技术资料

Maintenance of blood DC homeostasis is essential to preventing autoimmunity while controlling chronic infection. However,the ability of bacteremic pathogens to directly regulate blood DC homeostasis has not been defined. One such bacteremic pathogen,Porphyromonas gingivalis,is shown by our group to survive within mDCs under aerobic conditions and therein,metastasize from its oral mucosal niche. This is accompanied by expansion of the blood mDC pool in vivo,independently of canonical DC poietins. We presently know little of how this bacteremic pathogen causes blood DC expansion and the pathophysiological significance. This work shows that optimum differentiation of MoDCs from primary human monocytes,with or without GM-CSF/IL-4,is dependent on infection with P. gingivalis strains expressing the DC-SIGN ligand mfa-1. DC differentiation is lost when DC-SIGN is blocked with its ligand HIV gp120 or knocked out by siRNA gene silencing. Thus,we have identified a novel,noncanonical pathway of DC differentiation. We term these PDDCs and show that PDDCs are bona fide DCs,based on phenotype and phagocytic activity when immature and the ability to up-regulate accessory molecules and stimulate allo-CD4(+) T cell proliferation when matured. The latter is dependent on the P. gingivalis strain used to initially educate" PDDCs. Moreover we show that P. gingivalis-infected conventional MoDCs become resistant to apoptosis and inflammatory pyroptosis as determined by levels of Annexin V and caspase-8 -3/7 and -1. Taken together we provide new insights into how a relatively asymptomatic bacteremia may influence immune homeostasis and promote chronic inflammation." View Publication

With increasing worldwide demand for safe blood,there is much interest in generating red blood cells in vitro as an alternative clinical product. However,available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply,and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach,immortalizing early adult erythroblasts generating a stable line,which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature,functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level,and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture. View Publication

Recently,many hurdles and limitations for production of clinically applicable iPSC derivatives have been overcome. Transgene-free iPSCs can be efficiently derived from easily accessible cell sources such as blood. Here we describe the generation of transgene-free hiPS cells from cord blood derived CD34+ cells,reprogrammed using CytoTune™ Sendai reprogramming vectors. CD34+ cell isolation,cultivation,reprogramming and establishment of resulting hiPSC lines were performed under the exclusive usage of animal-derived component-free (ADCF) materials and components. View Publication

In recent years,immunotherapy has shown considerable promise in the management of several malignancies. However,the majority of preclinical studies have been conducted in rodents,the results of which often translate poorly to patients given the substantial differences between murine and human immunology. As the porcine immune system is far more analogous to that of humans,pigs may serve as a supplementary preclinical model for future testing of such therapies. We have generated the genetically modified Oncopig with inducible tumor formation resulting from concomitant KRAS(G12D) and TP53(R167H) mutations under control of an adenoviral vector Cre-recombinase (AdCre). The objective of this study was to characterize the tumor microenvironment in this novel animal model with respect to T-cell responses in particular and to elucidate the potential use of Oncopigs for future preclinical testing of cancer immunotherapies. In this study,we observed pronounced intratumoral T-cell infiltration with a strong CD8$\beta$(+) predominance alongside a representation of highly differentiated $\gamma$$\delta$ T cells. The infiltrating CD8$\beta$(+) T cells displayed increased expression of the cytotoxic marker perforin when compared with the peripheral T-cell pool. Similarly,there was robust granzyme B staining localizing to the tumors; affirming the presence of cytotoxic immune cells within the tumor. In parallel with this antitumor immune response,the tumors displayed enrichment in FOXP3-expressing T cells and increased gene expression of indoleamine 2,3-dioxygenase 1 (IDO1),cytotoxic T-lymphocyte-associated protein 4 (CTLA4),and programmed death-ligand 1 (PDL1). Finally,we investigated the Oncopig immune system in mediating antitumor immunity. We observed pronounced killing of autologous tumor cells,which demonstrates the propensity of the Oncopig immune system to recognize and mount a cytotoxic response against tumor cells. Together,these findings suggest innate and adaptive recognition of the induced tumors with a concomitant in vivo suppression of T-cell effector functions. Combined,the data support that the Oncopig may serve as a valuable model for future preclinical testing of immunotherapies aimed at reactivating tumor-directed cytotoxicity in vivo. View Publication

Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs,but is also targeted by binding,non-neutralizing antibodies. Env-based immunogens tested so far in various animal species and humans have elicited binding and autologous neutralizing antibodies but not bNAbs (with a few notable exceptions). The underlying reasons for this are not well understood despite intensive efforts to characterize the binding specificities of the elicited antibodies; mostly by employing serologic methodologies and monoclonal antibody isolation and characterization. These approaches provide limited information on the ontogenies and clonal B cell lineages that expand following Env-immunization. Thus,our current understanding on how the expansion of particular B cell lineages by Env may be linked to the development of non-neutralizing antibodies is limited. Here,in addition to serological analysis,we employed high-throughput BCR sequence analysis from the periphery,lymph nodes and bone marrow,as well as B cell- and antibody-isolation and characterization methods,to compare in great detail the B cell and antibody responses elicited in non-human primates by two forms of the clade C HIV Env 426c: one representing the full length extracellular portion of Env while the other lacking the variable domains 1,2 and 3 and three conserved N-linked glycosylation sites. The two forms were equally immunogenic,but only the latter elicited neutralizing antibodies by stimulating a more restricted expansion of B cells to a narrower set of IGH/IGK/IGL-V genes that represented a small fraction (0.003-0.02{\%}) of total B cells. Our study provides new information on how Env antigenic differences drastically affect the expansion of particular B cell lineages and supports immunogen-design efforts aiming at stimulating the expansion of cells expressing particular B cell receptors. View Publication

The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease. View Publication

The human brain is composed of a complex assembly of about 171 billion heterogeneous cellular units (86 billion neurons and 85 billion non-neuronal glia cells). A comprehensive description of brain cells is necessary to understand the nervous system in health and disease. Recently,advances in genomics have permitted the accurate analysis of the full transcriptome of single cells (scRNA-seq). We have built upon such technical progress to combine scRNA-seq with patch-clamping electrophysiological recording and morphological analysis of single human neurons in vitro. This new powerful method,referred to as Patch-seq,enables a thorough,multimodal profiling of neurons and permits us to expose the links between functional properties,morphology,and gene expression. Here,we present a detailed Patch-seq protocol for isolating single neurons from in vitro neuronal cultures. We have validated the Patch-seq whole-transcriptome profiling method with human neurons generated from embryonic and induced pluripotent stem cells (ESCs/iPSCs) derived from healthy subjects,but the procedure may be applied to any kind of cell type in vitro. Patch-seq may be used on neurons in vitro to profile cell types and states in depth to unravel the human molecular basis of neuronal diversity and investigate the cellular mechanisms underlying brain disorders. View Publication

We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse,we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding,at the molecular level,how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here,we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally,we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization,as well as T cell migration in vitro. Altogether,our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility. View Publication

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5,an immune-regulatory transcription factor. We show that the protein kinases IKK$\alpha$,IKK$\beta$,IKK$\epsilon$,and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization,thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets,we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs,linking IRF5 with immune regulatory and proinflammatory gene expression. Thus,TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome. View Publication

The developmental pathways of broadly neutralizing antibodies (bNAbs) against HIV are of great importance for the design of immunogens that can elicit protective responses. Here we show the maturation features of the HIV-neutralizing anti-V1V2 VRC26 lineage by simultaneously sequencing the exon together with the downstream intron of VRC26 members. Using the mutational landscapes of both segments and the selection-free nature of the intron region,we identify multiple events of amino acid mutational convergence in the complementarity-determining region 3 (CDR3) of VRC26 members,and determine potential intermediates with diverse CDR3s to a late stage bNAb from 2 years prior to its isolation. Moreover,we functionally characterize the earliest neutralizing intermediates with critical CDR3 mutations,with some emerging only 14 weeks after initial lineage detection and containing only {\~{}}6{\%} V gene mutations. Our results thus underscore the utility of analyzing exons and introns simultaneously for studying antibody maturation and repertoire selection. View Publication

CRISPR pools are being widely employed to identify gene functions. However,current technology,which utilizes DNA as barcodes,permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities,we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate {\textgreater}100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies,we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR,we simultaneously analyzed multiple phenotypic markers,including phospho-signaling,on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes,the immunoproteasome component Psmb8 and a chaperone Rtp4,are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution. View Publication

PURPOSE Tumor-associated macrophages (TAMs) and the hyperactivation of phosphoinositide 3-kinase(PI3K)/AKT pathway are involved in the pathogenesis of Hodgkin lymphoma (HL) and affect disease outcome. Since the $\delta$ and $\gamma$ isoforms of PI3K are overexpressed in Hodgkin/Reed-Sternberg (HRS) cells and the tumor microenvironment (TME),we propose that the PI3K$\delta$/$\gamma$ inhibitor RP6530 might affect both HRS cells and TME,ultimately leading to an enhanced antitumor response. EXPERIMENTAL DESIGN HL cell lines (L-540,KM-H2 and L-428) and primary human macrophages were used to investigate the activity of RP6530 in vitro and in vivo in HL cell line xenografts. RESULTS In vitro,RP6530 besides killing and inhibiting the proliferation of HL cells,downregulated lactic acid metabolism,switching the activation of macrophages from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state. By RNA sequencing,we define tumor glycolysis as a specific PI3K$\delta$/$\gamma$-dependent pathway implicated in the metabolic reprogramming of cancer cells. We identify the metabolic regulator Pyruvate Kinase M2 (PKM2) as the main mediator of tumor-induced immunosuppressive phenotype of macrophages. Furthermore,we show in human tumor xenografts that RP6530 repolarizes TAMs into pro-inflammatory macrophages and inhibits tumor vasculature,leading to tumor regression. Interestingly,HL patients experiencing objective responses (CR and PR) in a phase 1 trial using RP6530 showed a significant inhibition of circulating MDSCs and an average mean reduction in serum TARC levels of 40{\%} (range,4-76{\%}). CONCLUSIONS Our results support PI3K$\delta$/$\gamma$ inhibition as a novel therapeutic strategy that targets both malignant cells and the TME to treat HL patients. View Publication