberrations,identify biomarkers and assess pharmacodynamics,and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here,B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions,fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes. View Publication

Hypertrophic cardiomyopathy (HCM) is an inherited cardiomyopathy characterized by left ventricular hypertrophy ≥15 mm in the absence of loading conditions. HCM has a prevalence of up to one in 200,and can result in significant adverse outcomes including heart failure and sudden cardiac death. An induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells obtained from the whole blood of a 38-year-old female patient with HCM in which genetic testing identified the well-known pathogenic p.Arg403Gln mutation in myosin heavy chain 7. iPSCs express pluripotency markers,demonstrate trilineage differentiation capacity,and display a normal 46,XX female karyotype. This resource will allow further assessment of the pathophysiological development of HCM. View Publication

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here,we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF,a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30{\%} engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF,as evidenced by up to 18{\%} HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells,defined by the markers CD34+CD90+CD45RA-,allowing for a 10-fold reduction in the number of transplanted target cells,thus considerably reducing the need for editing reagents. The frequency of engrafted,gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases. View Publication

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis,with controversy regarding diagnosis in the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult,TP53 mutated; NPM1 mutated; KMT2A mutated/rearranged; adult,DDX41 mutated; and pediatric,NUP98 rearranged. Genomic features influenced outcome,with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis and TP53,FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45{\%} of cases and included recurrent mutations of ALK and NTRK1,the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition. This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease,and the rationale for testing targeted therapies in this high-risk leukemia. View Publication